Effect of phorbol ester on the regulation of LHhCG receptors

Life .Schces, Vol. 61, No. 14, pp. 1435-1443,1!297 copyright 0 1997 Elrcvier science Inc. Printed in the USA. AU rights seumd am3205/97 $17.00 + .oo

PII Sooz4-3205(97)oo6s9-9

EFFECT OF PHORBOL ESTER ON THE REGULATION Kazuto Nakamura,Takashi

Mine&hi,

OF LWHCG RECEPTORS

Mari Tano, Hiroshi Kishi, Takashi Kameda and Kaoru Miyamoto*

Department of Obstetrics and Gynecology, Gunma University School of Medicine, and *Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gumna University, Maebashi, Gunma 371, Japan (Received in final form June 26,lW)

Summary Granulosa cells have been used to study the regulation of LWhCG receptor protein and mRNA expression. Phorbol 12-myristate 13-acetate (PMA) dose-dependently attenuates the increases in LH/hCG receptor mRNA and protein induced by FSH and forskolin (FSK). The presence of PMA caused a decrease in CAMP production stimulated by FSH and FSK. These results suggest that PMA-mediated decreases in CAMP are a major factor in PMA-mediated decreases in LI-I/hCG receptor mRNA. On the other hand, in the presence of 8Br-CAMP, PMA significantly increased LI-I/hCG receptor mRNA and protein, with maximal stimulation between PMA concentrations of 3 to 30 nM (1.5 fold) with 8-Br-CAMP. These findings suggest that activation of protein kinase C by PMA attenuates the increase in CAMP accumulation induced by FSH but enhances the effect of CAMP on LI-hhCG receptor expression, and that the inhibitory and stimulatory effects of PMA on LWhCG receptor content are correlated with regulation of LWhCG receptor mRNA levels. Since the half-life study revealed no change in the stability of the LHihCG receptor mRNA following PMA treatment, a change in the rate of LH/hCG receptor gene transcription must be responsible for the change in the LWhCG receptor mRNA levels.

Key Words: phorbol ester, LH/hCG receptor mRNA, gene transcription, CAMP

Ovarian granulosa cells undergo a complex differentiation process during the growth and maturation of ovarian follicles. Primary cultures of rat granulosa cells obtained from hypophysectomized or immature female rats pretreated with estradiol (1,2) are a widely used model system for studying this phenomenon. In this defined system, the ability of FSH to stimulate the induction of LWHCG receptor has been shown to be mediated by CAMP, since exogenously added CAMP or other agents that increase intracellular levels of CAMP mimic the actions of FSH (3-6). Molecular cloning of the cDNA encoding the FSH and LHlhCG receptor has substantially facilitated our understanding of the mechanisms underlying these phenomena (7-9). The cloning of gonadotropin receptor cDNAs has indicated that LH/hCG receptors belong to theG-protein-coupled Address for correspondence: Takashi Minegishi, Department of Obstetrics and Gynecology, University School of Medicine, Maebashi, Gunma 371, Japan.

Gunma

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Phorbol Ester on LH/hCG Receptors

receptor family, with seven tmnsmembrane receptor (10).

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domains, and that they are closely related to the TSH

Two major signal transduction pathways activated by G-protein-coupled receptors are the pathways that stimulate phospholipase C and adenylyl cyclase, mediating cellular effects by activation of the effector kinases protein kinase C and protein kinase A, respectively. Many reports have indicated that cross-talk occurs between these two signaling pathways. Activation of one of the pathways has been shown to have a stimulatory or inhibitory effect on the other pathway (11). It is possible that the stimulating effect of FSH on both adenylyl cyclase and phospholipase C is mediated by the same receptor.The purpose of this study was to examine whether activation of protein kinase C signaling pathways could regulate LHlhCG receptor mRNA and protein expression and to elucidate the mechanism of the regulation of LH/hCG receptors by protein kinase C . Materials

and Methods

Hormones and reagents

Rat FSH (NIH, FSH, I-8) and hCG (CR-127) were provided by the National Pituitary Agency. Dlethylstilbestrol (DES), gentamicin sulfate, 8-bromo-adenosine 3,5cyclic monophosphate (8-Br-CAMP), phorbol 12-myristate 13-acetate (PMA), 3-isobutyl-1-methylxanthine (IMX) and forskolin (FSK) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). Dulbecco’s modified Eagle (DMEM) medium, Ham’s F-12 medium, and fungizone were purchased from GIBCO Laboratories (Grand Island, NY). The RNA labeling kit and nucleic acid detection kit were purchased from Boehringer Mannheim (Mannheim, Germany). Rat Granulosa Cell Culture

Immature female Wistar rats (21 days old) were obtained from Imai Experimental Animal Farm (Saitama Pref., Japan). Since estrogen treatment in vivo results in the formation of multiple preantral follicles and provides a large number of relatively homogeneous cells at the same stage of development, rats were given daily subcutaneous injections of 2 mg of DES in 0.1 ml sesame oil for 4 days. The rats were killed at 26 days, and their ovaries were aseptically excised. All procedures I-ollowed NIH guidelines. Granulosa cells were obtained by puncturing ovaries from DE-S-treated immature rats. The granulosa cells were then cultured in Ham’s F-121DMEM (1: 1 vol/vol) medium supplemented with 1.1 g/liter NaHCQ, 40 mg/liter gentamicin sulfate, 1 mglliter fungizone, and 100 mg/liter BSA in a humidified atmosphere containing 5% COz, 95% air at 37 C. Receptor binding assay

Highly purified hCG was iodinated by the chlommine-T method. For Scatchard analysis, after culture with or without treatment, medium was aspirated at selected times, and the cultures were quickly frozen at -70 C with hypotonic buffers until used. To prepare the membranes, cells were thawed and centrifuged at 20,000 x g for 30 min, and the pellets were resuspended in 1,2 ml PBS containing 0.1% BSA (pH 7.4). Then loo-u1 aliquots were incubated with 1251-hCG at 22 C overnight in the presence and absence of excess unlabeled hCG. After incubation, 1 ml of cold PBS containing 0.1% BSA was added, and bound and free hormones were separated by sedimentation at 3,000 x g for 10 min, and pellet-bound radioactivity was determined. Data from competition binding studies were analyzed by the Scatchard method (12). Preparation of cRNA Probe

Rat LH/hCG receptor cDNA was subcloned into the EZcoRI site of the Bluescript KS (+) vector and linearized with BglII (13). Digoxigenin-labeled FSH- receptor cRNA probe corresponding to bases 440-2560 was produced by in vitro transcription with 17 RNA polymerase and an RNA

Vol. 61, No. 14, 1997

labelmg kit (Boehringer Mannheim). the same method.

Phorbol Ester on LH/hCG Receptors

A digoxigenin-labeled

1431

1%actin cRNA probe was obtained by

RNA Isolution and Analysis Granulosa cells were cultured in 60-mm dishes containing 5x1@ viable cells in 5 ml of medium, and reagents were added to the medium after 24 h of cell culture. The granulosa cells were furthei in the guanidium incubated, and the cultures were stopped at the times Indicated The final RNA pellet was dissolv,ed in acid-thiocyanate-phenol-chloroform method (14). ðyl-pyrocarbonate-treated H20, and total RNA was quantified by measuring the absorbance 01. samples at 260 nm. For Northern blot analysis, 15 pg of total RNA from each dish was separated b!, electrophoresis on denaturing agarose gels and then transferred to a nylon membrane (Biodyne, ICN). Northern blots were prehybridized for 6 h at 68 C and then hybridized overnight at 68 C wrth digoxigenin-labeled cRNA probes. In accordance with the standard protocol for the nucleic aud detection kit (Boehringer Mannheim), Kodak X-Omat film (Eastman Kodak, Rochester, NY) was then exposed to the membranes. Luminescence detection was quantified with a LKB 2202 Unit&can Laser Densitometer (LKB Produkter AB, Bromma, Sweden), normalized against a corresponding relative amount of l.S-actin mRNA in each sample, and expressed as relal~\ c dcnsitometnc units. The data are presented as means &SE of measurements from triplicate sepal-ltc c\penments. Differences between control and treated cells were assessed by Student’s /-test I’oIindependent samples. Cyc.lic AMP ussuys The cells (1.5 x 105 cells/culture dish) were washed with warm growth medium and then preincubated for 15 min at 37 C in 1 ml growth medium in the presence of 0.5 mM MIX. Purified hormones were added to the dish, and the incubation was continued for 30 min at 37 C. After incubation, the medium was removed, and the cells were rinsed twice with phosphate-buffered Aliquots of the resulting Iysate wcrc saline (PBS) at 4 C and lysed with 1 ml 95% (v/v) ethanol. centrifuged at 4 C at 15,000 g for 15 min. The supernatant was dried and resuspended in 0.3 M imtdaz.ole buffer, pH 6.5. Intracellular CAMP levels were determined by the double-antibody radioimmunoassay method (15). Triplicate plates were analyzed for each data point. Trunscription stubiliv unulysis Cells were preincubated with FSH or FSH plus PMA for 48 h before the addition of 5 ti M actmomycin-D, to arrest new RNA synthesis. Cells were harvested 0, 3, 6, 9, and 12 h after and Northern blot analysis was performed as addition of actinomycin-D for RNA extraction. described above

Results

We previously demonstrated that FSH induces LHlhCG receptor mRNA, peaking at 72 h of incubation (16). We therefore investigated the effect of PMA on the induction of LH/hCG receptor mRNA by FSH, FSK, and 8-Br-CAMP at 72 h of incubation. As shown in Fig. la and lb, PhlA dose-dependently reduced the level of LWhCG receptor mRNA induced by FSH and FSK. On the other hand, PMA significantly increased LH/hCG receptor mRNA, with maximal stimulation between PMA concentrations of 3 to 30nM ( 1.5-fold ) when 8-Br-CAMP was used (Fig. 1~).

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Phorbol Ester on LH/hCG Receptors

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lA

kb

K FSH

30

30

30

PMA

0

3

10

200

2.3-

(nshnl) 30

60

12345

WI

1B I

FSK

10

10

10

10

10

PMA

0

3

10

30

60

CUM) 1 ( nM )

1c

2

3 4 5

kb

5.4

-

2.3-

I-Br-CAMP

1

1

1

1

1

PMA

0

3

10

30

60

Fig.

(mM) ( nM 1

1 2

3

4 5

1

Effects of PMA on induction of LHlhCG receptor mRNA by FSH, FSK, and &Br-CAMP. Granulosa cells were incubated for 72 h in serum-free medium containing FSH (30 nglml) (A), FSK (10 LLM) (B), and 8-Br-CAMP (1 mM) (C) with increasing doses of PMA. Northern blots and densitometric scannning were performed as described in Materials and Luminescence detection was quantified with a LKB 2202 UnitroScan Laser Methods. Densitometer (LKB Produkter AB, Bromma, Sweden), normalized against a corresponding relative amount of B-actin mRNA in each sample, and expressed as relative densitometric unitsEach bar represents the mean + SE of four different experiments.

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1439

Since PMA had a significant effect on the LHlhCG receptor mRNA level, we examined the effect Since, as we of PMA on LHlhCG receptor formation induced by FSH, FSK, and 8-Br-CAMP. showed in a previous work (16), FSH, FSK, and 8-Br-CAMP induce LH/hCG receptor, reaching a peak level at 96 h of incubation, we examined the effect of PMA on the induction of LH/hCG As shown in Table 1, LWhCG receptor expression was induced by receptor at 96 h of incubation. FSH , FSK, and 8-Br-CAMP, and FSK was much more potent in inducing LH/hCG receptor than Our results showed that PMA inhibited the effects of FSH and FSK in FSH or 8-Br-CAMP. increasing LWhCG receptor number, without any change in affinity, but enhanced the increase in LHihCG receptor number induced by 8-Br-CAMP, without any effect on affinity. These results suggested that the effect of PMA on LWhCG receptor protein is correlated with its effect on LHihCG receptor mRNA. It is unknown whether the above phenomenon is associated with changes in CAMP production. It has already been demonstrated that FSH and FSK stimulate adenylate cyclase to synthesize CAMP, We examined the effect of PMA on which in turn induces LHlhCG receptors in granulosa cells. cAMP synthesis in rat granulosa cells. As shown in Fig. 2, PMA dose-dependently decreased These results are consistent with LWhCG cAMP accumulation stimulated by FSH and FSK. receptor expression in granulosa cells cultured with FSH and FSK in the presence of PMA, suggesting that a PMA- mediated decrease in CAMP is a major factor in the PMA-mediated decrease In LHihCG receptor mRNA. Table

1.

Effects

of PMA on the expression

granulosa treatment

FSH (30ngIml) +PMA (1 OnM ) FSK (lo-5M

)

+PMA (1 OnM ) 8-&-CAMP

(1 mM )

+PMA (1 OnM )

cells treated lW-hCG

of LH/hCG

by FSH,Forskolin,or binding

receptor

in

8-Br-CAMP

Kd value (xl O-1 1)

21.3f1.5

3.5kO.3

14.6k3.1

3.71to.7

27.1+2.1

3.2+0.4

8.9k3.1

2.9f0.5

11.4k2.2

3.8kO.3

15.5f1.7

3.5f0.5

Effects of PMA on LH/hCG receptor expression in response to FSH, FSK, and 8-Br-CAMP. Granulosa cells were incubated for 96 h in serum-free medium containing each hormone to assay for equilibrium [ 12sI]hCG binding as described in Materials and Methods. The binding data were analyzed by Scatchard analysis. Values are means + SE of five different experiments. After the preincubation period, 5fiM actinomycin D was added to arrest new RNA synthesis. Cells were harvested at 0, 3, 6, and 9 h after addition of actinomycin D, and LH/hCG-receptor mRNA levels were quantitated by Northern analysis. The amount of LH/hCG-receptor mRNA at time 0 ( the time of addition of actinomycin D) in each treatment group was assigned a value of lOO%, and all other values in each treatment group at different time points were expressed as a percent of the time 0 value. As shown in Fig. 3, the decay curves for the 5.4 kb LH/hCG-receptor mRNA transcript in primary granulosa cells cultured with FSH in the presence and absence of PMA were not significantly different. Nor could we detect any difference between granulosa cells cultured with 8-Br-CAMP alone and 8-Br-CAMP plus PMA treated cells (data not shown). We interpreted these results as meaning that PMA had no effect on LWhCG receptor mRNA stability in our culture system.

Phorbol Ester on LH/hCG Receptors

1440

FSK 10~~

FSH 30ng/ml PMA

0

3

10

30

60

0

Fig.

2

3

10

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M 30

60

(nt-4)

Intracellular CAMP accumulation in granulosa cells. After preincubating with serum-free assay medium containing 0.5 mM 3-isobutyl-1-methylxanthine for 30 min, the cells were exposed to each hormone for 30 min. Cellular CAMP was extracted and measured by RIA as described in MateriaZsand Methods. The level of intracellular CAMP without treatment was 1.5+ 0.4. Values represent the means + SE of five different experiments.

10

’ 0

3

9

6

12

( hr1 Fig.

3

Effect of FSH and PMA on LWhCG receptor mRNA transcripts.

Cells were treated with

FSH or FSH plus PMA for 48 h, and actinomycin D (5 NM) was added. The cells were harvested at 0, 3, 6, 9, and 12 h after the addition of actinomycin D to the culture medium to extract RNA, which was subsequently used for Northern blot analysis, as described in Materials and Methods, The mRNA levels at time zero were assigned a relative value of lOO%, and the mRNA levels at all other times are expressed as percentages of the time 0 value. The ordinate is plotted on a logarithmic scale. Discussion Recent studies by our laboratory and others have described an association between increased levels of LHlhCG receptor in granulosa cells and increased amounts of LWhCG receptor mRNA. Consistent with previous studies, our data show that FSH, FSK, and S-Br-CAMP are capable of dramatically increasing LHlhCG receptor and LWhCG receptor mRNA in the granulosa cells of DES-primed immature rats from nearly undetectable basal levels. The binding experiment suggest

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1441

that the induction of the LH/hCG receptor by FSH, FSK, and 8-Br-CAMP results primarily from an increase in LH/hCG receptor mRNA levels. Although the details concerning regulation of the size of LHihCG receptor mRNAs remain to be determined, it is clear that changes in the total amount of LH/hCG receptor mRNA parallel changes in LH/hCG binding activity. The results of the run-on e\;periments (17) clearly demonstrated that FSH and 8-Br-CAMP significantly stimulate the rate of transcription of the LH/hCG receptor gene in the granulosa cells of DES-primed immature rats. These data showed that FSH increased LH/hCG receptor gene transcription through the CAMP/A kinase pathway (17). In contrast to granulosa cells, Leydig cells express LHlhCG receptors constitutively. Furthermore, LHlhCG receptor levels in Leydig cells are negatively modulated by S-Br-CAMP (l&19). 8-Br-CAMP has been shown to cause a decrease in the rate of transcription of the LHlhCG receptor gene in MA-10 Leydig tumor cells, resulting in decreased levels of LH/hCG receptor mRNA (20). In contrast to the very long lag time observed in FSH- and 8-Br-CAMP- treated cells, decreased LWhCG receptor gene transcription is observed within 30 min of addition of 8-Br-CAMP to MA- 10 cells. Moreover, in our previous experiment, the levels of LH/hCG receptor were found to be transiently and negatively modulated by 8-Br-CAMP and FSH within 6 hr of addition of FSH or cAMP to granulosa cells (21). Therefore, CAMP negatively regulates at short incubation, and positively regulates at longer incubations LHihCG gene transcription in granulosa cells. Direct stimulation of PKC with PMA resulted in a dose-dependent decrease in the steady-state levels of LH/hCG receptor mRNA in granulosa cells treated with FSH and FSK. A marked decrease in LWhCG receptor was also seen after 96 h. One mechanism that must be considered is FSH-dependent phosphorylation of FSH receptors. The FSH receptor also interacts with other G proteins leading to an increase in inositol phosphates and intracellular calcium (22,23). These events could ultimately result in protein kinase C-catalyzed phosphorylation of the glycoprotein hormone receptors at one or more of the protein kinase C consensus sequences present in their intracellular regions (24,2_5). While it is clear that phosphorylation is responsible for the agonist-induced uncoupling of catecholamine receptors from their respective effecters, a cause-effect relationship between agonist-induced phosphorylation and uncoupling of the FSH receptor has yet to be established. To determine whether the decreases in receptor number and mRNA level were accompanied by uncoupling of the receptor following PMA treatment, the ability of FSH receptor to Increase CAMP levels was measured. Intracellular CAMP concentrations are principally controlled at the synthesis level by hormonal regulation of adenylyl cyclase, the enzyme catalyzing the conversion of ATP to CAMP. Both FSH and forskolin treatment had been interfered by PMA on the CAMP accumulation, indicating interference of adenylate cyclase activity by PMA. Some level of adenylyl cyclase regulation has been suggested by the effects of phorbol esters on adenylyl cyclase activity initially reported by Yoshimasa et al. (26) and recent reports of phorbol ester effects on expressed adenylyl cyclase isoforms (27-29). The half-life study revealed no change in the stability of the LH/hCG receptor mRNA following PMA treatment. This suggests that a change in the rate of LH/hCG receptor gene transcription is responsible for the reduction in the steady-state levels of LH/hCG receptor mRNA. Although the decreased LHlhCG receptor mRNA elicited by PMA suggested that PMA mediates this effect through a decrease in CAMP levels, PMA augmented the effect of 8-Br-CAMP on the LHhCG receptor. These results suggest that protein kinase C has the ability to increase the respcnsiveness of adenylyl cyclase. Previous studies have revealed that the 5’flanking region of the rat LH/hCG receptor gene is characteristic of “housekeeping genes” in that it apparently contains no TATA or CAAT boxes, is rich in G and C residues, and there are multiple potential transcriptional sites (30). cAMP and PMA induce the transcription of the LH/hCG receptor gene in rat granulosa cells, and it \vill be important to characterize the cis and trans factors that are involved in this phenomenon.

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Acknowledgments

We thank Dr. K. Wakabayashi (Hormone Assay Center, Institute of Endocrinology, Gunma University) for CAMP antibody and the National Hormone and Pituitary Agency, National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases, University of Maryland School of Medicine for rat FSH and hCG (CR-127). This work was supported by a grant from the Ministry of Education, Science and Culture of Japan (08671867). References

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