Gonadotropin-releasing hormone agonist activates protein kinase C in rat Leydig cells

53

Molecular and Cellular Endocrinology, 55 (1988) 53-59 Elsevier Scientific Publishers Ireland. Ltd.

MCE 01775

Gonadotropin-releasing

hormone agonist activates protein kinase C in rat Leydig cells

Hannu Nikula and Ilpo Huhtaniemi Department

of Physiology,

(Received

Key words

Testis; Testosterone;

Protein

University of Turku, Turku, Finland

30 April 1987; accepted

kinase C; Phorbol

17 August

ester; Gonadotropin

1987)

releasing

hormone

Gonadotropin-releasing hormone (GnRH) has specific receptor sites in rat Leydig cells and has direct effects on their steroidogenesis. The purpose of the present study was to examine whether activation of the calcium- and phospholipid-dependent protein kinase C (PK-C) is involved in GnRH effects on rat Leydig cells, as has been shown in pituitary gonadotrophs. Testosterone production of Percoll-purified Leydig cells was similarly stimulated (about 50-100%) by a GnRH agonist (buserelin, maximum effect at concentration of lOA mol/l and above) and a tumor promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, maximum effect at lo-* mol/l), which is known to activate PK-C. In contrast, a GnRH antagonist (lop5 mol/l) and an inactive phorbol ester, 4a-phorbol-12,13-didecanoate (lop6 mol/l), were without effect on testosterone. None of these substances had clear effects on CAMP production. The maximum steroidogenic effects of GnRH agonist and TPA were the same whether used separately or together, suggesting that they share a common mechanism of action. TPA translocated the cytosolic proportion of Leydig cell PK-C activity to a membrane-associated form almost instantaneously, within 0.5-l min. A similar translocation, though less complete, was observed in the presence of buserelin in l-4 min. Inclusion of a loo-fold excess of a potent GnRH antagonist completely prevented the translocation of PK-C. These results provide evidence that GnRH agonist activates PK-C also in the testis tissue, and this may be the mechanism whereby it affects Leydig cell endocrine function.

Introduction

The calcium- and phospholipid-dependent protein kinase C (PK-C) is ubiquitously distributed in mammalian tissues, the nervous tissue being the most abundant source (Kuo et al., 1980, 1984; Kikkawa et al., 1982; Davis and Clark, 1983; Ohno et al., 1987). Purified rat Leydig cells also Address for correspondence: ment of Physiology, University SF-20520 Turku, Finland. 0303-7207/88/$03.50

Dr. Ilpo Huhtaniemi, Departof Turku, Kiinamyllynkatu 10,

0 1988 Elsevier Scientific

Publishers

Ireland,

contain this enzyme activity (Lin, 1985a; Nikula et al., 1987), but clearly less (about 10%) than present in the rich sources of PK-C. Activation of PK-C by phorbol esters (e.g. TPA) has clear effects on Leydig cell basal and gonadotropinstimulated steroidogenesis (Mukhopadhyay et al., 1984; Welsh et al., 1984; Lin, 1985a, b; Moger, 1985; Mukhopadhyay and Schuhmacher, 1985; Papadopoulus et al., 1985; Themmen et al., 1986; Dix et al., 1987; Nikula et al., 1987) suggesting a role for PK-C in regulation of these cells. However, nothing is yet known about the physiological Ltd.

54

ligands that could act through this mechanism. The importance of paracrine interactions between testicular cell compartments has recently been emphasized (Parvinen et al., 1984; Papadopoulos et al., 1986; Sharpe, 1986; T&k& 1986; Verhoeven and Cailleau, 1987), and a testicular GnRH-like factor was considered a likely candidate for such regulation. Although detectable in the testis (Sharpe and Fraser, 1980; Dutlow and Millar, 1981; Bhasin and Swerdloff, 1984; Hedger et al., 1985), the physiological significance of this particular factor may be limited (Clayton and Huhtaniemi, 1982; Huhtaniemi et al., 1987). However, GnRH and its agonists provide a useful model for studies on mechanisms of paracrine interactions within the testis. Since GnRH activates PK-C in the pituitary (Conn et al., 1985; Hirota et al., 1985; Naor and Eli, 1985) we found it of importance to explore whether the same mechanism of action is functional in the acute effects of this peptide on the rat Leydig cells. Materials and methods material

Phosphatidylse~ne (PS, bovine brain), calf thymus histone (type III-s), 1,Zdiolein (DG), bovine serum albumin (BSA), l-methylisobutylxanthine (MIX), collagenase (type I), 12-O-tetradecanoylphorbol-13-acetate (TPA), 4a-phorbol12,13-didec~oate, EGTA, EDTA, Tris, dithiothreitol (DTT), phenylmethylsulphonylfluo~de (PMSF), cyclic adenosine-3’,5’-monophosphate (CAMP), N6,2’-0-disuccinyladenosine-3’,5’-cyclic monophosphate (disuccinyl-CAMP), 8-bromo cyclic AMP and ATP were purchased from Sigma Chemical Comp. (St. Louis, MO). [Y-~~P]ATP (> 5000 Ci/mmol) and [ ‘251]testosterone (2000 Ci/mmol) were purchased from The Radiochemical Centre, Amersham, U.K. The GnRH agonist (buserelin; D-Ser-(tBu)6-des-Gly’o-GnRH N-ethylamide) was donated by Hoechst (Frankfurt am Main, F.R.G.) and the GnRH antagonist (RS 68439; N-Ac-D-Nal(Z)‘,o-pCl-Phe*,D-Trp3,DhArg(Et,)6,cr-Ala’o-GnRH) was a gift of Syntex (Palo Alto, CA). hCG (CR-121,130OO IU/mg by bioassay) was prepared by Dr. R. Canfield (Columbia University, New York, NY) and provided

by NICHD (Bethesda, MD). Medium 199 was purchased from Gibco (Paisley, Scotland, U.K.). Animals

Adult (80- to 100-day-old) W&tar-derived male rats from our own colony were used. The animals were kept ten per cage in constant conditions of light (14 h on/l0 h off) and temperature (21-22”(Z), and allowed food and water ad libiturn. The animals were killed by decapitation, the testes were removed, placed in ice-cold Dulbecco’s phosphate-buffered saline (PBS, pH 7.4) and processed immediately (see below). Isolation of Leydig cells

To isolate Leydig cells, decapsulated testes were incubated for 5-10 min in Medium 199 (+O.lW BSA) in the presence of 0.1 g/l collagenase (Dufau and Catt, 1975). The harvested interstitial cells were further purified on a cont~uous gradient of Percoll (Pharmacia, Uppsala, Sweden), essentially as described before (Schuhmacher et al., 1978; Parvinen et al., 1984). The purity of the Leydig cells obtained was 65-75% (proportion of 3/?-hydroxysteroid dehydrogenase-positive cells of all nucleated cells). Leydig cell incubations

For measurement of CAMP and testosterone production, Leydig cell suspensions (2-4 x 10’ cells/ml) in Medium 199-0.1% BSA, cont~~ng 0.1 mmol/l MIX, were incubated in the presence and absence of buserelin or TPA. Total incubation volumes were 300 ~1. The tubes were incubated for 3 h in a shaking water bath at 34” C in an atmosphere of 95% 0, and 5% CO,. A fraction of the media was diluted 1: 1 with 2 mmol/l theophylline, heated at 100” C for 5 min, and used for CAMP measurements (see below). The rest of the media was kept at -20°C until measured for testosterone (see below). For the tram&cation experiments of PK-C activity, 2 x lo6 Leydig cells were incubated in Medium 199 + 0.1% BSA in a final volume of 5 ml in the absence and presence of IO-’ mol/l of buserelin or TPA (conditions as above). The incubation times varied between 0.5 and 30 min. After incubation, the samples were cooled on ice.

55

~eas~$e~ent of PK-C actioify The incubated and cooled cells were centrifuged (600 X g, 5 min, 4°C) and the supernatant discarded. The cells were washed in 0.5 ml of a buffer containing 20 mmol/l Tris-HCl, pH 7.5, 2 mmol/l EDTA, 2 mmol/l EGTA, 0.1 mmol/I PMSF, 5 mmol/l DTT and 250 mmol/l sucrose (buffer A), reconstituted in 0.25 ml of buffer A, and homogenized using an Ultra-Turrax homogenizer. The homogenate was centrifuged at 4” C for 10 min at 100000 x g (Beckman Airfuge, 30 psi). The resulting supematant (soluble enzyme fraction) and pellet were collected. The pellet was reconstituted in the original volume of buffer A containig 0.3% Triton X-100 gently rehomogenized by repeated suction through a thin pipette, and incubated at 4” C for 60 min, followed by similar cent~fugation in the Airfuge. The supernatant (particulate enzyme fraction) was collected and the pellet discarded. Proteins of the soluble and particulate fractions were determined according to Bradford (1976). PK-C was assayed essentially as described before by Naor et al. (1985) and Nikula et al. (1987). In short, incorporation of 32P from [Y-~~P]ATP into calf thymus histone was measured. The reaction mixture (total volume 300 ~1) contained 33 mmol/l of Tris-HCl, pH 7.5, 8 mmol/l MgCl,, 333 mg/l histone, 17 pmol/l of [Y-~~P]ATP (5-10 X lo4 cpm/nmol), 50 mg/l of PS, 3.1 mg/l of DG, 333 pmol/l CaCl,, and the enzyme preparation (added in a volume of 25 ~1, containing lo-15 pg protein). Matched samples were incubated in the absence of PS and DG. Reaction was carried out for 3 min at 30 * C, and samples of 100 ~1 were removed in duplicate and transferred on 2.5 X 2.5 cm squares of phosphocellulose paper (Whatman, No. 3) and immersed in ice-cold 10% trichloroacetic acid (TCA). After five changes of TCA, the papers were washed with ethanol and diethylether, dried, and the radioactivity measured by liquid scintillation spectrometry. The difference of phosphorylation measured in the presence and absence of PS -t DG was regarded as PK-C activity. Hormone rne~~rernen~s CAMP was measured by radioimmunoassay as described before (Harper and Brooker, 1975). The

tracer, disu~inyl-CAMP, was radioi~nat~ with Na[1251]iodine as described before (Brooker et al., 1979). The testosterone radioimmunoassay has been described by us before (Huhtaniemi et al., 1985). statistical analysis The statistical analysis of the data was carried out using Duncan’s multiple range tests and twotailed Student’s t-tests. A P value below 0.05 was chosen as the limit of statistical significance. Rest&s

Fig. 1 shows the stimulability of rat Leydig cell testosterone production by varying concentrations of buserelin and TPA. Basal unstimulated production of testosterone in our cell prep~ations was of the order of 30, and that of CAMP 5 pmol/106 Leydig cells . 3 h. The maximum stimulating effect on testosterone was attained at about 10e9 mol/l of buserelin and lo-* mol/l of TPA. When buserelin and TPA were incubated together (Fig. 2), the maximum stimulating concentration of both of the substances yielded similar stimulation as either of them alone. The GnRH antagonist reversed the buserelin effect, and the inactive phorbol ester 4cY-phorbol-12,13_didecanoate (PDD) had no effect on testosterone production (Fig. 2). TPA decreased slightly in some but not all experiments CAMP formation, but buserelin, the GnRH antagonist and the inactive phorbol ester had no effect on this parameter (results not shown). The PK-C activities in non-incubated Leydig cells were 622 f 71 and 907 f 102 (mean f SE, n = 9) pmol 32P/mg protein . min in the soluble and membrane-associated fractions, respectively. When to total amounts of soluble and particulate protein per cell were taken into account, 60% of Leydig cell PK-C activity was present in the soluble, and 40% in the membrane-associated form (Nikula et al., 1987). Fig. 3 shows a typical experiment of the translocation of PK-C activity from the soluble to the membrane-bound form during GnRH agonist and phorbol ester stim~ation. When Leydig cells were incubated in the presence of lo-’ mol/l TPA, the soluble PK-C activity was lost almost instantaneously (within 0.5-l min),

56

t

i

: 0.01

Buserelin

1

0.1

(nmol/l)

+

c

6

TPA

TPA +

Ant. +

B

B

PDD

Fig. 2. Leydig cell testosterone production in controls (C) and in the presence of 10V9 mol/l buserelin (B), 10-l mol/l TPA, both of the former compounds, B and lo-.’ mol/l of the GnRH antagonist (Ant.), or 10m6 mol/l of 4a-phorbol-12,13didecanoate (PDD). A representative experiment is presented (mean& SE, n = 3). All the phenomena shown were reproduced in 2-3 other experiments. The bars indicated by asterisks are significantly different from those without asterisks (P < 0.05). 10

100

1000

TPA (nmolll) Fig. 1. Dose-response of Leydig cell testosterone production to stimulation with buserelin (upper panel) and TPA (lower panel). Meanf SE of data from three individual experiments with buserelin and four with TPA are presented.

and a respective increase occurred in the particulate activity. Similar distribution of the activities was maintained at least 30 mm in the presence of TPA (result not shown). A similar shift of PK-C from soluble to particulate form was seen in incubations with buserelin. The shift was less complete, and its timing varied between individual experiments (maximum shift between 1 and 4 mm). The transition also appeared to be somewhat slower than with TPA, and it was not maintamed when the incubations were continued beyond 8-10 mm (Fig. 4). Simiiar incubations in the presence of hCG (100 pg/l) or 8-bromo cyclic AMP (0.43 mmol/l) did not affect the subcellular distribution of Leydig cell PK-C activity (results not shown). The compiled data of six individual translocation experiments with buserelin are shown in Fig.

TPA-S / 0

2

4

0 6

8

Minutes

Fig. 3. PK-C activity of the soluble (S) and membrane (P) fractions of Leydig cell homogenates after varying times of incubation in the presence of lo-’ mol/l buserelin (B) or TPA. A representative example of six independent experiments is presented. Each point is the mean of duplicate incubations.

57

antagonist (10e5 mol/l) were incubated together, the ratio stayed at 1.44 f 0.13. Discussion

C B 0.5

CB

2

C

B 4

C B 8

Mmutes Fig. 4. The ratios of membrane-associated vs. soluble enzyme protein. mm) of activities (calculated as pmol 32P bound/mg PK-C in homogenates of Leydig cells incubated for 0.5, 1, 2, 4 or 8 min alone (C) or in the presence of 1OU’ mol/l buserelin (B). Each bar represents the mean&SE of 4-7 independent experiments. The asterisks indicate significant differences between the activity ratios of the control cells and those incubated in the presence of buserehn (*P < 0.02; **P < 0.01).

4. The transition of the PK-C activity from the soluble to the particulate fraction is shown as a change in the ratio of the membrane-bound/soluble activity. No changes in these ratios were seen in control cells and those incubated in the presence of buserelin at 0.5 and 8 min of the incubation. However, the proportion of the membranebound/soluble PK-C activity was significantly higher (P -c 0.02-0.01) in the buserelin samples at times 1, 2 and 4 min of the incubations. When a loo-fold excess of a potent GnRH antagonist was incubated together with buserelin, the change in the particulate/soluble PK-C activity ratio at 2 n-tin was completely inhibited. The control ratio was in this experiment 1.58 + 0.14 (mean + SE, n = 3), that in the presence of buserelin (lo-’ mol/l) increased to 3.80 f 0.52 (P < 0.01). and when buserelin and the GnRH

A number of hormones and effecters activate PK-C as a part of their transmembrane signal transmission (Kikkawa and Nishizuka, 1986). In the testis, the functions of Leydig (Lin, 1985; Dix et al., 1987; Nikula et al., 1987) and Sertoli cells (Galdieri et al., 1986; Monaco and Conti, 1987) are affected by PK-C activating phorbol esters. However, the physiological ligands employing this signalling system in the testis tissue are still largely unknown. The translocation of Leydig ceil PK-C from soluble to membrane-bound form, regarded as a part of activation of this enzyme, was near complete with TPA. This is understandable due to the potent action of phorbol ester; it bypasses the ligand-receptor interaction and directly activates PK-C. The buserelin-induced translocation of PK-C was less complete and of shorter duration, and can be explained by the purity (65575%) of the Leydig cell suspension. Only the Leydig cells contain GnRH receptors (Clayton and Catt, 1981), but probably most testicular cells have PK-C activity (Kimura et al., 1984; Galdieri et al., 1986; Nikula et al., 1987). Only the Leydig cell part of PK-C can be activated by buserelin but all of it by TPA. The short duration of PK-C activation by buserelin is similar to that observed with some other hormonal effecters (Durst and Martin, 1985; Farrar and Anderson, 1985; Fearon and Tashjian, 1985). Despite the differences in time courses, its relation to the slow steroidogenic response is possible due to the long duration of PK-C-induced protein phosphorylations (Nishizuka, 1986). The present stimulations of testosterone by buserelin and TPA are clearly distinct from the gonadotropin effects. The latter had no effect on Leydig cell PK-C activity. In contrast, it involves activation of CAMP production which is not directly related to GnRH and TPA effects (Cooke and Sullivan, 1985; Kikkawa and Nishizuka, 1986). Furthermore, TPA inhibits gonadotropin-stimulated CAMP production by Leydig cells (Nikula et al., 1987). Since TPA increases basal testosterone

58

production but appears to inhibit the gonadotropin-stimulated steroidogenesis, it most likely has several loci of action. The LH receptors-CAMP part of the cascade seems to be inhibited by PK-C activation whereas the basal steroidogenesis obviously independent of CAMP action - seems to be activated by this enzyme. It is possible that PK-C could phosphorylate some steroidogenic enzymes resulting in their partial activation, but the details of such effects still remain unknown. PK-C activation is involved in GnRH action at the pituitary level (Conn et al., 1985; Hirota et al., 1985; Naor and Eli, 1985). It is therefore not surprising that the same mechanism is involved in action of this peptide in the gonad. On the other hand, since the gonadal GnRH-like material may not be identical with the h~othal~c peptide (Dutlow and Millar, 1981; Bhasin and Swerdloff, 1984; Hedger et al., 1985), its mechanism of action in the gonad need not be identical. Some previous studies have shown similarities between GnRH and TPA actions in the Leydig cells (Lin, 1985b; Moger, 1985; Then-men et al., 1986) but to our knowledge the direct involvement of PK-C in the GnRH agonist action has not been shown before at the gonadal level. GnRH, or the putative testicular GnRH-like factor, may be of limited importance in the regulation of Leydig cell function. Besides Leydig cells, PK-C activity is also present in Sertoli and germinal cells (Kimura et al., 1984; Galdieri et al., 1986; Nikula et al., 1987). Further studies are needed to explore the spectrum of ligands and effects that are involved in the PK-C-mediated regulation of testicular function. Acknowledgements

The skillful technical assistance of Ms. Aila Metsavuori and Ms. Tarja Laiho is gratefully acknowledged. This study was supported by grants from the Academy of Finland and The S&rid Juselius Foundation. References Bhasin, S. and Swerdloff, R.S. (1984) B&hem. Biophys. Res. Commun. 122, 1071-1075. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.

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