Differential Activation of Protein Kinase C delta  and epsilon  Gene Expression by Gonadotropin-releasing Hormone in alpha T3-1 Cells
AUTOREGULATION BY PROTEIN KINASE C*

(Received for publication, September 26, 1996, and in revised form, February 13, 1997)

Dagan Harris , Nachum Reiss and Zvi Naor Dagger

From the Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The effect of gonadotropin-releasing hormone (GnRH) upon protein kinase C (PKC) delta  and PKCepsilon gene expression was investigated in the gonadotroph-derived alpha T3-1 cell line. Stimulation of the cells with a stable analog [D-Trp6]GnRH (GnRH-A) resulted in a rapid elevation of PKCepsilon mRNA levels (1 h), while PKCdelta mRNA levels were elevated only after 24 h of incubation. The rapid elevation of PKCepsilon mRNA by GnRH-A was blocked by pretreatment with a GnRH antagonist or actinomycin D. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA), but not the Ca2+ ionophore ionomycin, mimicked the rapid effect of GnRH-A upon PKCepsilon mRNA elevation. Additionally, the rapid stimulatory effect of GnRH-A was blocked by the selective PKC inhibitor GF109203X, by TPA-mediated down-regulation of endogenous PKC, or by Ca2+ removal. Interestingly, serum-starvation (24 h) advanced the stimulation of PKCdelta mRNA levels by GnRH-A and the effect could be detected at 1 h of incubation. The rapid effect of GnRH-A upon PKCdelta mRNA levels in serum-starved cells was mimicked by TPA, but not by ionomycin, and was abolished by down-regulation of PKC or by Ca2+ removal. Preactivation of alpha T3-1 cells with GnRH-A for 1 h followed by removal of ligand and serum resulted in elevation of PKCdelta mRNA levels after 24 h of incubation. Western blot analysis revealed that GnRH-A and TPA stimulated (within 5 min) the activation and some degradation of PKCdelta and PKCepsilon . We conclude that Ca2+ and PKC are involved in GnRH-A elevation of PKCdelta and PKCepsilon mRNA levels, with Ca2+ being necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH-A response. A serum factor masks PKCdelta but not PKCepsilon mRNA elevation by GnRH-A, and its removal exposes preactivation of PKCdelta mRNA by GnRH-A which can be memorized for 24 h. PKCdelta and PKCepsilon gene expression evoked by GnRH-A is autoregulated by PKC, and both isotypes might participate in the neurohormone action.


INTRODUCTION

The protein kinase C (PKC)1 family is a family of serine/threonine protein kinase isoforms, which play key roles in signal transduction (1-3). Conventional PKCs (alpha , beta I, beta II, and gamma ) are activated by Ca2+, diacylglycerol (DAG), and phospholipid such as phosphatidylserine (PS) and are tightly coupled to phosphoinositide turnover (1-3). Novel PKCs (delta , epsilon , eta , and theta ) are Ca2+-independent but DAG- and PS-activated isoforms. Atypical PKCs (zeta  and lambda /iota ) are Ca2+- and DAG-independent but PS-activated isoforms and are also stimulated by other lipid-derived mediators (1-4). PKCµ takes an intermediate position among the novel PKC and atypical PKC isoforms and is a Ca2+- and DAG-independent isoform. Whereas relatively much is known about regulation of PKC at the protein level including cofactor requirements, translocation to the membrane, substrate phosphorylation, and degradation (1-9), very little is known about ligand regulation of PKC gene expression (10-12). Previous work has implicated PKC in gonadotropin-releasing hormone (GnRH) action upon gonadotropin secretion and gonadotropin subunits gene expression in pituitary and alpha T3-1 cells (5, 6, 12-26). Recently, while examining conventional PKC regulation, we have shown that GnRH-A increases the levels of PKCbeta , but not PKCalpha , mRNA levels in alpha T3-1 cells, while PKCgamma is not expressed in the cells (12). Since PKCdelta and PKCepsilon of the novel PKC group are major subspecies in the pituitary (26), we decided to investigate the effect of GnRH-A on the mRNA levels of both isotypes in the alpha T3-1 cell line. Here we demonstrate that GnRH-A directs differential autoregulation of PKCdelta and PKCepsilon gene expression, which is dependent upon growth conditions and Ca2+, and reveals a memory mechanism, which might participate in PKCdelta autoregulation.


EXPERIMENTAL PROCEDURES

Materials

alpha T3-1 cells were kindly provided by Dr. P. Mellon (University of California San Diego, La Jolla, CA). The GnRH analog [D-Trp6]GnRH (GnRH-A) was a gift from Dr. R. Millar (University of Cape Town Medical School, Cape Town, South Africa). A potent GnRH antagonist [D-Glu(P)1,ClPhe(P)2,D-Trp3,6]GnRH was kindly provided by Dr. D. Coy (Tulane University School of Medicine, New Orleans, LA). Ionomycin was purchased from Boehringer (Mannheim, Germany). The PKC-selective inhibitor bisindolylmaleimide (GF 109203X) (27) was purchased from Calbiochem (Laufelfingen, Switzerland). Bovine serum albumin, TPA, and other chemicals were purchased from Sigma (Rehovot, Israel). Media and sera for cell culture were from Biological Industries (Kibbutz Beth Ha'Emek, Israel). [alpha -32P]dCTP was purchased from Rotem (Beersheba, Israel). PKCdelta and PKCepsilon cDNAs were kindly provided by Dr. H. Mischak (Institute of Clinical Molecular Biology, GSF, Munich, Germany) and Dr. F. Mushinsky (NIH, Bethesda, MD) (27), and the respective antibodies were obtained from Sigma.

Methods

Cell Culture

alpha T3-1 cells were subcultured into 60-mm tissue culture dishes (Sterilin, Hounslow, United Kingdom). Cells were grown in 5 ml of Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum, 5% horse serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. After 3-4 days, when cells were 70-80% confluent, the cultures were washed three times with fresh DMEM, and stimulants were added in 5 ml of DMEM at the indicated concentrations for the given length of time. For short period incubations (up to 1 h) 10 mM Hepes was added to the medium. When the stimulation period was longer than 9 h, the medium was supplemented with 0.1% bovine serum albumin.

RNA Extraction and Analysis

At the end of the stimulation period, total RNA was isolated from cells by extraction in guanidium thiocyanate containing 8% 2-mercaptoethanol by the LiCl method as described by Cathala et al. (28). For Northern blot analysis, total RNA (15 µg) was fractionated on 1.2% denaturing agarose gel and transferred to GeneScreen membranes (DuPont NEN). Alternatively, RNA samples (8 µg) were slot blotted onto GeneScreen using a slot blot manifold (Schleicher & Schüll). Following baking and prehybridization, the membranes were hybridized overnight with the specific cDNA probes labeled to high specific activity using a random primer labeling kit (Boehringer). Half of each lane was hybridized with a PKC cDNA, and the second half was hybridized with glyceraldehyde-3-phosphate dehydrogenase cDNA as an internal control. Thereafter, filters were washed at high stringency and were autoradiographed at -70 °C. Steady state levels of mRNAs were quantified with densitometric scanning of autoradiograms. The data were corrected for variability in loading by calculation as a ratio to glyceraldehyde-3-phosphate dehydrogenase.

PKC Translocation

Following ligand treatment, cells were washed with ice-cold Tris-buffered saline, pH 7.2, harvested with rubber policemen, and pelleted by short spin (1200 rpm for 5 min at 4 °C). Cells were resuspended in 10 mM EGTA, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 0.5 mM iodoacetic acid, and lysed by 10 strokes of a 25-gauge syringe. Following removal of nuclei (1200 rpm for 5 min at 4 °C), cytosol and membrane fractions were obtained by ultracentrifugation (100,000 × g for 2 h at 4 °C). The proteins were separated on 7.5-18% SDS-polyacrylamide gels (ratio of acrylamide to bisacrylamide, 30:0.5) and electrotransferred to nitrocellulose papers in 50 mM glycine, 50 mM Tris-HCl, pH 8.8 (100 V for 2 h at 4 °C). The papers were blocked for 60 min in 1% bovine serum albumin and 0.5% Tween 20 in Tris-buffered saline and treated overnight with the respective rabbit anti-PKC antibodies (Sigma). The signals were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG and the ECL method.

Statistical Analysis

The hybridization signals for PKC subtypes mRNA in each group were normalized to the hybridization signals for the housekeeping gene for glyceraldehyde-3-phosphate dehydrogenase. An arbitrary unit of 1 represents the control values. Statistical comparisons between control and treatment groups were performed using Student's t test; in the figures, a single asterisk indicates p < 0.05, a double asterisk indicates p < 0.01, and a triple asterisk indicates p < 0.001.


RESULTS

We first studied the cellular redistribution of PKCdelta and PKCepsilon following GnRH-A and TPA stimulation, since it is a criterion for PKC activation by extracellular signals (5-8). Both GnRH-A and TPA stimulated an increase in the molecular weight of cytosolic PKCdelta within 5 min (Fig. 1), consistent with the size shift reported for PKCdelta phosphorylation by Src (29), and with our own finding that GnRH-A and TPA stimulate protein-tyrosine phosphorylation in alpha T3-1 cells.2 PKCdelta in the membrane fraction is already of the high molecular weight form and is further elevated by GnRH-A and even more by TPA. In addition, translocation of PKCdelta to the membrane fraction by GnRH-A and TPA is further validated by the appearance of degradation products of 70 and 42 kDa (apparently PKM; Refs. 6-8) in the membrane fraction in the ligand-treated groups (3-4-fold stimulation by GnRH-A and TPA; Fig. 1). PKCepsilon activation is manifested by translocation to the membrane fraction and the appearance of 50- and 42-kDa bands (apparently PKM) in the ligand-treated groups (2-fold; Fig. 1). Consistent with our previous reports that TPA-mediated down-regulation of endogenous PKC in alpha T3-1 cells reduced cellular PKC activity by 90% (12, 21, 23), prolonged incubation with TPA (100 ng/ml, 24 h) resulted in loss of most of PKCepsilon (60 and 90% of the membrane and soluble enzyme, respectively), and all of the detectable soluble and membrane-bound PKCdelta (Fig. 1).


Fig. 1. Effect of GnRH-A and TPA on PKCdelta and PKCepsilon cellular distribution. alpha T3-1 cells were serum-starved (0.5% serum) for 24 h in the absence (lanes 1-3 and 5-7) or the presence of TPA (100 ng/ml, lanes 4 and 8). The cells were washed several times and stimulated for 5 min with GnRH-A (10 nM, lanes 2 and 6) or TPA (100 ng/ml, lanes 3 and 7). Cytosol (lanes 1-4) and membrane (lanes 5-8) fractions were prepared and Western-blotted (right panels) as described under "Methods" using anti-PKCdelta (upper panel) and anti-PKCepsilon (lower panel) antibodies. Right panels show the separation (right arrows) of PKCdelta and PKCepsilon and their degradation products (left arrows indicate the size shift of PKCdelta ). Left panels show the densitometric quantitation of PKCdelta and PKCepsilon and their degradation products (insets, p42 and p50, respectively) in the membrane fractions.
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The regulation of PKCdelta and PKCepsilon mRNA levels was determined by treatment of alpha T3-1 cells with [D-Trp6]GnRH, a stable GnRH analog. Addition of GnRH-A to the cells for 1 h elevated PKCepsilon but not PKCdelta mRNA levels in a dose-related fashion, with maximal response obtained at 10 nM analog (Fig. 2A). Stimulation of PKCepsilon mRNA levels by GnRH-A was rapid, with a peak at 1 h, declining thereafter to basal levels (Fig. 2B). On the other hand, significant elevation of PKCdelta mRNA levels was detected only after 24 h of incubation with GnRH-A (3-fold, p < 0.001; Fig. 2B).


Fig. 2. Effect of GnRH analog on PKCepsilon and PKCdelta mRNA levels. alpha T3-1 cells in triplicate were treated with or without GnRH-A at the indicated concentrations for 60 min (A) or for the time indicated (B). PKC mRNA levels were determined as described under "Methods." An arbitrary unit of 1 represents the control values. Results are mean ± S.E. (n = 6). A lane of the dot blot is shown in the inset. *, p < 0.05; **, p < 0.01; ***, p < 0.001 .
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The effect of GnRH-A on PKCepsilon mRNA levels was investigated further, since its rapid nature suggested that it more likely represents a physiological response to the neurohormone. Pretreatment of the cells with a potent GnRH antagonist (Fig. 3A) or with actinomycin D (Fig. 3B) abolished the stimulatory effect of GnRH-A upon PKCepsilon mRNA levels, indicating a receptor-mediated effect apparently at the transcriptional level (Fig. 3).


Fig. 3. Inhibition of GnRH-A-induced PKCepsilon mRNA elevation by a GnRH antagonist (A) and by actinomycin D (B). alpha T3-1 cells were treated with or without GnRH agonist [D-Trp6]GnRH (GnRH-A, 10 nM), GnRH antagonist [D-Glu(P)1,ClPhe(P)2,D-Trp3,6]GnRH (100 nM), or both for 1 h (A). alpha T3-1 cells were pretreated with actinomycin D (1 µg/ml) for 60 min followed by GnRH-A (10 nM) for an additional 60 min (B). PKCepsilon mRNA levels were determined as described under "Methods." Northern blot analysis for the treatment groups of B is shown in the inset. Results are mean ± S.E. (n = 6). *, p < 0.05; **, p < 0.01.
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The potential role of PKC and Ca2+ in mediating the GnRH response upon PKCepsilon mRNA levels was investigated since both messengers were implicated in GnRH action upon gonadotropin release and gonadotropin subunits gene expression (5, 6, 12-26, 30). Addition of the PKC activator TPA to alpha T3-1 cells for 1 h resulted in elevation of PKCepsilon but not PKCdelta mRNA levels, while the Ca2+ ionophore, ionomycin, had no effect (Fig. 4, A and C, and data not shown). Elevation of PKCepsilon mRNA levels by TPA was rapid, with a peak at 1 h and a return to basal levels (Fig. 4B). The similar time responses elicited by GnRH-A and TPA suggest that PKC is involved in GnRH-A stimulation of PKCepsilon gene expression.


Fig. 4. Effect of TPA and ionomycin on PKCepsilon mRNA levels. alpha T3-1 cells were incubated with increasing concentrations of TPA for 60 min (A), or with 100 ng/ml TPA for the time indicated (B), or ionomycin (C) for 60 min. Results are mean ± S.E. (n = 6). *, p < 0.05; **, p < 0.01. In this and some of the subsequent figures, a lane of the dot blot is shown in the inset.
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This notion was further supported by inhibition and depletion of PKC. Addition of the selective PKC inhibitor GF 109203X (27, 31) to the cells resulted in a dose-related inhibition of the GnRH-A stimulated PKCepsilon mRNA levels with half-maximal inhibition (IC50) observed at 0.8 µM of the drug, in good agreement with IC50 values of PKC inhibition in cellular systems such as Swiss 3T3 fibroblasts (31) (Fig. 5A). The drug alone (1 µM) reduced the basal level by about 50%, suggesting that PKC is also involved in the maintenance of basal PKCepsilon gene expression. We also used down-regulation of endogenous PKC by prolonged incubation with TPA. Pretreatment of the cells with TPA (100 ng/ml, 24 h) reduced cellular PKC activity by 90% as measured by enzymatic activity assay and Western blot analysis (Fig. 1 and Refs. 12, 21, and 23). The stimulatory effect of GnRH-A and TPA upon PKCepsilon mRNA levels was abolished in the down-regulated cells (Fig. 5B). In addition, we observed no additivity between GnRH-A and TPA upon PKCepsilon mRNA levels (Fig. 6), lending further support to the role of PKC in mediating the GnRH-A effect on PKCepsilon gene expression. The Ca2+ ionophore, ionomycin, had no effect on basal PKCepsilon mRNA levels or on the stimulatory response elicited by GnRH-A or TPA (Fig. 6). On the other hand, transfer of alpha T3-1 cells to Ca2+ free medium, in the presence or absence of EGTA, abolished stimulation of PKCepsilon mRNA levels by GnRH-A (Fig. 7). It therefore seems that Ca2+ is necessary but not sufficient for mediation of the GnRH-A response.


Fig. 5. Effect of PKC inhibition and depletion on GnRH-A -induced PKCepsilon mRNA levels. A, alpha T3-1 cells were preincubated with the indicated concentrations of GF 109203X for 5 min and further incubated with GnRH-A (10 nM) for 60 min. B, alpha T3-1 cells were pretreated with TPA (100 ng/ml) for 24 h. Cells were then washed and further stimulated with GnRH-A (10 nM) or TPA (100 ng/ml) for 1 h. Results are mean ± S.E. (n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Fig. 6. Lack of additivity between GnRH-A and TPA upon PKCepsilon mRNA elevation. alpha T3-1 cells were treated with or without GnRH-A (10 nM), TPA (100 ng/ml), or ionomycin (1 µM) or in combinations, for 60 min. Results are mean ± S.E. (n = 6). *, p < 0.05; **, p < 0.01.
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Fig. 7. Effect of Ca2+ removal upon GnRH-A-induced PKCepsilon mRNA levels. alpha T3-1 cells were transferred to DMEM (control), to Ca2+-free DMEM (Ca2+ free) or to Ca2+-free DMEM + 250 µM EGTA (EGTA) for 10 min. Cells were then incubated with (striped bars) or without (empty bars) GnRH-A (10 nM) for 60 min in the respective medium as indicated. PKCepsilon mRNA levels were analyzed as described under "Methods" (n = 6). **, p < 0.01.
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Since GnRH stimulated PKCdelta mRNA levels only after 24 h of incubation in medium without serum (Fig. 2B), it was possible that growth conditions are involved in PKCdelta gene expression. We therefore examined the role of serum in PKCepsilon and PKCdelta gene expression. Transfer of the cells to medium with low serum (0.5%) for 24 h had no effect on GnRH-A-stimulation of PKCepsilon mRNA levels (Fig. 8). On the other hand, serum starvation advanced the stimulation of PKCdelta mRNA levels by GnRH-A to 1 h of incubation that could not be observed in serum-grown cells (Fig. 8). Time course of PKCdelta mRNA levels in serum-starved cells revealed a rapid effect of GnRH-A at 1 h of incubation with no effect at 24 h, as seen in non-starved cells (Fig. 9A). Similarly, serum starvation exposed a rapid response (peak at 30 min) of TPA on PKCdelta mRNA levels (Fig. 9B), suggesting a role for PKC in mediating PKCdelta gene expression. Indeed, down-regulation of endogenous PKC by prolonged incubation with TPA abolished GnRH-A and TPA stimulation of PKCdelta mRNA levels in serum-starved cells (Fig. 10). Transfer of the cells to Ca2+ free medium, in the presence or absence of EGTA, abolished the rapid stimulation of PKCdelta in serum-starved cells (Fig. 11).


Fig. 8. Effect of serum starvation upon GnRH-A stimulation of PKCepsilon and PKCdelta mRNA levels. alpha T3-1 cells were preincubated with 10 or 0.5% serum for 24 h. Cells were then washed and incubated in serum-free medium with GnRH-A (10 nM) for 60 min. Results are mean ± S.E. (n = 6). **, p < 0.01; ***, p < 0.001.
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Fig. 9. Effect of GnRH-A and TPA upon PKCdelta mRNA levels in serum-starved cells. alpha T3-1 cells were serum-starved (0.5% serum) for 24 h, washed, and incubated with GnRH-A (10 nM, A) or TPA (100 ng/ml, B) for the indicated time periods. Results are mean ± S.E. (n = 6). **, p < 0.01; ***, p < 0.001.
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Fig. 10. Effect of PKC depletion upon GnRH-A and TPA-induced PKCdelta mRNA levels in serum-starved cells. alpha T3-1 cells were serum-starved (0.5% serum) in the presence or absence of TPA (100 ng/ml) for 24 h to deplete endogenous PKC activity. Cells were then washed and incubated with or without GnRH-A (10 nM) or TPA (100 ng/ml) for 60 min. Results are mean ± S.E. (n = 6). ***, p < 0.001.
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Fig. 11. Effect of Ca2+ removal upon GnRH-A-induced PKCdelta mRNA levels in serum-starved cells. alpha T3-1 cells were preincubated with 0.5% serum for 24 h. Cells were then transferred to DMEM (control)), to Ca2+-free DMEM (Ca2+ free) or to Ca2+-free DMEM + 250 µM EGTA (EGTA) for 10 min. Cells were then incubated with (striped bars) or without (empty bars) GnRH-A (10 nM) for 60 min in the respective medium as indicated. PKCdelta mRNA levels were analyzed as described under "Methods" (n = 6). ** p < 0.01.
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As shown above, when cells are transferred to serum-free medium and exposed to GnRH-A, elevation of PKCdelta mRNA levels is observed at 24 h, but not at 1 h of incubation (Fig. 12, columns 1-3). On the other hand, when cells are first serum- starved (24 h) and later exposed to GnRH-A, elevation of PKCdelta mRNA levels is observed after 1 but not 24 h of incubation (Fig. 12, columns 4-6). We therefore exposed normal cells to GnRH-A for 1 h, washed the cells several times to remove serum and GnRH-A, and further incubated the cells for 24 h. As seen in Fig. 12 (columns 7 and 8), PKCdelta mRNA levels were elevated at 24 h by pretreatment (1 h) with GnRH-A. Thus, the late effect of GnRH-A on PKCdelta mRNA levels (Fig. 12, column 3) is due to generation of a rapid signal (1 h, column 5), which is "memorized" during the long starvation period (t1/2 congruent 12 h) required for manifestation of the early signal by means of removal of the inhibitory effect of the serum.


Fig. 12. Effect of pretreatment with GnRH-A on PKCdelta mRNA levels in serum-starved cells. alpha T3-1 cells grown in serum (10%) were washed and stimulated with GnRH-A (10 nM) for 1 or 24 h in serum-free medium (columns 1-3). The second group was serum-starved (0.5% serum) for 24 h, washed, and incubated with GnRH-A (10 nM) for 1 or 24 h (strv=treat, columns 4-6). The third group was grown in 10% serum, washed and stimulated with GnRH-A (10 nM) for 1 h, and then serum-starved for 24 h (treat=strv, columns 7 and 8). Results are mean ± S.E. (n = 6). **, p < 0.01; ***, p < 0.001.
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DISCUSSION

Whereas much has been learned concerning the regulation of PKC and its subspecies at the protein level (1-9), very little is known about ligand regulation of PKC subtypes gene expression (10-12). Differentiation regulators of the human promyelocytic leukemia cell line (HL-60) such as 1alpha ,25-dihydroxyvitamin D3, retinoic acid, and dimethyl sulfoxide, were shown to increase the expression of PKCalpha and PKCbeta mRNA levels (10, 11). Furthermore, transcriptional activation of PKCalpha and PKCbeta expression was reported to result in increased PKC enzymatic activity (10, 11, 32). Here we demonstrate that GnRH-A, which does not promote growth or differentiation, is capable of activating differential nPKC isoforms gene expression. To the best of our knowledge, this is the first demonstration of a natural ligand stimulation of PKCdelta and PKCepsilon mRNA levels.

Activation of PKCdelta mRNA levels, but not that of PKCepsilon , is dependent upon growth conditions, suggesting the presence of a serum factor that is involved in regulation of PKCdelta gene expression, possibly via a serum response element.

The differential activation of PKCdelta and PKCepsilon mRNA levels by GnRH-A suggests that the isoforms might specialize in different functions. PKCdelta is the major subspecies in the 6-day-old rat pituitary and is markedly reduced in the 3-month-old pituitary (26). The opposite is observed for pituitary PKCepsilon , which increases with age (26). Therefore, PKCdelta and PKCepsilon might play different roles during pituitary development. It was also shown that while PKCdelta is involved in exocytosis, PKCepsilon participates in feed-back inhibition of phospholipase C activity in rat basophilic RBL-2H3 cells (33). In a recent study, GnRH was shown to translocate PKCepsilon and PKCzeta in alpha T3-1 cells while PKCdelta was not detected (30). We report here that both the PKCdelta and PKCepsilon isoforms are expressed in the alpha T3-1 cells at the mRNA and protein levels and that they are translocated to the membranes and activated in response to GnRH-A, as also validated by the apparent formation of the PKM species (6-8). The differences between the reports are most likely due to the use of different PKC type-specific antibodies.

While overexpression of PKCdelta resulted in inhibition of growth rate in NIH 3T3 cells, overexpression of PKCepsilon increased growth rate, and the transformed cells (NIH 3T3 or Rat 6 cells) formed tumors in nude mice (27, 34). Since GnRH affect differentiated responses, it is possible that PKCdelta and PKCepsilon are involved in separate functions such as gonadotropin release and gonadotropin subunit gene expression during the hormone action. Elevation of mRNA of a given PKC isoform by ligands in general and by GnRH-A in particular might be a step in the life cycle of PKCs during hormone action to replenish the enzymes after translocation and degradation as shown in Fig. 1.

The rapid effect (peak at 60 min) of GnRH-A upon PKCepsilon and PKCdelta (in serum-starved cells) mRNA levels might be physiologically relevant, since GnRH is released from the hypothalamus in a pulsatile manner at intervals of 1-2 h according to the species and its half-life is about 2-4 min (35-37). Thus, prolonged responses such as those observed in Fig. 2 (24 h) are more difficult to interpret, since it is not clear whether multiple pulses of GnRH are capable of eliciting a similar response. Hence, we investigated in more detail the rapid effects of GnRH, which prompted us to identify the second messengers involved in the neurohormone action. Indeed, the PKC activator TPA mimicked the GnRH-A rapid responses and stimulated PKCepsilon mRNA levels in serum-grown cells and PKCdelta mRNA levels in serum-starved cells with a similar time course. Additionally, the stimulatory effect of GnRH-A on PKCdelta and PKCepsilon mRNA levels was abolished in PKC-down-regulated cells or by the use of the selective PKC inhibitor GF 109203x (27, 31) (present results and data not shown). We therefore suggest that GnRH-A stimulation of PKCdelta and PKCepsilon gene expression is autoregulated by PKC.

While we show here positive regulation of PKCdelta mRNA levels by GnRH-A and TPA, others have recently reported that PKCdelta mRNA is down-regulated by TPA (4 h) in the mouse B lymphoma cell line A20 (38). The difference between the results might be due to the presence of a soluble destabilizing factor, which specifically accelerates degradation of PKCdelta mRNA in A20 cells (38).

Removal of Ca2+ abolished the effect of GnRH-A upon PKCdelta and PKCepsilon mRNA levels, but Ca2+ ionophore had no stimulatory effect. We therefore conclude that Ca2+ is necessary but not sufficient, while PKC is both necessary and sufficient to mediate the GnRH response. Furthermore, since removal of extracellular Ca2+ per se is not sufficient to block Ca2+ mobilization in pituitary cells (39), the data suggest that GnRH-A-induced PKCdelta and PKCepsilon gene expression is mainly mediated by Ca2+ influx, apparently via L-type voltage-sensitive channels (39). The data also suggest that a Ca2+-dependent PKC isoform might be involved in GnRH-A action. Since PKCdelta and PKCepsilon are differentially regulated by GnRH-A, it is unlikely that one regulates the expression of the other during the neurohormone action. Furthermore, it is unlikely that PKCepsilon is involved in the process since its membrane-bound form (the active form) was reduced only by 60% by down-regulation, whereas the effect of GnRH-A was abolished. Further studies are required to identify the PKC isoforms and the site of Ca2+ action in the GnRH-A response.

In addition to mediation by Ca2+ and PKC, elevation of PKCdelta but not PKCepsilon mRNA levels by GnRH-A and TPA required removal of a serum factor. Therefore, although PKCdelta and PKCepsilon gene expression share some common mechanisms, which are mediated by Ca2+ and PKC, they differ in sensitivity to the serum factor. Changes in the concentrations of the serum factor under physiological conditions might therefore enable preferential activation by GnRH of the two isotypes. Moreover, it was possible to first stimulate the cells with GnRH-A, and remove the hormone and serum for 24 h, at the end of which elevation of PKCdelta mRNA levels by GnRH-A was detected. The observation is in contrast to previous observations, in which GnRH-stimulated LH release in perifused pituitary cells was terminated immediately after removal of the neurohormone (Ref. 40 and data not shown). Since PKC is the main mediator of GnRH actions (5, 6, 12-26, 30), it is likely that the half-life of the phosphoproteins involved in exocytosis is very short (t1/2 congruent  8 min; Ref. 40), while those mediating the neurohormone effect on PKCdelta gene expression is relatively long (t1/2 congruent  12 h). The presence of the serum factor does not block the formation of downstream effectors involved in PKCdelta gene expression, but only masks the effectors activity. Since growth conditions also affected TPA stimulation of PKCdelta gene expression, it seems that the site of action of the serum factor is downstream to PKC activation. Since both Ca2+ and PKC participate in GnRH-stimulated PKCdelta and PKCepsilon gene expression, it is likely that transcriptional regulation of both isotypes by GnRH-A involves similar transcription factors but different coactivators and composite response elements (41). Future analysis of the gene structure of both isotypes will reveal the different response elements involved in ligand regulation of PKCdelta and PKCepsilon gene expression.

The present report demonstrates differential activation of PKCdelta and PKCepsilon mRNA levels by GnRH-A that is dependent upon growth conditions and Ca2+ influx, and is autoregulated by PKC. Since both isotypes are shown here to be translocated by GnRH from the cytosol to the membrane (an index of PKC activation; Refs. 1-3 and 5-8), PKCdelta and PKCepsilon are therefore likely candidates to participate in GnRH action, the first key hormone of the reproductive cycle.


FOOTNOTES

*   This work was supported by the United States-Israel Binational Science Foundation, the Israel Academy of Sciences and Humanities, the Eisne Foundation Center-Biology Research Center, and Tel Aviv University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Fax: 972-3-6415053.
1   The abbreviations used are: PKC, protein kinase C; PKCs, PKC subtypes; GnRH, gonadotropin-releasing hormone; GnRH-A, GnRH analog; TPA, 12-O-tetradecanoylphorbol-13-acetate; DMEM, Dulbecco's modified Eagle's medium; DAG, diacylglycerol; PS, phosphatidylserine; PKM, catalytic moiety of PKC.
2   N. Reiss, D. Harris, and Z. Naor, manuscript in preparation.

ACKNOWLEDGEMENTS

We thank Drs. P. Mellon for the alpha T3-1 cell line, H. Mischak and F. Mushinsky for PKC subtype cDNAs. We also thank Sharon Shacham for help during the studies and Angela Cohen and Erica Vallenci for editorial assistance.


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