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11p-Hydroxysteroid Dehydrogenase 2 (1 1 fbHSD2):

Molecular Structure and Regulation

by

Laura E. Pereira

Department of Physiology

Submitted in partial fulfillment

of the requirements for the degree of

Masters of Science

Faculty of Graduate S tudies

The University of Western Ontario

London, Ontario

December 1997

OLaura E. Pereira 1997

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ABSTRACT

1 1 B-hydroxysteroid dehydrogenase 2 ( 1 1 p-HSD2) is a microsomal enzyme which

catalyzes the inactivation of glucocorticoids. It is believed to protect mineralocorticoid

target tissues and the fetus from high levels of cortisol.

The ovine 1 1 B-HSD2 gene and its 5'-flanking region were cloned and characterized.

This gene consists of 5 exons. spanning over 4kb, and encodes a protein of 404 amino

acids.

In cultured JEG-3 cells, retinoic acids and NDGA (a known inhibitor of lipoxygenases)

stimulated while EGF and prostaglandins inhibired 1 1 p-HSD2 enzyme activity.

However. under the conditions of the present snidy. sex steroids. dexarnethasone. thyroid

hormone. insulin and TNFa had no effect.

These are novel findings which advance our understanding of the structure. hinction and

regulation of 1 1 $-HSD2.

Keywords: 1 1 p-hydroxysteroid dehydrogenase 2. 1 1 B-HSDZ. gene structure. regulation.

choriocarcinoma cells, EG-3.

At the outset. I express my deepest gratitude to Dr. Kaiping Yang, my supervisor, for his

guidance, support and advice throughout the last few years. Specid appreciation's are due for his C

attention to detail and constmctive criticism. without which this thesis would not have k e n

possible.

1 sincerely thank the members of my advisory cornmittee Dr. Trevor Archer. Dr. Don Killinger.

and Dr. Andy Watson. for their advice and guidance throughout my studies. Their input has

proved to be very usehl in this study.

I profoundly thank al1 the members of my Iaboratory for their help and suppon dong the way.

especiaily Andrew Damel. Dr. Sarnpath Kurnar. Helen Pu. Dr. Min Yu. Kathenne Shearman.

Jessica Dy and Steve Lam.

I aiso thank other members of the Lawson Research Institute. namely Dr. Edith Arany. Dawn

K i i k e ~ y . Patricia De Los Rios. Dr. David Hill, Brenda Strutt, Dr. Wahid Khaiil. Daniel Whang.

Karen Nygard. Jin Hayatsu and Dr- Tom DrysdaIe for their help and advice.

Tyrone. my husband. has helped me get through the svessfÙ1 tirnes with his emotional support.

His example of hard work and perseverance in life h a been an inspiration.

My parents. Judy and Bruce Campbell. have dways encouraged me to funher my education and

to work hard. 1 thank h e m for al1 their help and advice. My sister. Cindy Buzadi. has also

encouraged me in my studies.

Bob. Gwen and Stephanie Pereira, my "in laws" have provided me with much emotionai suppon

throughout this project.

The financial suppon for the study came from the MRC Research ~ a n t awarded to Dr. Kaiping

Y mg.

TABLE OF CONTENTS

TïïLEPAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

CERTIFTCATE OF EXAMINATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ii . -.

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

DEDICATION.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ACKNOWLEDGMENTS v

TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v i

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF TABLES xii * - -

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii i

LIST OF PHOTOGRAPHIC PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv

ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

CHAPTER 1 - INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

CHAPTER 2 -LITERATURE REVTEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

. . . . . . . . . . . 2.1 Introduction to 1 1 $-Hydroxysteroid Dehydrogenase ( 1 1 $-HSD) 3

2.1.1 Definition and Historical Perspectives . . . . . . . . . . . . . . . . . . . . . . . 3

2.1.2 Short Chain Alcohol Dehydrogenase (SCAD) Farnily . . . . . . . . . . . 6

2.2.1 11B-HSDI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2.1.1 Molecular S tnrcture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2.2.1.2 Tissue Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.2.1.3 Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.2 116-HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.2.1 Molecular Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5

2.2.2.2 Tissue Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.3 Physiology of 1 1 FHSD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

. . . . 2.3.1 Synthrsis and Metabolism of Cortisol (F) and Cortisone (E) 23

. . . . . . . . . . . . 2.3 -2 Glucocorticoid and Mineraloconicoid Receptors 26

2.3 -2.1 Binding of Cortisol to the Glucocorticoid and

. . . . . . . . . . . . . . . . . . . . Minedocorticoid Receptors 26

2.3.2.2 Response of Activated Receptors . . . . . . . . . . . . . . . . . 27

2.3.3 Protection of the Mineralocorticoid Receptor From Cortisol

by 1 1 p-HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -27

2.3 -4 Protection of the Fetus from Materna1 Conisol b y Placental

1 1 b.HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.4 Regulation of 1 1 P-HSD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.4.1 Regdation of 1 1 PHSD 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.4.2 Regulation of 1 lpHSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

2.5 Pathogenesis of 1 1 kHSD2 Deficiency . . . . . . . . . . . . . . . . . . . . . . 34

. . . . . . . 2.5.1 Apparent Mineralocorticoid Excess (AME) Syndmrne 34

2.5.2 Dielary Induced AME-like Effects . . . . . . . . . . . . . . . . . . . . . . . 35

2.5.3 Fetal Exposure to Excess Cortisol Due to Reduced Placental

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 la.HSD2 37

vii

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Scope of the Present Study 47

CHAPTER 3 . MOLECULAR STRUCTURE OF THE OVINE 1 1 p-HSD2

GENE AND 5'-FLANKING REGION . . . . . . . . . . . . . . . . . 39

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Materials and Methods 40

. . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Screening of the Cosmid Library 40

3.2.2 Preparation and Analysis of Cosmid Li brary DNA . . . . . . . . . . 42

3.2.3 Sequence Analysis of the Sheep 1 1 P-HSD2 Gene and 5'- ank king

Region . . . . . . . . . . . . . ... . . . . . . . . . .. . . . . . d

3.3.1 Structures of the Ovine 1 1 P HS D2 Gene and its Deduced

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Protein 47

. . . . . . . . . . . . . . . . . . . . 3.3.2 Structure of the 5'-Fianking Region 60

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Discussion -68

. . . . . . . . . . 3.4.1 The Ovine 1 lfbHSD2 Gene and the Deduced Protein 68

3 .4.2 The 5'-Flanking Region of the Ovine 1 l PHSD2 Gene . . . . . . . -69

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Conclusions 72

CHAPlZR 4 . REGULATION OF l lp-HSD2 LN EG-3 HUMAN

CHORIOCARCINOMA CELLS . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

4.2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.1 Culture of JEG-3 Cells . . . . . . . . . . . . . . . . . . . . . . . .

4.2.2 Treatment of JEG-3 Celis with Various Compounds . . . . . . . . . . 80

4 2.3 Assay of 1 1 PHSD2 Enzyme Activity: Radiometric Conversion

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AsSay 81

4.2.4 Andysis of the 1 1 kHSD2 Enzyme Activiw Data . . . . . . . . . . . . -81

4.3 Inhibition by Carbenoxolone: Possible involvement of the Protein Kinase APathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

4.3.1 Background and Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

4.3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

4.3.2.1 Carbenoxolone Dose Responx . . . . . . . . . . . . . . . . . - 8 8 4.3.2.2 Carbenoxolone and Forskolin Interaction . . . . . . . . . . . . . 88

4.3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

. . . . 4.4 Repulation by Retinoic Acid and Epidennal Growth Factor (EGF) 95

4.4.1 Background and Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

4.4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

. . . . . . . . 4.4.2.1 Retinoic Acid Time Course and Dose Response 96 4.4.2.2 EGF Dose Response . . . . . . . . . . . . . . . . . . . . . . . . . -103

. . . . . . . . . . . . . . . 4.4.2.3 Rehoic Acid and EGF Interaction 103

4.4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Regulation by Eicosanoids 112

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1 Background and Rationaie 112

4.5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

. . . . . . . . . . . . . . . . . . . . . . . 4.5 .2.1 Effects of Prostaglandins 115

4.5 .2.2 Effects of hdomethacin . . . . . . . . . . . . . . . . . . . . . . . 116

4.5.2.3 Effects of NDGA and NDGA plus Indomethacin

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . interaction 116

4.5.3 Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

4.6 Non-Regulating Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.1 Background and Rationale L32

4.6.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

4.6.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

4.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

CHAPTER 5 . GENERAL DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . 136

5.1 Overview of 1 1P-HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

. . . . . . . . . . . . 5.2 The Ovine 1 1 pHSD2 Gene and its 5'-Fianking Region 137

5.2.2 The 5'-Fianking Region . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

. . . . . . . . . . . . . . . . . . . . . . 5.3 Regdation of 1 I &HSD2 in JEG-3 Ceils 138

5.3.1 Human Choriocarcinoma CeU Line (JEG-3) . . . . . . . . . . . . . . . 138

5.3.2 Regulation of 1 L PHSDZ by Carbenoxolone: Possibly via

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PKA Pathway 139

5.3.4 Reguiation of 1 1 PHSD2 by Eicosanoids . . . . . . . . . . . . . . . . . . 140

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Conclusions 141

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

CURRICULUM VITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

LIST OF TABLES

Table Description Page

. . . . . . . . . . . . . . . 1 Cornparisonof 11FHSDland llkHSD2 21

2 Km value for 1 1 pHSD2 enzyme in various tissues . . . . . . . . . . 22

. . . . . 3 Mutations in the 1 1 pHSD2 gene idrntified in AME patients 36

4 b e r s used in sequencing reactions . . . . . . . . . . . . . . . . . . . . 46

5 Transcription regulatory elements and binding factors . . . . . . . -63

6 Compounds used in ceii treaunenrs . . . . . . . . . . . . . . . . . . . . 82

7 Eicosanoid receptors and their transduction systems . . . . . . . . . . 123

LIST OF FIGURES

Figure Description Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l l PHSD activity 5

Shon chah alcohol dehydropnasc: (SCAD) family . . . . . . . . . . . . - 8

Schematic of the human I LBHSDl gene . mRNA and primary protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . structure 12

Schematic of the tl-iree 1 1 BHSD 1 mRNA vanscripts . . . . . . . . . . 14

Schematic of the hurnan 1 lkHSD2 p n e . mRNA and primary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . protein . . . . l 8

. . . . . . . . . . . . Synthesis and metabolism of cortisol and cortisone -25

1 1 p-HSD2 protection of the mineralocorticoid receptor . . . . . . . . . . 29

Selection of the positive clone containhg the 1 lPHSD2 gene for . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fui-therstudy 49

. . . . . . . . . . . . . . . . . . . . . . . . . Strategy for sequencing reactions 51

Schematic of the ovine 1 1 PHSDZ p n e . . . . . . . . . . . . . . . . . . . . 53

The sequence of the ovine 1 1 ~ H S D 2 gene and the deduced arnino acid sequence of the 1 1 B-HSD2 polypeptide . . . . . . . . . . . . . . . . . - 5 5

Cornparison of the deducsd shtxp . hurnan . rabbit rat and moux 1 1 P-HSD2 amino acid sequences . . . . . . . . . . . . . . . . . . . . . . . . . -58

Schematic view of the ovine 1 l&HSD2 gene with an expanded . . . . . . . . . . . . . . . . . . . . . . . . . . . . view of the 5'-flanking region 61

Aligned sheep and hurnan 5'-flanking regions of the 1 lp-HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . gene 66

Morphological structure of the human placenta and its chorioniç villi . . 76

Dose dependent inhibition of 1 IP-HSD2 enzyme activity in JEG-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . celis by carknoxolone 90

Repression of the stimulatory effecu of forskoiin on 1 1 P-HSD2 enzyme activity by carbenoxolone in JEG-3 cells . . . . . . . . . . . . . . . 92

. . . . . . . . . . Actions of carbenoxolone and forskolin within the ceIl 94

The çffect of AT-RA on 1 1 PHSD2 enzyme activity in EG-3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . as a function of time 98

Dose dependent stimulation of 1 1 bHSD2 enzyme activity in E G - 3 ceus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . by AT-RA .100

The effects of 9C-RA and AT-RA on 1 1 bHSD2 enzyme activity in . . . . . 102

Dose dependent inhibition of L I &HSD2 enzyme activity by EGF in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . JEG-3 celis 105

Possible attenuation of the stimulatory effects of retinoic acid by EGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . in JEG-3 celis 107

. . . . . . . . . . . . . . . . . . Actions of retinoic acid and EGF within the ce11 1 I 1

S ynthesis of eicosanoids fro rn arachidonic acid released h m membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . phospholipids - 1 14

The inhibitory effects of lpglml PGE2 or lpg/ml PGF2a on 1 18-HSD2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . enzyme activity in JEG-3 ce11 . I l 8

Dose-dependent inhibition of I 1 PHSD2 enzyme activity in JEG-3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . by indomethach 120

The effects of 10pM indomediacin. lOOpM NDGA. and 10pM indornethacin+ lûûpM NDGA on 1 IPHSDZ enzyme activity in

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KG-3 celIs 122

Eicosanoid regdation of 1 18-HSD2 by stimulation of calcium releast: via . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . inositol triphosphate 127

Proposed mode1 of PGE2 inhibition of 1 1 pHSD2 by a decrease in . . . . . . . . . . . . . . . . . . PKA activity via inhibition of adenylyl cyclase 129

LIST OF PHOTOGRAPHIC PLATES

Plate Description Page

. . . . . . . . . . . . . . . . . 1 Hurnan chonocarcinoma JEG-3 c e k in cdtuie - 7 8

2 Human chonocarcinorna EG-3 c e k in culture . . . . . . . . . . . . . . . . . .78

ABBREVIATIONS

A A ACTH ADP Ala AME

Asn A ~ P ATCC AT-RA

BLT bp BSA

C C C CaM-kinase CAMP CBX cDNA CHO cpm CYS cys LT 1 cys LT2

D dATP DEX dCTP dGTP d m

3 fb hydroxysteroid dehydrogenase 7 a - hydroxysteroid dehydrogenase 5-hydroxyeicosatetraenoic acid 12- hydroxyeicosaetraenoic acid 15- hydroxyeicosateuaenoic acid 5-hydroperoxyeicosatetraenoic acid 9 cis retinoic acid 1 L Bhydroxysteroid dehydrogenase 1 1 fi-hydroxysteroid dehydrogenase type 1

1 1 p h ydrox ysteroid dehydrogenase type 2 L 7 &hydroxysteroid dehydrogenase

20B hydroxysteroid dehydrogenase

adenine alanine adrenocorticotropic hormone adenosine dip hosphate alanine apparent minerdocorticoid excess arginine asparagine as partate Amencan Type Culture C o i l x tion al1 tram retinoic acid

leukotriene Bq receptor base pair bovine s e m albumin

cytosine cysteine celcius calmodulin-de pendent pro teh kinase cyclic 33'-adenosine monophosphate carbenoxolone complementary deoxyribonucleic acid Chinese hamster ovary cells counts per minute cysteine cysteinyl leukotriene receptor 1 cysteinyl leukotriene receptor 2

aspartate deoxyadenosine triphosphate dexamethasone deoxycytosine triphosphate deoxyguanosine triphosphate deoxythymidine triphosphate

DNA DP m E E E2 E. coli EDTA EFl: EGF EP 1 EP2 EP3 EP4

F F FP FSK

GC-fich GE Gln Glu G ~ Y GR

H 3~ H2O HBSS HC1 His

1 IGF- 1 Ile IP IP3

deoxyribonucleic acid prostagandin D2 recep tor dithiothreitol

glutamate cortisone estradio 1 Escherichia coli ediylenediaminetetra-amtate epoxyeicossatetraenoic acid epidermal growth factor prostaglandin E?_ receptor 1 prostaglandin E2 receptor 2 prostaglandin E2 receptor 3 prostaglandin E2 recep tor 4

phenylalanine cortisol prostaglandin F2a receptor forskolin

guanine glycine e- glycyrrhizic acid guanindc ytosine ric h glycyrrhetinic acid glu tamine glutamate glycine glucocorticoid receptor

histidine tritium water Hanks buffered salt solution hydrochloric acid histidine

isoleucine insulin Like growth factor 1 isoleucine prostaglandin 12 receptor inosi tol triphosphate

isoprop y1 thio-g D-galac toside interferon-stimulated and v ins response elemcnts

lysine ki Io base

xvi

kDa kilocialton Km Michaelis constant Ko Ac potassium ace tate

leucine liire lauria bertania long chain aicohol dehydrogense leuche leukotriene Aq leukotriene Bq leukotriene C4 leukotriene D4 leuko triene Eq

methionine molar methionine rnilli1.i~ millimolar miUimeter mineralocorticoid recep tor

Cr!? microgram

W micromolar

N N NaCI NAD NADH NADP NADPH NaHsP04-H20 nM NaOH MXiA nt

asparagine normal sodium chloride nicotinamide adenine dinucleo tide dihydronicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate dihydronicotinamide adenine dinucleotide phosphate sodium dihydropn onhophosphate nanomolar sodium hydroxide nordih ydroguaiaretic acid nucleotide

pro line statistical pro bability progesterone polyoma enhancer site A3 prostaglandin D;! prostaglandin E2 prostaglandin Fza prostaglandin G2

xvii

PGH2 PGI2 Phe PKA PKC PMA Pro PVP

R RAR RPrRE RXR RNA RNase A

S SCAD SDS Ser SSC

T T T3 TE TEMED THE THF Thr TNFa Tris m 2

v Val

prostaglandin H2 prostaglandin 12 phenylalanine protein kinase A protein kinase C phorbol 12-m yristyryl 13-acetate prohe pol yvinylp ymlidone

arginine retinoic acid receptor retinoic acid response elemen t retinoid X receptor ribonucleic acïd ribonuclease A

serine short chah alcohol dehydrogenase sodium dodecyl sulphate serine sodium chloride/sodium citrate solution

thymine threonhe thyroid hormone tris/EDTA solution N,N, N',N', -Tetramethylethylenediamine te trah ydrocortkone teaahydrocortisol tfi~onine twnor necrosis factor a Tris(hydmxymethyl)methylamine throm boxane A2 tryptophan tyrosine

tryptophan

S-brorno-4-chloro-3-indolyl-~D-gdac toside

tyrosine

CHAPTER 1 - INTRODUCTION

1 1 Btfydroxysteroid dehydrogenase 2 ( 1 I fi-HSD2) is a unidirectional enzyme responsible

for the conversion of biologically active cortkoVcorticosterone to biologicdy inactive

cortisone/ 1 1-dehydrocorticosterone (Zhou et al.. 1995; Naray-Fejes-Toth and Fejes-Toth.

1995; Agamal et al.. 19%). The inactivation of conisol by 1 lpHSD2 protects the

mineraloconicoid receptor (MR) by prevenling cortisol from binding to the MR in place of

the rnineralocorticoid aldosterone (Whorwood et al.. 19%; Seckl. 1993). Mutations in the

human gene coding for 1 1 kHSD2 result in Apparent Mineralocorticoid Exces (AME)

Syndrome (Mune et al.. 1995; Wilson et al.. 1995). This is an autosomal recrssive disorder

which results in severe hypertension in young children (Milford et al.. 1994).

Glucocorticoids play a key role in fetal organ maturation and in the endocrine rnechanisms

leading to parnirition in sheep (Challis and Brooks. 1989). Because 1 lPHSD2 regulates

the intracellular level of bioactivr glucocorticoids. it is proposed to be a major factor in fetal

development Inhibition of placental 1 lkHSD2 in the rat is correlated with low bkth

weight and eventuai hypertension in the adult offspring.

The expression of 1 lPHSD2 is bodi tissue specific and developrnentally regulated. It is

expressed predominantly in mùieraloconicoid target h u e s (Whorwood et al.. 19%) and

the matemal-fetal exchange site within the placenta (Pepe et al.. 1996). These are tissues

which require protection from high levels of biologicaiîy active cortisol. Since decreased

expression of 1 LPHSD2 in these tissues has adverse effects on the health of the individual

(Milford et al.. 1994; Biglieri et al.. 1994). it is critical that the regulation of 1 I bHSD2 be

investigated. Moreover. errors in the published ovine 1 LPHSD2 cDNA sequence were

suspected because of the discrepancy in the deduced protein sequence between the sheep

and other mammals.

The present investigation was conducted to clone and charactrrize the gene encoding ovine

1 lBHSD2 to ver@ the cDNA-deduced ptimary stmcture of this enzyme. The primary

structure of this enzyme was shown to be highly conserved with that of the other species

known to date. The 5'-flanking region of the gene was also analyzed to identify potential

replatory elements. Several putative transcription factor binding sites were located within

this sequence. In addition. the regdation of 1 lfbHSD2 activity. a very t 3s t step in

identifying regdatory factors. was studied using the human choriocarcinoma (EG-3) ce11

line as a model. A range of physioiogical homones/factors were tested. and they were

chosen because they are either produced locaily in the placenta or known to have regdatory

effects on other members of the short chah alcohol dehydropnase farnily, to which 1 lP-

HSD2 belongs.

CHAPTER 2 - LITERATURE REVIEW

2.1 INTRODUCTION TO 11B-HYDROXYSTEROID

DEHYDROGENASE (11P-HSD)

2.1.1 DEFINITION AND HISTORICAL PERSPECTIVES

The enzyme 1 1 khydroxysteroid dehydrogenase ( 1 1 P-HSD. EC 1.1.1.146). as its name

implies. catalyzes the interconversion of a hydroxy goup (-OH) to a keto group (=O) at

C- 1 1 position in the glucocorticoids. cortisol (in human. sheep. and others) and

corticosterone (in rat and mouse) (Fig. 1). Since the biological activity of glucocorticoids is

dependent upon the presence of this hydroxy group, 1 lb-HSD modulates the intracellular

level of bioactive glucocorticoids. As illustrated in Fig. 1. the conversion of the potent

glucocorticoid cortisol to its weak metabolite cortisone is cataiyzed by 1 1 kdehydrogenase

activity with NAD(P) as its cofactor. while the reverse conversion is carried out by 1 1-

oxoreductax activity using NAD(P)H as cofactor.

The existence of an 1 IPHSD was first inferred from studies of metabolites of cortisol and

cortisone in urine by Mason (1950) and Burton et a1 (1953). The activity of 110-HSD was

demonstmted subsequently in isolated tissue preparations (homogenates, slices and ce11

cultures) from a range of organs. most notably the liver, kidney and placenta. This enzyme

was the focus of intense studies in the 1960s and 1970s. The interest in 1 1P-HSD was

revived in 1988 when two groups of investigators. led independently by John Funder

(Funder et aL.1988) and Chxistopher Edwards (Edwards et al.,1988), made the seminal

findings that 1 1 I&HSD in mammalian kidney helps to confer the specificity of aldosterone

for the rend rnineralocorticoid receptor (MR) by rapid local conversion of cortisol to

Figure 1 1 I p-HSD activity. Cortisol is inactivatrd to cortisone by NAD(P)-dependent 1 l~dehydrogenase activity. Cortisone is activated to cortisol by NAD(P)H-dependent 1 1- oxoreduc tase ac tivi ty .

cortisone. The work of the late Car1 Monder. who was the first to purify this enzyme from

nt liver microsomes (Lakshmi and Monder. 1988). laid the foundation for the subsequent

molecular cloning of this elusive enzyme. To date. two distinct isozymes of 1 1 BHSD

(known as 1 1 PHSD 1 and -2) have been idenufied, characterized and cloned; numerous

reviews have also k e n published (for exarnple. Whonvood et al.. 1993a; Monder and

White. 1993; Seckl. 1993; White et al.. LW%). The remaining portions of this chapter are

intended to provide a brief overview of the current state of knowledgt: and gaps with

respect to the structure. iunction and regulation of 1 1 FHSD 1 and 2. In doing so, the

rationale for the present investigation will be formulated. Prior to the presentation of these

topics. it is important to discuss briefly the phylogenesis of thesr: enzymes.

2.1.2 SHORT CHAIN ALCOHOL DEHYDROGENASE (SCAD) FAMILY

1 1 fi-HSD 1 and 2 belong to the short chah alcohol dehydrogenase (SCAD) farnily (Fig. 2).

whose members also include 3kHSD (Krozowski. 1992); L7P-HSD (Krozowski, 1992;

Tsigelny and Baker. 1995); 2OPHSD (Krozowski. 1992); 7a-HSD (Krozowski. 1992);

15-hydroxyprostaglandin dehydrogenase (Krozowski. 1992: Tsigelny and Baker, 1995);

and Drusuphila alcohol dehydrogenase (Krozowski. 1992) and others. These enzymes

exert their affects on a range of substrates. such as steroids. prostaglandins. sugars.

an tibio tics. aromatic hydrocarbons and ni trogen metabolizing compounds (Krozowski.

1994). They convert their substrates to an activated or inactivated smte (Krozowski, 1992;

Krozowski, 1994).

SCADs are genedly -300 amino acids in length. and are easily distinguished from the

long chah alcohol dehydrogenase (LCAD) family of enzymes which are usuaily -700

arnino acids (Krozowski. 1992). SCADs are capable of functioning in the absence of metal

ions (Krozowski, 1994).

7

Figure 2 Short chah alcohol dehydrogenase (SCAD) farnily.

Short Chain Alcohol Dehydrogenase (SCAD) Family

i

Prostaglandin Dehydrogenase

Members of the SCAD family show highly homologous domains (Persson et al., 1991).

There have b e n six domains (A to F) identified, but not every member contains a!l six

domains (Krozowski, 1992). The A domain is the cofactor binding region, where the

cofactor NAD(P) binds to the enzyme (Krozowski, 1992; Krozowski, 1994). The B and C

domains are involved in the stnictural configuration of the enzyme (Krozowski. 1992). The

D domain is where the hydride radical is transferred from the steroid to the cofactor, and is

known as the active site (Krozowski, 1992). The E and F domains are frarnework sites

around which amino acids that confer the specificity of the enzyme are located (Krozowski.

1992). The arnino acids surroundhg the E and F domains are highly variable between

SCAD rnembers (Persson et al,, 199 1).

2.2 ISOFORMS OF 11P-HSD

2.2.1.1 Molecular Structure

The 1 1 &HSD 1. also known as 1 1 PHSDL or hepatic 1 1 P-HSD. represrnts the enzyme

first purified and cloned from the rat b e r (Agaiwal et al..1989). Following the cloning of

rat 1 1 P-HSD 1 cDNA, die cDNA was cloned from a number of other marnmalian species

including the mouse (Opperman et al.. 19%; Rajan et ai., 1995), sheep (Yang et al.. 1992).

squirrel monkey (Moore et al.. 1993) and human (Tannin et d.,199 1). The cloned cDNAs

are highly homolopus. and contain open reading h m e s ranging from 864bp to 879bp.

The cDNA deduced proteins are 287-292 amino acids in size with a predicted mass of

-32kDa. The p n e for 1 1 PHSD I has been cloned and characterized for several species

including rat (Moisan et al., 1992b), squirrel monkey (Moore et al., 1993). baboon (Davies

et al.. 1997) and human (Tannin et al.. 199 1). The 1 1PHSDl gene is composed of 6 exons

and spans over 9kb (Fig. 3). in the human genome, this gene resides on chromosome 1

(Tannin et aL.1991).

Three different transcnpts arïse from the 1 l&HSDl gene. they are referred to as 1 Lb HSD 1 A. 1 L PHSD 1 B and 1 1 P-HSD 1C (Fig. 4). 1 1 PHSD LA mRNA is the full length

vanscript which is - 1.7kb in length (Krozowski et al.. 1992). and encodes an active protein

(Mercer et al., 1993). 1 1 BHSD 1B mRNA (1.5kb) is a truncated version lacking sequences

corresponding to exon 1. It is the product of an altemate use of 1 1PHSD 1 p n e promoter

(Krozowski et al.,1992). 1 1P-HSD 1C mRNA is the product of altemate exon splicing in

which exon 5 is spliced out but with no shift in the open reading frame (Yang et al.. 1995).

Ln vitro transfection studies demonstrated that both of these shorter forrns of mRNA encode

proteins lacking 1 1 P-HSD activity (Krozowski et al., 1992; Yang et al., 19%).

2.2.1.2 Tissue Distribution

The full length tmscript (1 1 P-HSD 1A mRNA) and its resulting 1 I P-HSD 1 enzyme

activity are widely expressed (Tannin et al.. 199 1) in glucocorticoid target tissues (Roland et

al.. 1995; Stewart et al.. l995b). The mRNA and activity are predominantly in the liver. as

well as in the h g . hypothalamus. pituitary. spleen. kidney, brain, testis myorneüiurn.

decidua and placenta of several mammalian species (Agarwai et ai.. 1989; Burton et

al., 1996; Moisan et al.. 1990; Moisan et ai.. lW2a; Nicholas and Lugg, 1982; Rajan et

al., 19%; Stewart et al., 1994b; Stewart et al., l99Sb; Sun et d., 1997; Tannin et al., 199 1;

Whonuood et al.. 19%; Yang, 1992; Yang et al.. 1992; Yang et al.. 1997b).

The 1 1 B-HSD 1 B mRNA displays a restricted pattern of expression, and has only k e n

found in the kidney (Mercer et al., 1993; Yang et aL. 1992; Yang, 1992). The expression of

Figure 3 Schematic of the human 1 IP-HSD 1 gene. mRNA and primary protein structure. Open boxes represent exonic sequence. Dashed h e indicam a break in the intronic sequence. Adap ted from Penning. 1997.

Figure 4 Schematic of the three variants of 1 1BHSD 1 mRNA. 1 1 PHSD 1A mRNA is the full length transcnpt which encodes a functional enzyme. L 1BHSD 1B mRNA is the product of a different start site of transcription and encodes a non-fwtional enzyme. 1 lp HSD 1C mRNA is the product of altemate exon splicing. It encodes a non-funchonal enzyme.

1 1 $ -HSD1 A mRNA

1 1 p-HSD1 B mRNA

1 1 p-HSD1 C mRNA

this vanscript has k n suggested to be a mechanism of dom-regulating the level of 1 1&

HSD 1 activity (Mercer et ai., 1993). as this transcript encodes a nonfunctional enzyme.

The 1 1 PHSD 1C transcript is widely expressed, being found in aU of the tissues

expressing the full length 1 1 P-HSD 1 A vanscript but at a lower level (Yang et al.. 19%).

The biological significance of this transcript is unknown. since Like 1 1 PHSD 1B mRNA.

1 l&HSD IC mRNA rncodes an enzyme devoid of any activity.

2.2.1.3 Characteristics

1 IP-HSD 1 is a NADP(H)-dependent enzyme which has low affinity for endogenous

glucocorticoids (Km in the micromolar range) (Whonvood et aL.1994; Yang and Yu,

1994; Yang. 1994). This enzyme is reversible in that it possesses both 1 1 P-dehydrogenase

and 1 1-oxoreductase activities (Yang and Yu. 1994: Aganval et al.. 1989). 1 1 PHSD 1 is a

glycoprotein (Ozols. 1995). in the rabbit protein, carbohydrates are attached to amino acids

Asn- 122. - 16 1. and -206 of the enzyme (Ozols, 1995). 1 1 P-HSD 1 is enriched in the

rnicrosomal fraction of the ceU (Yang and Yu. 1994; Opperman et al., 1995; Ozols, 1995).

and ment protein topology studies have revealed that the main portion of the polypeptide

chah is extended into the lumen of the endoplasrnic reticuium (Ozols, 1995).

2.2.2.1 Molecular Structure

The 1 lkHSD2 represents the enzyme identifed more recently from mammalian kidney.

and thus it is ako known as the rend 1 l&HSD (1 1PHSDK). The cDNA encoding 1 1 b

HSDZ was first cloned from human (Albiston et al., 1994; Brown et ai.. 1996a) and sheep

( A g w a i et d., 1994). and shortly thereafter from rat (Zhou et al.. 1995) and rabbit (Naray-

Fejes-To th and Fejas-Toth. 1 995). The cDNA-deduced pro teins from these species contain

397-406 amino acids wirh a predicted molecular m a s of -44kDa (Albiston et al.. 1994;

Brown et ai.. 1996a; Zhou et al.. 1995; Naray-Fejes-Toth and Fejes-Todi. 1995) except for

the shrep which predicts a protein of 427 amino acids (Agarwai et ai.. 1994). The arnino

acid sequences for 1 lBHSD2 show higher homology to the sequences for 17PHSD2

(>35%) thm they do for the 1 If3-HSDl sequences (40%) (Naray-Fejes-Toth and Fejes-

Toth. 1995; Albiston et al.. 1994). The gene for 1 IPHSDZ has k e n cloned and

characterized from the human (Aganval et ai.. 1995a) and ) and moux (Cole. 1995). The

gene is composed of 5 exons spanning over 5kb (Fig. 5) (Aganual et ai..1995a; Cole.

1995; Agarwai et al.. 199%). and is localized to chromosome 16q22 in the human

(Aganval et al.. l995a; Krozowski et al., 1995b: Agmal et al.. 1995b). In both species. the

gena dots not contain a consensus TATA box (Agarwal et a1..1995a; Agarwal et

al.. 1995b). and is very GC-rich (-708 G+C) (Cole, 1995; Agarwal et d.. 1995 b).

Figure 5 Schematic of the human I lP-HSD2 gene, mRNA and primary protein structure. Open boxes represent exonic sequence. Dashed h e s indicate a break in the intronic sequence. Adapted from Penning. 1997.

2.2.2.2 Tissue Distribution

Unidce i 1 p-HSD 1. 1 1 &HSD2 mRNA and enzyme activity are expressed in a tissue

restricted pattern (Cole. 1995; Brown et al.. 1996b). In dl species studied. 1 IfbHSD2 is

predominantly CO-localized with the mineraiocorticoid receptor in aldosterone mget tissues

(Whowood et al., 1995; Naray-Fejes-Toth and Fejes-Toth. 1995; Cole, 1995; Krozowski

et al.. 1995~; Naray-Fejes-Toth et al., l994; Stewart et al.. 1995b: Lombes et d.. 1995;

Krozowski et al., 1995a; Brown et al.. 1996a; Brown et al., 1996b; Roland and Funder.

1996).

in the adult. 1 1 P-HSD2 is most abundantly expressed in the kidney, within the distal

convoluted tubules and collecting ducts. 1 1 &HSD2 is also highly expressed in the distal

colon. parotid gland. placenta. and the adrenal gland of some species. but not found in the

mouse adrenal gland. In some spcxies extremely low levels are deirctable by reverse

transcription polyrnerase chah reaction in heart, lung. ovary. prostate. tesùs. spleen,

pancreas, hypothalamus. hippocampus and midbrain (Aganval et al.. 1994; Agarwal et

al.. 1995a; Albiston et al.. 1994; Brown et al.. 1996a; Brown et al., 1996b; Bujalska et

al., 1997; Cole. 1995; Condon et al., 1997; Krozowski et alJ995a; Krozowski et

ai.. 1995~; Kyossev et al.. 1996; Li et al.. 1996; Lombes et al.. 1995: Mercer and

Krozowski. 1992; Naray-Fejes-Toth and Fejes-Toth. 1995; Naray-Fejes-Toth et al.. 1994;

Roland et al.. 1995; Roland and Funder. 1996; Shimojo et al.. 1997; Slight et al.. 1996;

Smith et al.. 1996; Smith et al.. 1997; Stewart et a1.,1995a; Stewart et al., 1995b; Sun et

ai.. 1997; Whonvood et al.. 1994; Whowood et al., 1995; Yang and Matthews. 1995; Zhou

et al.,1995.

The expression of 1 l p-HSD2 has been shown to be developmentdly regulated. 1 1 BHSD2

mRNA and activity are present in the sheep fetus by embryonic day 85. and their level of

expression subsequently increases with advancing gestation (Langlois et al.. 1995). In the

mouse fetus. 1 1 P-HSD2 mRNA is widely expressed between embryonic days 9.5 and

12.5, after which it reverts to a tissue specitlc pattern as seen in the adult (Brown et

al.. 1996b).

2.2.2.3 Characteristics

Similar to 1 1 P-HSD 1. 1 lPHSD2 ha been localized to the microsornal fraction of the celi

(Yang and Yu, 1994; Reeves. 1995; Stewart et al.. 1994a; Brown et al.. 1996a; Naray-

Fejes-Toth and Fejes-Toth. 1996; Monta et ai., 1996; PetreUi et al.. 1997). The deduced

1 1 bHSD2 arnino acid sequence contains conserved regions of cytoplasmic translocaûon

signais. suggestive of it being anchored to the endoplasmic reticulurn (Krozowski et

al.. 1995a). The predicted 1 1BHSD2 amino acid sequence also contains five (A-E) of the

six conserved domains of the SCAD family (Albiston et ai., 1994). these are illustrated in

Chapter 2 (Fig. 12). The human 11BHSD2 enzyme has recently k e n purified from the

placenta (Krozowski et ai.. 1995~). This purified protein has a molecular weight.

determined by western blot analysis. of -4 1kDa which is smaller than that predicted from

the cDNA sequence (Krozowski et al.. 1995~). This discrepancy in the size of 1 1 pHSD2

between the predicted (44kDaj and native protein (4 1kDa) is indicative of post-

transcriptional modification (Krozowski et ai.. 1995~).

In marked contnst to 11PHSD 1. 1 lkHSD2 has a much higher affinity for its substrates

cortisol and corticosterone. with a Km in the nanomolar range ((Zhou et al.. 1995; Naray-

Fejes-Toth and Fejes-Toth. 1995; Yang and Matthews. 1995: Brown et al.. 1993; Albiston

et a1..1994; Yang and Yu. 1994; Yang, 1994; Reeves. 1995; Brem et aJJ993; Leckie et

ai.. 1995; Monta et ai.. 1996; Gomez-Sanchez et ai.,1997; Fermi et aL.1996b). Specific

Km values for this enzyme in various species and tissues are listed in Table 2. Moreover.

Table 1 Cornparison of human 1 l p-HSD 1 and 1 ID-HSD2. Taken from (Yang, 1997)

I Characteristic

Primary Structure

Co-Factor Preference NAD

Direction Cortisol ++ Cortisone Cortisol .-> Cortisone

Affinity for Cortisol Low (Km in micromolar) High (Km in nanomolar)

Dexame thasone Metabolism Yes

Sites of Expression Widespread

Gene Structure 5 Exons (6kb) 6 Exons (>9kb)

1 Gene Locus Chromosome 1 Chromosome 16

Yes 1 Mutations in AME

Table 2 Km value for 1 1P-HSD2 enzyme in various tissues. B represents corticosterone and F represents cortisol.

1 Tissue 1 Km Reference

(Gomez-Sanchez e t al., 1 996; Pasquarette e t al., 1996)

(Brown e t al., 1993; Pepe and Albrecht, 1984)

Human choriocarcinoma (JEG-3) cells

Human placenta

1 6nM for B nM for

14.1nM for B 55nM for F 14nM for F

(Brown e t al., 1996) I Human placental cDNA transfected into CHO cells

12.4nM for B 43.9nM for F

Ferrari e t al., 1 996) Human cloned placental enzyme in CHO cells

5.1 nM for B 6 1 nM for F

(Reeves, 1 995)

- - -

Human colonic epithelial (T84) cells

v n i c rnucosa 1 35.3nM for F

1 1.3nM for B 79.8nM for F

(Whorwood e t al., 1 994

Human fetal kidney microsomes 13nM for B 60nM for F

(Stewart e t al., 1994)

(Gornez-Sanchez e t al., 1997)

Sheep kidney 3.8SnM for 8 21.3nM for F

p h e e p kidney microsornes 1 68.7nM for F (Yang and Yu, 1 994)

1 Sheep placentz 1 I l O n M f o r F (Yang, 1994) - - -

(Yang and Matthews, 1995)

1 Sheep adrenal cortex 1 41.4nM for F

(Pepe and Albrecht, 1984)

1 Baboon placent; I 10nMforF

v R a b b i t & & a l collecfing duct 1 6.6nM for B Naray-Fejes-Toth and Fejes-Toth, 1995)

1 Rat kidney 1 1O.lnMforB (Zhou e t al., 1995)

(Whonivwd e t al., 1994

1 Rat cDNA transfected into CHO cell: 1 9.6nM for B (Morita e t al., 1996)

(Leckie e t al., 1995) I Pig kidney epithelial (UC-PU1 ) cells

1 Toad bladder 1 163nM for B

34.4nM for B 89.7nM for F

Brem e t al., 1 993)

the activity of 1 1 PHSD2 is NAD-dependent (Zhou et aL.1995; Naray-Fejes-Toth and

Fejes-Toth. 1995; Yang and Matthews, 1995; A g m a l et al.. 1994; Brown et al.. 19%;

Reeves, 1995; Leckie et al., 1995; Stewart et al.. 1994a) and under physiological

conditions. it exhibits only 1 lpdehydrogenase activity (Fig. 1) (Zhou et al.. 1995; Naray-

Fejes-Toth and Fejes-Toth. 1995; Agarwal et al.. 1994; Albiston et al.. 1994; Leckie et

al.. 1995; Stewart et al.. MMa; Monta et al.. 1996: Li et al.. 1997). Recentiy. 1 1 P-HSD2

has ken shown to be capable of L 1-oxoreductase activity. but only with the synthetic

glucocorticoid 1 1 -dehydrodexamethasone as its substrate. (Li et al.. 1997).

2.3 PHYSIOLOGY OF 11P-HSD

2.3.1 SYNTHESIS AND METABOLISM OF CORTISOL (F) AND

CORTISONE (E)

Cortisol (also known as F) is synthesized in the zona fasciculata of the adrenal cortex from

cholesterol (reviewed in White et al.. 1992: Margions et al.. 1994). Adrenocorticotropin

hormone (ACTH) regdates the synthesis and secretion of F fiorn the adrenal cortex by

stimulahg the expression of enzymes involved in this process (reviewed in White et

al.. 1992; Margions et al., 1994). High levels of F exen negative feedback effects. resulting

in decreased ACTH levels and subsequently decreased F production (reviewed in Vander et

al.. 1 994).

There are several enzymatic steps involved in the metabolic conversion of cholesterol to F

(White et al., 1992)- these are iilustrated in Fig. 6. First the cholesterol side chah must be

removed to produce pregnenolone (White et al., 1992). Pregnenolone is metabolized by 3P

HSD to produce progesterone (White et ai.. 1992). Progesterone is hydroxylated at the

Figure 6 Synthesis and metabolism of cortisol and cortisone. The synthesis of cortisol from cholesterol takes place in the adrenal cortex. The formation of cortisone from cortisol can take place in any tissue expressing 1 IPHSD. The final products (THF, aiio-THF and THE) are the urinary me tabolites of cortisol and cortisone.

tetrahydrocort isol a 1 Ietetrahydrocortisol tet rahydrocortisone

1 7-hydroxylase

+. 17odH-Pregnenolone , 4 3J3;ol-deh~drogenase 384-dehydrogenase A -~sornerase d * ~ ~ s m e r a ç c

Progesterone > 17a-OH-Progesterone 1 7-hydroxylase

17a-. 2 1-. and 1 @-positions by three different cytochrome P450 enzymes to finally

produce cortisol (White et al.. 1992).

Cortisone (also known as E) is produced from F by 1 IPHSD (Fig. 6). Both F and E are

enzymaticdy broken down to their urinary metabolites (Fig. 6) before they are excreted. F

is metabolized to ietrahydmconisol (THF) and do-tehahydrocortisol (aIio-THF) and E is

metabolized to tetrahydrocortisone (THE) (Walker and Edwards, 199 1; Biglieri et

al.. 1994).

2.3.2 GLUCOCORTICOID AND MINERALOCORTICOID RECEPTORS

2.3.2.1 Binding of Cortisol to the Glucocorticoid and Mineralocorticoid

Recep tors

It is widely accepted that cortisol enters the ceil by passive diffusion through the plasma

membrane and binds to glucocorticoid receptors (GRs) in the cytoplasm (Gustafison et

al.. 1987; Margions et aL.1994). Binding of F activates the GR, which is then uanslocated

into the nucleus so that it may bind to the DNA as a homodimer (Gustafsson et aL.1987;

Margioris et al., 1994).

The mineralocortïcoid receptor (MR) is highly homologous to the GR and functions

through a similar mechanism (Evans. 1989). The intended ligand for the MR is the

minerdocorticoid aldosterone. although the MR has the sarne affinity for F in vitro (Seckl.

1993: Margions et aL.1994). Cortisone has negligible affinity for either the GR or the MR

(Margions et al.. 1994).

2.3.2.2 Response of Activated Receptors

Activation of the GR results in several physiological effects (Todd-Turla et al.. 1993). h

acts to increase both glycogenesis and gluconeogenesis; decrease osteoblast formation and

activity; decrease production of interleukins. prostaglandins and cytokines; decrease

prolifenlion and migration of macrophages and lymphocytes: decrease absorption of

calcium from the gastmintestinal trac$ and decrease secretion of thyroid-stimulating

honnone (Margioris et al.. 1994). Stimulation of the GR also accelerates lung maturation in

the fetus by hcreasing the pulrnonary surfactant on the alveolar surface (Lugg and

Nicholas, 1978).

The activated MR is the primary regulator of sodium and potassium concentrations within

the cell (Margions et al.. 1994). This replation of ions occurs mainly in the rend distal

convoluted tubule and coilecting duct, resulûng in sodium ion reabsorption and excretion of

potassium and hydrogen ions (Todd-Turla et al.. 1993; Margions et al.. 1994).

2 .3 .3 PROTECTION OF MINERALOCORTICOID RECEPTOR FROM

CORTISOL BY 11P-HSD2

The MR binds both F and aldosterone with equal affinity in vitro (Seckl. 1993; Margioris et

al.. 1994). In vivo. however the MR shows a selectivity for binding aldosterone in the face

of 1000 tirnrs excess of F over aldosterone in circulation (Seckl. 1993). This selectivity has

bren attributrd to the presence of I 1P-HSD2. which is expressed pnmarily in

mineralocorticoid target tissues (Whorwood et al.. 1995). 1 L p-HSD2 protects the MR by

inactivating F to E. which is unable to bind to the MR, thus leaving the MR free for

aldosterone to bind (Fig. 7) (Whorwood et d.. 1995; Seckl. 1993). The 1 lBHSD2 enzyme

has been found to colocalize with the MR in the rend nephron (Krozowski et alJ995c;

Figure 7 Cortisol, which circulates at a much higher concentration than the mineralocorticoid aldosterone, binds to the mineralocorticoid receptor with high affuiity . This prevents the aldosterone from binding to its own receptor. 1 lkHSD2 prevents cortisol from binding to the rnineralocorticoid receptor b y converting cortisol to i ts bac tive form cortisone. This d o w s aldosterone to bind to its receptor. Modified from Seckl, 1993.

Naray-Fejes-Toth et al.. 1994) within the principal and fhtercalated cells (Naray-Fejes-

Toth et aL.1994).

2.3.4 PROTECTION OF THE FETUS FROM MATERNAL CORTISOL BY

PLACENTAL Ilfi-HSD2

The exposure of the fetus to appropriate physiological levels of F is essential for normal

organ growth. maturation and development (Monder and White. 1993). Fetal exposure to

excessive g,lucocorticoids in animals and humans results in retardation of fetal growth

(Seckl. 1997). The ratio of F to E is much higher in the materna1 plasma and arnniotic Huid

than in the cord plasma, and this difference increases with advancing gestation (Monder

and White. 1993; Pasqualini and Kincl, 1985). The majority of the matemal F passing

through the placenta to the fetus is converted to E before reaching the cord blood (Murphy

et a.L.1974). thus the placenta provides a barrier to protect the fetus from the biologically

active F (Monder and White. 1993; Stewart et d.. 1995a). It is the 1 @-HSD expressed

within the placental tissues which inactivates the F (Benediktsson et ai.. 1993; Krozowski et

al.. 199%: Seckl et al.. 19%). 1 lPHSD2 is idealiy located within the placenta to serve this

function. it is f'ound in the syncytiotrophoblast cek. the site of matemal-fetal exchange

(Pepe et al.. 1996).

2.4 REGULATION OF 11B-HSD

2.4.1 Regulation of 11P-HSDI

Much is known about the regulation of 1 1b-HSDl. Numerous studies have examined the

effect of steroids. cytokines and intracellular messengers as well as some dietary

components. on the level of 1 1 PHSD 1 mRNA and enzyme activity. The foliowing is a

sumrnary of these studies-

Dexamethasone. a synthetic glucocorticoid. has a stimulatory effect on 1 1 P H S D 1 mRNA

and activity in human skin fibroblasts (Hammami and Siiten. 199 1). 2s FAZA heptoma

cells (Voice et al.. 1996) and fetal sheep Iiver (Yang et a1..1994) and an inhibitory effect in

the adult sheep liver (Yang et al.. 1994). Thyroid homone (T3) decreases both liver and

pituitary 1 1 PHSD 1 mRNA and activity in the adult rat (Whonvood et al.. 1993b).

Estradiol matment resulted in a decrease in 1 1PHSD 1 activity and mRNA in the rat liver

(Low et al.. 1993). 1 1 a- and 1 L ~hydroxyprogesterone also had an inhibitory effect in rat

liver microsornes (Souness et al.. 1995). In human endometrial stroma1 cells.

rnrdroxyprogesterone acetate stimulated 1 1 BHSD 1 activity and the addition of rstradiol

potentiated this effrct (Arcuri et a!.. 19%).

Insulin decreased 11 PHSDI activity and mRNA in hurnan skin fibroblasts and 2 s FAZA

cells (Voice et al.,1996; Hamrnami and Süteri, 1991). IGF-1 also inhibited both the mRNA

and activity in 2s FAZA cells (Voice et al.. 1996). Interleukin- 1 P and Tumor necrosis

factor-a both stimulated 1 1 PHSD 1 activity in glornerular mesangial cells (Escher et

al.. 1997).

Agonists of both the protein kinase A and C ( P U and PKC) pathways have been

demonstrated to have an inhibitory effect on 1 1 PHSD 1. 8-bromo-CAMP (PKA) decreases

1 lp-HSD 1 activity in human skin fibroblasts (Hammami and Süteri. 199 1). Forskolin

(PKA) inhibits 1 lp-HSDl mRNA and activity in 2S FAZA cells (Voice et aL.1996).

Phorbol ester (PKC) decreases 1 1 FHSD 1 activity in hurnan skin fibroblasts (Harnrnami

and Siiten, 199 1).

nie licorice mot denvative glycyrrhizic acid decreased both 1 l&HSD 1 mRNA and enzyme

activity in rat liver and pituitary (Whowood et al.. l993a). Carbenoxolone. the synthetic

analog of glycynhizic acid, potently inhibited this activity in human males (Walker et

al., 1995) and rat Iiver microsornes (Latif et al., 1992).

Naringenin, and other dietary flavonoids found in grapefruit juice. inhibit NADP-

dependent 1 1P-HSD 1 activity in guinea pig kidney cortex microsornes (Lee et aL.1996:

Zhang et al.. 1994) with a magnitude comparable to that observed for glycyrrhizic acid

(Zhang et al.. 1994). Grapefruit juice consumption by normal healthy men was

demonstrated to decrease the unnary ratio of cortisone:cortisol metabolites. which suggests

that 1 1 BHSD activity may be inhibited in vivo by dietaiy flavonoids (Lee et al., 1996).

2.4.2 Regulation of I lP -HSD2

Relatively less is known about the regulation of 1 1 PHSDZ. Fewer studies have been

conducted. and many of which show inhibition of 1 lbHSD2 enzyme activity. It is

possible that these inhibitory effects could be due to simple cornpetitive inhibition of the

enzyme. The following is a summary of the results from these studies.

In adrenalectomized rats. the glucocorticoids dexamethasone. deoxycorticosterone and 9a-

fluorocortisol have b e n found to increase rend 11pHSD2 enzyme activity. This

stimulatory effect on enzyme activity was suggested to be a resuit of activation of latent

1 I kHSD2, since there was a decrease in 1 1 P-HSD2 mRNA (Li et al., 1996).

Estradiol stimulates 1 IPHSD2 activity in the rat kidney (Low et al.. 1993). and at low

doses stimulates the activity in pig kidney LLC-PKl cells (Leckie et al.. 1995). At hi@

d o s . estradiol inhibits 1 1 BHSD2 activity in LLC-PK 1 cells (Leckie et ai.. 1995). 1 1 a- HSD2 enzyme activity is inhibited by progesterone matment in the baboon placenta (Pepe

and Albrecht, 1984). LLC-PK1 cells (Leckie et a1.,1995), E G - 3 ceils (Gomez-Sanchez et

a1..1996) and CHO cells transfected with rat 1 1&HSD2 cDNA (Morita et aL.1996). Both

1 la - and 1 1 B-hydroxyprogesterone decrease 1 1 p-HSD2 activity in JEG-3 cells (Souness

et al.. 1995) and CHO cells transfected with rat 1 1 PHSDZ cDNA (Monta et al.. 1996). In

JEG-3 cells 1 1 PHSD2 activity is inhibited by both 5a- and SP-dihydroprogestemne

(Gomez-Sanchez et al.. 1996). In human endometrial stroma1 cells. medroxyprogesterone

acetate stimulated 1 1 PHSDZ activity and the addition of estradiol potentiated this effect

(Arcuri et ai.. 1996).

In E G - 3 cells, the PKA agonists. forskolin and dibutyryl CAMP stimulate both 1 1 pHSD2

mRNA and enzyme activity (Pasquarette et ai.. 1996). The PKC agonist phorbol ester had

no effect (Pasquarette et al.. 1996).

The Licorice root extracf glycyrrhetinic acid inhibits 1 1 P-HSD2 activity in EG-3 cells

(Gomez-Sanchez et al., 1996) and CHO cells transfected with rat 1 lp-HSD2 cDNA (Morita

et al.. 1996). Carbenoxolone, it's synthetic anaiog, aiso decreased 1 1P-HSD2 activity in

JEG-3 (Gomez-Sanchez et al., 1996) and LLC-PK 1 ceIls (Leckie et al., 1995).

As with 1 I P-HSD 1. diefary flavonoids from grapefruit juice inhibit 1 1 bHSD2 enzyme

activity in guinea pig kidney cortex microsornes (Lee et al.. 1996). Consumption of

grapefruit juice by normal healthy males led to a decrease in the urinary ratio of

cortisone:cortisol metabolites. indicating that I IBHSD2 enzyme activity may be inhibited

in vivo by dietary flavonoids (Lee et al.. 1996).

Because of their structural similarity to glucocorticoids the identified factors involved in the

replation of 1 lkHSD2 in the literature could conceivably act as competitive inhibitors

without affecting the expression of 1 IPHSM. In the present study. a range of

physiological hormonedfactors were tested in JEG-3 ceus. in order to identify potential

regdators of 1 IP-HSD2 in vivo. Many of these hormones/factors were chosen because

they had been known to have regdatory effects on other members of the SCAD famiiy.

such as 1 1PHSD 1 and I7PHSD. Others were chosen because they are produced locally

by the placenta.

2.5 PATHOGENESIS OF 11B-HSD2 DEFICIENCY

2.5.1 APPARENT MINERALOCORTICOID EXCESS (AME) SYNDROME

Apparent minrralocorticoid excess (AME) syndrome is a rare form of endocrine

hypertension (Milford et al.. 1994) which is inheriod in an autosomal recessive fashion

(Stewart et al., 1996; Mantero et al.. 1996; White et ai.. 1997b). Of the 30 cases described to

date, 29 occur in children (MiIford et alJ994; White et al,,1997b). Patients have normal to

subnormal levels of mineralocorticoids. but exhibit symptoms such as sodium retention.

hypokalemia and decreased renin levels. which are normally attributed to high amounts of

aldosterone (Mantero et al.. 1996; White et al.. I997b). It is common for patients of AME to

have experienced developmental obstacles such as inmuterine growth retardation. low birth

weight and failure to thrive postnatally (White et al.. l997b).

Other symptoms observed in AME patients are nephrocalcinosis. nephrogenic diabetes

insipidus and rhabdomyolysis. caused by the abnomally low potassium levels (White et

al.. 1997b). It is not uncommon for sufferers of this syndrome to die before the end of

adolescence due to a stroke or other cerebrovascular accidents (White et al.. 1997b).

Patients with AME possess extraordinarily high levels of intrarenal cortisol (Funder et

al..199û). The ratio of the u ~ a r y metabolites of cortisol (THF + do-THF) to cortisone

(THE) are also abnormally high, indicative of a deficiency in rend 1 1 PHSD2 (Mune et

ai,, 1995; Walker and Edwards. 199 1; Mantero et al., 1996; White et al,, 1997b).

indeed. AIME syndrome is due to reduced or absent 1 1 PHSD2 enzyme activity (Funder et

al.. 1990; Millord et al., 1994; Mantero et al., 1996) as a result of mutations in the 1 1 P HSD2 gene (Mune et ai.. 1995; Wilson et al.. 1995; Ferrari et al.. 1996a; White et

aJJ997b). Several mutations, but no gros deletions, have been identified (Table 3)

(Mune et al.. 1995). The gene for 1 I P-HSD I is normal in AME patients (Nikkila et

al., 1993).

Blockage of the MR with the dmg spironolactone and the irnplementation of a low sait-diet

improves the AME patients blood pressure, but treaunent with ACTH or hydrocortisone

intensifies the hypertension (White et al., 1997b).

2.5.2 DIETARY INDUCED AME-LIKE EFFECTS

Chronic ingestion of substantial arnounts of licorice or licorice containing products can

result in AME-like symptoms by inhibiting 1 LbHSD2 activity (Biglieri et al., 1994). A

recently reported case of this affliction. involved a 38 year old woman who consumed 200g

of ficonce per day. She was hospitalized for hypertension and hypokalaemic alkalosis

(Seelen et aI., 1996).

Recently consumption of dietary flavonoids from grapefruit juice was speculated to inhibit

1 1 pHSD2 activity in humans. Excessive iniake of grapefmit juice may result in AME-like

Table 3 Mutations in the 1 L BHSD2 gene identified in AME patients (Mune et al., 1995; Wilson et al,. 1995; Ferrari et al,. 1996; White et al-, 1997).

1 Mutation [ ~ocation 1 F to E activity

1 ~ t o l I intron 3 I ?

symptoms, as it has k e n shown to cause a decrease in the ratio of cortisone:cortisol

metabolites in the urine (Lx et al., 1996).

2.5.3 FETAL EXPOSURE TO EXCESS CORTISOL DUE TO REDUCED

PLACENTAL 11P-HSD2

Fetal origins of adult disease States has k e n extensively studied by the British scientist, Dr.

David Barker. He has found that small babies. in relation to their placental size. have an

increased risk of developing cardiovascular disease and non-uisulin-dependent diabetes as

adults (Barker et al., 1993).

One causal factor of this low birth weight may be decreased levels of placental 1 1 bHSD2.

In rats. the level of placental 1 1 PHSD2 activity is positively correlated with fetal weight

and negatively correlated with placental weight (Benediktsson et al.. 1993). inhibition of

1 1P-HSD2 in the rat placenta is associated with a reduced birth weight and subsequent

hypertension in the adult offspring (Seckl et aL.1995). This correlation in humans has

remained inconclusive. as there have been contrasting observations reported. One group

reported a positive correlation between birth weight and placental 1 1 B-HSD2 activity

(Stewart et ai.. 1995a). while another group reported that no such correlation existed

(Rogerson et al.. 1 997).

2.6 SCOPE OF THE PRESENT STUDY

1 1 bHSD2 is expressed in a tissue specific and developmentally regulated fashion. It is

localized specifically to tissues which require protection from high levels of cortisol. such

as the rnineralocorticoid target tissues. During human pregnancy, 1 1 P-HSD2 is highly

expressed in the placenta where it serves as a barrier to matemal glucocorticoids. Variations

in the expression of 1 1 kHSD2 in these tissues have k e n proven to be detrimental to the

healdi of an individual. and to normal fetal developrnent It is therefore irnpetative that the

regulation of 1 lPHSD2 gene expression be eiucidated. The present snidy was designed

with two objectives in mind. Firsf as one of the initial steps in studying the regulation of

1 1 PHSD2 gene expression at a molecular level. I wanted to clone and characteriz the

ovine 1 lp-HSD2 grne and its 5'-flanking region. Second. I wanted to idenUfy endogenous

hormones/factors which are capable of regulating 1 IPHSDZ activity. The human

choriocarcinoma (EG-3) cells were chosen as a mode1 for this study for the following

reasons. First, JEG-3 cells had been shown to express 1 @-HSD2 activity and mRNA. and

there was no sheep ce11 line available which was known to express 1 lPHSD2. Second.

the structure and sequence of 1 lbHSD2 gene and its 5'-flanking region as well as the

characteristics of 1 lkHSD2 enzyme activity are very similar between the sheep and

human. Third. regulatory mechanisms of a given gene are often highly conserved between

mammalian species.

CHAPTER 3 - MOLECULAR STRUCTURE OF THE OVINE 11P-HSD2 GENE AND 5'-FLANKING REGION

3.1 INTRODUCTION

The regdation of genes in eukaryotic oqanisms is essential for the function and

differentiated development of distinct tissues (Passarge. 1995). Most celis express only a

very srnail portion of their pnes. This portion may change during development or in

response to internai. and/or extemal signals from other ceils (Alkm et aL.1994)

To understand how a gene is regulated at a molecular level. one of the frst sstps is to clone

and characterize the gene of interest and its 5'-flanking region. This chapter deals with the

cloning of the ovine 1 1P-HSD2 gene and its 5'-flanking region. It should be noted that at

the o u w t of this study. the gene encoding 1 lPHSD2 had not been cloned in any species.

During the course of this study the sequences of the hurnan (Agarwal et al.. 1995a) and

mouse (Cole. 1995) 1 lPHSD2 genes were published.

The prirnary structure of 1 lkHSD2 protein has b e n deduced from sheep (Aganval et

ai.. 1994). human (Aibiston et ai.. 1994). rabbit (Naray-Fejes-Toth and Fejes-Toth. 1995).

rat (Zhou et al., 1995) and mouse (Cole, 1995). Sequence analysis of the five proteins

reveais that they are well conserved, except for the sheep which diverges from the others at

residue 358. Furthemore. it has a C-terminal extension of 22 amino acids when compared

with the hurnan protein. This has raised the possibility that there may be an error in the

reported cDNA sequence (Agarwal et al.. 1994). The sequence of the ovine 1 1 PHSD2

p n e and the correctly predicted arnino acid sequence. resulting from this snidy have been

published (Campbell et al.. 1996).

The characterization of an 1.8kb fragment correspondhg to the 5' Banking region of the

human 1 1 kHSD2 gene (Aganval and White. 1996) was recently published. It was found

that the region from -2 to -330 nt. relative to the translation start site (ATG) is essential for

basal transcription of the gene. This region contains two GC-boxes which are consensus

binding sites for the transcription factor Spl (Agarwal and White. 1996).

Transcriptional control of a gene is the most important mode of regulation of gene

expression (Alberts et al.. 1994). The 1 lPHSD2 gene is known to be expressed in a tissue

specific (Cole. 1995; Brown et al.. 1996b) and developmentally reguiated fashion in ail

(Langlois et al.. 1995) species studied to date . The Sp 1 transcription factor is a ubiquitous

one which is normally involved in the regulation of house-keeping genes (hckler, 1996).

Therefore. factors other than Sp l may be involved in the differentiai regulation of the 1 1 p-

HSD2 gene. In this study. the 5' flanking region of the ovine l l B-HSD2 gene was also

cloned and sequenced so that consensus binding sites for other well-known transcription

factors could be identified.

3.2 MATERIALS AND METHODS

3.2.1 SCREENING OF THE COSMID LIBRARY

AU chernicals and supplies were obtained from the following companies: BDWVWR.

Mississauga ON; Bio Rad. Mississauga ON; Boehringer Mannheim. Laval PQ; Carnation.

Markham ON; CLONETECH Laboratories, Inc., California; Difco Labontories, Detroit

MI; Gibco BRL. Burlington ON; KimberlyClark, Mississauga ON; Kodak, Rochester

NY; New England Nuclear, Boston MA; Pharmacia Biotech, Quebec; Schleicher &

Schuell. Keene NH; SeaKem. Rockland. ME; Sigma. St. Louis MO; Stratagene, La Joila

CA; U.S. Biochernical Corporation. Cleveland OH; VWR. Mississauga ON;

A sheep liver Cosmid library was plated out onto 150 mm nitrocellulose fdters on 10 LB-

Ampiciiiin plates [IL dJ320; 25g LB Broth mix; 15g agar; 1mM NaOH; 5Oj@ml

ampicillin], at a density of -M 000 colonies per filter. They were then incubated for 16

hours at 37°C to allow the bacteria to grow. Repiica fdters were then made of the master

filters by placing a new filter on top of the master filter, marking the two filters with

waterproof ink for later re-alignment-

Both the replica and the master filters were incubated on LB-Ampicillin plates for 5 hours at

37'C to allow the bactena to grow. The master filters were then stored at 4°C for later use.

The replica filwrs were transferred to LBÇhlorarnphenicol [IL dH20; 25g LB Broth mix;

15g agar, ImM NaOH; 50pdml chloramphenicol] plates and incubated for 10 hours ar

37'C to arnpiify the Cosmids.

The bacteria on the repiica fdters were lysed by incubating in 0.5N NaOH for 5 minutes.

They were then neutralized for 5 minutes in 1M Tris-HCl pH 7.5 and 0.5M Tris-HC1 pH

7 3 1.5M NaCl, respectiveiy. The filters were air dried for 30 minutes prior to baking at

80°C in a vacuum oven for 1 hour.

The replica filters were incubated for 5 hours at 42'C in prehybridization buffer [50%

formamide; 5xSSC; 1xDenhardt1s solution [0.5g Ficoll; 0.5g PVP; 0 S g BSA in 50ml

dH201; 1 0 8 dextran sulfate ; 0.1% SDS]. Human 1 1 p-HSD2 cDNA was randorn primed

labeled using an Oligo-Labeling Kit. Approximately 200ng of the cDNA was incubated for

10 minutes in a boiling water bath. followed by cooling in an ice water bath for 5 minutes.

The denatured DNA was mixed with LO pl Reagent Mix. 5 pl IOpCi/pl [ a - 3 2 ~ ] d C T P

(30ûûCi/mmol) and 1p1 Klenow fragment This mixture was incubated at 37'C for 1 hour

and then purified on a NICK Column. Two million counts per minute per ml buffer were

added to fresh prehybridization buffer and the fdiers were hybridized in this buffer

ovemight at 42'C with gentle rocking. The filters were then washed in 2-0.2XSSUO. 1%

SDS and exposed to X-OMAT film.

Positive colonies were selected by king up the master füters with the replica filters. Small

circles (0.5cm in diameter) containing these positive colonies were then cut out of the

master filters and placed in LB broth [ IL dH20; 25g LB Broth mix; ImM NaOHJ. The

medium containing individual positive colonies was plated out ont0 100 mm nitrocellulose

filters after diluting the number of colonies - k20 000 for a second round screening. The

above procedure was repeated to isolate individual colonies. The isolated positive colonies

were picked and grown up for 12-16 hours in LB-AmpicilIîn broth [IL dH20; 25g LB

Broth mix; ImM NaOH; 50pg/ml ampicillin]. A total of 12 colonies were seIected for

further analysis.

3.2.2 PREPARATION AND ANALYSIS OF COSMID LIBRARY DNA

The Cosmid library DNA. from the 12 isolated clones, was extracted by a standard

miniprep pIasmid DNA protocol (Sambrook et al., 1989). Iml of each liquid bacterial

culture were pelieted by microcentrifugation for 2 minutes at full speed. The pellets were

resuspended in lûûpl ice cold GTE [50mM glucose; lûmM EDTA; 25mM Tris-HCl pH

8-01 buffer and incubated for 5 minutes. The bacteria were lysed by 5 minute incubation in

200pi 0.2N NaOWl Q SDS followed by neutralization in 150 pl of ice cold KoAc solution

[6ml SM KoAc; 1.15ml glacial acetic acid; 2.85m1 sterile dH20]. The cellular debris was

pelleted by centrifugation and the Cosmid DNA was then retrieved from the supernalant by

phenoVchloroform+isoamyl alcohol extraction. The DNA was ethanol precipitated and then

resuspended in TE containing RNase A [IOmM Tris-HCl. pH 7.5; 1rnM EDTA. pH 8.0;

20pgmi RNase].

The purifïed miniprep DNA was digested with the restriction endonuclease BamHI. Four

diffmnt banding patterns resulted from the digestions when electrophoresed on a 1%

agarose gel. One preparation from each of the four groups were chosen for further

analysis.

The four sarnples of purifed rniniprep DNA were then digested with the restriction

endonucleases BamHI and EcoEU. The digested DNA was electrophoresed on a 1%

agarose gel. The DNA was then subjected to Southem blotting. transferring it from the gel

to a Zeta probe membrane. This was done by soaking the gel twice for 15 minutes in each

of the following solutions. 0.5N NaOH; 1 SM NaCl + 0SN NaOH; and 1 S M NaCl +

0.5M Tris-Cl respectively. The processed gel was placed on a platforni covered with wick

soaked in lOXSSC with the membrane placed on top of the gel. Above the membrane three

sheets of bloning paper paper and -5cm of Kirnwipes were placed. The DNA was allowed

to migrate from the gel to the membrane ovemight by capilliary transfer.

After the Southern transfer was cornplete, the membrane was allowed to air dry on the

bench and then baked in a vacuum oven at 80°C. The dried membrane was incubated for 3-

6 hours in prehybridization buffer 150% formamide; 4xSSPE [3M NaCl. 0.2M

NaH2P04d-I20,0.02M EDTA; pH to 7-41; 1% SDS; 0.5% Blotto; OSmg/ml denatured

sperm DNA]. The hybridization was carried out in fresh prehybridization buffer containhg

2x106 cprn/ml human 1 1 pHSD2 cDNA probe ovemight at 42'C. The blot was then

washed sequentially in 2-0.2XSSUO. 1% SDS and exposed to X-OMAT film.

Each of the clones gave an different banding pattern on the agarose gel, but shared some

common bands. There was two different hybndization patterns on the Southem blot after

incubation with the 1 1 BHSD2 cDNA probe (Fig. 8). The third clone. of the four in Figure

8. was chosen for further analysis because it displayed the strongest hybridization with the

probe.

The idenùried positive DNA fragments from clone number three, were subcloned into

pBluescript II KS by Ligation with T4 DNA Ligase and transformed into E. coli Subcloning

Efficiency DH5a Competent C e k by heat shocking the bacterial c e k at 37'C for 20

seconds. then allowing the cells to recover in SOC media for 1 hour at 37'C. The

transformed bacterid cells were grown up on LB-Ampicillin plates (see above) with X-gal

and IPTG. These compounds allow for identification of colonies containing the plasmid

which has the DNA insert. Ligation of the DNA insert into the plasmid results in an

interruption of the Pgal gene on the plasmid and therefore inhibits the formation of

galactosidase. Colonies which contain plasmids unable to produce Bgalactosidase wili also

be unable to rnetaboiize X-gai and therefore will appear white in colour. as opposed to

those producing bgalactosidase which appear blue in colour after metabolizing X-gal

(Sam brook et al., 1989). The white colonies were selected for further analysis because they

contain plasrnids which incorponted the DNA insen. The selected colonies were culnired

in LB-Ampicillin media (see above). To facaciliiate sequencing analysis. these clones were

further subcloned into srnaller fragments using the restriction enzymes BarnHI. PstI and

SacII.

A 1. lkb fragment containing approximacly 1 kb of the sequence 5' of the translational start

site (ATG) of the 1 1 &HSD2 p n e was subcloned into the plasmid vector pBluescript. This

DNA fragment was further subcloned into smaller fragments by digestion with SacII.

3.2.3 SEQUENCE ANALYSIS OF THE 1lB-HSD2 GENE AND ITS 5'-

FLANKING ICEGION

Subcloned fragments were sequenced by the dideoxy chain termination method using a

Quick-Denature Sequenase 2.0 K i t The subclones were initiaiiy sequenced using forward

and reverse primes (Table 4) which bind to pBluescript KS flanking the DNA clone insert.

To facilitate sequencing of the inner portion of sequence of these fragments. primers were

designed (Table 4) from sequence obtained from the initial sequencing reaction. The

binding sites of these primers is indicated in Figure 9.

The DNA was denatured by incubating in boihg water for 5 minutes in a 0Sml tube

containing Plasmid Denaturing Reagent and the appropnate primer. The denatured DNA

was then chilled in an ice-water bath for 5 minutes. Plasmid Reaction Buffer was added to

the mixture. which was then incubated at 37'C for 10 minutes to dlow for annealing of the

primer to the template. The anneded primer-template was chilled on ice for 5 minutes. The

ice cold annded DNA was then incubated for 5- 10 minutes at room temperature in the

presence of DTT, Labeling Mix. [%]~ATP and Sequenase 2.0 Modified T7 Polymerase.

The sequencing reaction was terminated by splitting up the reaction into four tubes, each

containing one of dideoxy ATP, ïTP. CTP or GTP. and incubating at 37'C for 5 minutes.

Remanire termination caused by pausing in GC-nch regions was reduced by incubating at

37°C for 30 minutes with 1 AU Terminal Transferase and 0.2mM dNTPs. The reactions

were stopped by adding Stop Solution.

The quencing reactions were electrophoresed on a denaturing urea-polyacrylarnide gel

[23.5g Urea: 14.5ml302 Bis-Acrylamide; 5.51111 1OX Glycerol Tolerant Buffer; 35ml

dH2O: 85pl TEMED; lûûpi 25% ammonium persulphate. The gel was dried and the

sequence bands were visualized by exposing the gel to Bio Max f im.

Table 4 Primen used in sequencing reactions.

Sequence Source Primer

Fo rw ard U.S. Biochemical Coq Cleveland, OH

Reverse U.S. Biochemical Coq Cleveland, OH

Hammond Laboratory LRCC, London ON









Exodintron boundaries of the 1 1 bHSD2 gene were identified by cornparison to consensus

splice sequence. and to the ovine 1 lfbHSD2 cDNA sequence. using the DNAStar*

Alignment Program.

The sequence of the 5'-flanking region was aligned with its human counterpart. also using

the DNAStar* Alignment Rogram. The sequence was further subjected to a cornputer

search using MatInspector (Quandt et al.. 1995) to identify common potential transcription

factor consensus binding sites.

3.3 - RESULTS

3.3.1 STRUCTURES OF THE OVINE 11B-HSD2 GENE AND ITS

DEDUCED PROTEIN

One positive clone was selected by Southem blot anaiysis for further analysis (Fig. 8).

This clone was sequenced using the primers listed in Table 4. which bind to the ovine

1 lfbHSD2 gene at the positions indicated in Figure 9. The positive clone isolated from the

sheep liver cosmid library was found to contain the entire ovine 1 lpHSD2 gene. This

gene is composed of 5 exons spanning over >4kb. and its first exon is separated by a large

intron of > 1.7kb from the rest of four exons clustered over 1.9kb (Fig. 10). The ovine

gene contains an open reading frarne of 12 15bp which codes for a protein of 404 amino

acids with a predicted molecular weight of 44kDa (Fig. 1 1). The deduced ovine 1 lPHSD2

protein displays over 78% Sequence identity to those of the human, rabbit rat and mouse

(Fig. 12).

Exonic sequence analysis of the ovine 1 LkHSD2 gene revealed that it is very similar to the

published ovine kidney 1 lbHSD2 cDNA sequence except for two single nucleotide

Figure 8 Selection of the positive clone containhg the 1 1 pHSD2 gene for further analysis. The upper panel displays the Southem blot analysis of the four clones. Each clone had been digested with BarnHI and EcoRI. The third clone was used for further studies. The lower panel displays the ethidium bromide s h e d agarose gel electrophoresis of the third clone chosen from the above Southem blot analysis. This clone was digested with BarnHI, EcoRI and BamHI+EcoRi.

Figure 9 Suategy for sequencing reactions. Vertical lines represent restriction enzyme cut sites. open boxes represent exons. and the numbers below the exons indicate the nucleotide position in relation to the cDNA sequence. Dotted Line represents a break in the exonic sequence. The mows indicate the primer annealing site within or flanking the ovine 1 lp HSDS gene.

Eco RI

Barn HI

Pst I

Pst I

Pst 1 Pst I

Barn HI

Figure 10 Schematic of the ovine 1 lpHSD2 gene. mRNA and pnmary protein structure. Vertical lines represent restriction enzyme cut sites. open boxes represent exons. and the numbers below the exons indicate the nucleot.de position in relation to the cDNA sequence. Dotted line indicates a break in the intronic sequence.

Barn HI

Pst I

Pst I

Pst Pst

Sac I

P s t I

Barn HI

Sac I

Figure 11 The sequence of the ovine 1 lkHSD2 gene and the deduced amino acid sequence of 1 LB-HSD2 polypeptide. Differences in the published ovine 1 1 PHSD2 cDNA sequence are shown in parentheses. and the two single nucleotide omissions are indicated by #. Spiice donar and acceptor sites are underlinrd and the intronic sequence is shown between these sites.

. . . g g aaggg gtgcgggcag agaagtaaag gggctcttta aaagctcggc ccgagggcga acagagaaag cgagtatccc c t c c c g C C C C TGGTGGTGTC CTGCTGCACC CCGCGTCCCA GCCCCGAGTC CCAGTCCCTG CTCTCCAGCC CYGTCCCTGC CCCGCCCCGC CCCGGCCGCC

M e t G l u Ser T r p Pro T r p P r o Ser G l y G l y A l a T r p L e u L e u 1 4 ATG GAA AGC TGG CCC TGG CCG TCG GGC GGC GCC TGG CTG CTC

Val A l a A i a A r g A h L e u L e u G l n L e u L e u A r g A h A s p L e u 2 8 GTG GCG GCC CGT GCG CTG CTG CAG CTG CTG CGC GCA GAC CTG

A r g L e u G l y -4rg Pro L e u L e u A l a A l a L e u A l a L e u L e u A h 42 CGT CTG GGC CGC CCG CTG CTG GCT GCG CTG GCG CTG CTG GCC

A 1 a L e u A s p T r p L e u C y s G l n A r g L e u L e u Pro Pro L e u A l a 56 GCG CTC GAC TGG CTG TGC CAG CGC CTG CTA CCC CCG CTG GCC

A h L e u A i a V a l L e u A h 3-la T h r G l y T r p I l e V a l L e u Ser 7 0 GCA CTT GCC GTG T T G GCC GCC ACC GGC TGG ATC GTG TTG TCC

Airg L e u A i a A r g P r o G l n Arg L e u P r o V a l A h T h r Arg A l a 8 4 CGC CTG GCG CGC CCG CAG CGC CTG CCC GTG GCG ACT CGC GCG

V a l L e u I l e T h r G l y C y s A s p 9 1 GTG CTC ATC ACC G GTGAGTGC..>1.7kb..CTACCC& GC TGT GAC

Ser G l y P h e G l y A s n A l a T h r hla L y s L y s L e u A s p A l a M e t 105 T C T GGT T T T GGC ÀAC GCG ACG GCC AAG AAG CTT GAC GCC ÀTG

G l y P h e T h r V a l L e u A l a T h r V a l L e u A s p L e u A s n Ser Pro 1 1 9 GGC TTC ACA GTG T T G GCC ACC GTG TTG GAT CTG M T AGC CCT

G l y A l a L e u G l u L e u A r g A l a C y s C y s S e r Ser A r g L e u G l n 133 GGC GCC CTA GAG CTG CGT GCC TGC TGT TCT TCT CGT CTG CAG

Leu L e u G l n M e t A s p L e u T h r L y s P r o A l a Asp Ile Ser A r g 1 4 7 CTG CTG CAG ATG GAC CTG ACC M G CCA GCA GAC ATT AGC CGT

Val Leu G l u P h e T h r Lys V a l His T h r A i a S e r T h r GTG CTG GAG TTC ACC AAG GTC CAC ACC GCC AGC ACT G

GTCAGTAAGC TCAACTTCGC CAGGAAAGGG CATGGCTGGG CAGATGTGAG GGGGCCAGGA TTGGAGGTGT GCGGTGGGAC TGACCCGCAG CTTCCTCTGG

G l y L e u T r p G l y L e u V a l A s n A s n A l a G i y 169 CTCCATGCCC TC= G T CTG TGG GGC CTG GTC AAC M T GCG GGC

G i n Asn I l e P h e V a l A l a A s p A h G l u L e u C y s P r o V a l A h 183 CAG 2 C ATC T T T GTG GCG GAT GCA GAG CTG TGT CCG GTG GCC

T h r P h e A r g T h r C y s M e t G l u V a l A s n P h e P h e G l y A l a L e u 1 9 7 ACT TTC CGC ACC TGC ATG GAG GTG AAC TTC T T T GGT GCA CTA

G l u M e t T h r L y s G l y L e u L e u Pro L e u L e u A r g A r g Ser Ser 211 GAG ATG ACC XiA GGC CTC TTG CCA CTG CTG CGT CGT TCG AGT

G l y A r g I le V a l T h r V a l Ser Ser Pro A h GGT CGC ATT GTG ACC GTA AGC AGC CCA GCA G GTGAGGGATC

TCCTCCCGCA CTGGAGTCCC

CTGGAGCAGA CGGCCCAGGC

AAÀGATGCCT TGAGGCATCC

CTGCTGGGCA GCCACTCCCA

GGGAGAGATA ATCCTATCCC

Gly A s p M e t P r o P h e Pro C y s L e u A i a A l a T y r G l y C A G E GA GAC ATG CCG T T T CCA TGC TTG GCT GCC TAT GGG

T h r Ser L y s 41a A l a L e u A l a Leu L e u M e t G l y A s n P h e Ser 2 4 7 ACC TCC A M GC-9 GCC TTG GCA TTG CTC ATG GGC FAT T T T AGC

C y s G l u Leu L e u P r o T r p G l y V a l L y s V a l Ser I l e I l e G l n 2 6 1 TGT GAA CTT CTG CCC TGG GGT GTC AAG GTC AGC ATC ATC CAG

( T l

P r o A l a C y s P h e L y s Thr 267 CCT GCC TGC TTC AAG ACA G GTGAGGCT CAGGGATTTG GGGGTGGCCT

GTCGGGGAGA GGAGGAAGGC AGGGTGGGTG TGGTCTTCTC TGGAGTGGGA TTGGGTCTGA GGMTGGGCC TGACTTTGGC TGGGGCGGGT CTCAGGTTGT GAGTTAGAGA CCTGGGTCTG GGTTTGGCTT CCCTCTCTGA CCCCTTTCTG

G l u Ser V a l L y s A s p V a l H i s 2 7 4 GCCTTGGCAC T C C T T C C G C S AG TCA GTG AAG GAC GTG CAC

G l n T r p G l u G l u Arg L y s G l n G l n Leu L e u A l a T h r L e u P r o 288 CAG TGG GAA GAG CGC AAG CAG CAG CTG CTG GCC ACC CTG CCG

G l n G l u Leu L e u G l n Ala Tyr G l y G l u A s p Tyr I l e G l u H i s 302 CAA GAG CTG CTG CAG GCC TAT GGT GAG GAC TAC ATC GAG CAC

L e u A s n G l y G l n P h e L e u His Ser L e u Ser G l n A l a Leu P r o 316 TTG M T GGG CAG T T C CTG CAC TCT CTG AGC CAG GCC CTG CCA

A s p L e u Ser Pro V a l V a l A s p A l a I l e T h r A s p A l a L e u L e u 330 GAC CTC AGC CCG GTG GTA GAT GCC ATC ACC GAT GCG CTG CTG

A i a A l a G l n Pro A r g Arg Arg Tyr Tyr P r o G l y H i s G l y Leu 3 4 4 GCG GCC CAG CCÀ CGC CGC CGC TAT TAC CCA GGT CAT GGC CTG

Gly L e u I l e Tyr P h e I l e His Tyr Tyr Leu Pro G l u G l y Leu 358 GGG CTC ATA TAC T T C ATC CAC TAC TAC CTG CCT GAG GGC CTG

( # 1

A r g G l n A r g P h e L e u Gln Ser Phe P h e 11e Ser P r o Tyr Val 372

P r o Arg A l a L e u G l n A l a G l y G h Pro G l y L e u T h r Ser A l a 386 CCA AGA GCA CTA CAG GCT GGC CAG CCT GGC CTT ACC T C T GCC

( *

A r g Asp I l e A i a G l n A s p G l n G l y Pro A r g L e u A s p Pro Ser 400 CGG GAC ATA GCC CAG GAC CAA GGC CCC AGA CTG GAC CCC T C T

P r o T h r A h Gln s t op 4 0 4 CCC ACT GCC CAG TGA GCAGGGCACA TGTAGAGCAG CTCCAGCAGA

GGAGGTTCCT TCTGCCCTAG CACAGGCCAG TCCTGCTTCA GCAGCTTGCA GAGCCTTAGC TTAGGTTGGT AGTGCTGTGA AGGGCCCTGA ACTCACCTGA GAATGGGGGA GACCAGAACT

TGTGCCCTTG AGCCTGGCCC GCCTGGTGAG TGAGCCCAAA GAACCTAGCT TAGGATCCTA TGGGGACCCC TTTGAAGGGT CCACCCACCC TTGACACTTT AATGCTCTGT G C T . . . .

CTCTTCTTCC AFAGGACCCC GTGAAGGCTT CAGACCCTCC GGATGGGAAG CAGACAGTGC CTCAGGATTA GAATCCTGTT CAAACCCCTC TTGAATAAAG TTGCCTAGTG

AGGTATTCTG ACCCCCMGG AACCCATGCA CTGCCGATGC TCCAGTGAAC TCTGGGCCTC TGGGCACAAG GCTCCACCAT TTTAGTTCAA GGCTTAACTA CTCTGCAIWC TAAGGCGAGA TTTCTTGGCA CCAGTGCCTC TCTTGACTGG CTCAAGGATT AAGCCACAGG GAGGCTACAT ATAAATTTTT ATTTCTCCTA ACAGGCTGTG GCGTGTGACA

Figure 12 Cornparison of the deduced sheep. human. rabbit, rat and mouse ll&HSDZ amino acid srquences. Hyphens indicate identical amino acids with mptrt to the sheep sequence. and # indicates gaps that are required to align the sequences. Differences in the published cDNA-deduced sheep 1 IP-HSD2 sequence are shown in parentheses. The conserved SCAD domains are underlined,

(VSCSPSSSVPMCQEHYRLPAWPYLCPGHSPGPRPQTGPLSHCPVSRA) SHEEP RFLQSFFISPYVPRALQAGQPGLTSARDIAQDQGPRLDPSPT++~AQ 4 0 4 H U ~ - - - -A----HcL-----P---- T-PPQ-A---gii-N-S-G-SPAV-R 4 0 5 BIT - - - - - - - - I -cL--- -R~--- -A-P-P-T---Na-NPN-D-SLVG-R 4 0 6 FAT - - - -N- - - -HLL- - - -R~- - - - %gPVH-TT--gi-NPS-T##+VS-L 4 0 0 MOUSE C---N---NHLL----RP--H--LLLKTHsi#g#iiie##g-SPVA-L 3 9 6

differences. and two single nucleotide omissions between nucleotides 1165- 1 166 (C). and

1228- 1229 (G) in the published cDNA sequence. The omissions caused a shift in the open

reading frame at the codon for residue 358. As a result, the published cDNA predicted a

protein of 427 amino acids. The other two differences lie within the codons for residues

26 1 (CTG to CAG; Leu to Gln) and 36 1 (GGT to CGT: Gly to Arg) (Fig. 1 1).

1 1P-HSD2 is a member of the shon chah alcohol dehydrogenase (SCAD) family

(Krozowski. 1992). The predicted ovine 1 lBHSD2 protein contains five of the six SCAD

farnily conserved domains. Dornain A. the cofactor binding region (residues 82- L 11);

domains B and C, structural domains (residues 160- 170 and 2 12-222); domain D, the

cataiyuc site (residues 232-249); and domain E. a framework site (residues 255-269)

(Krozowski, 1992) (Fig. 12).

3.3.2 STRUCTURE OF THE 5'-FLANKING REGION

The lkb 5'-tlanking region (Fig. 13) of the ovine 1 1 &HSD2 gene contains no TATA or

CAAT consensus sites (Fig. 14). This region of DNA is very G+C-nch, having a G+C

content of approximately 70%. This is consistent with the human 5'-fianking region

(Aganual and White. 1996). The sheep S'-tlanking region is highly conserved with the

human xquencr. with the 325bp immediately upsveam of the start site of translation

(ATG) being approximately 75% identical.

Using MaiInspector (Quandt et al.. 1995) two GC-boxes (possible Sp 1 binding sites) were

detected in the 5'-tlanking region of the sheep 1 1P-HSD2 gene. The position of these GC-

boxes coincides with the Sp 1 binding sites identified in the human 1 1 PHSDZ 5'-flanking

region (Aganval and White. 1996). and the consensus sequences are iden tical between the

two sprcies.

Figure 13 Schematic view of the ovine 1 LpHSD2 gene with an expanded view of the 5'- flanking region. Vertical lines represent restriction enzyme cut sites, boxes represent exons. The numbers on the full view indicate the nucleotide position in relation to the cDNA sequence, whereas the numbers on the expanded view indicate the nucleotide position in dation to the start site of translation (ATG). Dotted line indicates a break in the intronic sequence.

Eco RI + Barn HI

Pst I

Pst I

Pst I Pst I

Barn HI

Sac l

Pst l

Barn HI

Sac I

ATG

Pst 1 (+60)

Nco I (+1)

- Sma 1 (-1 64)

- Sac 11 (-278)

- Sac Il (-51 5)

- Dra 11 (-922)

- Bst XI (-940)

- Barn HI (-990)

Table 5 Transcription regdatory elements and binding factors (Quandt et al.. 1995; Lockler, 1996)

(Class 1 enhancer) AP2 -124 to-132, -149 to-757

C/EBP site (AFP box) liver, fat cell C/EBPB - 799 t~ -206

6EF1 site (Polyoma Enhancer site A3 - PEA3) 6 EF1 -234 to -24 7

E2A site (Polyoma Enhancer site A3 - PEA3) €47 -233 to -24 7 , -467 to -479

E2F site General, ceil cycle DP1 :E2F -767 to -774

Elkl site (Polyoma Enhancer site A3 - PEA3) Elkl -534 to -538

Ets site (Polyoma Enhancer site A3 - PEA3) cEts1 P54 -299 to -307

GATA site Red cell, T ceIl developrnental; GATL -702 to -707

Family

defined, but not characterized

leucine zipper dornain

homeobox domain/zinc finger domain

basic domains; helix-loophelix; ubiquitous (class A) factors

basic domains; helix-loophelix

helix-turn-helix; tryptophan clusters; Ets-type

helix-turn-helix; tryptophan clusters; c-Ets- 1

zinc finger domain

TPA, PKC, CAMP & ras gene inducible; frequentiy next t o other enhancer elements

family o f mostly liver- and fat-cell-enriched factors

Factor that regulates lensspecific transcription of the 6 crystallin gene

bind as heterodirners t o E boxes in the lg gene enhancers

heterodimeric cell cycle regulators; form complexes with Rb inhibit transactivation

immediately upstrearn (3 bases) o f SRF binding site (CA& box); SRF dependent

found as compound with AP1 site in PEA3 and several other genes

in promoters and enhancers o f erythroid-specific genes

Table 5 continued

EJement/Factor location in sheep

Famiiy Description

GC- box SPI -188 to -797, -264 to -273

. --

zinc finger domain; Kru ppel

- . - -.

activator found in many promoters & enhancers; linking distant elements

IRF2 Interferon- stimulated and virus response elements - 7 70 to -122

helix-tum-helix; tryptophan clusters; interferon- regulating factors

reg date interferon genes & [RF-responsive genes; constitutive repressor

LyFAkaros site B cell, T cell LyF, lkl-5 -777 to -782

regulate numerous lymphocyte-specific genes

Muscle E box (Poly- oma Enhancer site A3 - PEA3) MyoD -233 to -238,

basic domains; helix-loophelix factors; rnyogenic transcription factors

regulate myogenesis; bind as heterodimers to E box (central motif binding site)

element (MRE) I tryptophan clusters; l product; essential rote in cMy b Myb; Myb-factors hernatopoietic proliferation

-469 to -474 ~ y b recognition

-485 to -490 MZFl sites Myeloid, kidney MZF1, Kid 1 , PLZF -93 to - 1 05 NFKB site (Enhancer core AP3 site,) NF KB - 7 93 to -202 S8 site, developmental S8 -884 to -893

zinc finger domain

helix-tum-helix;

pscaffold factors with minor groove contacts; Rel homolog region; ReVanky rin

nuclear protooncogene

homeobox domain factor (variant)

kidney ischemia and developmentally regulated gent and promyelocytic leukemia

Re1 homology domain factors are activated by many extracellular stimuli

murine; variant domain, resembles the prd class

~

USF site (Polyoma Enhancer site A3 - PEA3) USF -462 to -474, -557 to -569

basic domains; helix-loophelix/leucine zipper factors; ubiquitous bHLH-ZIP factors

common upstream activator involved in DNA looping tetramerization of USF dimers

Figure 12 Aligned sheep and human 5'-flanhg regions of the I l BHSDZ gene. Sequences which are conserved between the two species are indicated by hyphens and # indicates nucleo tide gaps that are required to align the sequences. Possible transcnptio n factor binding sites are indicated by a single underline and their consensus binding sites are shown below. The Sp 1 binding sites ( c o d m e d for the human sequence) are indicated by a double underline. The numbering of the nucleotides is in relation to the start site o f translation (ATG). Transcriptional start sites for the human 1 LfLHSD2 gene are indicated by T.

----A ---- - C CTGG (USF)

ATTGI'03GAA G G T F E I G CAACCCAGGG -7 5 8 CC---CGC '333%--CE -T----C -G--T-C-C- hiari;iri -771

(LyFI GGGAA AT

(E2F) TTTCGCG C

TGXKTDC C w aheep -560 G-C-GCPIC- -GA--- -*-A-## if------- < h~man -579

(US F ) CCGGTGCAC

GCCNNNG GC ( A P 2 ) -Y

(MZF 1 ) T T C C C C C T C N C C C G ( I RFS) GAAAAGCT GAAAGC

sheep +3 human +3

Several other possible transcription factor binding sites cornmon to both the sheep and the

human sequences were also identified. These sites were identified using a MatInspector

filter with a core similarity of at least 80% and a ma& similarity of at least 85%. The

location of the identified sites (PEA3: USF, Elkl. E47[E2A]. cEts 1 P54; MyoD and 6EF1:

CM yb; NF& [Enhancer Core AP31; CIEBPP: AP2; MZF l and RF2 [Interferon-stimulated

response rlement]) are illustrated in Figure 14 and a description of each is given in Table 5.

Less probable transcription factor binding sites found only in the sheep sequencr. using the

same MatInspector füter are as follows: E2F; GATA; LyF [Ikaros site]; and S8. The

position of these possible factors are d s o indicated on Figure 14 and a description of each

is provided in Table 5.

3.4 DISCUSSION

3.4.1 THE OVINE 1lP-HSD2 GENE AND THE DEDUCED PROTEIN

This study reports the characterization of ovine 1 lPHSD2 gene. This gene predicts a

protein of 404 amino acids, similar to the deduced human (Aibiston et al., 1994), rabbit

(Naray-Fejes-Toth and Fejes-Toth. 1995), rat (Zhou et al.. 1995) and mouse (Cole, 1995)

protein but distinct Crom the 427 amino acid protein predicted by the published ovine 1 lp-

HSD2 cDNA (Aganval et al., 1994). This discrepancy is attributed to two single nucleotide

omissions in the pubiished cDNA sequence which resulted in the utilization of a different

reading frame at the 3'-end of the coding sequence. Therefore, the present resulis have

provided conclusive evidence that the primary structure of 1 1 P-HSD2 protein is well

conserved beween the sheep and the other four rnammals.

The ovine 1 1 fbHSD2 gene consists of 5 exons and spans over 9 k b . Two reports have

been published describing the cloning of 1 lkHSD2 gene from the human and mouse

(Agarwal et a1..1995a; Cole. 1995). and both genes show identical structure to the ovine

gene descnbed here.

The predicted m i n o acid sequence of ovine 1 L PHSD2 is only 18% identical to that of

1 1 BHSD 1 (Yang et al.. 1995). When the sequences of ovine 1 1 pHSD2 gene and human

1 1 BHSD 1 gene (Tannin et al.. 199 1) were aiigned. it becarne clear that these two genes

belong to different Families because their introns do not correspond in number or location.

3.4.2 THE 5'-FLANKING REGION OF THE OVINE 1lP-HSD2 GENE

Previously most molecular biologists believed that the majority of promoters for eukaryotic

protein-coding genes contained a TATA box approxirnately 25 base pairs upstream of the

start site of transcription. This theory is now being dispeiled. as a growing number of

promoters for higher eukatyotes are k ing discovered which lack the consensus TATA

box, and instead have a highly GC-rich region immediately upstream of the transcription

start site (Beebee and Burke, 1992).

Consistent with its human counterpart, the 5'-flanking region of the ovine 1 lPHSD2 gene

(Figure 10) dws not contain a consensus TATA box. and if is very G+C-rich

(approxirnately 7 0 8 ) ( Aganval et al.. l995a). Within this G+C-rich region two GC-boxes

were identified using the MatInspector program (Quandt et al.. 1995). These potential Sp 1

binding sites are iocated at - 188 to - 197 and -264 to -273 with respect to the start site of

translation. The location and the sequence of the GC-boxes are absolutely conserved

between the sheep and human (Agarwal and White. 1996). Thus. it is Wtely that Sp 1 is

involved in the regulation of the ovine 1 LkHSD2 gene in the same manner as it is for the

human 1 l&HSD2 gene. The Sp l trdnscription lactor is a ubiquitous one which is usually

involved in the regulation of housekeeping genes (Beebee and Burke. 1992). This renders

it unlikely to be the sole regulating factor involved in 1 lBHSD2 gene expression.

In addition to the TATA and GC boxes. the 5'-flanking region of a gene contains upsveam

regulatory sequences which can sometimes be located several hundreds or even diousands

of base pairs upstream of the start site of transcription. These upstream regulatory

sequences are bound by specific transcription factors which either enhance or repress the

expression of a given gene. A commonly recognized upsüeam regulatory sequence is the

CAAT box, which is usually located 70 bases upstream of the transcriptional start site.

Many p n e s which lack the TATA box also lack the CAAT box (Beebee and Burke. 1992).

Like its human counterpart, the ovine 1 lbHSD2 gene lacks a CAAT box in its 5'-flanking

region (Aganval et al.. 1995a). Sequence analysis with the MatInspector program (Quandt

et al.. 1995) identified 12 putative transcription factor binding sites which could be involved

in the regulation of both the sheep and human I lkHSD2 genes. These sites include AP2

sites - 124 to -132 and -149 to - 157, to which the uncharacterized AP2 protein commonly

binds (Quandt et al.. 1995); CEBPP site - 199 to -206 to which the leucine zipper protein

ClEBPB commonly binds (Quandt et aL.1995; Lockler. 1996); 6EFl sites -234 to -241 to

which the horneobox/zinc finger protein 6EF1 commonly binds (Lockler. 1996); E2A sites

-233 to -241 and -467 to -474 to which the helix-loophelix protein E47 commonly binds

(Lockler. 1996); Elkl site -534 to -538 to which the helix-turn-helix protein Ek I

commonty binds (Quandt et a1..1995); Ets site -299 to -307 to which the helix-tum-helix

protein cEul P54 commonly binds (Quandt et al.. 1995); [RF site -1 10 to - 122 to which the

helix-mm-helix protein IRF2 commonly binds (Quandt et al.. 1995; Lockler. 1996); Muscle

E boxes -233 to -238 and -469 to 474 to which the helix-loophelix protein MyoD

commonly binds (Quandt et al.. 1995; Lockler. 1996); Myb site -485 to -490 to which the

helix-turn-helix protein cMyb commonly binds (Quandt et ai., 1995; Lockler, 1996); M E 1

site -93 to - 105 to which the zinc finger protein MZFl commonly binds (lockler, 1996);

NFicB site -193 to -202 to which the B sheet protein NFKB commonly binds (Quandt et

al., 1995) and USF sites 462 to -474 and -557 to -569 to which the helix-loop-helix

protein commonly binds (Quandt et al.. 1995; Lockler. 1996). These identified transcription

factor binding sites may be involved in the regulation of 11 P-HSDZ gene expression, as

thcy are conserved between the two species (Fig. 14).

Four additional consensus elements. which are not present in the human pne, have been

identified in the ovine gene. using MatInspector (Quandt et al.. 1995). These elements are

potential binding sites for E2F (E2F site -767 to -774) a helix-loophelix protein (Lockler.

1996); GATA (GATA site - 102 to - 107) which contains a zinc finger motif (Lockler.

1996); Lyf (LyFmtaros site -777 to -782) also containing a zinc finger domain (Lockler.

1996); and S8 (S8 site -884 to -893) a homeobox protein (Lockler. 1996). These four

elements are unlikely to be involved in the regulation of the 1 IpHSD2 gene, since they are

not conserved across species.

Eukaryotic transcription factors fa11 into 6 main categories. al1 of which are represented in

the above summary of putative transcription factors and binding sites. These are the helix-

tum-helix motifs. homeodomains, zinc finger motifs, sheets, leucine zipper motifs and

helix-loop-helix motifs (Albe= et al.. 1994). The helix-tum-helix motifs are composed of

two a helices which are joined together by a short extension of amino acids. This amino

acid chah represents what is known as the "tum" (Hanison and Agarwal. 1990: Laughon

and Scott, 1984; Sauer et al., 1982). The homeodomains contain the helix-turn-helix motif,

but also contain a stretch of 60 arnino acids which is almost identical in all homeodomain

proteins (Scott et al.. 1989; Wuthrich and Gehring. 1992). This region resembles the

homeoboxes in Drosophila DNA-binding proteins (Weaver and Hendrick, 1992). The zinc-

tinger motifs contain fmger-iike structures which utilize a zinc molecule within each of the

finprs (Coleman. 1992; Miller. 1985). The P sheet proteins are composed of sheet-like

structures (Knight and Sauer. 1992; Scwabe. 1992). The leucine zipper motif proteins

interact with the DNA regulatory regions as a dirner. This dimer is made up of two a-

helical monomers which are joined together in a zipper-like formation (Landschuitz et al..

1988; Lamb and McNight, 1991). The helix-loop-helix motif is related to the leucine zipper

motif. This motif is distinct from the helix-tum-helix motif. and contains a short and a long

oc-helix connected by a flexible arnino acid loop ( G m l l and Campuzano, 199 1).

3.5 CONCLUSIONS

Both 1 1 PHSD2 gene and its 5'-flanking region are highly conserved between the ovine

and human. The two sequences show greater than 70% homology in the 325 bp

irnmediately upstream of the translational start site. This similarity in sequence tends great

support to the notion that the two 1 1P-HSD2 genes are iikely to be regulated by common

mechanisms/factors. The enzyrnatic properties of 1 1 fbHSD2 are identical between the two

species. For example. the Km values for cortisol in human tissues range from 14.4nM-

79.8nM (Brown et al., 1993: Pepe and Albrecht. 1984; Brown e t d.. l996a: Reeves. 1995;

Whorwood et ai.. 1994; S tewan et ai.. l994a; Ferrari et al., 1996b) and those in sheep

tissues from 2 1.3nM- 1 lOnM (Gomez-Sanchez et aL.1997; Yang and Yu. 1994: Yang.

1994; Yang and Manhews. 1995) (Table 2 - Chapter 1). The Km for cortisol of 1 lp-HSD2

in the human choriocarcinoma (JEG-3) cells has been determined to be 3 1nM (Pasquarette

et al.,1996). which is consistent with the values for both human and sheep tissues.

After detemining the structure, sequence and characteristics of the ovine 1 1 &HSD2 gene

and its 5'-flanking region. the next step in understanding how the expression of this gene is

regulated is to identify the factors which regdate 11 kHSD2. An attempt was made to

prepare a reporter gene constnrct to measure the promo terlenhancer ac tivity of the cloned

5'-flanking region and to identify which of the putative transcription factors are involved in

regulation. The initial step of this construction involved removal of the fnmcriptional start

site by restriction enzyme digestion with NcoI foilowed by S 1 nuclease digestion. After

several unsuccessful trials with S 1 nuclease. which failed to digest the cut DNA. the

approach to studying 1 lp-HSD2 regulation was shifted to identifying factors which

regdate 1 1 bHSD2 enzyme activity in the human chonocarcinorna ceil Line JEG-3.

Additional reasons for shifting the focus of this study to regulation of 1 1 PHSDZ enzyme

activity are ( 1) the promoter region was mapped for the human 1 1 PHSD2 gene (Aganuai

and White. 1996) during the trials with S 1 nuclease; and (2) there is a very littie known

about the regdation of 1 l&HSD2 in any species or ceil line.

The JEG-3 ce11 line was chosen for this study for several reasons. First, E G - 3 ceiis had

b e n shown to express 1 lbHSD2 activity and mRNA., and there was no sheep cell line

availabk which was known to express 1lP-HSD2. Second, tlle structure and sequence o l

1 IP-HSD2 gene and its 5'-flanking region as well as the chmcteristics of 1 IP-HSD2

enzyme activity are very similar between the sheep and human. Third. regulatory

mechanisms of a given gene are highly conserved between marnmalian species. Finally. the

ultimate goal of studies using an animal model. such a s the sheep. is to help understand the

human 1 18-HSD2 gene and its regulation.

CHAPTER 4 - REGULATION OF 11P-HSD2 IN JEG-3

HUMAN CHORIOCARCINOMA CELLS

4.1 INTRODUCTION

The human choriocarcinoma cell line JEG-3 was estabLished h m one of six clonai lines in

197 1 from a Woods strain of Emin-Turner tumor. It was in its 387th passage in the

hamster cheek pouch before king harvested for culture (Kohler and Bndson. 197 1). In

1975. a culture of these cells at passage 103 was supplied to Amencan Type Culture

ColIection (ATCC. Rockville MD).

JEG-3 ceils have the phenotypic characteristics of syncytiotrophoblast cells (Deutsch et

al.. 1990: Tuan et al.. 199 1; nekis and Benveniste. 1989). They grow a s large,

multinucleated cells (Tuan et al.. 199 1) which secrete human chorionic gonadotropin.

chorionic sommatouopin. and progesterone (Kohler and Bridson. 197 1; Moise et al.. 1986:

Tuan et al,. 199 1 ; Ilekis and Benveniste. 1989).

The syncytiotrophoblast is a thin layer of cells which grow on top of the cytotrophoblast

cells on the chorionic villi of the placenta (Tuan et al.. 199 1) (Fig. 15). This ce11 type has

b e n reported to be the primary site of 1 lPHSD2 expression in the placenta (Stewart et

ai.. 1995a; Krozowski et al.. 1995~; Pepe et al.. 19%). This is dso the site of maternai-fetal

exchange (Pepe et al.. 1996; Tuan et d.. 199 1).

JEG-3 cells are large multinucleated epithelial-like cells which grow in large clusters.

eventuaily connecting with one another (plate 1). These clusters contain well-defined

borders outlining the cellular mass. In the sub-confluent culture, fibroblast-like extensions

appear when the clusters are expanding (plate 1). After reaching confluence. the cells tend

Figure 15 Morphological structure of the human placenta and its chononic villi. The upper illustration diagrams a cross-section, showing the location of the chorionic villi within the placenta. The middle illustration diagrams the structure of the chorionic villi, taken from the area indicated by a box in the upper illustration. The lower illustration diagrans an expanded version of the chorionic villi taken from the area indicated by a box in the middle iIlustration. Modified from Benirschke and Kaufmann, 1992 and Eaton and Contractor, 1993.

inte~illous space

chorionic villous tree

syncytiotrophobla (multi-nucleated)

intervillous space

basement

intervillous space

syncytiotrophoblast

basement

endotheliurn

fetal capillary lumen V

Plate 1 Human choriocarcinorna EG-3 ceils in culture magnïfied 100X. Cells are approximately 50% confluent and are therefore stiU expanding. Note the fibroblast-like extensions.

Plate 2 Human choriocarcinorna JEG-3 cells in culture magnitied 100X. CeUs have xached contluency.

to grow on top of each other (plate 2). These observations are consistent with those

described by others who culture this cell line (Tuan et al.. 199 1 ).

The KG-3 ceils were found to grow at a much faster rate and showed an increased

adherence to the plastic when the culture ware was changed from Falcon to Coming-Costar

dishes. This phenornenon has been docummted by others (Tuan et al.. 199 1).

As stated previously. relatively Little is known about the regulation of 1 lBHSD2. This

study was designed to examine the cffects of a range of physiological hormones and factors

on the activity of 1 lbHSD2. The factors used in this study are forskolin. carbenoxolone.

EGF. 9-cis-retinoic acid. dl-mans-retinoic acid. PG&. PGF,. indomethacin, nordihydro-

guaiaretic acid. PMA. insulin. IGF- 1. dexamethasone. esîradiol. progesterone, thyroid

hormone testosierone and TNFa. Many of these factors were chosen because they are

known to have regulatory effects on other members of the SCAD family. such as 1 1 B-

HSD 1 and 17p-HSD. Others were chosen because they are produced locally in the

placenta. The experiments described below represent a very important first step in

rlucidating the regulation of the 1 1 P-HSD2 gene.

4.2 MATERIALS AND METHODS

4.2 .1 CULTURE OF JEG-3 CELLS

EG-3 cells (ATCC. Rockville MD) were grown as monolayers in Minimal Essential Media

containing non-essential amino acids, sodium pyruvate, penicillin/streptomycin and 7.5%

fetal bovine serurn (Gibco BRL, Buriington ON) in Coming T-25 llasks (Fisher Scientifc.

Toronto ON). The cells were kept in a 5% Cm incubator at 37°C. The medium was

changed rvery other day and the cells were passed. using trypsin (Gibco BRL. Burlington

ON). at a 1:6 dilution 1-2 tirna per week.

Cells were Frozen in Iml aliquots of culture medium containing 5% dimethyl sulphoxide in

Nalgene cryovials (VWR. Mississauga ON) and stored in Liquid nitrogen vapour in a

cryoprexrvation tank.

4.2.2 TREATMENT OF JEG-3 CELLS WITH VARIOUS COMPOUNDS

E G - 3 ceils were passed to Corning 12-well plates and grown to confluence in medium

containing serum. The celis were then cultured for 24 houn in s e m free medium pnor to

katment.

The ceils were treated with the following compounds (Table 6) in semm free medium for

12-48 hours: iOpM Forskolin [positive conuol]; 0.1 - 1 O p M Carbenoxolone; 1Op.M

Forskolin + 0.5- lOpM Carbenoxolone; 1 - lûûng/ml EGF; 1 pM 9-cis-Retinoic Acid; 1 pM

9-cis-Retinoic Acid + 5ûnglml EGF; O. 1nM- LpM ali-nm-Retinoic Acid; lpM dl-nans-

Retinoic Acid + 50ng/ml EGF; lpg/ml Prostaglandin E, (PGE,); 1 pdml Prostaglandin F, -a

(PGF, ); 1-100 pM Indomethach; LOOpM NDGA; 1OpM Lndomethacin + 1OOp.M NDGA; -a

5ûnM PMA (phorbol ester) [negative controll; 5pM Insulin; 50ng/ml IGF- 1; lûnM

Dexamethasone; lOnM Estradiol (E2); lpM Pmgesterone (Pq); lOnM Estradio1 + 1 p M

Progesterone; 10 nM Thyroid Hormone (T3); lûnM Testosterone; and 10 ng TNFa. Each

set of ce11 treatmenls included conuol cells which were untreated. Compounds used in this

study were purchased from Cayman Chemicals. Ann Arbor MI; Hoffmann-LaRoche

insutute. Fort Worth TX; ICN. Costa Mesa CA; Lilly Pharmaceuùcals. Independence OH;

R&D Systems. Minneapolis MN; Sigma. St. Louis MO; Steraloids. Wilton NH.

4.2.3 ASSAY OF 1IP-HSD2 ENZYME ACTIVITY: RADIOMETRIC

CONVERSION ASSAY

At the end of the treatment, the cells were washed 3 times with HBSS to remove the

compounds. in an attempt to exclude their having a possible cornpetitive inhibition. The

level of 1 I P-HSD2 activity in the washed intact ceUs was determinrd by a standard

radiometric conversion assay. as descrikd previously (Yang et al., 1997). Bnefly. the

cells were incubated for 4-7 hours at 37'C in serum free medium containing - 100 000 cpm

[3~]-conisol plus 5nM unlabeled cortisol. At the end of incubation. the medium was

removed tiom the crils. and the steroids were extracted with ethyl acetate containing 40pg

mixture of non-radioactive cortisol and cortisone as cmier steroids. The extracts were dried

down under air and then resuspended in 1 0 p l ethyl acetate. A fraction of the resuspension

was spotted on a Whatrnan siiica p l TLC tlex-plate (VWR. Mississauga ON) which was

developed in chloroform/methanol(9: 1. vfv). The non-radioactive carriers allowed for

identification of the bands containing the labeled cortisol and cortisone under UV light

T h w bands were cut out into scintillation vials and counted in Scintisafe (Fisher Scientitk,

Toronto O N scintillation cocktail. From the s p ~ i f i c activity of the labrled cortisol and the

ndioactivity of cortisone. the rate of conversion of cortisol to cortisone was calculated as a

percentage. and expressed as 1 1 P-HSD2 enzyme activity.

4.2.4 Analysis of the 11P-HSD2 Enzyme Activity Data

The value reprwnting the 1 lpHSD2 enzyme activity was converted to a percentage of the

control value pnor to graphing. These graphs include error bars which represent the

standard error of the mean which is calculated by dividing the standard deviation (average

distance each value is from the mean) by the square rwt of the number of tirnes the

Table 6 Compounds used in ceii matrnents

I Compound

Carbenoxolone (CBX)

Epiderrnal Growth Factor (EGF)

Description

CioHsoOi: MW 570.77; 1 8P-glycyrrhetic acid hydrogen succinate; anti-infiammatory glucocorticoid related t o enoxalone ( 1 8fbglycyrrhetinic acid/ glycyrrhetic acid)

CzzHzsFOs; MW 39 2.47; synthetic glucocorticoid "\ON

O

6 kDa polypeptide cytokine; binds t o the EGF receptor, which is a transmembrane glycoprotein; binding of EGF t o it's receptor stimulates tyrosine kinase activity of the receptor; levels of EGF in humans: urine 100ng/ml, milk 80ng/ml, plasma Zng/ml. saliva 1 Zng/ml, amniotic fluid 1 ng/ml

CrsH240< MW 272; derived f rom cholesterol; serum concentrations: adult male 73-1 84pM adult female 73pM (early fo1licular)- 1 285pM (luteal)

C2~f-bO7; MW 4 1 0.5; a diterpene from the roots of Coleus forskohlii; a hypotensive agent; stimulates CAMP formation by activating adenylate cyclas~ CHJ

H3C CH3

Table 6 continued

Compound

lndomethacin

Insulin

Insulin-Like Growth Factor 1 (IGF- 1 )

Nordi hydroguaia- retic Acid (NDGA:

Description

Ci&i6CfN04; MW 357.79; blocker of prostaglandin biosynthesis; non-steroidal anti-infiammatory agent; irreversi bly inhibits cyclooxygenase pathway of arachidonic acid metabolism

MW 5808; 55 arnino acid protein composed of 2 peptide chains (A & B); chains are joined by a disulfide bridge; circulatory half-life of 3-5 minutes; normal fasting level in human serum is 69pM; normal level in human serum after a meal is 690pM; binds to receptor on the ceIl surface of the target cell membrar

70 amino acid protein; structuraliy similar to preinsulin; binds to IGF-1 cell membrane receptor which stimulates tyrosine kinase activity and autophosphorylation of tyrosine molecules; normal adult senrrn levels are 90-3 1 8p g/L

Crdirz04 MW 302.4; plant lignan; phenolic antioxidant; known inhibitor of OH

the lipoxygenase pathway of arachidonic acid OH

meta bolism; inhibits leukotriene production HO

Protein kinase C agonist; a phorbol ester

Table 6 continued

Cornpound

Progesterone (P4)

Prostaglandin E; (PGEz )

Prostaglandin FZ (PûFz a)

9-CieRetinoic Acid (SC-RA)

Al 1- Trans- Retinoic Acid ( AT-RA)

Description

CtiH300< MW 3 1 4.47; derived from cholesterol; serurn concentrations: adult male 0.3-0.9nM adult female 1 nM (folIicular phase) - 64nM (luteal phase)

CmHazOs MW 352.47; O

most common and biological potent prostaglandin in rnamrnals; an eicosanoid; . product of arachidonic acid "" :

HO metabolism by cyclooxygenase; binds to €Pi, EP2 and EP3 receptors; can increase or decrease CAMP levels dependent on receptor

CmHs40~ MW 354.49; closely related to PGE2; HO

an eicosanoid; product of arachidonic acia metabolism by e #

cyclooxygenase; 0

HO . HO

binds to FP receptor

- -

CzoHzsO4 MW 300.44; binds to both the retinoid X receptor (RXR) and the retinoic acid receptor (RAR), but with higher RXR and affinity RAR bind to the to retinoic RXR; acid HH

COOH response elements (RARES) in DNA as heterodimers

CzoHzeO< MW 300.44; HSC

binds to retinoic acid receptor (RAR); circulates in blood at a concentration of a pproximately 4 . 3 ~ 1 O ~ M

Table 6 continued

Cornpound

Testosterone

Thyroid Hormone (T3)

Tumor Necrosis Factor a (TNFa)

Description - -.

CisHzeO< MW 288.43; derived from cholesterol; serum concentrations: adult male 1 0.4-34.7nM adult fernale 1.04-2.43nM

CisHtaNI3 Q MW 657.03; L-3, 5,3'-Triiodothyronine; binds t o the thyroid receptor which in tum binds to the thyroid-hormone response element (TRE) in DNA; adult serum levels 95-1 90ng/ml I

a cytokine; involved in the immune inflamrnatory response; can kill cells outright; stimulates inflammation; mediates several of the systemic acute phase response

experiment was conducted (Altman. 1995). The standard deviation was calculated using

Microsoft Excel Version 5.0a.

Statisticai analysis was performed on the 1 lPHSD2 enzyme activity resdts in the form of

an Analysis of Variance (ANOVA) followed by a Dunnea's test. Dumett's test is often

used to analyze the mlans of a set of samples to determine the probability (P) that the

means are s~tistically different from the control value. It is a pairWise multiple cornparison

t-test. It adjusts the P values for the multiple comparisons. thereby eliminating the false

increase in probability observed when conducting multiple t-tests. The results are

statistically signiticant if the P value is below 0.05. and if the P value is below 0.01 the

results ÿre highly significant (Altman. 1995). The P value was calculacd for the mean of

rach value compared to the mean. It was calculated using a two tailed, two sample

assuming equal variance (homoscedastic) t-test on Microsoft Excel Version 5.0a

4.3 INHIBITION BY CARBENOXOLONE: POSSIBLE

INVOLVEMENT OF THE PROTEIN KINASE A PATHWAY

4.3.1 BACKGROUND AND RATIONALE

The protein kinase A (PKA) pathway has b e n irnplicated in the upregulation of the I l p-

HSD2 gene in E G - 3 cells (Pasquarette et al.. 1996). The enzyme activity and mRNA level

of 1 1 P-HSD2 were both increased when the cells were treated with forskolin. but were

unchanged when treated with the protein kinase C agonis^ phorbol ester. Therefore the

protein kinase A but not C pathway is involved in upregulating 1 1 p-HSD2 expression

(Pasquarette et al.. 1996).

Forskolin is a diterpene from the roou of Coleus forskohlii (Seamon et al.. 198 1). It

functions to increase the intracellular level of CAMP available for use by the reglatory

subunits of PKA. The forskolin-induced increase in the Ievel of CAMP is a result of

upregulation of adenylyl cyclase (Seamon et d.. 198 1).

Licorice is derived from the roots of the Glvcyrrhizia glabra plant. which is a legurne

commonly found in Asian and Mediterranean countrïes (Baker, 1994). Liconce has been

used as an herbal medicine for more than 2000 years (Baker, 1994). These ancient cultures

used it as a treatment for ulcers. to quench thirst and as a flavoring agent (Baker. 1994).

During the 1950's it was discovered that licorice has anti-intlammatory effects and can lead

to fluid retention (Baker. 1994). Licorice extract contains the two active compounds

glycyrrhizic acid (GA), which is a glycosylated triterpene and glycyrrhetinic acid (GE). an

aglycone of GA (Baker. 1994). Carbenoxolone is a synthetic analog of GE. It is more

soluble and has a longer haif life (Baker. 1994).

Both GA and CBX are known to inhibit I 1P-HSDl enzyme activity and mRNA

(Whonvood et al.. 1993a; Yang et al.. L997a). and they are also potent inhibitors of 1 lp

HSD2 enzyme activity in JEG-3 ceIls (Gomez-Sanchez et al., 1996). Since the cells were

CO-incubated with the inhibitors and the substrate HI-cortisol or H HI-corticosterone) it

rernains possible that this inhibitory effect codd be due to simple cornpetitive inhibition of

the 1 1 B-HSD2 enzyme.

Both 18a- and 1 8PGE have been shown to be powerful inhibitors of the PKA catalytic

subunit (Wang and Polya. 1996). The expression of 1 lkHSD2 is known to be

upregulated by the PKA pathway. Thus. it is possible that CBX inhibits 1 lp-HSD2 via the

PKA pathway. This experiment was designed to test this hypothesis.

4.3.2 RESULTS

4.3.2.1 Carbenoxolone Dose Response

As shown in Fig. 16, carbenoxolone inhibits 1 1 pHSD2 enzyme activity in KG-3 ceils in

a dose dependent manner with a maximal effect of 90% inhibition at 10p.M. This inhibition

is statistically significant for each of the values of carbenoxolone used.

4.3.2.2 Carbenoxolone and Forskolin Interaction

As expected. forskoiin increased 1 1 PHSD2 enzyme activity in IEG-3 cells by

approximately four-fold when compared to the control (Fig. 17).

Carbenoxolone completely elïminated the stimulatory effect of forskolin on 1 lkHSD2

enzyme activity in EG-3 celis, with =ch combined marnent resulting in an activity level

similar to or below that of the unueated cells (Fig. 17).

4.3.3 DISCUSSION

Protein kinase A, in the inactive state. is composed of 2 regulatory subunits which possess

CAMP binding sites and 2 inactive catalytic subunits (Fig. 18). When the regulatory

subunits bind CAMP they undergo a conformational change in structure. This change in the

regulatory subunit structure allows for the release of the catalytic subunits which are now

activated. The activated cata ipc subunits are now free to phosphorylate their appropriate

substrate proteins (Krebs. 1989; Taylor et al.. 1990).

Figure 16 Dose dependent inhibition of 1 lfLHSD2 enzyme activity in JEG-3 celis by carbenoxolone. The ceiis were treated for 24 hr with increasing concentrations of CBX (0.1- LOpM). At the end of rreatment, the cells were washed 3 times in HBSS pnor to the assay of L I pHSD2 activity in intact aek. as described in the Materials and Methods. Error bars represent the standard error of the mean. ** represents Pc0.01 for indicated sample cornpared to the control.

Carbenoxolone (PM)

Figure 17 The effect of CBX on forskolin-induced 1 lkHSD2 enzyme activity in IEG-3 ceiis. The cells were treated with increasing concentrations of CBX (0.5- 1 O W ) in the absence and presence of FSK (1w) for 24 hours. At the end o f the treatment, the celis were washed 3 times in HBSS prior to the assay of i IkHSD2 activity in intact celis. as dacribed in the Materials and Methods. Error bars represent the standard error of the mran. ** represents Pc0.O 1 for indicated sample compad to the control.

1 lOpM FSK-

Carbenoxolone (PM)

Figure 18 Proposed mode1 of actions of carbenoxolone and forskoh within the ceil. Forskolin stimulates CAMP production an hence activates PKA Carbenoxolone inhibits the catalytic subunit of PKA. and subsequently inhibits the PKA stimulation of 1 lEHSD2. The PKA pathway may be regulating 1 1fbHSD2 activity by phosphorylation of a as yet unidentifled protein which in tum rnay increase 1 lBHSD2 activity by having stimulatory effects on I 1 BHSD2 transcription. mRNA stability or translation.

\ inhibition of PKA 1

activation of the catalytic subunit

Forskoiin activates the catalytic subunit of PKA by increasing the levels of CAMP (Seamon

et al., 198 1) (Fig. 18). The catalytic subunit of PKA is known to be potently inhibited by

glycyrrhizic acid and its derivatives (Wang and Polya 1996). Thus. powerful inhibition of

this catalytic subunit by carûenoxolone (Wang and Polya 1996) may render the increase in

CAMP levels inconsequential in the activation of PKA. This could possibly result in no

PKA activation by forskoh with the addition of carbenoxolone. and a possible decrease in

PKA activation. from basai levels, with increwd amounts of carbenoxolone.

The ability of carbenoxolone to completely block the stimulation of 11bHSD2 by the PKA

agonist. forskolin. suggests that the PKA pathway may be involved in carbenoxolone-

induced inhibition of 1 1 FHSDZ.

4.4 REGULATION BY RETINOIC ACID AND EPIDERMAL

GROWTH FACTOR (EGF)


17p-HSD 1 is a member of the short chah alcohol dehydrogenase (SC AD) famdy. All-

ms-retinoic acid (AT-RA) and 9-cis-retinoic acid (K-RA) exert stimulatory effwts on

both 17PHSD 1 mRNA and enzyme activity in JEG-3 celis (Piao et aL.1997). The gene

for 17P-HSD 1 possesses a retinoic acid response element (RARE) at -503 to -487 of the

5'-tlanking region with respect to the start site of translation (initial ATG). This RARE is

responsive to retinoic acid treatrnent in JEG-3 ceils (Piao et aL.1995).

17BHSD 1 enzyme activity and mRNA in IU3-3 ce& are also siimulated by epidermal

growth factor (EGF) (Piao et al.. 1997). This stimulatory effect is potentiated b y AT-RA

and 9C-RA (Piao et al., l997).

As mentioned previously. 1 lkHSD2 is also a member of the SCAD family. It is possible

that common replatory mechanisms may exist between 17PHSD and 1 1PHSD. This

axperimrnt was therefore designed to study the effects of RA and EGF on the activity of

1 lBHSD2 in E G - 3 cells.

4.4 .2 RESULTS

4.4.2.1 Retinoic Acid Time Course and Dose Response

When IEG-3 cells were treated with 1pM AT-RA for variable amounts of time ranging

from 12 to 48 hours. increases in the level of 1 1 P-HSD2 activity were evident at 12 hous

with maximal increases at 24 houn (Fig. 19). The level of stimulation &ter 48 houn of

treatrnent did not diverge from that observed after 24 hours.

AT-RA resulted in a dose dependent stimulation of 1 l&HSD2 enzyme activity in JEG-3

cells. with significant stimulation (- 175%) at lOnM and maximal stimulation (-300%) at

lOOnM (Fig. 20).

To better understand which receptors for retinoic acid are involved in the stimulation of

1 lPHSD2 activity in JEG-3 cells, the ceils were treated with 9C-RA. AT-RA and 9C-RA

preferentially use different retinoic acid receptors, which will be described in more detail in

the discussion of this section, and therefore much c m be deduced from the actions of the

two forms of retinoic acid. 9C-RA resulted in a stimulation of 1 lb-HSD2 enzyme activity

(-320%). similar to that observed with AT-RA matment (Fig. 21).

Figure 19 The effect of AT-RA on 1 l&HSD2 enzyme activity in EG-3 ceils as a function of time. The celis were treated with LpM AT-RA for 12.24 and 48 hours. At the end of the treatment, the cells were washed 3 times in HBSS prior to the assay of 1 lp HSD2 activity in intact ceils, as described in the Materials and Methods. Error bars represent the standard error of the mean. ** represents P d 0 1 for indicated sample compared to the control.

Incubation Time (hours)

Figure 20 Dose dependent stimulation of 1 LBHSD2 enzyme activity in JEG-3 cells by AT-RA. The celk were treated for 24 hours with increasing concentrations of AT-RA (O. 1- 500 nM}. At the end of the ueatrnent, the cells were washed 3 thes in HBSS prior to the assay of 1 I &HSD2 activity in intact cells. as descnbed in the Materiais and Methods. Error bars represent the standard error of the rnean. ** represents Pcû-O1 for indicated sample compared to the control.

All Trans Retinoic Acid (nM)

Figure 21 The effects of 9C-RA and AT-RA on 1 1 P-HSD2 enzyme activity in JEG-3 ceils. The ceHs were ûeated for 24 hours with 1 p M retinoic acid. At the end of the treatment. the ceils were washed 3 times in HBSS prior to the assay of 1 1FHSD2 activity in intact cells. as descnbed in the Materials and Methods. E m r bars represent the standard error of the mean. ** represents P4.O 1 for indicated sample compared to the conuol.

Control AT-RA

4.4.2.2 EGF Dose Response

When EG-3 ceils were treated with increasing concentrations (1-lûûng/ml) of EGF. there

was a concentration-dependent inhibition of 1 lPHSD2 enzyme activity that achieved

statistical significance at greater than Iûng/ml EGF concentrations.

4.4.2.3 Retinoic Acid and EGF Interaction

In an attlmpt to study possible interaction between RA and EGF. the combined treatment

was cmied out in JEG-3 ceils (Fig. 23). Although there was an apparent attenuation by

EGF on RA-induced stimulation of 1 1 PHSD2 activity, it failed to reach the statistical

significance (P>0.05).

4.4.3 DISCUSSION

There are two known receptor classes for retinoic acids. The retùioic acid receptors (RAR-

a. RAR-P. and RAR-y) and the retinoid X receptors (RXR-a, RXR-P, and RXR-y)

(Mangelsdorfet al.. 1994; Rochette-Egly et a1.,1995; Heery et aL.1994; Pfahl, 1996). Both

9C-RA and AT-RA bind with high affinity to the RARs (Heery et al.,1994; Rochette-Egly

et al.. 1995; Pfahl, 1996; Mangelsdorf et ai.. 1994). The RXRs can bind both 9C-RA and

AT-RA but with 40 times greaar affinity for the former (Hwry et al., 1994; Mangelsdorf et

al.. 1994). The activation of RXRs by AT-RA is concentration-dependent in that at

concentrations less than 50nM, AT-RA is unable to bind to the RXRs (Roulier et al., 1996).

This activation of the RXRs at higher concentrations is beiieved to be due to invacellular

interconversion of AT-RA to 9C-RA (Roulier et al., 1996). This interconversion between

AT-RA and 9C-RA is common and the direction of which is ceIl-specific (Mangelsdorf et

al.. 1994).

Figure 22 Dose dependent inhibition of I 1 pHSD2 enzyme activity in JEG-3 ceiis by EGF. The cells were treated for 24 hr with increasing concentrations of EGF (1-100ng/ml). At the end of treatment, the cells were washed 3 times in HBSS prior to the assay of 1 lfb HSD2 activity in intact ceiis. as described in the Materials and Methods. Error bars represent the standard error of the mean. ** represents Pd.01 for indicated sample compared to the control.

I 1 I I 1

O 1 10 50 1 O 0

EGF (ng/ml)

Figure 23 Possible attenuation of the stimulatory effects of retinoic acid by EGF in JEG-3 ceiis. The cens were treated with EGF, RA and EGF+RA for 48 hours. At the end of marnent, the cells were washed 3 times in HBSS prior to the assay of 1 lPHSD2 activity in intact ceik, as described in the Materials and Methods. Error bars represent the standard error of the mean.

Cellular retinoid binding proteins are responsible for transporthg retinoic acids from the

cytosol into the nucleus. where the unbound RARs and RXRs are localized (Mangelsdorf

et al.. 1994). Upon binding by their ligands. the activated receptors c m form homodimers

(RAR-RAR or RXR-RXR) but more often form heterodirners (RAR-RXR) (Pfahl. 1996;

Manplsdorf et ai.. 1994). It is the heterodirners which are then able to bind to the

appropriate response element of a target gene (Rochette-Egly et al.. 1995; Mangelsdorf et

al.. 1994; Chambon, 1996).

The response elements to which these ligand bound heterodirners bind are known as the

retinoic acid response elements (RAREs) (Rochette-Egly et al., 1995; Mangelsdorf et

al., 1994; Chambon. 1996). The RARES contain the consensus motif AGGTCA. which in

the RARES can be arranged as direct repeats (DR-1 AGGTCAnAGGTCA; DR-2

AGGTCAnzAGGTCA; DR-5 AGGTCAnmmAGGTCA; or ER-8 TGACCTngAGGTCA)

or complex elements (AGGTCAnxAGGTCAnx AGGTCA ...). The consensus sequence

may have variations in one of the nucleotides in any of the arrangements. The direct repeat

configuration has a greater binding affmity for the Ligand-receptor complex than does the

complex element (MangeIsdod et d.. 1994).

Retinoic acid treatrnent of JEG-3 cells has k e n demonstrated to exert no effect on

intracellular CAMP levels or cell number (Kato and Braunstein. 1991).

Revious studies have demonstrated that JEG-3 cells contain retinoid binding proteins

(Kato and Braunstein. 199 1) and constitutively express high levels of RXR-a but

negligible levels of RAR-fl (Roulier et ai.. 1996). Although JEG-3 cells have been shown

to lack the RAR-p receptor (Roulier et ai.. 1996). they may possess RAR-a or RAR-y

receptors which could. in combination with a RXR receptor, be involved in the regdation

of 1 1 P-HSD2 (Fig. 24).

This study demonstrates for the f i t time that retinoic acids stimulate I 1 bHSD2 enzyme

activity in E G - 3 cells. and both 9C-RA and AT-RA are capable of doing so.

As discussed in the previous chapter. an Ikb 5'-flanking region of the ovine 1 lp-HSD2

gene was analyzed. In this region no consensus sequence for RARE was identiîïed. These

response elements can occur several thousands of bases upstream of the start site of

transcription, and may therefore occur out of the range analyzed in this study. Indeed,

sequence analysis of the 5'-flanking region of the human 1 lkHSD2 gene (Genebank

U273 17) revealed a putative RARE from -2306 to -2287 relative to the s t m site of

translation (AGGTCAngAGGTCA). It is therefore conceivable that this RARE is

responsible for mediating the observed effects of RAS on 1 lp-HSD2 expression in JEG-3

cel1s.

EGF exerts physiological effects in its targef cells through a tram-membrane receptor

(Pimentel. 1994; Heath. 1993). This receptor is a tyrosine kinase specific protein which

becomes autophosphorylates upon ligand binding (Heath. 1993). Once phosphorylated, the

EGF receptor becomes activated and can then phosphorylate a variety of cellular proteins.

which in turn become activated (Pimentel, 1994).

A large variety of cell types are known to express the EGF-receptor (Pimentel, 1994). The

EGF-receptor h a b e n found to be expressed in the placenta, rxclusively in the

syncytiotrophoblast cells (Pimenal, 1994). It is also expressed in E G - 3 cells (Pimentel,

1 994).

This study is the first demonstration that EGF inhibits 1 lbHSD2 enzyme activity, up to

5 5 8 of the control value in E G - 3 cells. This inhibition is likely mediated by the EGF-

Figure 24 Proposed mode1 of actions of retinoic acid and EGF within the cell. The two compounds appear to have separate pathways in their regdation of 1 1 pHSD2. EGF interacts with the tyrosine kinase receptor and possibly phosphorylates an unidentified protein which in tum decreases 1 1 PHSDZ activity by having inhibitory effecls on 1 1 B- HSD2 transcription. mRNA stability or translation. 9C-RA and AT-EU bind to cellular rethoid binding proteins which transport RA in the cell to its site of action. RA may increase 1 lkHSD2 activity by having stimulatory effects on 1 lkHSD2 û-anscription (via the RAR-RXR heterodimer). mRNA stability or translation.

receptor. which is known to be present in JEG-3 celis (Fig. 24) (Pimentel. 1994; Heath.

1993).

Previous studies have demonstrated that in some instances EGF can potentiate the effects of

retinoic acids (Piao et al.. 1997; Roulier et al.. 1996). lhis potentiation has ben attributed to

the EGF-induced increases in the level of RXRa protein and mFWA (Rouiier et al.. 1996).

The stimulatory effects of both 9C-M and AT-RA on 1 lBHSD2 enzyme activity in EG-

3 cells may be attenuated by EGF. This apparent attenuation may be due to their opposite

effects via distinct pathways (Fig. 24).

4.5 REGULATION BY EICOSANOIDS


Eicosanoids (mainly prostaglandins and leukotrienes) are intimately involved in the

maintenance of pregnancy and parturition (Schafer et al.. 1996). They are known to be

produced in the placenta. decidua, myometrium. and the fetal membranes amnion and

chorion (Behrman and Romero. 199 1). The syncytiotmphoblast has b e n shown to

express both synthesizing and metabolizing enzymes for prostaglandins from early in

gestation (Cheung et al., 1992).

The eicosanoids are synthesized from amchidonic acid (Fig. 25) which is liberated from

fany acid stores in the phospholipid membrane of the cell by the hydrolytic actions of

phospholipase (Behrman and Romero. 199 1 ; KirnbaLl and Kirton. 1986). Arachidonic acid

is rnetabolized by cyclooxygenase to produce the prostanoids (prostaglandins and

thromboxanes): 5-lipoxygenase to produce leukotrienes. 5-hydroperoxyeicosatetraenoic

acid (5 -HPETE) and 5- h y drox yeicosa tetraenoic acid (5-HETE); 1 2- and 1 5- li poxy genases

Figure 25 S ynrhesis of eicosanoids from arachidonic acid released from membrane phospholipids. Indomethacin biocks the cyclooxygenase pathway. NDGA blocks the Lipoxygense pathways. Adapted from Piomeili. 1996.

1 Cell Membrane Phosphollplds 1

) Arachidonlc cid di a 1

S-HPETE

PGD2, PûEZ, PGF 2u. PûI

to produce iipoxins, HPETEs and HETES; and cytochrome P450 monooxygenases to

produce DHTs. EETs and 12-HETE (Behrman and Romero. 1991; Piomelli. 1996).

hdomcthacin. a potent inhibitor of the cyclooxygenase pathway (Ottino and Duncan, 1997;

Gerrard, 1985). is a non-steroidal anti-inflammatory dmg (Ottino and Duncan, 1997). It

inhibits the synthesis of prostanoids by binding to cyclooxygenase and causing a

confornational change in the enzyme which enhances the bond between the two (Omno

and Duncan. 1997). therefore resulting in competitive inhibition (Gemard. 1985).

inhibition of the cyclooxygenase pathway by indomethacin may lead to a shift in

anchidonic acid metabolism to the other pathways. the Iipoxygenases and cytochrome

P450 monooxygenases (Fig. 25) (Behrman and Romero. 1991).

NDGA is one of the weU-known inhibitors of the Lipoxygenase pathways (Fig. 25)

(Behrman and Romero. 1991; Bach. 1984: Fitzsirnmons and Rokach. 1989). It is a plant

lignan (Aganval et al.. 1991) and is widely known for its antioxidant properties (Behrmm

and Rornero, 199 1 ; Bach, 1983).

There are no published studies involving the regdation of 1 1 P-HSD by eicosanoids or their

inhibitors.

4.5.2 RESULTS

4.5.2.1 Effects of Prostaglandins

N E , resulted in an inhibition of 1 1 pHSD2 enzyme activity to approximately 70% of the

conuol level (Fig. 26). PGF, resuited in a similar inhibition of 1 lBHSD2 enzyme activity

to approxirnately 65% of the control level (Fig. 26). The inhibition of I1&HSD2 by

prostaglandins is statistically signircant and is not statistically different for the two

prostaglandins used.

4.5.2.2 Effects of Indomethacin

Indomethacin was used to block the synthesis of prostanoids in an attempt to reverse the

inhibitory affects of prostaglandins. Unexpectedly. it resulted in a dose-dependent

inhibition of 1 lBHSD2 enzyme activity. This inhibition may be a result of an increased

production of other eicosanoids (Fig. 27).

4.5.2.3 Effects of NDGA and NDGA plus Indomethacin Interaction

Due to the unexpected inhibition of 1 LPHSD2 by indomethacin, N M j A was used to

determine if products of the lipoxygenase pathways are involved. NDGA resulted in a 2.5-

fold stimulation of 1 I&HSDZ enzyme activity (Fig. 28). To further study possible

interactions betwean the products of cyclooxygenase and those of lipoxygenases. NDGA

was used in combination with indomethacin. The= was an apparent potentiation of NDGA-

induced stimulation of 1 1fbHSD2 by indomethacin. although this effect failed to reach

s tatis tical si gnificance.

4.5 .3 DISCUSSION

The prostanoid receptors are G-protein coupled receptors. which transverse the plasma

membrane of the ce11 (Piomelli. 1996). Al1 of the prostanoids, except PGE2. bind to only

one receptor subtypr (Table 7) (Piomelii, 1996; Asby, 1994; Coleman, 1996). Ligand

binding of these receptors results in an increase or decrease in CAMP levels via adenylyl

cyclase activity andor an increase in intraceilular calcium levels via inositol triphosphate

Figure 26 The inhibitory effects of 1pg/ml PGE2 or lpg/ml PGFza on 1 lPHSD2 enzyme activity in JEG-3 cells. The cells were treated for 48 h o u a At the end of the treatment, the cells were washed 3 times in HBSS prior to the assay of 1 lfbHSD2 activity in intact cells, as dwribed in the Materiais and Methods. Error bars represent the standard error of the mean. ** represents Pcû.01 for indicated sample compared to the conuol.

Control PGE2 PGF2a

Figure 27 Dose-dependent inhibition of 1 lb-HSD2 enzyme activity in JEG-3 ceiis by indornethacin. The cells were treated for 36 hours with increasing concentrations of indomethacin ( 1 - 1ûûpM). At the end of the mtment . the cek were washed 3 tirnes in HBSS pior to the assay of 1 l&HSD2 activity in intact cells, as descnbed in the Materials and Methods. Error bars represent the standard error of the mean * represents Pc0.05 and ** represents Pc0.01 for indicated sampie compared to the control.

lndomethacin (PM)

Figure 28 The e f f ~ t s of 1Op.M indomethacin. 1OOpM NDGA. and 10pM indomethacin + 1OOpM NDGA on 1 LPHSD2 enzyme activity in EG-3 ceils. Ceils were incubated for 36 hours. At the end of treatment, the cells were washed 3 times in HBSS pnor to the assay of 1 1 kHSD2 activity in intact ceils. as described in the Materials and Methods. Error bars represent the standard error of the mean. * represents Pc0.05 and ** represents Pe0.O 1 for indicated sarnpIe compared to the control.

Table 7 Eicosanoid receptors and their transduction systems (Asby, 1994; PiomeLli. 1996; Coleman, 1996)

Ligand -

Transduction System

increase CAMP, via adenylyl cyclase

increase intracellular calcium, via inositol triphosphate

PGEz

increase CAMP, via adenylyl cyclase PGEz

decrease CAMP, via adenylyl cyclase; increase intracellular calcium via inositol triphosphate

PGEz

increase CAMP, via adenylyl cyclase PGEt


increase CAMP, via adenylyl cyclase





pg (Asby. 1994; Piornelli. 1996; Coleman. 1996). The leukotnene receptors are also

membrane proteins coupled with the G-proteins (Table 7) (Piornefi. 1996). Activation of

these recep tors results in an increase in intracellular calcium levels also via P3 (Piornelli.

1996). Receptors for other eicosanoids appear to remain as yet undetermined. as no

information on them could be found.

This study describes the f int observation that prostaglandins inhibit 1 1 PHSD2 enzyme

activity. Both PGE, and PGF, inhibit this activity by at least 30%. As the PKA pathway ,a

has previously been shown to upregulate 1 lPHSD2 activity in JEG-3 cells. it c m be

assumed that any prostaglandin receptor which functions via stimulation of adenylyl

cyclase to increase CAMP. is not involved in the inhibition of 1 lkHSD2 activity. This

leaves the EPI and the EP, receptoa to be possible candidates for mediating the effect of

PGE, and the FP receptor for mediating that of PGF, . ,a

The mechanism by which the EP,. EP, and FP receptors work is outlined beiow in Fig.

27. Ligand binding to these receptors results in an interaction with the membrane bound Gq

protein complex (Houslay and Milligan. 1990). This protein complex. cornprised of the

subunits b a n d q. stimulate production of IP3. IP, binds to IP,-gated calcium channels

located on the endoplasmic reticuiurn. allowing the channels to open and intraceiluiar levels

of calcium to rise (Sekar and Kokin. 1986; Fems and Snyder, 1992). The increased Ievel

of calcium activates the protein CaM-kinase, allowing it to phosphorylate an unidentified

protein which is involved in the inhibition of I lPHSD2.

The EP3 receptor can also function via another pathway. This involves coupling of the

receptor to the Gi protein complex. which is another membrane G protein (Fig. 30)

(Houslay and Milligan. 1990). The Gi protein is cornposed of the same bsubunit and the

unique ai subunit The Gi protein is an inhibitory protein which exerts its actions on

adenyly 1 c yclase. resulting in a decrease in production of CAMP. A decrease in the level of

CAMP would result in decreased activity of PKA and therefore a s u b q u e n t decrease in

1 I &HSD2 activity.

Indomethacin. a blocker of the cyclooxygenase pathway and therefore a blocker of

prostanoid synthesis from arachidonic acid, resulted in an unexpected 40% inhibition of

1 IkHSD2 enzyme activity in JEG-3 cells. This is beiieved to be due to the increased level

of arachidonic acid left available for metabolism by lipoxygenases and cytochrome P450

monooxygenases into leukotrienes. iipoxins and HETEs. This i nc~ased level of these

other eicosanoids may be inhibiting the 1 IPHSDZ enzyme activity (Fig. 29).

To suengthen the notion that eicosanoids, other than the prostanoids. may also be involved

in the inhibition of llBHSD2, the Lipoxygenase inhibitor NDGA was used. NDGA

resulted in a stimulation of 1 lPHSD2 enzyme activity to approxirnately 2508 of the

control value in JEG-3 cells. This strongly suggests that endogenous products of the

lipoxygenase pathways are potent inhibitors of 1 1 BHSM activity.

If the lipoxygenase products involved in 1 lBHSD2 regdation are leukotrienes. then they

could inhibit I l PHSD2 activity much in the sarne way as the prostaglandiris. kukotrienes

bind to their appropriate receptor (BLT for LTB, and cysLT, and cysLT, for LTC,, LTDI - and LTEJ which activates the receptor and resulu in their interaction with the same Gq

protein described above and detailed in Fig. 29.

In a recent study it has k n shown that metastatic tumor ce11 lines, such as JEG-3, result in

more metabolism of arachidonic acid via the lipoxygenase pathway than the cyclooxygen-

ase pathway (Darntew and Spagnuolo. 1997). At least one of the leukotrienes. namely

LTB, is shown to be produced at a rate of 14 +/- 6ng per 107 cells and 5-HETE is

Figure 29 Proposed mode1 of eicosanoid regdation of 1 1 pHSD2 activity within the cell by stimulation of calcium release via inositol triphosphate. The released calcium activates CaM-kinase which phosphorylates an h o w n protein which decreases 1 1 PHSD2 enzyme activity by inhibiting 1 lbHSD2 transcription. mRNA stability. or translation.

Eicosanoid ( Receptor 1

Figure 30 Additional proposed mode1 of EGE2 inhibition of 11f3-HSDZ. PGE2 bound to the EP, receptor c m ais0 interact with the Gi protein which results in a decrease in PKA activity via inhibition of adenylyl cyclase. The decreased level of PKA activity resdts in a decreased amount of 1 1 bHSD2 activity by inhibiting 1 1pHSD2 transcription. mRNA stability, or translation.

produced at a rate of 2n%lo7 ceUs in JEG-3 cells. It remains to be detemined if LTB,

andor 5-HETE is responsible for the proposed down-regdation of 11P-HSD2 in these

cells.

Blockage of both Lipoxygenase and cyclooxygenase pathways with M X i A + indomettiacin

resulted in a funher stimulation of 1 1 P-HSD2 enzyme activity to approximately 3508 of

the control value. This suggests that both endogenously produced prostanoids and

iipoxygenase products may be inhibiting I l bHSD2 enzyme activity. and that the latter

rnay have a greater effect than the former.

Eicosanoids play a major role in the maintenance of prepancy. parturition and replation

of placental function (Schafer et al.. 1996). They are produced in the amnion. chorion.

decidua. myornetriurn and placenta (Behman and Romero. 199 1). The physiologicd

significance in pregnmcy and placenta of prostanoids is far better understood than the role

played by the lipoxygenase products (Schafer et al.. 1996; Langlois et al., 1993; Walsh,

199 1). Prostaglandins are intimately involved in the onset of labor. effecting rnyometrial

contractility. cervical dilation and effacement (Behnnan and Romero, 1991; Cheung et al..

1990; Walsh. 199 1). Lipoxygenase products stimulate contractions in strips of human

rnyomeirium. but l e s effectively than pmstaglandins (Bennett et al.. 1987). Both 5-HETE

and LTC, have been shown to induce uterine contractions in rhesus monkeys (Walsh.

199 1).

Sheep placental amnion, chorion and placenta convert arachidonic acid to prostaglandins.

leukotrienes and HETES from day 78 of gestation to terni (day 145) (Langlois et ai.. 1993).

During the fuial third of gestation in sheep. lipoxygenase activities are evident in matemal

and fetal cotyledon. myometrium and fetal membranes (Mitchell et al.. 1987). The primary

product of the iipoxygenase pathway in fetal sheep membranes and cotyledon is LTB, and

in maternai cotyledon and myometrium is 12-HETE (Mitchell et al.. 1987). From day 78

until &y 140 the amnion produces more prostaglandins than the chorion and placenta. but

at term the production of prostaglandins in the placenta rises to a level that is two-fold

higher than that of the amnion and ten-fold higher than that of chorion (Langlois et ai.,

1993). The ratio of cyc1ooxygenase:lipoxygenase products inmeases at term in both amnion

and placenta in the sheep (Langlois et al.. 1993).

Both lipoxygenase and cyclooxygenase products are formed in human intrauterine tissues

at term (Mitchell. 1986; Mitchell and Grzyboski. 1987; Rees et al.. 1988: Rose et al.. 1990;

Pasetto et al.. 1992). The predorninant product of the lipoxygenase pathway in human

amnion prior to term is LTB, but after labor becomes 12-HETE (Mitchell and Grzyboski.

1987). Human amniotic Buid displays elevated levels of leukotrienes and HETEs at terrn

(Romero et ai.. 1989). Throughout pregnancy the most prevalent lipoxygenase product in

human chorion laeve and decidua vera is 15-HETE and in placenta is 12-HETE (Mitchell

and Grzyboski. 1987).

in early pregnancy the sheep placenta expresses prostaglandin El, synthase (PGHS) in the

matemal syncytial layer (Challis. 199 1). This expression is diminished in mid-prepancy

and then reappears at day 1 15 of gestation in the monlayer trophoblastic epithelium

(Challis. 199 1).

The main source ofcirculating &ta1 PG& is the placenta (Mitchell. 1987). It is secreted

from the sheep placenta into the fetal circulation in increasing amounts during late

pregnancy (Challis. 199 1).

15Hydroxyprostaglandin dehydrogenase (PGDH) is involved in the inactivation of PG-

and PGF?, (Cheung et al., 1992). in early pregnancy, human placental PGDH has been

localized to the syncytiotrophoblast cytotrophoblast and intermediate trophoblast (Cheung

et al.. 1992). By weeks 23-30 and at term this expression is Limited to the syncytiotropho-

blast and intermediate trophoblast (Cheung et al.. 1992). This pattern of expression of

PGDH is klieved to be involved in the maintenance of low prostaglandin concenuations in

the fetal membranes for the majority of gestation (Cheung et al.. 1992).

My present finding suggest that Iocaily produced eicosanoids rnay regulate the spatial and

temporal expression of 1 lPHSD2 in the placenta. Thus. complex interactions rxist in the

placenta between 1 1 PHSDZ and other locally produced hormones/factors. such as

prostaglandins and leukotrienes. These interactions may play an important role in the

maintenance of prepancy and the process of labor.

4.6 NON-REGULATING FACTORS


This study was drsigned to test the regdatory effects on 1 1 kHSD2 enzyme activity of

seved compounds which have been demonstrated to influence 1 1 PHSD 1 enzyme activity

and/or mRNA. Since both of these enzymes belong to the same family and they are known

to share at bast one common regdator. carbenoxolone. there is a possibility that they may

be regulated by some other common factors.

The factors involved in the replation of 1 LbHSDl are listed below. (1) Both insulin and

(2) IGF- 1 exert inhibitory effects on 1 IPHSD 1 mRNA and enzyme activity in 2s FAZA

hepatoma celis (Voice et al.. 1996). (3) TNF-a has a stimulatory effect on 1 I P-HSDl

reductase activity in glomemlar mesangial ceils (GMC) (Escher et al.. MV). (4)

Dexamediasone has stimulatory effects on both 1 IPHSD 1 mRNA and enzyme activity in

2 s FAZA cells (Voice et aL.1996). (5) Injection of rats with 40pg of thyroid hormone (T3)

for up to 7 days resulted in a decrease of 1 IPHSD L enzyme activity and mRNA in borh

the Iiver and the pituitary. The kidney and distal colon 11 PHSD activity was unaffected.

suggesthg that T3 had no effect on 1 @-HSD2 expression (Whonvood et al..1993b). (6)

Treatrnent of gonadectomized rats with estrogen resulted in a decrease in 1 @HSD 1

mRNA and enzyme activity in the liver while the kidney showed an increase in 1 lPHSD

activity with no detection of 1 1 PHSD 1 mRNA (Low et al.. 1993). The stimulated 1 1 fb

HSD enzyme activity in the kidney was iikely due to 1 1 PHSD2. (7) Treatment of

gonadectomized rats with testosterone resulted in no change in 1 1 FHSD enzyme activity

and 1 ID-HSD 1 mRNA in the liver (Low et al., 1993).

One other factor used in this study is progesterone. This factor was shown in EG-3 ceUs

to result in a complete inhibition of 1 1PHSD2 enzyme activity (Gomez-Sanchez et

aL.1996). Due to the fact that the cells were CO-incubated with [3~]corticosterone and

1OOp.M progesterone. it is difficult to discem whether this inhibition was due only to

simple competitive inhibition of I 1 BHSD2.

4.6.2 RESULTS

Negligible effecrs on 1 1 BHSD2 enzyme activity were demonstrated in JEG-3 cells treated

with the following compounds: 50nM PMA (negative control); lOnM estradio1 (E2); 1w progesterone (P4); lOnM estradiol + 1pM progesterone; IO-50nM testosterone; 10-50nM

thyroid hormone (T3); 10ng/ml TNFa; 50nglml IGF- 1 ; 5pM insulin; 1 - 100nM

dexamethasone (data not shown).

4.6.3 DISCUSSION

Estradiol. progesterone. testosterone. thyroid hormone. TNFa. IGF- 1. insulin,

dexamethasone. and PMA have no effect at the concentrations employed. on 1 lbHSD2

enzyme activity in IEG-3 cells.

Given the similarity in the structure between progesterone and glucocorticoids. the

published study in which progesterone inhibits 1 1 PHSD2 enzyme activity in JEG-3 c e h

(Gomez-Sanchez et al.. 1996) is liliely due to simple cornpetitive inhibition, as no such

inhibition was observed in this study.

The lack of effecls of the compounds used which are known to regulate 1 IPHSDI can be

rationalized by at Ieast two possible explanations. One explanation may be that since there

is very low homology between the g e n s for 1 1 PHSD 1 and 2. as outlined in chapter 1. it

is iikely that the 1 IP-HSDZ gene lacks the required correspondhg response-elements for

these cornpounds. Alternately. KG-3 cells may lack the appropriate receptors for these

compounds.

4.7 CONCLUSIONS

This chapter detailed the novel discovery of severai factors which regulate 1 1 PHSD2

enzyme activity. These factors are AT-RA, 9C-RA, EGF. PGE2, ffiF2a. Indomethacin

(via inhibition of prostanoid synthesis. possibly resulting in increased lipoxygenase and

cytochrome P450 monooxygenase metabolism of arachidonic acid). and NDGA (via

inhibition of lipoxygenase activity). The regulatory actions of a well known inhibitor of

1 lPHSD2 activity. carbenoxolone, were also further characterized.

Regulation of enzyme activity c m occur at any one of the six steps involved fmm gene

expression to manifestation of its activity. These steps include: (1) rate of transcription; (2)

mRNA p ~ e s s i n g (splicing and polyadenylation); (3) mRNA transport out of the nucleus;

(4) stability of the mRNA; (5) rate of translation; and (6) the activity of the protein (Dm&

1982; Derman. 198 1). Each of these steps are important in the regulation of an enzyme, but

the most important step at which regulation cm occur is at a transcriptional level (Alberts et

al., 1994). because without transcription none of the other regulatory steps cm occur.

The regulatory factors identified in this chapter can act at any of these steps. This study has

laid the foundation for launching future studies directed at elucidating the mechanisms of

their effects. The F i t step in such undertakùigs may be to determine if conesponding

changes occur in the level of 1 1 &HSD2 mRNA in these cells after various treatments. A

further sep couid be taken to determine if changes in the mRNA are due to alterations in the

rate of transcription andor in the stabiiity of the m R N k If the rate of transcription is

altered. studies involving the preparation of a reporter gene consmct containing the 5'-

Flanking region of the 11P-HSD2 gene fused upstrearn of a reporter gene (a gene whose

product is easily detected but not endogenously produced in the celi system used) in an

expression vector cm be designed. This consuvct would be transfected into a ce11 system

which is known to express 1 Ip-HSD2 (such as JEG-3 cells). and the level of its

expression would be measured after treatment with the regulatory compounds outlined

above.

CHAPTER SGENERAL DISCUSSION

5.1 OVERVIEW OF 11P-HSD2

1 lBHSD2 is a microsornal enzyme which catalyzes Ihe conversion of cortisol and

corticosterone to their inactive metabolim. cortisone and 1 1-dehydrocorticosterone

(Reeves. 1995; Stewart et a1..1994a). It is NAD-dependent and has a high &fmity for

endogenous glucocorticoids (Zhou et al.. 1995; Naray-Fejes-Toth and Fejrs-Toth. 1995;

Yang and Matthews, 1995; Reeves, 1995)-

The expression of' 1 1 P-HSD2 is regulated in a tissue specifc (localized to mineralocorticoid

target tissues and the placenta) and developmentally prograrnmed manner (Cole. 1995;

Brown et al., l996b). Relatively Little is known about the regulation of 1 1 P-HSDî in the

îitenture.

In rnarnrnais. 1 1 PHSD2 serves two important physiologicai functions. It protecu the

rnineralocorticoid receptor by inactivating cortisol (Whonvood et al.. 1995; Seckl. 1993).

leaving the receptor free for aldosterone to bind. It aiso protects the fetus from the

teratogenic effects of high levels of materna1 cortisol (Benediktsson et al.. 1993; Krozowski

et a.., 1995c; Swkl et al., 1995).

Deficiency in 1 1 PHSD2. either inherited (AME) or acquired (excessive licorice ingestion)

results in severe sodium retention and potassium secretion. leading to hypertension and

often suoke (White et al.. 1997b; Biglien et al.. 1994). Deficiency of placental L 1 &HSD2

rnay result in a lower birth weight (Seckl et aL.1995) which has been linked to higher

incidence of adult diseases such as hypertension and non-insulin-dependent diabetes

(Barker et al., 1993).

5.2 THE OVINE 11P-HSD2 GENE AND ITS 5'mFLANKING

REGION

5.2.1 STRUCTURE OF T m GENE

The svucture/sequence of the ovine 1 IFHSD2 gene is highiy conserved with its hurnan

(Agarwal et al.. 199%) and mouse (Cole. 1995) counterparts. The 1 1 PHSD2 gene for al1

three species contains 5 exons spanning >4kb, with a large first intron. They ail encode

proteins with a predicted length of -400 amino acids. The deduced ovine 1 lPHSD2

protein displays over 78% sequence identity to those of the human. rabbit rat, and mouse.

However. this differs from the published sequence of ovine kidney 1 1 PHSD2 cDNA

which predicts a protein of 427 amino acids. Sequence alignment indicated that this

discrepancy is attributed to two single nucleotide omissions in the published cDNA

sequence which resulted in a shift in the open reading frame at the codon for residue 358.

Therefore. the present results have provided concIusive evidence that the prirnary structure

of I IP-HSD2 protein is weii conserved between the sheep and the other four rnammals

(Campbell et ai.. 1996).

5.2 .2 THE 5'-FLANKING REGION

The 5'-tlanking region of the ovine 1 1 PHSDZ gene is highly homologous to that of the

human (Agarwal and White, 1996). Like the hurnan. the sheep 5'-flanking region lacks the

consensus TATA and CAAT boxes (Agarwal et al.. 1995a). In both species this region has

an extremely high G+Ccontent (-80%) (Aganual et a1.,1995a) which is cornmon in genes

lacking a TATA box (Beebee and Burke, 1992). Two conserved GC-boxes are identified in

both species. In the human these boxes are functional Spl binding sites (Aganval and

White. 1996). The functional signifcance of these boxes in the sheep rernains to be

established,

Sp 1 is a ubiquitous transcription factor often involved in the regulation of housekeeping

genes (Beebee and Burke. 1992). Thus. it is highly conceivable that factors other than Sp L

are involved in the regulation of the 1 lPHSD2 gene. which is known to be expressed in a

tissue specific and developmentally regulated fashion.

This study identified several other putative transcription factor binding sias located in the

5'-flanking region of the 1 LpHSD2 gene. The identified binding sites include those for

AP2, CEBPB. 6EF1. E47. Ekl. cEts 1 P54. RF2. MyoD. cMyb. MZF1. NFKB and

USF.

5.3 REGULATION OF 11P-HSD2 IN JEG-3 CELLS

5.3.1 HUMAN CHORIOCARCINOMA CELL LINE (JEG-3)

The JEG-3 celi iine is a continuous line of human chonocarcinorna cells (Kohler and

Bridson, 197 1). JEG-3 ceUs are phenotypically iike the syncytiotrophoblast, grow as large

multinucleated ceîls. and secrete hurnan chorionic gonadotropin. chorionic sommatotropin

and progesterone (Tuan et al., 199 1 ; nekis and Benveniste, 1989).

The syncytiotrophoblast is the primary site of expression of 1 lkHSD2 in the placenta and

is also the site of matemal-fetal exchange (Pepe et alJ996). Thus. the use of JEG-3 cells

for studying the regulation of 1 1 bHSD2 in the placenta is appropriate and of physiologicai

5.3.2 REGULATION OF 1lP-HSD2 BY CARBENOXOLONE: POSSIBLY

VIA PKA PATHWAY

Carbenoxolone is a synthetic f o m of the PKA antagonist glycyrrhetinic acid (Baker, 1994;

Wang and Polya 1996). In this study it was found to inhibit 1 lBHSD2 enzyme activity in

a dose dependent manner in JEG-3 cells.

Forskolin. a PKA agonist, resulted in an expected four-fold stimulation of 1 1B-HSD2

activity in JEG-3 cells. This sùmulatory effect of forskolin was eliminated by the addition

of carbenoxolone. with the activity level decreasing below the level expressed in the control

cells.

Reversal of the PKA agonist forskolin induced stimulatory effects on 1 lPHSD2 activity

by carbenoxolone suggests that the PKA pathway may be involved in the inhibition of 1lP-

HSD2 enzyme activity by carbenoxolone. This is the f i t demonstration of interactions

behueen carbenoxolone and forskolin with regard to 1 1P-HSD2.

5.3.3 REGULATION OF 11p-HSD2 BY RETINOIC ACIDS AND EGF

NI-trans-retinoic acid stimulated 1 1 PHSD2 enzyme activity in a time dependent manner,

reaching maximal stimulation at 24 houn. The effect of dl-rrm-retinoic acid was also dose

dependent, with maximal stimulation at 1ûûnM. 9-cis-retinoic acid was aiso efCective in this

regard, and the level of its stimulation was sirnilar to that observed with dl-rrans-retinoic

ricid.

Treatment of JEG-3 cells with EGF resulted in a dose dependent inhibition of 1 1 p-HSD2

enzyme activity. This inhibition wched its maximum, -55% of the control value, at a

dosage of Lûûng/ml of EGF for 48 hours. EGF appears to attenuate the stimulatory effects

of retinoic acids on 1 1 PHSD2 enzyme activity in JEG-3 ceus.

These are novel Findings with respect to the regulation of 1 l&HSD2 in any biological

system.

5.3.4 REGULATION OF 11P-HSD2 BY EICOSANOIDS

This study demonstrated. for the f i s t time. that prostaglandins EGE2 and ffiFza resulted

in an inhibition of 1 1 kHSD2 enzyme activity in EG-3 cells to approximately 65-70% of

the control value.

Indomethacin. an inhibitor of prostanoid synthesis. resulted in an unexpected 40%

inhibition of 1 1 &HSD2 enzyme activity. This result was surprising given the inhibitory

effects of prostaglandins. The inhibition of 1 lPHSD2 by indomethacin is believed to be

due to an increase in the availability of arachidonic acid to be metabolired into other

eicosanoids. which may be potential inhibitors of 1 1 kHSD2.

To investigate whether products of the lipoxygenase pathways are inhibiting 1 1P-HSD2

activity in JEG-3 cells. the inhibitor of the three lipoxygenases. NDGA was used. NDGA

resulted in a 2.5 fold stimulation of 11B-HSD2 enzyme activity, indicating that one or more

of the products of lipoxygenases are potent inhibitors of 1 IkHSD2 enzyme activity.

Furthemore, combined treatrnent with indomethacin + NDGA resulted in a further increase

in 1 lPHSD2 enzyme activity to -350% of control. This is likely due to elimination of both

endogenous cyclooxygenase and lipoxygenase products. which may be inhibiting 1 1 p- HSD2. It appears that lipoxygenase products may exert a more potent effect on 11pHSD2

inhibition in JEG-3 cells than the endopnous prostanoids.

5.4 CONCLUSIONS

In the present study, the gene encoding sheep 1 lBHSD2 has b e n cloned and character-

ized In doing so. errors in the previously published cDNA sequence have k e n corrected.

Furthemore, an approximately lkb 5'-flanking region of the gene has also been analyzed.

and several putative consensus transcription factor binding sites have been identified

dthough they remain to be cont-med functionally. In addition, several important

physiologicai factors. including retinoic acids. EGF. PGs and the products of the

lipoxygenase pathways, have been identified as potent replators of 1 1P-HSD2 expression

in JEG-3 cells. These are novel findings which have certainly advancrd Our understanding

of the structure. function and ~gulation of 1 lpHSD2 in mammals. More importantly

perhaps. the present findings have laid the foundation for launching future studies aimed at

clucidating the function and regdation of this necessary enzyme in various biological

systems. It is important that we understand the mechanisms regulating 1 lPHSD2. as it is

crucial in the protection of the fetus from high levels of matemal cortisol as weil as the

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