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THE OURNAL
f BIOLOGICAL
H EMIS TRY
Vol.
257. NO.19,
Issue of October
10, pp. 11627-11632 1982
P o n i e d m U S.A.
Growth HormoneGene Transcription Is Regulated by Thyroid and
Glucocorticoid Hormones
n
Cultured Rat Pituitary Tumor Cells*
(Received for publication, April 28,1982)
Stephen R. Spindlerlg, Synthia
H.
Mellonnll, an d Jo hn
D.
Baxterl[**
From the Department o f Biochemistry, U niversity o f California, Riversid e, California 92521 and IJTheHoward Hughes
Med ical Institute L abora tories, Metabolic Research Unit, Department of Medicine, University
of
California, Sun Francisco,
California 94143
The effects of thyroid and glucocorticoid hormones
on th e transcription
of
the growth hormone ene were
measuredusingradioactively abeled cell-free tra n-
scription products of nuclei isolated from cultured rat
pituitary tumor cells (GC line). The amount of growth
hormone gene transcription as quantit ated y hybrid-
ization
of
the radioactivelyabeled transcripts o
cloned growth hormone gene sequences which were
immobilized on nitrocellulose filters. Cells were main-
tained for 7 days in culture medium containing 10%
serum from a hyroidectomized calf, and then incu-
bated in this medium supplemented with hormones.
After 4 h of hormone t reatm ent, hyroidhormone
3,3’,5’-triiodo-~-thyronine)roduced a 17-fold increase
in gene activity. Dexamethasone, a synthetic glucocor-
ticoid, produced an approximate doubling in gene ac-
tivity when
it
was administered alone to theells for
4
h. When dexamethasone was administered in combi-
nation with triiodothyronine,
t
enhanced gene activity
to abou t twice that seen with triiodothyronine alone.
These early hormonal effects were diminished by con-
tinued hormone treatment. After 72 h of triiodothyro-
nine stimulation, or stimulation with both triiodothy-
ronine and dexamethasone, transcription of the gene
decreased about 8-fold from the levels found after
4
h.
However, both the transcriptional responsef the gene
to triiodothyronine alone and the synergistic response
of the gene to the ombination of thyroid andglucocor-
ticoid hormones were still evident
fter
72 h.
In he presence of the protein synthesis nhibitor
cycloheximide, thyroid hormone and the combination
of
thyroid and glucocorticoid hormones induced gene
activity 9- and 31-fold, respectively, after 4 h of treat-
ment. These results suggest that annduced protein is
not required for the inductionf the gene. The presence
of 2 pg/ml of a-amanitin abolishes the cell-free tran -
scription of the growth hormone gene, indica ting ha t
the gene
is
transcribed by RNA polymerase 11.
The number of growth hormone mRNA molecules/
cell wasdetermined by RNA-driven hybridizations
with adioactively abeled growth hormone cDNA.
Growth hormone mRNA number/cell increased about
The costs of publication of this article were defrayed in part by
the payment of page chmges. Thi s article must therefore be hereby
marked
“aduertisernent”
in accordance with
18
U.S.C. Section
1734
solely to indicate this fact.
$Recipient of National Institutes of Health Grant AM 30412,
United States Public Health Service Biomedical Research Support
Grant P R 0701016, and research grants from the Academic Senate,
University of California, Riverside.
1Recipient of Nationa l Institutes of Health Postdoctoral Fellow-
ships
CA
06630 and AM 06588.
* * Recipient of National Ins titutes of Health GrantsAM 18878 and
AM 19997. Investigator
of
the Howard Hughes Medical Instit ute.
2.7-, 22-, and 83-fold after 72 h of stimulationwith
dexamethasone,riiodothyronine, ndriiodothyro-
nineplusdexamethasone, espectively.Because the
kinetics of the riiodothyronine and dexamethasone
stimulation of gene activity s not known, it is not
possible presently to correlate accurately the increases
in growth hormone mRNA content with the increases
in gene activities. However, the data demo nstrate
hat
glucocorticoid andhyroidhormonesncreasehe
growth hormone mRNA content of the GC line of rat
pituitary tumor cells at least in p ar t by directly and
rapidly increasing the amountf growth hormone gene
transcription. In addition, the effects
of
the hormones
are synergistic at the ranscriptional level.
Growth hormone production in the anter ior pituitary of
mammals appears tobe regulated by a number of hormones.
The
GH
clonal iines of rat pituita ry tumorcells produce
GH’
and prolactin n culture (1, 2) and provide model systems for
studying the expression of these genes under defined condi-
tions. Thyroid nd glucocorticoid hormones nduceboth
growth hormone production and
GH
mRNA activity in these
cells (3-5), and together the hormones areynergistic in their
effects under appropriate culture conditions (4,
6).
The hor-
mones apparently enhance the accumulationf both mature
and precursor growth hormone mRNA in these cells 7, 8).
When associated with their receptors, thyroid and gluco-
corticoid hormones interact specifically with the genome of
target tissues. Thus, it is possible that the hormone-receptor
complexes act directly to enhance the transcription of the
growth hormone gene. Indeed, glucocorticoids appear to in-
crease directly mouse mammary tumor virus (9) and mouse
metallothionein 1gene transcription (10).However, hormones
may
also
affect other aspects of RNA metabolism to increase
th e expression of a gene. Enhanced stabili ty of
GH
mRNA
and
its
precursor could account for the accumulationf these
molecules in hormonally stimulated cells. Estradiol and pro-
gesterone increase the stabilityof conalbumin mRNA, from a
calculated half-life of 2.9 h in the absenceof the hormones
o
at least 8 h in their presence (11).
We report here that both thyroid and glucocorticoid hor-
mones directly and rapidly stimulate the transcriptionof the
growth hormone gene in rat pituitary tumor cells GC line).
Together the hormones have synergistic effect on the tran-
scription of the gene.
MATERIALS AND METHODS
Cell Culture-Cells of the GC rat pituitary tumor line (1,
2)
were
I
The abbreviations used are: GH, growth hormone;
T,,,
3,3‘5’-
triiodo-L-thyronine.
11627
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11628 Growth Hormone Gene Transcription
cultured in monolayer in DME H-21 nutrient medium (University of
California, San Francisco, Tissue Culture Facility or GIBCO, Inc.)
containing 10% calf serum, 100 pg/ml of streptomycin sulfate, 60 pg/
ml (100uni ts/ml) of penicillin, 2 mM glutamine.
Preparation
of
Immobilized Probe DNA-A recombinant bacte-
rial plasmid containing the struc tural gene sequence (cDNA) for rat
growthhormone, prGH-1 (12), was cleaved by either estrict ion
endonuclease Bum H1, in 50 mM NaCI, 20 mM Tris-HCI, pH 7.5, 7
mM MgC12, 100 pg/ml of bovine serum albumin (nuclease-free), 1mM
dithiothreitol, or
Eco
RI, in 50 mM NaCI, 100 mM Tris-HCI, pH 7.9,
7 mM MgC12, 100pg/ml of bovine serum albumin, 1mM dithiothreitol.
Th e reactions were termina ted by heating to 70 "C for 10
min,
and
the DNA was digested to completion with exonuclease I11 at 37 "C.
After the addition of sodium dodecyl sulfate to 0.5% and protease K
to 50 pg/ml, the reaction mixtu res were incubated for 1 h at 45 "C
and extracted wice with equal volumesof phenol/chloroform (l :l, v/
v) ,
and the DNA was precipitated with 2 volumes of ethanol. DNA
was immobilized on 6-mmitrocelluloseilters (grade BA85,
Schleicher & Schuell) by either the methodf McKnight and Palmiter
(11) or Kafatos et al. (13). Fiiters containing pBR322 were similarly
prepared. The filters were washed
1
h a t 45 "C in Buffer A (0.3
M
NaCI, 2 mM EDTA, 10 mM Tris-HCI, pH 7.5) containing 0.1% sodium
dodecyl sulfate, 0.2% Ficoll, 0.2% polyvinyl polypyrrolidone, 0.01%
RNase-free bovine serum albumin, blotted with paper towels, air-
dried, and baked for 2 h at 80 "C in a vacuum oven. About 0.4 to 2
pg of prGH-1 or pBR322 DNA was bound/filter as judged by the
retent ion of radioactively labeled plasmid DNA on the filters, or by
diphenylamine DNA determinations (14). Sequential incubation of
prGH-1 with Bum H1 andexonuclease
I11
results in the digestion of
the sense strand of the cloned cDNA. Treatment of prGH-1
with
Eco
RI and xonuclease
111
results in the digestion of the antisense strand
of the cloned cDNA.
Preparation
of
"H-labeled GH cRNA-RNA complementary to
the GH oding sequence was prepared by transcribing polyacrylamide
gel-purified CH sequences excised from prGH-1 with HindIII. Tran-
scription reactions of 12 pl contained 0.1 pg of cloned GH cDNA, 2.3
pg of Escherich ia coli RNA polymerase, 20 mM Tris-HCI, pH 7.9, 1.6
mM MnCL, 10% glycerol (v/v ), 60 PM GTP, CTP , ATP, and [5'-3H]
UT P (24 Ci/mmol), 150 mM NaCI, 0.5 mM dithiothreitol. Reactions
were incubated at 7 "C for 2 h nd theRNA was purified by digestion
with
100
pg/ml of RNase-free DNase I for 10 min at 37 C. The
reactions were adjusted toinal concentrat ions of 0.5% sodium dodecyl
sulfate, 120 mM EDTA,
100
pg/ml of yeast RNA, 50 pg/ml of protease
K and incubated at 45 "C for
1
h. After two extractions with phenol/
chloroform l:l, v/v) the reacti ons were chromatographed on Seph-
adex G-50 or Bio-Gel P-60 columns.Th e purified ["HIRNA was heat -
denatured andhybridized t o nitrocellulose fdters containing either 50
pgof Bum HI or Eco RI cleaved, exonuclease 111 digested prGH-1
DNA. Hybridizations were performed in 300 mM NaCI, 20 mM 1,4-
piperazinediethanesulfonicacid, pH 7.0, 0.4%sodium dodecyl sulfate,
2 mM EDTA, 33% formamide a t 45 "C for 48 h. Afterextensive
washing in Buffer A, 0.1% sodiumdodecylsulfate, the RNA was
eluted from the filters by heating them separately to 00 "C for 5 min
in 2 mM EDTA, pH 7.9, 0.1% sodium dodecyl sulfate. Th e RN A was
recovered by ethanol precipitation.
Determination of Growth Hormone Gene Actiuities-Confluent
cultures of GC cells were maintained for 7 days in DME H-21medium
containing 10% serum derived from a thyroidectomized calf (hypo-
medium; Rockland, Gilbertsville, PA). Medium was changed daily.
Hormone nductionswere nitiated by adding freshhypo-medium
containing 1 PM dexamethasoneand/or 10 nM TJ. After various
lengths
of
incubation, the cell monolayers were removed by washing
twice with phosphate-buffered saline, once with calcium- and mag-
nesium-free phosphate-buffered saline,and incubating or 5 to 10min
at 37 "C in calcium- and magnesium-free phosphate-buffered saline,
3 mM EDTA. Subsequent procedures were conducted at 0-4 "C. Th e
dislodged cells were collected by centrifugation, washed one time in
lysis buffer (10 mM NaCI, 10mM 'iris-HCI, pH 7.9,3mM MgC12, 1mM
dithiothreitol), and resuspended in lysis buffer. Th e suspension was
adjusted to0.5% Nonidet P-40, and the cells were disrupted by to 8
strokes with the
A
pestle naDounce homogenizer. Nucleiwere
collected by low speed centrifugation and washed once in ysis buffer.
RNA was purified from the cytoplasmic fraction a s described below.
The nuclear pellets were resuspended with volume of transcription
cocktail to a final concentration of 60 mM Tris-HCI, pH 7.9, 6%
glycerol,
0.6
mM ATP and UTP,
3
mM MnCL, 35 mM ammonium
sulfate,
5
mM NaF, 9 p~ creatine phosphate,
18
pg/ml of creatine
phosphokinase, 0.5 mM dithio threitol, 250 pCi each of lol-32P1-CTP
and [,-3'P]-GTP (400 Ci/mmol). After 20 min of incubation a t 29 "C,
an equalvolume of DNase buffer (220 mM 4-(2-hydroxyethyl)- l-
piperazineethanesulfonic
acid, pH 7.4), 5 mM MgCI2,1 mM CaCI2,1
mM MnC12, and iodoace tate-treated RNase-free DNase I (100 pg/ml
final concentration) were added and thencubation continued for 5 o
10 minutes. An equal volume of 25 mM Tris-HCI, pH 7.9, 200 mM
NaCI, 10 mM EDTA, 100 pg/ml of protease K, 2% sodium dodecyl
sulfate was added, and the reaction was incubated
at
45 "C for 1 h.
After two extractions with phenol/chloroform ( Ll , v/v) the RNAwas
further purified by the method of Evans et
al.
(15). Using a modifi-
cation of the hybridization procedure of McKnight and Palmiterl l ) ,
RNA was hybridized to
growth hormone gene sequences or pBR322
DNA prepared and immobilized on nitrocellulose filters as described
above. Reactions contained, in a final volume of 30 pl, 0.5 M NaCI, 50
mM 1,4-piperazinediethanesulfonic cid, pH 7.0,33% formamide, 0.4%
sodium dodecyl sulfate, 2 mM EDTA, 2000 cpm of GH [3H]cRNA.
Incubations were at 45 "C for 3 days. The filters were washed twice
by vortexing briefly in an equal volume of chloroform and Buffer A
a t room temperature, followed by two 1-h washes with entle shaking
in Buffer A, 0.1% sodium dodecyl sulfate at 45 "C. T he filters were
further washed for 30 min a t 45 "C in 0.4% sodium dodecyl sulfate, 5
mM Tris-HCI, pH 7.5, 2 mM EDTA, 10 mM NaCI, followed by two
brief washes with Buffer A and digestion a t 37 "C with 10 pg/ml of
RNase
A
and 1 pg/ml of RNase TI n Buffer A. Each enzyme had
been previously incuba ted at 80 "C for10 min. Th e filters were inally
washed 2 times for
1
h each a t 45 "C in Buffer A, 0.1% odium dodecyl
sulfate. Radioactivity bound o the ilters was dete rmined as escribed
(11).
The efficiency of these reactions was about 15% using filters
prepared by the method of McKnight and Palmiter (11) and about
30% using filters prepared by the method of Kafatos et al. (13), as
judged by the hybridization of the cRNA internal standards. Th e
amount of GH RNA synthesis was determined by subtracting the
average radioactivity of two filters containing pBR322 DNA
(1
to
3
ppm) from the radioactivitybound to a prGH-1 DNA-containing
filter divided by both the tota l ["'PIRNA in the react ion and the
efficiency of the hybridization reaction, determined by t he hybridi-
zation of the ["HIcRNA internal standard. There was no significant
reduction in the hybridization efficiencies of the inte rnal standard y
the labeled nuclear RNA preparations.
Preparation
of
Growth Hormone ~"'P]cDNA-["P]DNA comple-
mentary to GH mRNA was prepared from prGH-1 as described (7).
Th e specific activity of the probe was about 4 X
lo7
pm/pg.
T4
DNA
polymerase was obtained from Bethesda Research Laboratories.
Determination
of
Growth Hormone mRNA Leuels-Cytoplasmic
fractions obtained as described above were adjusted to 0.5% sodium
dodecyl sulfate, 50 pg/ml of protease K and incuba ted at 45 "C for 1
h. Th e solutions were extracted twice with phenol/chloroform, fol-
lowed by ethanol precipitation of the RNA. The RNAwas collected
by centrifugation and hybridized a t various concentrations with GH
["PIcDNA. The tot al concentration of RNA n each reaction was
adjusted to 10mg/ml by the addition of yeast total RNA. Reactions
were performed sealed in siliconized 5-pl capillary pipettes in a final
volume of 5
pl
containing 0.4 M NaCI, 10 mM 4-(2-hydroxyethyl)-l-
piperazineethanesulfonic
acid, pH 7.4, 1 mM EDTA, and lo00 cpm of
growth hormone [J2P]cDNA. Th e percentage of hybrid formed was
determined by
S1
nuclease digestion as described by Maxwell et al.
(16), and he number of molecules of growthhormoneRNA/cell
calculated as described by Wegnez et al. (7).
RESULTS
R N A
Synthesis in Isolated Nuclei-To determine the ef-
fects of thyroid and glucocorticoid hormones on the activity
of the growth hormone gene, measurements were made of the
relative amount of transcription of the gene in the presence or
absence of thyroid and/or glucocorticoid hormones. To make
these measurements radioactively labeled R N A was synthe-
sized in nuclei isolated from rat pituitary tumor cells of th e
GC
cell line. Fig. 1 llustrates th e kinetics of the R N A synthetic
reaction. Synthesis proceeded rapidly during the fiist 10 min
of incubation, after which it declined steadily until at 60 min
no further incorporation of radioactivity was observed.
As
illustrated, no decrease was observed in the amount of radio-
activity incorporated into R N A after 1 additional h of incu-
bation. Similar results were obtained even when a 100-fold
excess of unlabeled nucleotide triphosphate was added at th e
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Growth Hormone Gene Transcription 11629
I I
MINUTES
6
20
FIG.
1. Rate
of
RNA synthesis in nuclei.
Reactions of
40 pl
contained
50
mM Tris-HC1,pH
7.9, 2
mM MnCL,
2
mM MgCL,
20
mM
(NH4)2S04,
0%
glycerol,
0.6
mM GTP, CTP, and ATP,
60
p~ UTP;
0.5
mM dithiothreitol;
2
pCi of ['HIUTP,
6
mM NaF,
10
p~ creatine
phosphate,
1
pg/ml of creatine phosphokinase, IO6 nuclei, and 20
units/ml of human placental ribonuclease inhibitor prepared by the
method of Blackburn (19) O),
r
no ribonuclease inhibitor 0).t
the indicated times
50
p g / d of iodoacetate-treated, RNase-free
DNase was added and incubation continued for an additional
5
min.
Sodium dodecyl sulfate was added to a final concentration of 0.5%.
and aliquots were transferred o
DE-81
ilters. The filters were washed
and counted as previously described
(26).
end of the
1st
h of synthesis. However, occasionally as much
as a 20%decrease in the maximum incorporated radioactivity
was found during the 2nd hour of incubation, suggesting that
some nuclease activity was present in the nuclear prepara-
tions. Therefore, transcription reactions were usually carried
out for only 20 to 30 min, so that the ratef RNA degradation
would be minimal with respect to the rate f RNA synthesis.
Nuclei prepared from the
GHa
line of rat pituitary tumor
cells were reported to be unable to support appreciable cell-
free RNA synthesis unless rat liver ribonuclease inhibitor was
present (17). Since the GC cell line is a clonal derivative of
the GH3 line (18), we investigated the effects of ra t liver
ribonuclease inhibitor on the inetics of RNA synthesis in
GC
cell nuclei. The commercial inhibitor used in the published
studies was no longer available. However, we prepared the rat
liver inhibitor according to the protocol obtained from the
original supplier, and also according to th e method of Black-
burn (19). We
also
tested th e effects of the human placental
ribonuclease inhibitor (19). Some stimulation in the rate and
extent of RNA synthesis was observed with rat liver extracts
prepared according to the ommercial protocol. However, the
inhibitorsprepared by themethod of Blackburn had no
measurable effect on the kinetics or extent of RNA synthesis
in isolated GC cell nuclei, although the inhibitors had high
specific activities and were nearly homogeneous as judged by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The effects of the humanplacental ribonuclease inhibitor on
nuclear transcription are shown in Fig. 1. This inhibitor had
little effect
on
the ra te or extent of RNA synthesis. Similar
results were obtained using nuclei prepared from our clonal
subline of
GHa
cells. Therefore, no RNase inhibitor wasused
in the transcription experiments described below.
The effects of ionic strength on nuclear transcription were
also examined (Fig. 2). A bimodal ionic strength-activity pro-
file wasfound for RNA polymerase I1 transcription in GC cell
nuclei. Because the integrity of the nuclei was disrupted at
the higher ionic strengths, a s judged by light microscopy, we
chose to work at the lower ionic strength optimum where
these disruptive effects appeared minimal. At
35
mM ammo-
nium sulfate RNA polymerase activity in isolated nuclei is
composed of 22% polymerase I, 70% polymerase 11, and 8%
polymerase 111activity, as judged by titra tion of transcription
reactions with cy-amanitin (data not shown).
Activity
of
the Growth Hormone
Gene-In order to deter-
mine the role of thyroid and glucocorticoid hormones in the
transcription of the growth hormone gene, confluent cultures
of GC cells were maintained for 7 days in medium containing
10% serum from a thyroidectomized calf. This medium, hypo-
medium, lacks measurable levels of thyroid hormones. Under
these culture conditions the amountof growth hormone pro-
duced by the cells falls to low levels 4,6). The rateof growth
hormone synthesis an be markedly increased by several days
of stimulation with glucocorticoid and/or thyroid hormones
(4, 6). For this reason we began our investigations by deter-
mining GH gene activity after
72
h of hormonal stimulation
with the synthetic glucocorticoid hormone, dexamethasone,
T:l,
or a combination of the two hormones. Nuclei were iso-
lated, and the NA polymerases which had initiated synthesis
in vivo
were allowed to continue RNA synthesis in the pres-
ence of radioactively labeled precursors. This RNA was iso-
OI
I /
/.'
0
/
I
100
2 300
mM NH4)2 s o 4
FIG.
2. Dependence ofnuclear RNA polymerase activity on
ammonium sulfate concentration. Reactions of
40
pl contained 50
m
Tris-HCI, pH
7.9,
2
mM
MnC12,
2
mMMgC12,
10%
glycerol,
0.6
mM GTP, CTP, and ATP,
60 PM
UTP,
0.5
mM dithiothreitol,
1
pCi of
[ HIUTP,
6
mM NaF, 10
~
creatine phosphate,
100
p g / d of creatine
phosphokinase,
2 X
I d nuclei, the indicated concentrations of
(NH&S04 plus
or
minus 0.5 pg/ml of a-amanitin. After
20
rnin,
synthesis was terminated and the amount of radioactive label incor-
porated determined as described in the legend to Fig. 1. Synthesis in
the presence of a-amanitin
0);
ynthesis in the absence of a-amanitin
minus synthesis in
i t s
presence
0).
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11630
Growth
Hormone Gene Transcription
lated and hybridized to growth hormone gene sequences im-
mobilized on nitrocellulose filters. DNA of ei ther the growth
hormone coding or noncoding genesequences was usedon the
filters.
Consistent with the diminished production of growth hor-
mone in unstimulated, deinduced cells, the level of transcrip-
tion of the gene was also found to be quite low. As shown in
Table I, itaveraged
2.8
ppmafter
a
total of 10 days of
incubation in hypo-medium. A omparably low level of growth
hormone gene transcription was found after 7 days of incu-
bation in hypo-medium (Table
11).
After incubation for72 h
in hypo-mediumcontaining
Tn,
a 2.6-fold increase in the
activity of the genewas observed. When T:,was administered
together with dexamethasone for 72 h, there was a 5.2-fold
increase in the activi ty
o f
th e gene. However, dexamethasone
alone resulted in less than
a
2-fold increase in growth hormone
gene activity. These results suggest that the hormones stim -
ulate the transcription of the
GH
gene, and t hat together the
hormones are synergistic in this stimulation. No significant
hybridization to filters containing only the noncoding strand
of the genewas found (data not shown),uggesting that ther e
is no transcription
of
the antisense strandof the gene.
Th e transcript.iona1 effects of the hormones are rapid, as
shown inTable 11.After
4
h of treatment with dexamethasone
alone, the activity of the gene approximately doubled. Incu-
bation with
T:l
alone for the same amount f time resulted in
a 20-fold increase in the activi ty of the gene, while Tn and
TABLE
Growth
hormone gene transcriptional activi ty and cellular mRNA
after 72 h of hormonal stimulation
Confluent cultures of
GC
cells were maintained in hypo-medium
for 7 days. Where ndicated, he hypo-medium was supplemented
with hormones. Medium was changed daily. Nuclear and cytoplasmic
fractions were prepared and assayedor growth hormone ene activity
and mRNA levels. RNA and protein determinations were performed
on aliquots
of
cells. In the determin ationsf gene activity, background
hybridization to pBR322-containing filters was o 3 ppm.Th e results
presented are theaverages of three separate determinations, and the
hybridizations were usually performed in duplicate. Th e values for
GH-mRNA number/cell were calculated using the data shown in this
table and in Fig. 3.
In-
crease
GH gene
in
GH-
crease Total
2
ctivity
gene
mRNA
in
GH-
R N A
tein
In-
activ-
mRNA
ity
pprn foldolcules/ fold pg/rellpg/cell
cell
Control 2.8 i .0 45 49 23
Dexamethasone 3.9 3.8 1.4 125 2.7 43 20
T.J 7.2 2.1 2.6 1030 22.0 492
TZJnd dexa- 14.5 0.6 5.2900 83.0 460
methasone
TABLE1
Growth hormone gene activity after 4 h of hormonal stimulation
Confluent cu ltures of GC cells were incubated 7 days n hypo -
medium and reatedwith hypo-mediumcontaining the ndica ted
hormones
for
4
h. Nuclear growth hormoneRNA ranscriptional
activities were determined. Th e activities presented are averages of
three or m ore separate dete rmination s. Hybridizations were usually
pBR322 DNA-containing filters were
1
o 3 ppm.
performed in duplicate or triplicate.
Background hybridizations to
Hormone treatment
of
cells
Increase
in
~ _ _
GH genectivity
gene act ivi ty
PPm
fold
None 3.3 1.8
Dexamethasone 5.8 1.6 1.8
T:l
55 4.5 17
Dexamethasonend
T.3
120& 346
TABLE
11
Znduction of growth hormone gene transcription in the presence
of
cycloheximide
Nuclei were prepared from cells which had been deinduced for 7
days in hypo-medium and where indicated induced with hormones
for
4
h. When present, cycloheximide (0.1 mM) was included in the
medium 30 min prior to, as well as during the hormonal stimulation.
Background hybridization to pBR322 DNA -containing fdters was 2
to 3 ppm in these reactions, and the hybridization efficiencies were 33
&
58 . Hvbridizations were oerformed in duulicate or tridicate .
Cyclohexi- Input
[, PI ,2pPlRNA GH
RNA
cells zation
SIX
Hormonal treatment of midereat-RNA pm
cellsent
of
in hybridi-
h:::id
synthe-
__
CPm w ppm
None + 45.8 X lo6 17.4 1.2
Dexamethasone + 29.8 X
10"
10.0 1.0
Dexamethasone and e
+ 15.9 X IOti
191.5 36.6
None
65.0
X
10''
9.0 0.6
Dexamethasone and TI
31.9 X
10~:
78.5
7 5
T.3
+ 18.1
X
10 66.0 11.0
dexamethasone togetherncreased growthhormone gene tran-
scription 36-fold. Th e effects of thyroid and glucocorticoid
hormones are clearly synergistic after 4 h of hormonal treat-
ment, and the amountof transcription obtained at this early
time is about 8 times greater than that bserved after
72
h of
stimulation. Thus, the initial transcriptional esponse
of
the
gene is greatly reduced by prolonged incubation of t he cells
with the hormones.
Hormonal Responsiveness of the Gene after Inhibition of
Pro tein Synthesis-The studies described to this point were
performed with serum collected from
a
single thyroidecto-
mized calf. After this serum source was exhausted, we used
serum from a second thyroidectomized calf. Using this serum
we found tha t th e maximum and minimum levels of gene
activity were significantly less than those found previously.
Th e deinduced levelsof gene activity averaged about
0.8
ppm,
and after
4
h
of
stimulation with
T:i
and dexamethasone evels
were typically about 9 ppm. The reasons for these changesn
gene activity are obscure, but serum can haverofound effec ts
on the amountf growth hormone mRNAwhich accumulates
in response to hormones
7),
and these ffects may be eflected
at the transcriptionalevel. Using th e new serum batches, the
studies described below were performed.
In order to determine whether thyro id or glucocorticoid
hormones act directly to induce growth hormone gene tran-
scription, the hormona l esponsiveness of the gene was inves-
tigated in theresence of cycloheximide. At the concentra tion
used, about 95% of protein synthesis
is
inhibited while total
nuclear RNA synthesis is reduced by only about 20% (dat a
not shown). After deinduct ion in hypo-medium for 7 days,
cells were pretreated with cycloheximide for 30 min and then
hormonally induced for 4 h in the presence of the inh ibitor .
The resultsof these studies are hown in Table 111.Cyclohex-
imide does not prevent the rapid transcriptional esponse of
the growth hormone gene to th e ormones. Both
he TR
ffect
and the glucocorticoid-T:l synergism were observed. In addi-
tion, gene activitywas about 4.5-fold greate r in the absencef
proteinsynthesis han n its presence. The absence of a
response todexamethasonealone in thisexperiment was
probably not due o he presence of cycloheximide, since
similar results occasionally occur in the presence of protein
synthesis. Furthermore,
a
clear dexamethasone-Tn synergism
was found in the presence of th e drug.
Effect of a-Amanit in on the ell-free Transcription of the
Growth Hormone Gene-To determine whether the growth
hormone gene is transcribed by RNA polymerase
11,
RNA
synthesis was carried out in the presence of 2 pg/ml of a
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Growth Hormone Gene Transcription
11631
TABLEV
Growth hormone gene transcription with a-amanitin present in the
cell-free nuclear
R N A
synthetic reaction
Nuclei were prepared from cells which had been deinduced for 7
days in hypo-medium and induced with T.1and dexamethasone for 4
h where indicated. In the indicated reaction 2 pg/ml of a-amanitin
was present in the cell-free RNA synthetic reaction. In these experi-
ments background hybridization to pBR322 DNA-containing filters
was
1
to 2 ppm. The hybridization efficiencies were 25
r
2%.
Hybrid-
izations were Derformed in dunlicate.
Hormonal treat- transcription
ddition to Input
["PI
[ PIRNA G H
ment of cellseactionNA in hY-$$d- synthesis
bridization
cpm cpm
w
Noneone 65 x lo6 9 0.6
Dexamethasone None 25 X
IOti
63 10.1
Dexamethasone a-Amanitin
90 X
10 15.5
0.7
~~ ~~
and
T.,
and
T.,
I I O '
I O 2
l o 3
RO
FIG.
3. Hybridization-kinetic analysis of growth hormone
mRNA levels in the cytoplasm of deinduced and hormona ly
induced cells. GC
cells were deinduced for
7
days in hypo-medium
and reinduced for 72 h by the addition of the indicated hormones.
Medium was changed daily. The cytoplasmic RNA was purified and
analyzed for growth hormone mRNA. RNA from deinduced cells
0 ) ,
nd from cells induced with dexamethasone
O),
T,?
A),
and the
combination of dexamethasone and
T.,( A ) .
amanitin.Thisconce ntration of th e toxin is sufficient to
inhibit RNA polymerase
I1
but not RNA polymerase
I
or
I11
activities (20). As shown in Tab le
IV,
a-aman itin abolished
cell-free transcription of the growth hormone gene.
Levels of Growth Hormone m RNA
in
Induced and Dein-
duced
Cells-Fig. 3 presents the hybridization analys is and
Table
I
the number of growth hormone mRNA molecules
found in the cytoplasm of GC cells afte r 72 h of hormonal
stimulation. After 10 days in hypo-medium, an averagef only
45 molecules of GH mRNA were found/cell. Thi s result is
consiste nt with the ow level of growth hormone gene activi ty
andproteinproductio n found under hese conditions.
A s
shown nTable I, thereareabout 3 timesmoregrowth
hormone mRNAmolecules/cell afte r 3 daysof treatment with
dexamethasone than found in untreate d cells. T:, increased
the num ber of mRNA molecules/cell 22-fold, and when the
hormones were administeredogether, growth hormone
mRNA levels increase about 83-fold. Recause the kine tics of
the changes hich occur in genectivity ispresently unknown,
it is not possible to correlate them ith the accumu lated evels
of GH-mRNA found a t 72 h. However, it is clear from the
dat a in Table I that the hormones rofoundly affect the evel
of accumulated GH-mRNA/cell without significantly chang-
ing the total amountof RNA or protein/cell.
DISCUSSION
In contrast to the results obtainedy others with GH cells
17), the GC cell nuclear transcription system employed in
these studies does not appear to contain any major RNA
nuclease activity. In addition, the system described here ap -
pears to be asfficient with re spect to bo th the rate a nd ex tent
of RNA synthesis as most published nuclear transcription
systems (21, 22). The reaso ns or t he differences between our
results and thosef Biswas
et
al. (17) are not nown. However,
our cell linesmayconta in less ibonuclease and/orRNA
processing enzyme activity, or these nucleases may be ess
active under the transcription conditions employed he re. In
addition, it appears that t least part of the increase n RNA
synthetic rate found by others using ribonuclease inhibitor
preparations was not due to an RNase inhibitor, but rather to
other factors present n the commercial preparations.
The studies presentedshow that T;3ha s a profound, rapid,
and direct effect on the transcriptionof the growth ho rmone
gene. S amuels and his co-workers (23) noted an inverse rela-
tionship between the levels of g rowth hormone production
and the depletion of
T:,
receptor in GH cells exposed to
T:).
The y suggested t ha t unoccupied receptor may repress he
transcription of th e GH gene, and recepto r depletion could
therefore activate theene. However, in heir studies receptor
was depleted only about 15% in 4 h, while we find th at th e
gene is very active by this time. The rapid ityof the transcrip-
tionalresponse to T3 suggests that recepto r depletion can
explain induction of gene activity only if there is a rapid and
prefere ntial deple tion of
a
subgroup of T:3 receptors involved
in epression of growth hormone ranscrip tion. I t appears
more likely that it is the occupied receptors which remain
chroma tin-bound that are involved in the transcriptiona l ac-
tivation of the gene.
Glucocorticoids were also shown to have rapid and direct
effect on growth hormone gene transcription. The transcrip-
tional effects of dexamethasone alone are somewhat evanes-
cent, and occasionally no effect
is
seen. However, on average
the hormone approximately doubles the transcriptional activ-
ity of the gene after 4 h. This approx imateoubling of activi ty
is always seen when exogenously added thyroid hormone is
present. Indeed, it isossible tha t th e ncreased transcription
observed after de xamethasone administration is dependent on
low levels of thyroid hormone or other serum factorsn the
hypo-medium.
In the presencef concentrations of cycloheximide sufficient
to inhib it 95% of cellular p rotein synthesis, the induction of
GH gene transcription by the hormo nes was similar to t ha t
observed in its absence. However, the magnitude of the re-
sponsewas about 4.5-fold grea ter in thepresence of the
inhibitor. These results uggest th at de nouo protein synthesis
is not requiredor the induction f the gene. These results
re
consistent with those of Samuels and Shapiro 24) who found
thatanRNA moleculewhich is rate-limiting forgrowth
hormone translation, most probablyrowth hormone mRNA,
continued oaccumulate in GH, cells in thepresence of
cycloheximide.
Th e effect on transcriptionof T;,,and perhapsof dexameth-
asone, also decreased with prolonged stimulation . The reason
for the diminution of the T:, effect, as well as th e kinetics of
the decrease, is presently unknown. However, in many hor-
monally responsive cells continued stimula tion with hormone
results in
a
diminution of the response (25). Thisffect, termed
partial desensitization, is prevented in a variety of hormone-
target cell systems by antimetabolites thatblock protein syn-
thesis (25). Such data suggest tha t one of th e responses of
target cells to many hormones is th e
de
novo
synthesis of at
least one protein which mediate s the desensitization. It may
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11632 Growth Hormone Gene Transcription
be significant th at the transcriptional effects of dexametha- Baxter, J . D. (1977) Proc.Natl.Acad. Sci. U S. A. 7 4 , 4 2 9 3
Sone and TBon growth hormone gene activity diminish with
7. Wegnez, M., chachter, B.
s.,
Baxter, J . D., and Martial,
J.
A.
(1982)
DNA
1, 145
kinetics of the transcriptional responses is not yet known, it is
possible that in the absence of cycloheximide the gene activ- 9.
Ringold, G. M., Yamamoto, K.
R.,
Bishop,
M.
J. , and Varmus, H.
(1981) Proc. Natl. Acad.Sci. U S. A. 7 8 , 2 2 3 0
ities which are measured at 4 h have already decreased from E. (1977)
Proc. Natl.Acad. Sci. U
S.
A.
74, 2879
an initial maximum. Cycloheximide may block this decrease, 10.
Hager, L. J., and Palmiter, R. D.
(1981)
Nature
2 9 1 , 3 4 0
and appear to
be
enhanced
by
cyc1oheximide.Since the 8 ,
Dobner,
p,
R., Kawasaki, E. S,, yu , L-y., and Bancroft, F, C,
resulting in the apparent stimulation in gene activity which is 11.
McKnight, G.
s.,
nd Palmiter, R. D.
(1979) J .
Biol. Chem.
2 5 4 ,
Observed
after in the combined presence
Of
the inhibitor 12.
Seeburg,
p,
H., Shine, J,, Martial,
J.
A., Baxter,
J D,,
and
9050-9058
and hormones.
and glucocorticoid hormones rapidly and directly increase
Acids
Res. 7, 1541
growth hormone gene transcription by RNA polymerase I1 in
14. Burton, K. (1956) Biochem.
J.
6 2 , 3 1 5
the GC line of rat pituitary tumor cells.
15.
Evans, M.
I.,
Hagar,
L.
J., and McKnight, G.
S (1981)
Cell
25,
16. Maxwell, I. H., Van Ness, J., and Hahn, W. E. (1978) Nucleic
insightful technicalassistance,Richard Imbra for performing the
17.
Biswas, D. K., Martin, R. F. J., and Tashjian,
A.
H., Jr.
(1976)
experiment shown
in
Fig.
3,
and StanleyMcKnight
for
communicating
Biochemistry 1 5 , 3 2 7 0
his nuclear RNA purification procedure prior to
its
publication.
18.
Bancroft, F. C., and Tashjian,A. H., Jr .
(1970)
In Vitro (Rockuille)
In summary, the data Presented demonstrate that thyroid
13. Kafatos, F. C., Jones, W. C., and Efstratiadis, A. (1979) Nucleic
Goodman, H. M.
(1976)
Nature
2 7 0 , 4 8 6
197
Acknowledgment-We thank Anh
P.
Nguyen for excellent andAcids Res.
5, 2033
6, 180
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