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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



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



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).



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



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



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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