Session V. Regulation of Gene Expression (Part 2)

Is Methylation of a CpG Rich Island in the Promoter Region Responsible for the Highly Tissue Specific Expression of the Interphotoreceptor Matrix Protein IRBP?

A. Albini, Zhen Zhu, J. Toffenetti, G. J. Chader and D.M.Noonan

National Institute for Cancer Research, Genova, Italy and NEI, NIH, Behesda, MD, USA.

Interphotoreceptor-retinoid-binding protein (IRBP) is a large glycoprotein (140 kDa) found in the extracellular photoreceptor matrix, between the neural retina and the pigment epithelium. It is thought to mediate retinoid trans­port in this extraceullar compartment. IRBP is highly con­served among vertebrates. Previous studies have demon­strated that the protein is a specific product of photorecep­tor cells and pinealocytes. It is also expressed in retinoblas­toma cells, which are derived from a primitive stem cell of the retina. In order to study the regulation of gene expres­sion we have cloned the gene for human IRBP from a human genomic AEMBL 3 library. The two clones ob­tained, A4 and A24 contained the entire translated region plus 6 kb of the 5' flanking region. A restriction fragment from the transcribed region hybridized to a 4.4-kb mRNA on northern blots of Y-79 retinoblastoma cells. Human lymphocytes did not express IRBP mRNA. We have exam­ined the genomic DNA methylation state in expressing (Y-79) and non-expressing (lymphocyte) cells using Hpa III Msp I and Hha I digestion of genomic DNA prepared from these cells. Probes to the 3' flanking DNA, the intronic regions and most of the exons did not show any significant differences in the digestion patterns. However, hypomethy­lation of sites from the immediate 5' flanking DNA was found only in the Y-79 cell genomic DNA. Sequence analy­sis of this region indicates these hypomethylation sites were within a CpG rich island at approximately -1500 in the promoter region and another segment within the beginning of the first exon. We suggest that hypomethylation is a requirement of IRBP expression and may regulate the bind­ing of nuclear factors to this region.

Characterization of the Murine Monoclonal Antibody (MoAb) NL07 Specific for the Human CD36 Receptor.

M. Alessio, F. Bussolino, S. Roggero, M. Saitta and F. Malavasi

Dept. of Genetic, Biology and Medical Chemistry; University of Torino, Italy. CNR and Children Hospital Regina Margherita Torino, Italy.

This work has been addressed to the molecular and func­tional characterization of a structure expressed on the sur­face of human platelets. The MoAb NL07 has been gener­ated using sonicate extracts of human platelets as immunizer. The molecule recognized by the NL07 MoAb is expressed by platelet, monocytes and endothelial cells, as well as by myelonomocytic lines (e. g. U93 7) and by some melanomas. Endothelial and melanoma cells express the NL07 epitope only during adhesion to a substrate.

The molecule recognized by NL07 MoAb after immunoprecipitation from NP-40 detergent extracts of surface [125I]-labelled platelets underwent an extensive biochemical investigation by SDS polyacrylamide gel electrophoresis and two-dimensional gel analysis. Our results indicated that the molecule recognized by the NL07 MoAb is a single chain structure featuring a molecular weight of 85 kDa under reducing conditions, with an acidic isoelectric point ranging from 5.2 to 5.5. The specific phenotype distribution as well as the biochemical charac­teristics suggested that NL07 MoAb could recognize the platelet gplV (CD36), a surface glycoprotein with a func­tional role of thrombospondin receptor. To investigate whether NL07 MoAb recognized the CD36 molecule, we performed an epitope competition test with the OKM5 MoAb (specific for the CD36 molecule). Preincubation of U937 cells with saturating concentrations of OKM5 inhi­bited the binding of NL07 MoAb directly labeled with FITe. These results indicated an epitope coincidence as well as molecular specificity. Further, the addition of NL07 MoAb in the medium inhibits the FGF-induced migration of human endothelial cells in a Boyden chamber. A further finding was that CD36 molecule is not constitutively phos­phorylated either on endothelial or U937 cells, nor CD36 molecule became phophorylated upon cell activation (e.g., after FGF treatment on endothelial cells or PMA treatment

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on U937). Finally the regulation of CD36 expression is gamma-IFN independent and the CD36 molecule was not induced in endothelial cells in suspension by treatment by gamma-IFN. Our efforts are presently addressed to deline­ate the functions of CD36 molecule on monocytes and endothelial cells.

Glutamine Increases Collagen Messenger RNA in Fibroblast Culture by Interfering with the Cellular Content of cAMP.

G. Bellon 1, A. Mauviel2, J. c. Monoboisse1, J. P. Pujol2 and J.P. Borel'

proteins (36 kDa and 92 kDa) were purified by DEAE­Sephacel, Heparan-Sepharose, salmon sperm DNA-sephar­ose and an A34-oligonucleotide affinity chromatography. The amino terminal sequence of the 92-kDa protein does not show sequence homology to the known proteins in Genbank and NBRF protein data base. In vitro transcrip­tion experiments confirmed that the 210-bp segment was required for the transcription of the a1(IV) gene. It also indicated that nuclear extracts from both undifferentiated and differentiated F9 cells, but not from NIH 3T3 cells were capable of transcribing the a1 (IV) gene. Competition analysis from in vitro transcription also suggests that an additional common nuclear factor(s) interacts with both promoter and enhancer sequences.

1 Lab. Biochem., URA CNRS 610, Univ. Reims, 51 rue Cognacq Jay, F-51095 Reims and 2 Lab. Connective tissue Biochem, IBBA, University of Caen, Novel AP-l Site Binding Factors. F-14033 Caen, France.

By increasing the concentration of glutamine from 0 to 10 mM in the culture medium of human dermal fibroblasts, a significant dose-dependent increase of neosynthesized collagen was observed. In the same culture conditions, the steady state amounts of al (I) pro collagen mRNA, al (III) pro collagen mRNA and fibronectin mRNA, as measured by dot blot hybridization, were significantly higher for 10 mM glutamine than without glutamine in the medium. In parallel, the intracellular cAMP levels were found signifi­cantly lowered in the presence of 10 mM glutamine, as measured by specific radioimmunoassay.

Regulation of Collagen IV Gene Transcription In Vivo and In Vitro.

Peter Burbelo, Makoto Sawada, Leslie Bruggeman, Paul Klotman, Gary Gabriel and Yoshihiko Yamada

Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, NIH, Bethesda, MD 20892, USA.

The expression of the mouse al(IV) and a2(IV) collagen genes is regulated by a bidirectional promoter and shared enhancer in the first intron of the al(IV) gene. Transient transfection assays indicated that a 210-bp segment in the first intron is important for the enhancer activity. DNase protection and gel retardation assays revealed that two regions within this fragment bound distinct nuclear factors from both differentiated F9 cells and EHS tumor cells. Mutations within one of these sites (A34) reduced nuclear factor binding activity as well as the enhancer activity. Two

Giovanna Buttice and Markku Kurkinen

Dept. of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

The AP-l site, TGAGTCA, binds the Fos and Jun family of transcription factors. Different proteins from the Fos and Jun family interact with each other to stimulate transcrip­tion. Using a 91-base pair fragment from the human stromelysin promoter containing the AP-l site (-70 to -64), we have studied by gelshift and DNase footprinting AP-l site binding factors from skin fibroblasts, Hela and HepG2 cells. Similar footprinting patterns were seen using different cell extracts. One protected site is centered around the AP-l element, and another site is upstream of the AP-l site. In gelshift, common complexes were present in all extracts. Three of them, denoted (A, Band C) were HepG2-specific and could be specifically competed by double­stranded oligonucleotide containing the AP-l sequence. None of the A, B, or C complexes comigrated with the complex formed using in vitro synthesized Fos and Jun proteins. Furthermore, antibodies against Fos or Jun had no effect on the A, B or C gelshift complexes. Inclusion of in vitro synthesized Fos and Jun proteins with the HepG2 cell extract competed for the formation of the A, Band C gelshift complexes. These data suggest that the A, Band C complexes represent novel AP-l site binding factors from HepG2 cells immunologically unrelated to Fos and Jun proteins.

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The AP-l Site is Required for Basal Expression but is not Necessary for TP A Induction of the Stromelysin Promoter.

Giovanna Buttice, Susan Quinones and Markku Kurkinen

Dept of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

Stromelysin is a major metalloproteinase that degrades connective tissue matrix components. The expression of the gene is affected by a variety of stimuli, including hormones, growth factors, cytokines and the tumor promoting phor­bol ester TPA. We have studied the role of the AP-l site, a target for Fos and Jun, which mediate transcriptional induction by TPA, in the regulation of the human strom ely­sin promoter (-1303 to +4). In transiently transfected cell cultures, point mutations in the AP-l sequence TGAGTCA reduce basal level promoter activity to 55%-5% of the wild-type activity, depending on the mutation and cell line used. In cotransfection experiments, antisense Fos mRNA expression plasmid reduces the basal promoter activity by 50% . The idea that the AP-l site and Fos and Jun are involved in the basal level control of stromelysin expression is also supported by the following data. First, in in vivo studies using F9 cells, which do not express c-Jun, cotrans­fection of Fos and Jun expression plasmid increased the basal promoter activity 60-fold; whereas no effect was seen with the mutant promoters. Second, stromelysin promoter fragments (-102 to -11) containing the mutated AP-l site all failed to bind in vitro synthesized Fos and J un proteins in gelshift assay. These data suggest that the Fos and Jun factors and the AP-l site regulate the basal activity of stromelysin promoter. Surprisingly however, the relative TPA inducibility was not affected by any of the mutations. Further, a mutant promoter from which the AP-l site had been removed could still be induced by TP A. We interpret these results to suggest that the AP-l site in the promoter context studied here is not necessary for TPA-induction.

The Genes Coding for Tropoelastin and Type ill Collagen are Localized on Different Regions of the Long Arm of Human Chromosome 2.

Angela M. Christiano!, SuEllen Toth-FejeJ2, Susan B. Deak l, Ellen Magenis2 and Charles D. Boydl

1 Department of Surgery, UMDNJ-Robert Wood Johnson Medi­cal School, New Brunswick, NJ 08903 and 2 Department of Medical Genetics, Crippled Children's Division, The Oregon Health Sciences University, Portland, OR 97201, USA.

Over the last few years, several investigators have shown that the genes coding for tropoelastin and proal (III) colla­gen are both localized on the long arm of human chromo­some 2. The tropoelastin gene was localized to the region 2q3l ~ 2qter and the gene coding for proal (III) collagen was mapped to the region 2q3l ~ 2q32. It is evident from this mapping data that a potential overlap exists in the chromosomal loci for these two genes. This raises the possi­bility that the genes coding for tropoelastin are not only syntenic on chromosome 2 but may also share a common chromosomal locus. By in situ hybridization analysis of normal metaphase chromosomes using cDNA clones con­taining DNA sequences coding for human tropoelastin and proal(III) collagen we have localized, however, the genes coding for these two extracellular matrix components to different regions of the long arm of chromosome 2. The tropoelastin gene was localized to 2q2l and the gene coding for proal(III) collagen was mapped to the q32 region of chromosome 2.

Signal Transduction Events which Mediate the Regulation of Collagen Gene Expression by Interleukin-l (IL-l) in Human Chondrocytes.

K. Fukuo, S. M. Krane and M. B. Goldring

Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA.

In human chondrocytes IL-l stimulates the synthesis of types I and III collagens. This effect is observed only when IL-l-induced PGE2 is blocked [e. g., with indomethacin (indo)]. In contrast, IL-l suppresses type II collagen synthe­sis even in the presence of indo. The signaling pathways by which IL-l mediates these effects have not been defined. To study the potential role of pertussis toxin (PT)-sensitive guanine nucleotide regulatory (G i ) inhibitory proteins in the regulation of collagen synthesis by IL-I, chondrocytes were pre incubated 24 h with PT (50-250 nglml) followed by 24 h treatment with IL-l. IL-l alone decreased the level

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of type! (al) collagen mRNA. When endogenous PGEz synthesis was blocked by indo, the stimulatory effect of IL­l on type I collagen mRNA levels was unmasked. PT reversed the inhibitory effect of IL-l alone and augmented significantly the IL-l-induced increase in type I collagen mRNA levels in the presence of indo. PT potentiated the IL­l-induced increase in PGEz synthesis (Exp. 1: Control, 4.9; IL-l, 438; PT, 16.2; PT+ IL-l, 513, and Exp. 2: Control: 11.1; IL-l, 4500; PT, 14.0; PT +IL-l, 8020pglml ofPGEz). The augmentation by PT of IL-l-induced type! collagen mRNA in the presence of increased ambient PGEz concen­trations is paradoxical, since PGEz suppresses collagen syn­thesis in these cells. Down-regulation of PGEz receptor con­centrations could account for these effects. In contrast, PT partially reversed the suppressive effects of IL-l on type II collagen mRNA levels, consistent with distinct mechanisms controlling expression of these different collagen genes. These results suggest that the effects of IL-l on collagen synthesis may involve aPT-sensitive Gj-protein.

Cloning and Analysis of the Promoter Region of the Human u2(V) Collagen Gene.

DanielS. Greenspan, Bum-Soo Lee, Seung-Taek Lee and Guy G. Hoffman

Department of Pathology and Program in Cell and Molecular Biology, University of Wisconsin-Madison, WI 53706, USA.

We have isolated a 17-kb genomic clone containing the 5'-portion of the human a2(V) collagen gene. Nucleotide sequence was determined for 1671 base pairs comprising the promoter region, first exon and 335 bp of the first inrron, and the transcription start site determined by primer extension and Sl nuclease analysis. Sequence comparison revealed the a2(V) promoter to be similar in structure to the promoter of the al(III) collagen gene. This is the first instance of such similarities between promoter regions of genes encoding different fibrillar collagen chains. Homol­ogy, in 5' flanking sequences, extends upstream to about nucleotide -120 in each gene and is particularly striking near the T ATTT A sequence (TAT A box) present in each promoter. Some homology also surrounds the two tran­scription start sites. The 5' -untranslated regions of the two genes also show strong homology, particularly around two small open reading frames upstream of the translation initi­ation codons. Chimeric chloramphenicol acetyltransferase constructs were prepared with various portions of a2(V) 5'­flanking, exonic and intronic sequences. Transient expres-5ion a5says, in human fibroblasts, localized the functional a2(V) promoter to the region of 5' flanking sequence con­served between the a2(V) and al(III) genes. Expression

assays also identified negatively acting element(s), which inhibit transcription.

Molecular Cloning of Chicken Galactosylhydroxylysyl Glucosyltransferase.

J. Heikkinen and R. Myllyla

Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland.

Galactosylhydroxylsyl glucosyltransferase (GGT, E.C.2.4.1.66) catalzyes the post-translational transfer of

. the carbohydrate unit from UDP-glucose to galactosylhy­droxlysyl residues in collagen. Polyclonal antibodies pre­pared against purified chicken GGT stained only one band when partially purified chicken GGT was studied in immunoblotting. The antibodies were shown to be inhibit­ory and species-specific, staining only the chicken GGT in immunostaining. When GGT was treated with endogly­cosidase H the mobility of the immunostained band shifted from a molecular weight of 74000 to about 68000 in SDS polyacrylamide gel electrophoresis, suggesting that GGT is a glycoprotein containing asparagine-linked oligosac­charides. An expressing chicken embryo library was screened with the poly clonal antibodies, and seven positive clones were identified among 1.3 X 105 recombinant phages. Five of the clones hybridizided to a mRNA species of about 3.7 kb in Northern blotting. The clones have been sequenced and found overlap so that they covered about 2. 7 kb of the corresponding mRN A. To confirm the identify of the clones, we are preparing the peptides from isolated chicken GGT in order to obtain internal amino acid sequences for the enzymes.

Regulation of Expression of Alternatively-Spliced Human Fibronectin IIICS mRNA Variants.

Richard P. Hershberger and Lloyd A. Culp

Department of Molecular Biology and Microbiology. Case West­ern Reserve University, Cleveland, OH 44106, USA.

Expression of the cell type-specific CS-l cell-binding domain within fihronectin (FN) polypeptides is regulated by alternative splice site choice within the type-III connect­ing segment (IIICS) exon. In order to examine the regula­tion of IIICS variant expression, a novel double primer extension (DPE) assay was developed that simultaneously identifies each of the five human IllCS mRNA splicing

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variants. Differences in IIICS expression patterns were observed among different cell types, among fibroblasts of different tissue origins, and between comparable normal and transformed cells. The most predominant cell type­specific differences were in the abundance of the one IIICS­mRNA variant (3%-57% of total FN mRNA) relative to the four IIICS+ variants. The relative abundance of tran­scripts containing the CS-1-encoding portion of the IIICS was substantially reduced in liver (pFN mRNA: 22% CS-1 +) relative to that in fibroblasts from various tissues (cFN mRNA: 56-86% CS-1 +) . Cell type-specific changes among the expression levels of the four IIICS+ mRNA variants are consistent with a proposed model in which regulation of alternative 3' splice site selection predomi­nates over regulation of 5' splice site selection in generating specific patterns of IIICS mRNA expression. Site-directed mutagenesis of transfected IIICS splicing substrates is being used to elucidate the pathway of IIICS splicing and its cell type-specific regulation.

Molecular Cloning of the Rat Prolyl 4-Hydroxylase a Subunit.

I. Hopkinson 1, S. A. Smith I, A. Donne l, ]. Rosamond I,

H. Gregory2, T.]. Franklin2 and M.E. Grant l.

Dept. of IBiochemistry & Molecular Biology, The Univ. of Man­chester, Manchester and 2Bioscience 1, l. C. I. Pharmceuticals p.l.c., Alderley Park, Cheshire, England.

The enzyme prolyI4-hydroxylase, the activity of which is essential for the normal biosynthesis of the extracellular matrix protein collagen, is a tetrameric structure of molecu­lar formula a2~2' The a subunit M2 = 64,000 contains most of the enzyme active site whilst the ~ subunit is one guise of a multifunctional 60,000-Da gene product. It is likely that the a subunit is important in the regulation of prolyl 4-hydroxylase activity and that the determination of the primary sequence of this subunit will facilitate the rational design of prolyl 4-hydroxylase inhibitors. Such inhibitors may well have a role in the therapy of common fibrotic and rheumatological disorders. Prolyl 4-hydroxylase was purified from neonatal rat skins using poly L-proline affin­ity chromatography followed by ion-exchange chromatog­raphy (1) . The enzyme subunits were resolved by SDS­PAGE, electrotransferred onto polyvinylidene difluoride membranes and then analyzed by amino acid microseque­nation (2). A 20-amino acid N-terminal sequence was determined for the a subunit and oligonucleotides were designed on the basis of this sequence. Several clones which hybridized with one of these oligonucleotides were isolated from a neonatal rat aorta cDNA library. DNA sequencing

of a 3-kb insert excised from one of these clones demon­strated that it encoded the 20-amino acid N-terminal sequence of the rat prolyl 4-hydroxylase a subunit deter­mined previously. Sequencing of this insert revealed remarkable sequence homology between the prolyl 4-hydroxylase a subunit purified from human (3), chick (4) and rat tissues.

(1) Kedersha, N. L. and Berg, R.A., Collagen Rei. Res. 1: 345,1981. (2) Matsudaira, P.,]. BioI. Chern. 262: 10035, 1987, (3) Helaakoski, T., Vuori, K., Myllyla, R., Kivirikko, K. I. and Pihlajaniemi, T., Proc. Natl. Acad. Sci. USA 86: 4392, 1989, (4) Bassuk, ].A., Kao, W., W.-Y., Herzer, P., Kedersha, N. L., Seyer, ]., DeMartino, ]., Daugherty, B. L., Mark, G. E. and Berg, R. A., Proc. Natl. Acad. Sci. USA 86: 7382,1989.

Regulation of Collagenase Gene Expression.

C.]onat, H.-P. Auer, P. Herrlich and H.]. Rahmsdorf

Kernforschungszentrum Karlsruhe, Institut fiir Genetik und Toxi­ko\ogie, P. O. Box 3640, D-7500 Karlsruhe, FRG.

Transcription of the human collagenase I gene is en­hanced by phorbol ester tumor promoters and repressed by glucocorticoids. Both positive and negative regulation are mediated through the major enhancer of the gene which is localized between positions -73 and -65 with respect to the start site of transcription. The enhancer suffices to control minimal promoter constructs carrying only the TAT A-box linked to the chloramphenicol-acetyl-transfer­ase gene. Positive and negative regulation of such minimal gene constructs is independent of ongoing protein synthesis suggesting posttranscriptional regulation of the transcrip­tion factor AP-1 that binds to this enhancer. Activation of the enhancer by phorbol esters is enhanced by sequences located 5' of the AP-l site: the presence of a PEA 3 like site (Wasylyk et aI., EMBO ]. 8: 3371, 1989) around position -90 augments phorbol ester inducibility 2-fold, the pres­ence of sequences up to position -110 (box 3, Angel et a!., Cell 49: 729, 1987) 8-fold.

The downregulation of collagenase enhancer activity by glucocorticoids depends on activated glucocorticoid recep­tor. A 10 times lower hormone concentration suffices for half maximal repression of the collagenase gene as com­pared to that required to activate the mouse mammary tumor virus long terminal repeat. Immunoprecipitates of the glucocorticoid receptor contain AP-l suggesting a direct interaction between both transcription factors. This interaction may represent the molecular basis of inactiva­tion. The targeting of phorbol esters and glucocorticoids onto AP-l places the factor in a key position in the regula­tion of matrix metabolism and cell growths.

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Deletion Analysis of the 5' -Flanking Region of the Human Elastin Gene: Delineation of Functional Promoter and Regulatory Cis-Elements.

Veli-Matti Kahari, MichaelJ. Fazio, Yue Qiu Chen, Muhammad Bashir, Joel Rosenbloom and Jouni Uitto

Departments of Dermatology, Biochemistry and Molecular Biol­ogy, Jefferson Medical College, and Center for Oral Health Re­search, University of Pennsylvania, Philadelphia, PA, USA.

Analysis of the 5'-flanking region of the human elastin gene has revealed several unusual features. The region from - 2260 to -1 was shown to contain several putative SP-1 and AP2 binding sites as well as putative glucocorticoid, cAMP and TPA responsive elements. We have developed several elastin promoter/CA T reporter gene constructs spanning from -2,260 to +2 by cloning 5'-flanking DNA into a promoterless Bluescript/CA T construct (pBSOCA T). Assay of promoter activity in transient transfections of rat aortic smooth muscle cells, human skin fibroblasts, HeLa cells or mouse NIH-3T3 cells allowed delineation of several functional subregions within 2.26 kb of the 5' -flanking sequences. The basic promoter element was found to reside within the region -128 to -1. In addition, two distinct up­regulatory and two down-regulatory regions were deline­ated. Deletion analysis of the up-regulatory region between -475 and -129 revealed a strong up-regulatory activity in the fragment extending from -198 to -129, which con­tains four putative AP2 binding sites. When this fragment was cloned in front of thymidine kinase (TK) promoter in the plasmid pBL2CA T it stimulated the activity of TK promoter 4-fold. Also, deletion of the fragment extending from -134 to -87, which contains three of the most 3' SP-1 binding sites decreased the promoter activity by 80%. These findings indicate that these putative cis-acting ele­ments are functional, and attest to the complexity of tran­scriptional regulation of the elastin gene expression in mammalian cells.

Specific Inactivation of Prolyl4-Hydroxylase and Inhibition of Collagen Synthesis by Oxaproline­Containing Peptides in Cultured Human Skin Fibroblasts.

K. Karvonen\ L. Ala-Kokko\ T. Pihlajaniemi\ T. Helaakoski1, S. Henke2, V. Gunzler2, K.l. Kivirikko1

and E.-R. Savolainen1

1 Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland and 2 Division of Pharmaceutical Research, Hoechst AG, Frankfurt, FRG.

Peptides containing the unphysiologic amino acid 5-oxaproline (Opr) of the general formula RrXaa-Opr-Gly­R2 were shown to be syncatalytic inactivators of prolyl 4-hydroxylase in vitro with the IDso of the most active sub­stances being below l!J.M after 1 h . Two oxaproline containing peptides, benzyloxycarbonyl-Phe-Opr-Gly­benzyl ester (I) and benzloxycarbonyl-Phe-Opr-Gly-ethyl ester (II) were found to inactivate prolyl 4-hydroxylase in cultured human skin fibroblasts, peptide I being about twice as potent as peptide II. Inactivation by 50% was observed after culturing with 20-40!J.M concentrations of peptide I for 48 h. The inactivation was specific, as no changes were found in the activities of two other intracellu­lar enzymes of collagen synthesis, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase. Synthesis of 4-hydroxyproline by the fibroblasts accumulated intracellu­larly; whereas no changes were found in the incorporation of e4C]leucine into protein after culturing of the cells with a 30 !J.M concentration of peptide I for 48 h. No changes were seen in the viability of the cells or the release of lactate dehydrogenase from them into the culture medium. No significant changes were found in the steady-state levels of the mRNAs for the proal chains of type I and type III procollagens or for the a and ~ subunits of 48 h. The data indicate that inactivation of cellular prolyl 4-hydroxylase has marked effects on cellular 4-hydroxyproline formation and collagen secretion, but no effects on the steady-state levels of mRNAs for type I and III procollagens or the two types of subunit of prolyI4-hydroxylase.

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Glucose Increases the Expression of Matrix Genes in Rat Neural Connective Tissue Cells In Vitro.

Paivi Muona, Sirkku Jaakkola, J uha Peltonen and Jouni Ditto

Departments of Dermatology, and Biochemistry and Molecular Biology, Jefferson Medical College and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA,USA.

The effects of hyperglycemic glucose concentrations on the expression of extracellular matrix genes were examined in primary cell cultures initiated from sciatic nerves of normal Sprague-Dawley rats. Confluent cell cultures con­sisting of a mixture of Schwann cells, perineurial cells and fibroblasts were incubated either under normoglycemic (5.5 mM of D-glucose), or hyperglycemic (15 mM or 25 mM of D-glucose) conditions for 3 or 14 days, and the expression of the genes encoding type I and IV collagens, fibronection and ~-actin was examined by Northern trans­fer analysis, followed by scanning densitometry of the auto­radiographic films. Increased mRNA levels of al(IV), a2(IV), al(I) and fibronectin (FN) were observed after 3 days of incubation in hyperglycemic glucose concentra­tions. Extension of incubation up to 14 days did not elicit an additional increase in the mRNA levels. The relative mRNA levels expressed as percent of mRNA levels in nor­moglycemic cultures (mean ± SE) are given in the following table:

15mM 25mM

al (IV)/ ~-actin 206 ± 37 252 ± 43

a2(IV)/ ~-actin 190 ± 40 227 ± 39

a1(I)/ ~-actin 246 ± 70 255 ± 49

FN/ ~-actin 142 ± 24 225 ± 64

The use of D-mannitol instead of D-glucose did not alter the mRNA steady-state levels. Consequently, the effect of glucose cannot be attributed to the change in the osmolality of the cell culture medium. Also, the apparent half-lives of mRNAs were unchanged in cells incubated with high glu­cose concentrations as judged by transcription inhibition studies. The enhanced expression of genes coding for the a­chains of type IV collagen in response to high glucose con­centration may contribute to the accumulation of basement membrane material in diabetic peripheral nerves, and thus playa role in the development of diabetic neuropathy.

The Complete Primary Structure of Two Distinct Forms of Human al(IX) Collagen Chains.

Y. Muragaki, T. Kimura, Y. Ninomiya and B. R. Olsen

Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, MA, USA.

The human al(IX) collagen gene, located on chromo­some 6, gives rise to two transcripts with different sequences in their 5' regions because of the alternative use of two transcription start sites and RNA splice patterns. Given the potential involvement of type IX collagen in human chondrodysplasias, the generation of probes and determination of the primary structure for the two tran­scripts is of considerable interest. We have therefore iso­lated and sequenced cDNAs encoding the mutually exclu­sive forms of the human al(IX) collagen chain.

Poly(A)-rich RNA isolated from human chondrocytes (provided by Dr. M. Goldring) was used as template for synthesis of cDNAs specific for the long form of al(IX). Both priming with oligo(dT) and specific oligonucleotides, as well as the polymerase chain reaction (PCR) were used to generate several overlapping cDNAs. The conceptual trans­lation product of the combined nucleotide sequence of the cDNAs contains 931 amino acid residues, which 268 residues encoding the signal peptide and the amino-termi­nal globular domain (NC4).

Human fetal RNA (provided by Dr. A. Henney) was used in conjunction with PCR to generate a cDNA specific for the short form of al(IX). The translation product did not contain the 268-residue sequence of the long form but contained instead an alternative signal peptide of 22-amino acid residues and 2 residues of non-triple-helical sequence. This 24-residue sequence is encoded by an alternative exon in the al(IX) gene.

We conclude that human fetal tissues contain two alter­native forms of al (IX) collagen chains, similar to the forms previously described in embryonic chick tissues.

cDNA Clones for Chicken and Human Lysyl Hydroxylase.

R. Myllyla, L. Pajunen, T. Hautala, T. Turpeenniemi­Hujanen, K. I. Kivirikko and T. Pihlajaniemi

Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland.

Lysyl hydroxylase (E. C.1.14 .11.4), an enzyme catalysing the hydroxylation of lysine residues in collagen, is charac­terized here at the cDNA level. Highly specific polyclonal antibodies were used to screen a chicken embryo cDNA

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library in Agtll bacteriophage. To establish the identity of the positive recombinants recognized by the antibodies, these recombinants were further studied by hybridization with oligonucleotides based on internal peptide sequence of isolated chicken embryo lysyl hydroxylase. A clone that was positive by both antibody and oligonucleotide screen­ing was used as a probe to obtain additional overlapping cDNA clones for lysyl hydroxylase. The cDNA clones encode a 730-amino acid residue polypeptide and cover about 110 nucleotides of 5' untranslated sequences and about 1600 nucleotides of 3' sequences. A 20-amino acid residue hydrophobic signal peptide precedes the aminoter­minal end of the chicken lysyl hydroxylase obtained by protein sequencing. The amino acid sequence derived from the cDNA clones fully matched six peptide sequences obtained from isolated chicken lysyl hydroxylase. Anti­serum to a synthetic peptide of IS-amino acids derived from the cDNA sequence was prepared in a rabbit. These antibodies stained the same protein band in immunoblot­ting as the antibodies prepared against purified chicken lysyl hydroxylase, and also bound the lysyl hydroxylase activity when examined with antibodies coupled to a pro­tein A-Sepharose column. The chicken lysyl hydroxylase mRNA is about 4 kb by Northern blotting, and Southern blotting data suggest that one gene encodes the enzyme. The cDNA clones for chicken lysyl hydroxylase were used to obtain clones for human lysyl hydroxylase, and charac­terization of these human clones is now in progress.

Human Nidogen Gene: Characterization of Nucleotide Sequences at the 5'-Flanking Region and Evidence for Functional Promoter Activity.

Joan O'Leary, Michael Fazio, Yeli-Matti Kiihiiri, Yue Qiu Chen, Biagio Saitta and Jouni Uitto.

Jefferson Med. College, Philadelphia, PA, USA.

Nidogen is a sulfated multifunctional glycoprotein pres­ent in basement membranes. We have recently delineated the entire primary sequence of human nidogen through cDNA cloning (DNA 8: 581-594, 1989). In this study, we have cloned the 5'-flanking region of the human nidogen gene. A -35-kb clone (NCos4) was isolated from a human cosmid genomic library by screening with a 5' fragment of the nidogen cDNA. Hybridizations of EcoRI-digested NCos4 with a 21-bp oligomer corresponding to the nido­gen signal sequence allowed isolation of a 3.7-kb fragment. This subclone contained -0.9 kb of 5' -flanking sequences of the gene. Nucleotide sequencing of the flanking DNA revealed the presence of two canonical CCAA T consensus sequences in the antisense strand and a variant of the TAT A

motif, TA TTT. One putative AP-2 and six SP1 binding sites were also present. A TPA-responsive element and two regions resembling a recently characterized retinoic acid responsive element were also identified. To test the func­tional promoter activity of the 5'-flanking genomic DNA, several nidogen promoter/CA T reporter gene constructs were developed and analyzed in transient transfections of human and mouse cell cultures. Clearly detectable CAT activity was present in cells transfected with constructs spanning the promoter region from -1 to -864. Thus, the results suggest that the 5'-flanking region of the nidogen gene contains cis-acting regulatory elements necessary for transcription. The nidogen promoter/CA T gene constructs provide a means to study the transcriptional regulation of nidogen gene expression by trans-acting factors, and in human diseases of the basement membrane zone.

Regulatory Sequences of the 3 'UTR of CO LlAl Gene.

Risto Penttinen 1, Arto Miiiittii 1 and Paul Bornstein2

1 Department of Medical Biochemistry, Univ. of Turku, Finland and 2 Department of Biochemistry, Univ. of Washington, Seattle, W A, USA.

Sequencing of human COLIAl gene 3' untranslated region (3'UTR) revealed two polyadenylation sites about 1.1 kb apart, a long T rich stretch and potential regulatory motifs. Nucleotides flanking both polyadenylation signals were extremely conserved and were separated by a less homologous region of 700 bp.

Sequences 3' from the last exon covering either one (0.3 kb) or both of the polyadenylation sites (2.3 kb) were cloned into expression plasmids containing a homologous COllA 1 promoter/enhancer plus a short segment of human growth hormone (hGH) gene and transfected into chick tendon cells. Plasm ids containing only the first poly­adenylation site yielded less hGH RNA than those having both sites suggesting that COllA 1 sequences 3' from the first polyadenylation site are important in the expression of collagen gene.

Supported by The Turku University Foundation.

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AP-l Site and Upstream Region(s) of Human Stromelysin Promoter Can Cooperatively Mediate IL-l Induction in Human Fibroblasts.

Susan Quinones, Giovanna Buttice and Markku Kurkinen

Department of Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.

Interleukin-1 (IL-1) is produced by monocytes and mac­rophages during the inflammatory response. It induces the synthesis of stromelysin, a metalloproteinase capable of degrading a wide variety of extracellular matrix compo­nents. IL-1 exerts its influence on stromelysin production at the level of transcription. We are delineating the regions of the human stromelysin promoter (1.3 kb) that are the prim­ary targets for IL-1 induction in human fibroblasts using transient transfection techniques. Studies with constructs containing promoter fragments progressively shortened from the 5'-end with Bal31 reveal that the stromelysin promoter remains inducible by IL-1 until the region between -102 and - 57 is deleted. This region contains the AP-1 sequence, TGAGTCA, that binds Fos/Jun proteins and thereby mediates the induction of many different genes. A reporter vector containing only 5 copies of the AP-1 sequence was also inducible by IL-1. However, 1.3-kb pro­moter mutants containing single nucleotide changes in the AP-1 site, that eliminate in vitro synthesized Fos/Jun bind­ing in gelshift, still exhibited relative IL-1 induction pat­terns similar to the wild-type promoter. The same was true for a mutant from which the AP-1 sequence was removed. These data suggest that the AP-1 site at -70 to -64 is only one of the regions of the stromelysin promoter responsive to IL-1. Preliminary experiments with a fragment of the promoter containing a mutated AP-1 site suggest that at least one IL-1 responsive region resides within 460 nucle­otides of the transcription start site.

Analysis of Transcriptional Enhancers of Mouse Collagen IV Gene.

Shizuko Tanaka\ Paul Kaytes2, Gabriel Vogeli2 and Markku Kurkinen 1

1 UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, and 2 The Upjohn Company, Kalamazoo, MI, USA.

Mouse a1(IV) and a2(IV) collagen genes are assembled head to head and separated by only 130-base pairs, which contain a bidirectional promoter for both genes. So the regulation of type IV collagen gene promoter can be an

essential factor to a tissue specific and a developmentally regulated expression of type IV collagen. To study the gene regulation, an activity of type IV collagen promoter of a2(IV) direction was monitored by a transient transfection assay with human growth hormone as a reporter gene. Several gene fragments covering 5.0 kb upstream and 8.0 kb downstream of the promoter were examined for their transcriptional enhancing activities. There are two remarkable enhancers: One is in the a1(IV) first intron and the other one is in the a2(IV) third intron, both of which could work synergistically.

The a1(IV) first intron enhancer (3.2-kb Hind III-Hind III fragment) not only had a larger enhancing activity in PYS-2 cells (partietal york sac lineage) than in 3T3 cells (fi­broblast lineage) but also showed a transcriptional augmentation in differentiated F9 cells compared to the undifferentiated F9 cells. This enhancer could be narrowed down to 0.3 kb, which still retained a substantial enhancing activity and all the specificity of the original 3.2-kb frag­ment. To further characterize the enhancing elements, the 0.3-kb fragment was analyzed by a DNAse footprinting using a PYS-2 nuclear extract and several protected areas were found. Gel shift assays with oligonucleotides corre­sponding to the protected areas confirmed a presence of specific binding proteins to them and some of the oligonuc­leotides could cross-compete each other for their specific binding to various degree. Based on these results we have identifed a family of enhancing elements with the core consensus sequence, AC(T/A)(T/A)(T/A)TC(TlA).

Characterization of Regulatory Elements of the Human Prolyl4-Hydroxylase B-Subunit Gene.

K. T asanen, J. Oikarinen, K.1. Kivirikko and T. Pihlajaniemi

Collagen Research Unit, Biocenter and Department of Medical Biochemistry, University of Oulu, Oulu, Finland.

ProlyI4-hydroxylase, an a2~2 tetramer catalyzes the for­mation of 4-hydroxyproline in collagens. The ~-subunit of this enzyme has proved to be a highly unusual, multifunc­tional polypeptide. In addition to being the ~-subunit of prolyl 4-hydroxylase, it is identical to the enzyme protein disulfide isomerase and a major cellular thyroid hormone binding protein, and highly homologous to a glycosylation site binding component of oligosaccharyl transferase.

The human gene coding for the ~-subunit is 16.5 kb in length and consists of 11 exons. The 5'-flanking region of the gene contains several potential control elements includ­ing a TAT A box and six CCAA T boxes. In order to eluci­date the regulatory elements of the ~-subunit gene we have constructed several promoter fragments which have differ-

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ent 5' ends and the same 3' end. These deletion fragments were fused to the human growth hormone (hGH) gene and the fusion genes were introduced into HeLa cells by trans­ient transfections. The transcriptional efficiency of each promoter fragment was determined by a radioimmunoas­say for hGH. All deletion fragments of the ~-subunit pro­moter were also linked to a plasmid vector containing the bacterial chloramphenicol acetyl transferase (CAT) gene and the transcriptional efficiency was determined by a CAT assay. The highest transcriptional efficiency was obtained with a 630-nucleotide fragment covering nucleotides - 5 63 to +67 of the ~-subunit gene. DNase I footprinting studies with nuclear extracts from He La cells have revealed several protected areas including all six CCAAT boxes and a puta­tive cyclic AMP-responsive element. Further studies to measure the effects of point mutations in the regulatory elements of the ~-subunit gene are now in progress.

Modulated Expression of Collagen IX During Endochondral Ossification.

Ronald W. Tracy, Bjorn R. Olsen and !chiro Nishimura

Harvard School of Dental Medicine and Harvard Medical School, Cambridge, MA, USA.

In the developing chicken embryo, collagen IV is ex­pressed in two distinct forms. In hyaline cartilage, colla­gen IX contains a large amino-terminal globule (NC4 globule) at the end of the short collagenous arm so that the NC4 globules decorate the surface of the collagen II fibrils. In the embryonic cornea, the NC4 globule is missing. This difference is due to the usage of alternative promoters in the al(IX) gene.

During endochondral ossification, chondrocytes become hypertrophic and the cartilage matrix is mineralized before it is degraded and replaced by bone and bone marrow. In the present study, the expression and the modulation of the al(IX) gene was investigated using in situ hybridization. 18 day-old embryonic chick sternal cartilage, the superior por­tion of which undergoes endochondral ossification, was chosen as the model for the study.

The al(II) probe (p YN509) stained both small and hypertrophic chondrocytes. The negative control probe (pGEM) stained neither. The long form al(IX) probe (IN321) labeled the small chondrocyte region; whereas the short form al(IX) probe (IN212) was localized only in the region of hypertrophic chondrocytes.

The nucleotide sequence of IN212 corresponds to the 5'

portion of al(IX) mRNA transcribed from a downstream promoter, also utilized in the embryonic cornea. Significant staining of the hypertrophic chondrocytes with the IN212 probe indicates that the downstream promoter becomes active in hypertrophic chondrocytes; while the upstream promoter, used for synthesis of the long form of al(IX) chains, is active in small chondrocytes.

These data suggest that the conversion of cartilage to bone may be regulated, in part, by the molecular alteration at the NC4 domain of collagen IX due to a promoter switch in the al(IX) gene. (Supported by NIH grant EY 8219)

Differential Induction of mRNAs for a and ~ Subunits ofProlyl4-Hydroxylase in Human Skin Fibroblasts by Hydralazine.

Heather N. Yeowell, Saood Murad and Sheldon R. Pinnell

Division of Dermatology, Duke University Medical Center, Durham, NC, USA.

Prolyl hydroxylase (PH), a key enzyme in collagen syn­thesis, is a tetra mer composed of nonidentical subunits a2~2> which have differently regulated rates of synthesis. The a subunit, probably the major component of the catalytic site, is incorporated into the tetramer immediately after its synthesis whereas the ~ subunit, which is virtually identical to protein disulfide-isomerase, is produced in excess and more slowly incorporated. Hydralazine has been shown to increase PH activity in human skin fibro­blasts whereas it reduces total collagen synthesis (A. B. B. 241: 356, 1985). To determine the mechanism of these effects, we measured mRNA levels for the a and ~ subunits of PH and al(I) collagen by hybridization analysis. Over a period of 0-96 h, 50 11M hydralazine induced aPH mRNA maximally by > 3-fold at 24 h whereas ~PH mRNA was not maximally induced until 72 h. In contrast, the 5.8- and 4.8-kb mRNAs for al(I) collagen were virtually eliminated by 48 hand 72 h respectively. Interestingly, a 3- to 4-fold induction of fibronectin mRNA by hydralazine was ob­served at 24 h which was maintained up to 72 h. This study demonstrates that the increased PH activity is a direct result of hydralazine-mediated increases in message for the a and ~ subunits of PH. Moreover, the earlier induction of aPH mRNA by hydralazine may indicate a central role for this subunit in expression of PH activity. In addition, the decrease in collagen synthesis effected by hydralazine results from suppression of both species of mRNA for al(I) collagen.