Vol. 131, No. 6 Printed tn U.S.A.

Phorbol Ester Activation of the Protein Pathway Inhibits Gonadotropin-Releasing Gene Expression*

Kinase C Hormone

JAN

E.

M.

BRUDERt,

WILLIAM

D.

KREBS,

TERRY

M.

NETT,

AND

MARGARET

WIERMANS

Department of Medicine, University of Colorado Health Sciences Center, and Research Service, Veterans Affairs Medical Center, Denver, Colorado 80220; and the Department of Physiology, Colorado State University, College of Veterinary Medicine and Biomedical Sciences (T.M.N.), Fort Collins, Colorado 80523 ABSTRACT The effects of the phorbol ester 12-0-tetradecanoyl phorbol 13acetate (TPA), an activator of protein kinase C (PKC), and the PKC inhibitor staurosporine on GnRH secretion and mRNA levels were studied in GTl-7 hypothalamic neuronal cells. Dose-response and time-course studies revealed that TPA (lo-@ M) acutely increased GnRH secretion 3-fold at 3-6 h, which then declined to baseline at 24 h, while it progressively decreased GnRH mRNA levels by 50% and 70% at 6 and 24 h, respectively. To ensure that these effects were due to activation and not down-regulation of PKC, cells were treated for 30 min with TPA (10-s M). This brief exposure to TPA also resulted in a decrease (60%) in GnRH mRNA levels at 6 h, with a 1.5- to 2-fold increase in GnRH secretion compared to control values, suggesting

that activation of PKC decreases the pretranslational expression of GnRH while increasing GnRH secretion. Additional studies measured PKC activity and documented a shift from a cytosolic to a membrane fraction after incubation with TPA, again supporting PKC activation. Exposure of GTl-7 cells to staurosporine (lo-@ M), a PKC inhibitor, resulted in no change in the level of GnRH mRNA or secretion at 6 h. However, incubation with both TPA and staurosporine prevented the decrease in GnRH mRNA levels and partially blocked the increase in GnRH secretion induced by TPA. We conclude that TPA, by activating the PKC pathway, acutely increases GnRH secretion, but dramatically decreases GnRH gene expression. The exact mechanism of these divergent effects on the synthesis and secretion of GnRH remain to be elucidated. (Endocrinology 131: 2552-2558, 1992)

G

messengersystemsare important in the pretranslational regulation of GnRH. In these studies we investigated the role of the PKC system (16-18) in GnRH gene expression. This pathway is initiated by a ligand receptor/G-protein-coupled complex that activates phospholipase-C (see Fig. 1). Phospholipase-C cleaves phosphatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-triphosphate. DAG then activates PKC by a mechanism by which the enzyme undergoes a translocation from the cytosol to the plasmamembrane. PKC is a family of enzymes, which, when activated, phosphorylates many proteins and results in the induction or activation of many nuclear transcription factors (17, 18). These transcription factors then may positively or negatively regulate gene expression. Tumor-promoting phorbol esters, such as 12-O-tetradecanoyl phorbol 13-acetate (TPA), mimic the action of the second messengerDAG to stimulate PKC. Like DAG, TPA increases the affinity of PKC for calcium, resulting in full activation of this enzyme at physiological calcium concentrations. However, a curious feature of TPA is that with prolonged exposure, down-regulation or depletion of PKC can occur (18, 19). In contrast, staurosporine is a substancethat directly inhibits PKC activity (20). Attempts to directly study GnRH gene expression have been difficult becauseof the paucity of experimental models. The recently derived neuronal cell line created by Mellon and colleagues (21) provides for the first time a uniform population of GnRH-secreting neurons in which to directly examine these issuesof hypothalamic GnRH gene expression.

is the hypothalamic releasing factor that regulates the expression of the pituitary gonadotropins and, ultimately, mediates gametogenesisand reproductive competence (1). The decapeptide directs both the biosynthesis of the pituitary gonadotropin subunits (cu,LHP, and FSHP) and the release of the intact dimeric hormones LH and FSH, which, in turn, stimulate the production of sex steroid hormones and gonadal peptides (2). Neurotransmitters, hypothalamic releasing factors, and various peptide hormones are thought to impact on GnRH expression by interacting at the membrane with their receptors to activate a second messenger signaling cascade. Recent immunocytochemical studies indicate that catecholaminergic (3, 4), dopaminergic (5), serotoninergic (6), y-aminobutyric acid-ergic (7), opioid peptidergic (8), substance-P (9), CRF-immunoreactive axons (lo), and other GnRH neurons (11) synapse on GnRH neurons. The mechanismsby which these substancesimpact on GnRH production via second messenger systems have not been elucidated. Although previous studies have suggestedthe importance of both the CAMP (12) and protein kinase C (PKC) pathways (12-15) on the releaseof GnRH, it is not known what second nRH

Received May 26, 1992. Address requests for reprints to: Dr. Margaret E. Wierman, Section of Endocrinology (11 lH), Veterans Administration Medical Center, 1055 Clermont Street, Denver, Colorado 80220. * This work was support by V.A. Merit Review 001 and Grant HD25275 (to M.E.W.) and Grant HD-0741 (to T.M.N.). t Associate Investigator, Veterans Affairs. $ Research Associate, Veterans Affairs.

2552

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TPA

ACTIVATION

OF PKC

In these studies we hypothesized that activation of the PKC system in these neuronal cells would increase GnRH secretion and be preceded or followed by an increase in the synthesis of GnRH mRNA. We then asked the following questions. 1) What are the dose- and time-dependent effects of the phorbol ester TPA on GnRH secretion and mRNA levels in hypothalamic neuronal cells? 2) Are the effects of TPA the result of activation of the PKC pathway? 3) What are the effects of the PKC inhibitor staurosporine on GnRH secretion and mRNA levels?

Materials

and Methods

INHIBITS

GnRH

mRNAs

2553

at a density of 1.5-3 X 10h cells/lo-mm plate and allowed to reattach. After 1 day (for 3 X 10h cells) or 3 days (for 1.5 X 10h cells), triplicate plates of cells were incubated with TI’A, TPA and/or staurosporine, or DMSO or ethanol in the medium as control vehicles and harvested at various times, as outlined in Resulfs.

GnRH RIA Concentrations of GnRH in medium were determined using antiserum R-42, as described previously (24). GnRH was radioiodinated using a glucose oxidase-lactoperoxidase procedure (25), and monoiodinated GnRH was separated on a 0.9 X 20.cm column of QAE-Sephadexeluted 0.01.~ Tris buffer (pH 9.2). In six assays, the sensitivity was 0.5 f 0.1 pg/tube. Intraassay variation was 5.3%, and interassay variation was 9.8%.

Materials

PKC assay

The GTl-7 hypothalamic neuronal cells were kindly provided by I’. Mellon (21). The GTl-7 cells were derived from a hypothalamic tumor above the optic chiasm in a transgenic mouse. The cells were targeted by coupling the rat GnRH promoter to the simian virus-40 T-antigen. The fusion gene was introduced into mouse embryos, and some transgenie mice expressing the fusion protein developed tumors. The tumors were subcloned to produce uniform populations of GnRH neuronal cells. The GTl-7 cells produce prepro-GnRH, GnRH, and GnRH-associated peptide mRNA and protein. They also express neuronal, but not glial, markers, as has been previously described (21-23). Tissue culture media and PBS were obtained from Gibco (Grand Island, NY) and the Tissue Culture Core Laboratory of the University of Colorado Cancer Center. The [a-32P]deoxy-CTP, [n-32P]lJTI’, and [y-?‘]ATI’ were purchased from ICN (Irvine, CA). The phorbol ester TPA, staurosporine, and dimethylsulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO). A kit to assay PKC activity was purchased from Amersham (Arlington Heights, IL). TPA and staurosporine were dissolved in DMSO at concentrations of 10m2 and lo-’ M, respectively, and stored at -20 C. The chemicals were diluted with medium to appropriate concentrations on the day of each experiment.

The PKC enzyme assay was performed in duplicate according to the instructions in the Amersham kit. The assay is based upon the PKCcatalyzed transfer of the 2-phosphate group of ATP to a peptide. The cells were suspended in ice-cold buffer A [50 rnM Tris (pH 7.5), 5 rnM EDTA, and 0.3% mercaptoethanol] and centrifuged at 1,000 rpm for 10 min, and supernates were decanted and sonicated for 30 sec. Cell homogenates were centrifuged at 35,000 rpm for 60 min to obtain the cytosol fraction (supernatant) and a crude membrane pellet. The pellet was sonicated in buffer A and 1% Triton X-100 for 30 set and spun at 35,000 rpm for 30 min. This supernatant was used as the membrane extract. The phosphorylated peptide was separated on binding paper. Both fractions were assayed for PKC activity, as measured by the extent of phosphorylation detected by scintillation counting.

Cell culture The hypothalamic neuronal cells were cultured at 37 C in Dulbecco’s Modified Eagle’s Medium supplemented with 5-10% fetal bovine serum, 100 U/ml penicillin, 100 pg/ml streptomycin, and 0.25 fig/ml fungizone. At a confluency of 80-90% (loo-mm culture plates), cells were replated

Radiolabeled

probes

A genomic fragment encoding 289 basepairs (bp) of the rat GnRH gene and a mouse P-actin cDNA were radiolabeled with [ru-3ZP]dexoyCTP by the random primer technique (26), which resulted in a specific activity of 0.5-l X 10’ cpm/Fg DNA. The GnRH genomic fragment encodes a portion of intron 1, exon 2, and a portion of intron 2 and is specific for GnRH (27). To create strand-specific RNA probes (riboprobes), the 289-bp fragment was also inserted into pGEM4 and linearized with HirrdIII or EcoRI. The fragment was radiolabeled by incorporating [~z-~~I’]UTI’, using either SF6 or T7 polymerase (Promega, Madison, WI) to make the sense or antisense RNA strand.

RNA isolation

-OR a 8

PHOSPHOLIPA8E

Total RNA was isolated from individual dishes after homogenization of cells in 0.6 ml 4 M guanidine isothiocyanate, layered over a 0.4.ml 5.7~M cesium chloride cushion, and centrifuged in a TL100.2 rotor at 68,000 rpm for 8-15 hours (28, 29). C

Northern

PKC

FIG. 1. Diagram

of the second messenger system in which DAG stimulates PKC to activate transcriptional factors that positively or negatively regulate gene expression. TPA activates PKC (+), while staurosporine inhibits (-) PKC.

analysis

Five or 10 rg total RNA (OD260) from each sample and X/Hind111 cold and radiolabeled markers were electrophoresed through a 1.4% (wt/vol) agarose gel and diffusion blotted on nitrocellulose, as previously described (28, 29). Ethidium bromide staining confirmed that the ribosomal RNAs were intact, and approximately equal amounts of RNA were loaded in each lane. Blots were prehybridized at 42 C, as previously described (29), and hybridized with the32P-labeled DNA or RNA strandspecific probe for 16-24 h. Conditions ensured probe excess. Blots were washed with 2 x SSC (1 X SSC = 0.15 M NaCl, 0.015 M Na citrate, pH 7.0)-0.1% sodium dodecyl sulfate (SDS) at 50 C for the DNA probes and with 2 x SSC-0.1% SDS at 52 C, followed by 0.1 X SSC-0.1% at 55 C for the strand-specific riboprobes, and then exposed to Kodak XAR-2 film (Eastman Kodak, Rochester, NY) for l-2 days at -70 C with intensifying screens. After hybridization, excess probe was removed by stripping the blot in 10 rnM Tris at 70 C, prehybridized, and then rehybridized with another radiolabeled probe. There is no preferential

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TPA ACTIVATION

2554

OF PKC INHIBITS

loss of RNA from blots using this technique (29). Autoradiographic bands were quantitated in the linear range of the Bio-Rad densitometer (Richmond, CA) and standardized against the @actin signal for each sample. mRNA ievels were expressedin arbitrary densitometric units relative to the control mRNA levels t- SEM.

Statistical

analysis

Analysis and within differences

of variance (30) was used to analyze the data sets over time different groups, and a t test (30) was used to determine between control and experimental groups of dishes.

23

m

Control

i%@#

IO-IlM

1992 No 6

2

Effects of TPA on PKC activity

In other sets of experiments, cells were incubated with TPA (lo-“, 10m9,lo-‘, and 10e7M ) or DMSO and harvested at 6 h to determine PKC activity (see Table 2). This activity

2

tzza

IO-8M

0.50 1 o-tbi

0.00

TPA

Triplicate plates were incubated in a final concentration of lo-“, 10e9, lo-*, or 10m7M TPA or DMSO (as the control vehicle). After 6 h, cells were harvested for RNA measurements, and medium was frozen at -70 C for GnRH RIA. Initial studies demonstrated that TPA (10m7M) resulted in dramatic decreasesof 70% (P < 0.05) in GnRH mRNA levels at 24 h (seeFig. 2). To determine the minimum dose needed for this suppressive effect of TPA, dose-response studies were performed. Exposure to increasing amounts of TPA for 6 h resulted in significant decreasesin steady state GnRH mRNA levels to a maximum of 50-60% at lo-* and 10m7M TPA compared to control values (P < 0.05; Fig. 3). These data are representative of three experiments showing similar suppressionof GnRH mRNA levels at lo-’ and 10m7M TPA. In contrast, GnRH secretion in the medium increased 5- to 6-fold by 6 h (P < 0.05; seeTable 1).

3

4

2. Northern blot analysis of GnRH mRNA levels after treatment with TPA. Ten micrograms of total RNA were electrophoresed, transferred to nitrocellulose, and baked. The blot was hybridized with a radiolabeled probe specific for GnRH, washed, and exposed to film for 1 day. Each lane represents RNA isolated from individual dishes of cells. Lanes 1 and 2 are controls (DMSO). Lanes 3 and 4 are from cells treated with 10e7 M TPA. The GnRH mRNA is approximately 600 bp.

FIG.

Endo. Vol131.

1 o-9M

effects of the PKC activator

1

mRNAs

1 .oo

Results Dose-response

GnRH

FIG. 3. Dose-response effects of the PKC activator TPA on GnRH mRNA levels. GTl-7 cells in triplicate dishes were treated with TPA (lo-“-lo-’ M), as indicated, or DMSO (C, control). Cells were harvested at 6 h. and total RNA was extracted. GnRH and &actin mRNA levels were determined by blot hybridization analysis. Each column represents the mean optical density + SEM of three autoradiographic bands. All data are standardized, such that the mean mRNA levels in control cells are 1.0 arbitrary densitometric units (ADU) of: SEM. Symbols represent the significance of comparisons of data points with values in control cells (*, P < 0.05). This figure represents data from one of three experiments showing similar effects of TPA in doses of lo-’ and lo-? M.

was measured in the cytosolic and particulate fractions and expressed as picomoles of phosphate incorporated per pg protein. A shift from the cytosolic to the particulate fraction documented activation of PKC. In individual experiments the raw data varied extensively (control dishes: cytosolic, 1.5-6.8; particulate, 0.4-3.7; experimental, 1.5-7.8 and 0.45.8, respectively). In each study, however, there was a shift in the ratio of cytosolic to particulate fractions in cells treated with TPA (lo-’ and 1O-7 M) compared to that in control dishes(P < 0.05). A lower ratio indicates stimulation of PKC enzyme activity. No significant stimulation was observed in cells treated with 10-l’ or 10m9M TPA (P = NS). Specificity

of effects of TPA on GnRH vs. SH mRNAs

Adelman and colleagues (31, 32) demonstrated that the DNA strand opposite that of GnRH encodes the SH gene. Its RNA is about 700 bp and is thought to be expressedin the heart and hypothalamus (31, 32), although its function is yet undefined. Since our GnRH probe was a double stranded DNA probe and included intronic sequencesfor GnRH that were exonic for SH, we wished to ensure that the changes in mRNAs detected by the 289-bp double stranded probe were due to changes specific to GnRH and not to the RNA encoded for by the opposite strand DNA, SH. Thus, blots were hybridized sequentially with specific sense(SH) and antisense(GnRH) riboprobes. In contrast to the dramatic changesin GnRH mRNA levels, there were no changes in SH mRNA levels after TPA treatment (see Fig. 4). These studies confirm the presence of SH in these neuronal cells, but suggest that the mRNA transcript is not regulated by our experimental manipulations. Effects of short term exposure to TPA

To further ensure that the inhibitory effects of TPA on GnRH mRNAs at 6 h were due to activation and not a down-

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TPA ACTIVATION TABLE

OF PKC INHIBITS

GnRH

mRNAs

2555

1. Dose-response effects of the PKC activator TPA on cumulative GnRH secretion TPA Control lo-”

M

10-g M

lo-’

M

10-T M

GnRH (rig/ml) 0.11 + 0.006 0.096 f 0.007 0.19 f 0.003 0.57 f 0.06” 0.65 + 0.02” GTl-7 cells in triplicate dishes were treated with TPA in various doses (lo-“-lo-’ M) or DMSO (control) in medium sampled after 6 h for cumulative GnRH levels. GnRH levels were determined as described in Materials and Methods. ’ Significant difference from control dishes (P < 0.05). TABLE

2. Effects of TPA on PKC activity TPA

Control lo-”

M

10-g M

lo-’

M

lo-’

M

0.48 f 0.06” 3.9 + 0.4 3.2 f 0.8 2.6 zk 0.7 1.2 + 0.07” PKC activity in response to various doses of TPA (10-11-10-7 M) for 6 h was determined (as described in Materials and Methods). Activity is expressed as picomoles of phosphate incorporated per pg protein in a ratio of cytosolic to membrane fraction. ’ Significant difference from control dishes (P < 0.05). [

C

]

[lo-llM]

[lo-9M]

[lo-8M]

a

1 .oo m

Control

m

TPA IO-EM 30 min

Q

2

E

0.50

[lo-7M] 3

6

9

24

HOURS

FIG. 4. Specificity of the effects of TPA on GnRH, but not SH, mRNA levels. Northern analysis of RNA from dishes treated with TPA (lo-“lo-’ M) or DMSO as controls. Radiolabeled RNA strand-specific probes encoding GnRH (top panel), SH (middle panel), and p-actin (bottom panel) were hybridized to a blot containing 10 pg RNA in each lane to confirm the specificity of TPA’s effects.

regulation of PKC, cells were incubated briefly with TPA (lo-’ M) and harvested at 3, 6, 9, or 24 h. After a 30-min exposure to TPA, GnRH mRNA levels decreasedto 64% and 34% of control values at 6 and 9 h, respectively (P < 0.05), with recovery at 24 h (Fig. 5). Cumulative GnRH secretion increased 1.5- to 2-fold over the time course (data not shown). Time-course

effects of TPA

We next examined time-dependent effects of TPA on GnRH expression. Triplicate plates were incubated with TPA (10m8M) or DMSO as a control and harvested at 1, 2, 3, 6, 9, and 24 h for determination of GnRH mRNA levels. Medium was saved for RIA of cumulative GnRH levels. Exposure of cells to TPA (lo-* M) resulted in no significant effects on GnRH mRNA levels at 1-3 h (P = NS), but in progressive decreasesat 6, 9, and 24 h of 57%, 65%, and 72%, respectively (P < 0.05; Fig. 6A). GnRH secretion increased 5- to 6fold in cumulative sampling (P < 0.05). In other experiments, medium was sampled at 3-h intervals and replaced with fresh medium with or without TPA for 24 h. In TPA-treated cells, GnRH secretion increased 3-fold more than in controls at 3 and 6 h (P < 0.05), then gradually decreased to the

FIG. 5. Effects of short term exposure to TPA (lo-’ M) on GnRH mRNA levels. Triplicate dishes of cells were treated with lo-’ M TPA or DMSO briefly for 30 min and harvested at 3, 6, 9, and 24 h, as indicated. Total RNA was extracted as described in the text. Each column represents the mean optical density + SEM of three autoradiographic bands. All data are standardized such that the mean GnRH mRNA level in the control dishes is 1.0 arbitrary densitometric unit (ADU) f SEM. Symbols represent the significance of comparisons of data points with the control values (P < 0.05). This figure represents data from one of two experiments with similar results.

control level at 24 h (P = NS), suggesting that the 5- to 6fold increasesin GnRH observed with cumulative sampling were the result of an early release of GnRH that declined with time (Fig. 6B). Effects of the PKC inhibitor

staurosporine

Studies were next performed to examine the effects of staurosporine, an inhibitor of PKC, on GnRH expression(Fig. 7). Cells were incubated with staurosporine (10m8M), TPA (10m8M), or both substancesand harvested at 6 h. Interestingly, there were no significant increases or decreasesin GnRH mRNA levels after exposure to staurosporine alone compared to values in the control group (P = NS). As before, TPA resulted in 50% decreasesin GnRH mRNA levels (P < 0.05). However, incubation with both staurosporine and TPA partially prevented the TPA-induced decreasesin GnRH mRNAs (P < 0.05 ZIS.TPA alone and controls). Staurosporine had no effect on GnRH secretion and partially blocked the TPA-induced increasesin GnRH secretion (P < 0.05 ZIS.TPA alone and controls; Table 3). Discussion

In the present study we examined the regulatory effects of an activator and an inhibitor of PKC on GnRH secretion

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TPA

ACTIVATION

a 1.oo a:

OF PKC

INHIBITS

: m Control

2

m

TPA

GnRH

mRNAs

Endo. Vol131.

1.oo

stauroIO-8M m

2

lo-DM

1992 No 6

TPA

050

IO-8M

Stauro+TPA

050

1

2

3

6

9

FIG. 7. Effects of the PKC inhibitor staurosporine (Stauro) on GnRH mRNA levels. Triplicate dishes of GTl-7 iells were incubated with staurosporine (lo-’ M) or in combination with TPA (lo-’ M) or DMSO, as described in the text. Each column represents the mean optical density f SEM of three autoradiographic bands. All data points are standardized, such that the mean GnRH mRNA level in the control cells is 1.0 arbitrary densitometric unit (ADU) + SEM. *, Significance of comparison of d&a with the control values (P < 0.05). Staurosporine with or without TPA was also significantly different from TPA alone (P < 0.05). This figure represents data from one of two experiments with similar results.

24

HOURS

-

Control

--t--

IPA

IO- AM

TABLE

3. Effect

of staurosporine

Control

GnRH (rig/ml) oonI 0

3

6

9

17

15

:Y

2 1

L 24

HOURS

6. A, Time-course effects of TPA on GnRH mRNA levels. GTl7 cells were treated with TPA (lo-” M) or DMSO (C, control) for 3, 6, 9, and 24 h, as indicated, and RNA was extracted, as described in Materials and Methods. Each column represents the mean optical density f SEM of three autoradiographic bands. All data are standardized, such that the mean GnRH mRNA level in the control cells is 1.0 arbitrary densitometric unit (ADU) + SEM. *, Significance of comparisons of data points with the control values (P < 0.05). This figure represents the data from one of two experiments with similar results. B, Time-course effects of TPA on GnRH secretion at 3-h intervals. GTl-7 cells were treated with TPA (lo-’ M; - - -) or DMSO (----). Aliquots of medium were sampled at 3-h intervals and assayed for GnRH levels by RIA (see Materials and Methods). *, Differences from control values (P < 0.05).

FIG.

and mRNA levels in hypothalamic neuronal cells. In vim, GnRH neurons make contact with a variety of other neurons that secrete catecholamines, dopamine, serotonin, y-aminobutyric acid, opioids, substance-P, CRF, and GnRH (3-11). Although this uniform population of neuronal cells is not truly representative of the in vim state, the cell culture model system allows us for the first time to directly study GnRH gene expression. Activation of the PKC system has been characterized as one of the major intracellular pathways involved in the secretion of many neuropeptide hormones (16-18). In addition, studies of other hypothalamic releasing factors, such as CRF, vasopressin, oxytocin, and somatostatin, also report an increase in the secretion of these peptides when cells are stimulated by TPA (33, 34). GH-releasing hormone levels,

0.13 + 0.016

Staurosporine (lo-’ M) 0.12 -I 0.006

on cumulative

GnRH

TPA (lo-’ M) 0.53 + 0.018”

secretion

Staurosporine TPA

+

0.41 c O.OOl”,*

GTl-7 cells in triplicate dishes were treated with TPA, staurosporine, both, or DMSO, and medium was sampled at 6 h for cumulative levels of GnRH. GnRH levels were determined as described in Materials and Methods. a Significant difference from the control dishes (P < 0.05). b Significant difference from TPA-treated dishes (P < 0.05).

however, are not increased with exposure to TPA (35). Although our studies involve an integrated assessment of GnRH secretion, the data are in agreement with those of previous studies showing that activation of the PKC pathway acutely increases GnRH secretion (13-15). The decline in GnRH secretion with continued exposure to TPA may reflect depletion of GnRH protein stores or effects of down-regulation of PKC. Future studies with perifusion systems will provide a more detailed picture of GnRH secretory dynamics after experimental manipulation of the PKC pathway. In contrast to our initial hypothesis, we observed divergent effects of activation of the PKC pathway on GnRH mRNA levels. Although many investigators have routinely used a dose of lo-’ M TPA, we observed significant changes in cell morphology with this dose, suggesting some toxicity to the cells (data not shown). Thus, dose-response studies were performed to demonstrate the minimal dose required for inhibitory effects on GnRH mRNA levels. TPA (lo-’ M) resulted in inhibition of GnRH mRNAs without marked changes in cell morphology. The effects of the time-course studies, brief exposure to TPA, and the PKC enzyme activity experiments support the concept that TPA inhibition of gene expression is due to activation of PKC. Little data are available concerning the effects of second messenger signaling systems on the pretranslational regula-

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TPA

ACTIVATION

OF

PKC

tion of hypothalamic releasing factors. Investigators have reported an increase in CRF mRNA levels but no change in somatostatin mRNAs in primary rat hypothalamic neuronal cell cultures treated with TPA for 24 h (36, 37). We speculate that the observed divergent effects on secretion and synthesis may reflect a mechanism by which these cells limit a secretory response by simultaneously turning off synthesis as secretion begins. Further studies will be necessary to clarify these results. Staurosporine, a microbial alkaloid with antifungal activity, is a noncompetitive inhibitor of PKC (20). Other compounds that inhibit PKC do so at micromolar concentrations and have cytotoxic effects on growth (20). Our studies show that staurosporine inhibition of PKC significantly blocks the effects of TPA on GnRH mRNA levels and partially blocks TPA-induced GnRH secretion. Staurosporine alone produces no effects, suggesting that the PKC pathway is not constitutively active in these cells and, thus, is not required for the basallevels of GnRH secretedor steady state GnRH mRNAs. The mechanism by which TPA regulates GnRH mRNAs has not been precisely delineated. Changes in GnRH steady state mRNAs could be due to altered gene transcription, changes in the turnover rate of the transcripts, or both. Preliminary studies in our laboratory using gene transfer experiments suggestthat the effects of TPA occur at the level of gene transcription (data not shown). Further studies, however, to assessgene transcription and mRNA stability will be needed to determine which of these mechanismsis responsible for these results. The steps by which activation of the PKC system may regulate gene transcription are not entirely understood. In many systems, phorbol esters activate PKC and result in increased transcription of the intermediate-early gene c-fos (38). The Fos protein is a component of a transcriptional regulatory complex and interacts in concert with membersof the Jun family of proteins, binding as heterodimers at the AP-1 DNA consensus to either stimulate or inhibit gene expression (39, 40). Currently, the distal mouse promoter sequence has not been published, but the rodent species appear quite homologous in their proximal promoters, in contrast to the human sequence.Of interest, an Al’1 consensus sequence(TGACTCA) is located at approximately -800 bp in the rat GnRH promoter. Whether these sequences mediate the actions of phorbol ester on GnRH expression remain to be elucidated. It is known, however, that GnRH neurons in rat brain coexpress c-fos and c-jun, and both protein and mRNA levels are regulated acutely by physiological manipulations (41-43). In addition, this family of nuclear transcription proteins may be regulated at posttranslational as well as transcriptional levels after phorbol ester treatment (44). Thus, changes in the phosphorylation state of specific nuclear proteins could impact on the transactivation or inhibition of GnRH expression. Finally, within the cell, various signal transduction pathways may interact with each other to impact on gene expression (45, 46). For example, induction of the CAMPprotein kinase-A pathway can secondarily activate or inhibit intracellular Ca2+ flux and/or DAG, which can, in turn,

INHIBITS

GnRH

2557

mRNAs

trigger the PKC pathway. Further studies will be required to delineate the precise mechanismsby which TPA activation of PKC acutely stimulates GnRH secretion but inhibits GnRH gene expression in neuronal cells. Acknowledgments We thank Gloria Smith for excellent secretarial Kepa, William Wood, and Boris Draznin for critiques.

support, and Jadwiga their suggestions and

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Phorbol ester activation of the protein kinase C pathway inhibits gonadotropin-releasing hormone gene expression.

The effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (PKC), and the PKC inhibitor staurospor...
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