Molecular Mechanisms of Phorbol Ester, Thyrotropin-releasing

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 260, No. 21, Issue of Sptember 25, pp. 11852-11858,1985 Printed in U.S.A.

0 1985 by The American Society of Biological Chemists,Inc.

Molecular Mechanismsof Phorbol Ester, Thyrotropin-releasing Hormone, and Growth FactorStimulation of Prolactin Gene Transcription* (Received for publication, January 10,1985)

Geoffrey H. MurdochSlI, Marian Waterman$, RonaldM. Evanss, and Michael G. Rosenfeld/ From the $Eukaryotic Regulatory Biology Program, “013, University of California, SanDiego, School of Medicine, La JoUn, California 92093 and the §Molecular Biology and Tumor Virology Laboratory, The Salk Institute, SanDiego, California 92138

The polypeptide thyrotropin-releasing hormone (TRH) and epidermal growth factor (EGF) stimulate, within secondstominutes,thetranscriptionofthe prolactin gene in a rat pituitary cell line (GHJ. Because a series of agents that act to stimulate prolactin secretion fail toalterprolactingenetranscription, it is suggested that secretoryevents are neither obligatory fornorcausalofhormone-inducedtranscriptional stimulation. Elevation of cytosolic-free calcium does not stimulate prolactin gene transcription; however, several agents that act to antagonize calcium-dependent processes inhibit or abolish bothTRHandEGF stimulation of prolactin gene transcription and a specific hormone-dependent nuclear phosphorylation. In contrast, inhibitors of theslow calcium channel exert minimal effects onTRH-stimulatedprolactingene expression, suggesting thatcalcium influx through membranechannels is notcrucialfortheobserved nuclear actions of TRH.Activation of proteinkinase C by phorbol esters mimics the nuclear actions of TRH. In the presence of increased intracellular calcium levels, the effects of 12-0-tetradecanoyl phorbol 13-acetate on prolactin gene transcription are quantitatively identical to those observed in responseTRH to or EGF.

Understanding the mechanisms by which polypeptide hormones and neuropeptides control gene expression in their targettissues is centralto defining the critical biological processes of differentiation, growth, and homeostatic regulation. While there are clearly many cellular levels where regulation of protein biosynthesis can occur (l), it has recently been established that polypeptide hormones and growth factors can exert rapid effects on the transcription of specific genes (2-5), comparable to steroid hormone effects on gene transcription. In the case of steroid hormones, considerable evidence supports amodel predicting that thecomplex formed by the binding of a steroid hormone to anintracellular receptor protein directly regulates gene transcription as a consequence of binding to specific sequences located near or within the regulated gene. In contrast, polypeptide hormone regulation of gene transcription requires a model of signal transmission, i.e. second messenger(s), that permits the binding of a hormone to discrete receptors located on the plasma mem-

* This work was supported by grants from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. ll Current address: Department of Pathology, Yale University School of Medicine, New Haven, CT 06510-8024.

brane to exert rapid, specific effects within the nucleus. Neither the identity of the predicted “second messengers” nor the putative DNA-binding proteins that might mediate the transcriptional effects of polypeptide hormones have been identified. In thehormone-responsive rat pituitary cell line (GH,), the tripeptide neurohormone, thyrotropin-releasing hormone (TRH’), and epidermal growth factor (EGF) stimulate both the release and synthesisof prolactin (reviewed in Refs. 6 and 7). We have recently demonstrated that the effect of TRH and EGF on prolactin biosynthesis is the direct consequence of a rapid 7- to 12-foldstimulation of prolactin gene transcription (2, 3) resulting in the accumulation of nuclear and cytoplasmic prolactin mRNA (8). In these cells, TRH exerts a series of additional effects, including stimulation of uridine uptake, alterationof cell shape, inhibition of growth hormone synthesis, reduction of growth rate (6-lo), and phosphorylation of a variety of proteins (3, 11, 12). The initial interaction of TRH with its receptor has been shown to influence several potential second messenger systems that areassociated with the activation of protein kinases, including elevation of cyclic AMP levels, increased cytosolic calcium concentration, and activation of the phosphatidylinositol cycle (11-17). TRH stimulates both an influx of calcium across the plasma membrane (13, 14) and a redistribution of cellular calcium pools (15, 16). Although the relative contribution of each process is controversial, the net effect is an increase in the cytoplasmic free calcium concentration as documented by the effect of TRH on the fluorescence of QUIN 2, an internalizable calcium indicator dye (17). Calcium ionophores and depolarizing agents mimic the effect of TRH on prolactin release (18) and reproduce some of the cytoplasmic phosphorylations stimulated by TRH (19). Recent studies have demonstrated that theextracellular calcium concentration can markedly influence both cytoplasmic and nuclear prolactin RNA levels(20). Finally, TRH has been shown to rapidly activate the phosphatidylinositol cycle (21-23). In this enzymatic cycle, which is regulated by a variety of hormones in many different tissues, phosphatidylinositol is sequentially cleaved to diacylglycerol and phosphoinositol and then converted first to phosphatidic acid and finally back to phosphatidylinositol (reviewed in Refs. 24 and 25). The intermediates that are transiently produced by the cycling of the pathway are believed to initiate a cascade of cellular effects. For example, phosphatidic acid is suggestedto act as calcium a ionophore (26), andphosphoinositol may signal the release of The abbreviations used are: TRH,thyrotropin-releasing hormone; EGF, epidermal growth factor; kb, kilobase pairs; EGTA, ethylene glycol bid@-aminoethylether)-N,N,N’,N’-tetraaceticacid TPA, 12-0-tetradecanoylphorbol 13-acetate.

11852

EGF and TRH Stimulate Prolactin Gene Transcription

11853

cellular calcium stores (27). However, a unique consequence resent the mean f S.E. of triplicate hybridizations (each with 8-20 of stimulating this cycle is the activation of the recently X lo6cpm input labeled RNA) in parts/million/kb probe size. Phosphorylation of Nuclear Basic Proteim-100-mm plates of GH, described calcium-phospholipid-dependent protein kinase recells were preincubated with [32P]H2P04 (0.5 mCi/ml) in phosphateferred to asprotein kinase C (28, 29,31, and reviewed in Ref. free Ham’s FlO with serum for 5-24 h prior to addition of hormones. 33). This kinase is maximally activated in the presence of Acid-soluble proteins were prepared and analyzed as previously dephospholipid by a combination of calcium and diacylglycerol. scribed on two-dimensional polyacrylamide gels (1st dimension: 5% The requirement for diacylglycerol can also be met by the acetic acid, 5 M urea, 0.38% Triton X-100; 2nd dimension: sodium various tumor-promoting phorbol esters (34). Because these dodecyl sulfate, 12.5% polyacrylamide) (3). Acid-soluble chromatin agents bindto andactivate protein kinaseC in the same rank proteins were prepared and analyzed as for in uiuo labeling (3). order as theireffects on the growth or differentiated function RESULTS of a variety of cell types, it has been suggested that protein kinase C itself may be a cellular receptor for phorbol esters. The Role of Calcium in TRH Actions on Gene ExpressionThis hypothesis issupported by the observation that the Both TRH and EGF rapidly stimulate prolactin gene trancellular phorbol ester receptor is initially cytoplasmic, but scription ( 2 , 3 , 8 ) producing measurable increases at 1-2 min becomes associated with the plasma membrane in the pres- and maximal stimulation at 20-40 min (see Fig. 1). The ence of phorbol esters (35), and by the recent observation rapidity of onset of the transcriptional effects suggests that that protein kinase C and phorbol ester-binding protein CO- new protein synthesis is not required for mediation of horpurify to homogeneity (36). In GH cells phorbol esters induce mone-regulated prolactin gene transcription. Consistent with many of the same effects as TRH, including stimulation of this observation, pretreatment with cycloheximide for 2-4 h prolactin release and synthesis (37). We have reported that prior to hormonal stimulation has no inhibitory effect on the prolactin gene contains specific DNA sequences that prolactin gene transcription. The hormone-induced transcripconfer regulation by peptide hormones (38). tional stimulation characteristically attenuates beginning at In thismanuscript, we demonstrate that regulation of pro- 1 h following hormone addition. Because of these “burstlactin secretion is independent of and separable from regula- attenuation” kinetics, analysis of the rapid hormonal effects tion of prolactin gene transcription. Although elevation of on prolactin gene transcription provides an assay critical for cytosolic free calcium does not itself influence prolactin gene definition of the initial mediation of TRH and EGF nuclear transcription, there appears to be a calcium-dependent step actions. S1 nuclease resistance mapping confirms that the required for TRH toexert its characteristic effects on prolac- identical prolactin mRNA CAP site isused in TRH- and tin gene transcription and protein. The activation of protein EGF-stimulated cultures, indicating that TRHand EGF regkinase C by phorbol esters mimics the rapid effects of TRH ulate the identical transcription unit (data notshown). on prolactin gene transcription. Because TRH has been demonstrated to increase intracellular calcium concentrations (13-16), experiments were perEXPERIMENTAL PROCEDURES formed to evaluate the potential role of calcium ions in initiating or mediating the TRH-induced stimulation of proMaterials lactin gene transcription. Quantitationof prolactin gene tranProteinase K, RNase TI, and DNA polymerase I were obtained scription avoids any potential problems of delayed or toxic from Boehringer Mannheim; RNase-free DNAse (DPRF) andRNase effects of the experimental manipulations because it permits A were obtained from Worthington. [32P]UTP(>400 Ci/mmol) and [32P]dCTP (2000-4000 Ci/mmol) were obtained from Amersham examination of the nuclear effects within 10-50 min following the addition of substances which alter cellular calcium metabCorp. [3zP]H2P04(carrier-free) was obtained from ICN. Methods Cell Culture and Hormonal Treatment-GH, cells were routinely cultured on 100-mm plates (Nunc) inHam’s F10 (Gibco) with 12.5% horse serum and 2.5% fetal calf serum, plated at 3 X IO6 cells/plate 6 days prior to harvest. Cultures were fed on alternate days. Hormones and chemical agents were added as 1000 X stocks in either phosphatebuffered saline or ethanol. All additions were made to plates in the incubator because manipulation of cultures, such as changing the media, was found to produce a transient (less than 2 h) stimulation of both prolactin gene transcription and protein phosphorylation, analogous to observations in other laboratories (12). Preparation and Quantitation of Prolactin mRNA-Cytoplasmic RNA was prepared by phenol/chloroform extraction oflow speed supernatants following lysis of cells in 0.4% Nonidet P-40, 20 mM NaCl, 5 mM MgC12, 20 mM Tris (pH 8.0). Prolactin sequences were quantitated by hybridizing aliquots of RNA immobilized to squares of diazotized paper (DBM) with nick-translated cloned prolactin cDNA as previously described (3). Values presented represent the mean of triplicate determinations (values routinely varied less than lo%), expressed as a percentage of control hybridization after correcting for background binding. Assay of Prolactin Gene Transcription-Gene-specific transcription rates were determined by quantitating nascent prolactin and growth hormone RNA transcripts in nuclei from treated cells rapidly harvested in phosphate-buffered saline supplemented with 0.5 mM EGTA. Elongating nuclear transcrlpm were labeled and isolated using a modification of the method of McKnight and Palmiter (39) as previously described (2, 3). Transcription rates are expressed as the percentage of labeled transcripts which hybridize to an excess of cloned specific DNA bound to nitrocellulose. Values presented rep-

+

+

TRH

TRH

FIG. 1. Comparison of effects of cobalt chloride and pimozide on prolactin gene transcription. The transcription of the prolactin gene (open bars) and growth hormone (stippled bars) were quantitated by nuclear run-off assay in both unstimulated GH cells and cells treated for 45 min with TRH (3 X lo-? M) as described under “Experimental Procedures.” Selected cultures were pretreated for 10 min with either cobalt chloride (1 mM) or pimozide (PZM) (1 p ~ as) indicated. Results are expressed as the mean k S.E. Similar experimental results were obtained in six additional experiments.

EGF a n d TRH Stimulate Prolactin

11854

olism. In GH cells, cobalt ions have been shown to block TRH-stimulated spike potentials (40), calcium influx (14), and prolactin release (41). As shown in Fig. 1,a10-min preincubation with 1mM cobalt chloride completely prevents the stimulation by TRH of prolactin gene transcription, while producing only a small inhibitory effect on the basal prolactin gene transcription rate. This inhibition of basal prolactin gene transcription appears to reflect the fact that GH, cells grown in serum-containing medium are not in a true basal state with respect to prolactin gene transcription. Thus, the prolactin gene transcription rate is 2-3.5 ppm/kb in serumcontaining medium, but 1-1.5 ppm/kb in serum-free medium. Therefore, TRH stimulation of prolactin gene transcription, but not basal prolactin gene transcription, is calcium-dependent. In addition, the inhibitory effect of cobalt appears to be gene-specific because cobalt treatment had no effect on the transcription of the growth hormone gene (Fig. 1) or of five other genes exhibiting constitutive expression in GH, cells (Table I).The rapid inhibitory effects of cobalt ongene transcription suggest the direct importance of a calcium-

Gene Transcription

dependent process in mediating TRH actions on geneexpression. A second group of compounds that exert inhibitory effects on calcium-dependent processes in the cell was also analyzed for their effects on prolactin gene transcription. Three such agents with distinct chemical structures, pimozide, chlorprothixene, and trifluperazine, have been shown to bind to calmodulin in a calcium-dependent manner and to inhibit calmodulin-mediated effects with EDso values similar to their affinities for calmodulin (typically 5-30 g M ) (42). At 10 p M final concentration, each of the three antipsychotic agents exert inhibitory effects on half-maximal TRH stimulation of prolactin mRNA accumulation (Fig. 2). At these concentrations, these compounds also interfere with other calciumdependent processes (43), presumably as a result of binding to other calmodulin-related proteins. The ability of these agents to antagonize hormonal actions therefore provides presumptive evidence that a calcium-dependent process mediates the hormonal effect (e.g. Ref. 44). At lower concentrations (half-maximal effects at 200 nM), pimozide appears to act as aselective inhibitor of slow, voltage-dependent calcium channels (45), andat these concentrations pimozide produces TABLE I a 45% inhibition of TRH-stimulated prolactin gene transcripEffect of cobalt o n specific gene transcription tion (Fig. 1)and acorresponding (50%)inhibition of prolactin The rate of transcription of a series of genes was quantitated as mRNA accumulation (Fig. 2). Pimozide and cobalt ions exert described under “Experimental Procedures” using unstimulated GH, cell cultures; cobalt chloride (1mM) was added 30 min prior to harvest. inhibitory effects on TRH-stimulated phosphorylation of an Probes used included five cDNA clones, varying in length from 540 acid-soluble protein in the nucleus (Fig. 3); cobalt ions comto 1640 base pairs, which exhibited “constitutive” expression in GH, pletely abolish the TRHeffects to levels even lower than that cells; that is, those which werenot regulated by TRH, EGF, or phorhol in control cultures, while pimozide produces a 50-60% inhiesters. Transcription rates are the average of triplicate determinations bition of TRH-induced phosphorylation. The similar effects k S.M. of these chemically unrelated agents, both of which disrupt cellular calcium metabolism on TRH-induced prolactin gene cDNA clonal rate Transcription probe -Cobalt +Cobalt transcription, suggest that a calcium-dependent process is involved in the normal pathway mediating the nuclear effects ppmlkb of TRH. Prolactin 2.8 f 0.4 1.5 +- 0.3 Prolactin Secretagogues Do Not Mimic the Nuclear Effects 1.8 f 0.4 1.2 f 0.2 Growth hormone of TRH-If an elevation of cytoplasmic calcium concentra40.3 f 3.2 38.2 f 4.2 Clone A Clone B 200.1 f 6.8 205.8 f 5.1 tion, whether produced by transmembrane calcium fluxes or 120.3 f 10.2 Clone C 110.8 f 2.7 redistribution of cellular calcium pools, was itself the essential 8.5 f 0.8 9.4 f 0.9 Clone D initial mediator of the transcriptional effects of TRH, then 32.6 f 2.3 29.7 f 3.6 Clone E pharmacologic elevation of cytosolic calcium should stimulate prolactin gene transcription. The effects of various ionophores I I and depolarizing agents on prolactin gene transcription is shown in Fig. 4. Although each agent elevates cytosolic cal300 cium in GH cells sufficiently to rapidly stimulate release of Y prolactin (18, 46), it is clear that none of them stimulate prolactin gene transcription (Fig. 4A) or prolactin mRNA accumulation (Fig. 4B);these data suggest that elevation of 200 cytosolic free calcium cannot be the sole determinant of hormone-induced prolactin gene transcription. In addition, because these compounds do not alter cytoplasmic prolactin mRNAlevels or prolactin biosynthesis (datanotshown), 100 release of prolactin stores appears to have no effect on prolactin mRNA stability or translation. This is consistent with the previous observation that stimulation of prolactin gene transcription by TRH can entirely account for its effects on IO” 10.6 10.~ IO a prolactin biosynthesis (3). [ A N T I P S Y C H O T I C ] (MI The Effect of Phorbol Esters and thePotential Role of FIG. 2. The effect of antipsychotic agents on TRH-stimu- Protein Kinase C-If the effects of TRH on calcium mobililated prolactin mRNA accumulation. GH cell cultures were pre- zation do not itself account for the stimulation of prolactin treated with the indicated concentrations of pimozide (0-O), triflu- gene transcription, it is possible that the increased intracelperazine (A-A), or chlorprothixene (0-0) for 15 min prior to the lular calcium levels act in concert with other biochemical addition of TRH (3 X M). After an additional 20 h incuhation, processes generated by theTRH-receptorinteraction. Beprolactin mRNA was isolated and quantitated as described under “Experimental Procedures.” The cytoplasmic prolactin mRNA levels cause activation of the phosphatidylinositol cycle by TRH have been normalized to levels present in control cultures. This results in the rapid generation of free diacylglycerol (22, 23), protein kinase C activation is one possible candidate for the experiment was repeated three times with similar results. I

I

I

11855

EGF and TRH Stimulate ProlactinGene Transcription

b

B

. IH1 m

H2A

C I

-

-u H2A

H3

“2A

FIG. 3. The effect of cobalt chloride and pimozide on TRH-stimulated acid-soluble protein phosphorylation. GH cell cultures, preincubated with [32P]HPOr, were either untreated (panel A ) , incubated with TRH (3 X lo-’ M) for 45 min (panel I?), or pretreated with pimozide (1p M ) (panel c) or CoC12 (1mM) (panel D)for 15 min prior to addition of TRH (3 X lo-’ M). The figure shows autoradiographs of acid-soluble chromatin proteins analyzedontwo-dimensional sodium dodecyl sulfate-polyacrylamide gels as described under“Experimental Procedures.” Similar results were obtained in five experiments of similar design.

calcium-dependent site in the pathway mediating the nuclear tion, acts synergistically with TPA-activated protein kinase effects of TRH. If this were the case, then other hormones C to produce a transcriptional stimulation characteristic of which stimulate the cleavage of phosphatidylinositol in GH, that induced by EGF or TRH. One possible explanation of cells should also stimulate prolactingene transcription. Bom- these data is that TPA fulfills onlythe diacylglycerol requirebesin, although reported not to significantly stimulate prolac- ment (34), and the extent of kinase activation by TPA detin levels in GH cells (47), is approximatelyhalf as potentas pends on the level of cytosolic calcium at the time of the TRH in stimulating phosphatidylinositol turnover (21) and induction. The cytosolic freecalcium concentration in unthe phosphorylationof certain cytoplasmic proteins (48). Pro- stimulated GH cells is reported to be approximately 0.1 p~ lactin gene transcription, 40 min following hormonal addition (17) which is well below the observed Kd for protein kinase C to GH, cultures, is 2.3 0.2 ppm/kb in unstimulated cells, activation in vitro (49). Therefore, because diacylglycerol and M), and calcium synergistically activate protein kinase C, even small 8.4 & 0.3 ppm/kbinbombesin-treatedcultures in TRH-treated cultures M). As changes in GH cell cytosolic calcium might produce signifi17.2 f 0.5 ppm/kb shown in Fig. 5, TPA addition rapidly stimulates prolactin cant changes in protein kinase C activity in the presence of gene transcription, generally producing 50-80% of the maxi- diacylglycerol or TPA. Cobalt chloride rapidlyand completely mal transcription rateachieved following addition of TRH or inhibits the transcriptional stimulationof the prolactin gene EGF. These results are consistent with the possibility that produced by TPA as well as that produced by TRH or EGF activation of protein kinase C is necessary to initiate tran- (Fig. 5). Thus, all three regulatory agents appear to exert their scriptional effects on the prolactin gene, but is itself not initialactionsonprolactin gene transcriptionin aCa2+sufficient to achieve maximalstimulation. Because of the dependent fashion. rapid effects of TRH on calcium distribution, we evaluated To further define whether calcium influx is a requirement the possibility that simultaneous elevation of intracellular for the stimulation of prolactin gene expression by TRH, freecalcium levels and protein kinase C activation would organic inhibitors of voltage-dependent and hormonally regmaximally stimulate prolactin gene transcription. When the ulated slow calcium channels (50), such as nifedipine and calcium ionophore A23187 is added simultaneously with TPA, D600 (the methoxy derivative of verapamil), were utilized. In the prolactin gene transcription rate becomes equivalent to GH cells these agentshave been shownto be as efficacious as that observed in EGF- or TRH-treatedcells (Fig. 5). cobalt ions inblocking TRH-stimulated calcium spike potenThese data suggest that elevation of intracellular calcium, tials (51,52),calcium influx, and therelease of stored prolacalthough itself exerting noeffect on prolactin gene transcrip- tin by hormonal stimulation (14) or depolarization (53). At

*

Stimulate TRHandEGF

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Prsolactin Gene Transcription 28 a”

z

I

0 c

T

zI-

o a

1 -

a

t -

t -

CNT

t

TAH

EGF

- +

-

-

TPA

Y A

Az3’e1

ImMCoCI,

423181

Ct

A23187 TRH

lono

K’

4AP

FIG. 5. The effect of cobalt chloride on prolactingene transcription. The transcription of the prolactin gene was quantitated in the presence (+) (stippled bars) and absence (-) (open bars) of 1 mM cobalt chloride (see “Experimental Procedures.”). Cell cultures were either unstimulated (CNT)or treated with TRH (3 X lo-’ M), M),A23187 (1.5 x 10” M), or TPA EGF (1 X lo-’ M),TPA (2 X (2 X lo-’ M) and A23187 (1.5 X 10“ M) for 30 min prior to harvest. Cobalt chloride was added to the cells 5 min before hormonal additions. Results are theaverage of triplicate determinations2 S.E., and similar results were obtained insix experiments of similar design, but varying in the time of hormonal addition from 2 min to 40 min. Transcriptional stimulation (>180%) by TRH, EGF, and TPA is consistently observed by 2 min. Prolactin gene transcription is 1.5 _t 0.3 ppm/kb in GH, cells cultured under serum-free conditions, in the presence or absence of cobalt chloride; a result confirmed in four experiments of identical design.

TABLE I1

Ct

TRH A23187 TPA

K‘

4AP

FIG. 4. The effect of prolactin secretagogues on prolactin gene transcription and prolactin mRNA levels. A , GH, cell cultures were incubated for 45 min in the absence of added agent ( C T )or with TRH (3 X M), the calcium ionophores A23187 (1.5 p M ) , or ionomycin (lono) (3 pM), KC1 (40 mM), or 4-aminopyridine ( 4 A P ) (0.3mM), and prolactin gene transcription was quantitated as described under “Experimental Procedures.” B, GH, cell cultures were incubated with the agents for 30 h, and prolactin mRNA was

Effect of organic calcium channel blockers on thes t i m ~ l ~ t i oofn prolactin mRNA by TRH and TPA GH cell cultures were treated with the indicated concentrations of either D600, nifedipine (NIF), pimozide (PIM), or trifluperazine M) or (TFP) for 15 min prior to the addition of either TRH (3 X M).After an additional 20-h incubation, cytoplasmic TPA (2 X RNA was prepared and assayed for prolactin mRNA as described under “Experimental Procedures.” The first three columns present the prolactin mRNA levels as a percentage of levels in unstimulated cultures. Each result is the average of triplicate determinationsvarying by less than 10%. The last two columns present the -fold stimulation Droduced bv TRH or TPA. ~~

quantitated as described under “Experimental Procedures.” All results are the average of triplicate determinations f S.E.; results are typical of four experiments of identical design.

Prolactin mRNA Control

TRH

~~~

Stimulation

TPA

TRH

% control

low concentrations which saturate the calcium channel, both nifedipine and D600 induce a proportional inhibitionof basal and TPA-stimulatedprolactin mRNA levels but actually have no effect on the “fold stimulation produced by TPA (see Table 11). A t concentrations used in these experiments,D600 and nifedipine do not inhibit protein kinase C activity (54). In contrast, TRH, which stimulates both diacylglycerol production and a redistribution of cellular calcium pools, actually produces a larger fold stimulation than in the absence of these agents, reflecting lower basal prolactingene expression (Table 11). Pimozide, at low micromolar concentrations where it acts as a calcium channel blocker, produces the same pattern of inhibition as D600 and nifedipine (Table 11). Therefore, calcium influx does not appear to be required for TRH-stimulated prolactin gene transcription. In contrast an agent, trifluperazine, which inhibitsthe activity of many calciumdependentproteins,entirelyinhibitsboth TRH and TPA effects on prolactingene expression. Trifluperazine is a potent

Experiment 1 No addition D600 1 p M 50 p M NIF.

1pM

50 p~ Experiment 2 No addition PIM 1 p M TFP 30 p M

TPA

-fold

100 85 45

332 329 102

206 177 76

3.3 3.9 2.3

2.1 2.1 1.7

63 56

261 180

126 110

4.1 3.21

2.0 2.0

loo

210 277 162 103

2.8

100 75

2.1 2.2 0.9

45 79

3.6 1.3

inhibitor of protein kinase C in vitro (54, 55), consistent with a potential role for protein kinase C in regulating prolactin gene transcription. DISCUSSION

In GH cells the peptide hormones TRH and EGFinduce a 10-fold stimulation of prolactin gene transcriptionwithin

EGF and TRH Stimulate Prolactin Gene Transcription minutes of binding to their plasma membrane receptor. This transcription stimulation and the resultant accumulation of cytoplasmic prolactin mRNAappear toaccount for the effects of TRH or EGF on prolactin biosynthesis. The experiments presented in this manuscript were designed to examine the cellular processes by which events at the plasma membrane in response to TRH-receptoractivation could transfer a signal to the nucleus in GH cells. Initial effects of TRH at the plasma membrane include a stimulation of transmembrane calcium fluxes, increased activity of the phosphatidylinositol cycle, a modest stimulation of cyclic AMP accumulation, and internalization of the hormone-receptor complex (e.g. Ref. 4, 11-17). Because elevation of cytosolic calcium levels produced by transmembrane calcium fluxes and mobilization of cellular calcium pools appear capable of mediating prolactin release (18)and because cells cultured in media with extremely low concentrations of calcium contain decreased prolactin mRNA levels (20), one plausible model is that a calcium-dependent process represents the rate-limiting step in the regulation by TRH of prolactin gene transcription. While there is no evidence that EGF stimulates phosphoinositol turnover in GH cells, EGF has been reported to stimulate protein kinase C activity in a varietyof cell types (30,55). The fact that rapid increases in cytosolic calcium stimulated either by membrane depolarization or calcium ionophores do not. activate transcription of the prolactin gene suggests that regulation of prolactin release and biosynthesis are independent processes. However, the ability of agents which disrupt cellular calcium metabolism to interfere with the regulation of prolactin gene transcription by TRH suggests that mobilization of calcium may be an importantcomponent. In contrast to the complete inhibition of TRH-induced prolactin gene transcription by cobalt chloride, organic calcium channel blockers (nifedipine, D600) exert only minimal effects on the stimulation of prolactin mRNA by TRH. These datasuggest that calcium influx through membrane channelsis not critical for the observed nuclear effects of TRH. Because of the ability of phorbol esters (TPA) to mimic the transcriptional effects of TRH, it is tempting tospeculate that activation of protein kinase C could be the proximal calcium-dependent step by which TRH exerts its nuclearactions. The activation of cellular protein kinase C is associated with nearly complete translocation from the cytoplasm to the plasma membrane, suggesting that the plasma membrane-bound calcium pool might represent the site critical to thenuclear effects exerted by TRH. Indeed,bothcobalt chloride and antipsychotic agents (40-45), but notorganic calcium channel blockers (5153), have been shown to displace calcium from this pool in GH cells, consistent with the observation that theformer, but not the latter, agents inhibit TPA induction of increased prolactin mRNA levels. If TPA, EGF, andTRH actually were to regulate prolactin gene expression by a common molecular mechanism, then one would predict that a single genomic sequence would transfer transcriptional regulation by all three agents to a normally unresponsive transcription unit. Analysis by DNAmediated gene transfer of fusion genes containing 5' flanking portions of the rat prolactin gene provided the evidence that specific genomic sequences can confer upon normally unresponsive gene transcriptional regulation by EGF and TPA (38). Further analysis has revealed that prolactin sequences conferring hormonal responsivity to TRH, EGF, and TPA co-localize to a small 5' flanking genomic sequence.* The nature of the regulatory factor(s) which bind to these se-

' H. Elsholtz, H. Mangalam, E. Potter, R. M. Evans, and M. G. Rosenfeld, unpublished data.

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