Control of steroidogenesis in Leydig cells

J SteroutBiochem Molec Bwl Vol 43, No 5, pp 445-449, 1992

0960-0760/92 $5 00 + 0 00 Copyright © 1992Pergamon Press Ltd

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CONTROL

OF STEROIDOGENESIS

IN LEYDIG

CELLS

B A CooicF.,* M C K CHOI, G DIRAMI, M P LOVF.z-RuIz and A P W~ST Department of Blochenustry, Royal Free Hospital School of Me&cme, Rowland Hill Street, London NW3 2PF, England Snmmary--Lutemlzmg hormone (LH) interacts with as plasma membrane receptor to stimulate steroldogenesls not only wa cychc A M P but also other pathways which include aracludomc acid and leukotnenes and regulation of chlonde and calcmm channels The same stlmulatory pathways may lead to desensmzatlon and down-regulation of the LH receptor and steroldogenesls The LH receptor exists In a dynamic state, being truncated, or mternahzed, degraded or recycled Desensmzatlon is controlled by protein hnase C (PKC) m the rat and by cychc A M P dependant protein kmase and PKC m the mouse Ley&g cells Using an adapted anti-sense ohgonucleotlde strategy we have shown that the cytoplasmic C-ternunal sequence of the LH receptor Is essentml for desensmzataon to occur In contrast, these sequences of the LH receptor are not rcqmred for the stimulation of cychc A M P and steroid producUon We have also shown that the extracellular domain of the LH receptor is secreted from the Ley&g cell and may act as a LH-bmdmg protein

INTRODUCTION

REGULATION OF STEROIDOGENESIS BY PROTEIN KINASE C (PKC)

It is well estabhshed that steroldogenesls m Ley&g cells is regulated by lutemlzmg hormone (lutropm, LH), wa the second messenger Y,5'cychc AMP (cAMP) It has also been shown that other second messenger systems may be revolved, including arachldomc acid and its metabohtes [1], calcmm [1] and efflux of chloride ions [2]. The release of arachldomc acid to the cytosol can occur by a hormone-med|ated protess, through the acUvaUon of phosphohpase A2 (PLA2), phosphohpase D (PLD) and/or phosphohpase C (PLC) followed by hydrolysis of diacylglycerol by dmcylglycerol hpase PLA2 and PLC can be coupled to a common membrane receptor by &street GTP-bmdmg proteins and may be activated by the same membrane active hormone [3] Arachldomc acid can be further metabohzed wa cyclo oxygenase and lipoxygenase pathways to prostaglandlns and leukotnenes, respecUvely Our previous results have shown that LH causes a rapid release of aracludomc acid from Ley&g cells, probably wa activation of PLA2 [4] and that aracludonlc acid hpoxygenase metabohtes are revolved m LHreduced steroldogenesls [5, 6] Proceedings of the XV Meeting of the International Study Group for Stermd Hormones, Rome, Italy, 28-30

November 1991 *To whom correspondence should be addressed 445

Actlvauon of PKC using the tumorpromoting phorbol ester, phorbol-12-mlnstate13-acetate (PMA), has been reported to regulate, both posmvely and negatively, steroldogenesls m Ley&g cells [7-9] In addmon to &acylglycerol, arachldomc acid has been shown to be a physlologncal regulator of PKC m vanous t~ssues, wa acUvat~on of the ~ PKC subumt [10] In rat Ley&g cells the ~ subunlt as well as the ~t and/~ subumt are present [11] and thus It Is theoretically possible that arachldonlc acid also regulates PKC m Ley&g cells Recently[12] we have demonstrated that m rat Ley&g cells arachldonlc acid has a dose- and time-dependent biphaslc effect on maximal and submaxlmal LH- and &butyryl-cAMPstimulated testosterone producUon The locus of the mhlbmon, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP producUon The same inhibitory effect was found vath the PKC acUvators, PMA and olelc acid, also w~th no change m LH-stlmulated cAMP production Arachldomc acid, PMA and dlolem, all sUmulated PKC actavlty m a dose-dependent fashon m Ley&g cell homogenates The arach~domc aod potentmt~on of LH- and &butyryl-cAMP-sumulated testosterone producUon, which occurred dunng 5 h, was also

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B A COOKE et al

mimicked by PMA PKC was down-regulated over 5 h (but not dunng 3 h) by pretreatmg Ley&g cells with PMA or arachldomc acid m the presence of LH The sUmulatory effect on stero~dogenesls was specific for arachldonlc acid, since olelc and elm&c acid did not have any effect on LH-stlmulated testosterone production Llpoxygenase and cyclooxygenase mh~bltors &d not alter the stimulation effects of arachldomc acid We concluded that the short-term inhibitory effect of arachldonlc acid is wa actwatlon of PKC, but when PKC is down-regulated, arachldonlc acid potentmtes steroldogenesls in response to LH One possible explanation for the st~mulatory effect of arach~domc acid is because of the down-regulation of PKC actlv~ty, the rat testis Ley&g cell may normally be under tonic inhibitory control by this enzyme Th~s may be by phosphorylatlon of regulatory proteins revolved m the modulation of steroldogenesls When PKC IS down-regulated th~s mhlbmon ~s removed and the cells become more actwe stero~dogenlcally In accordance with this hypothes~s we have shown that following downregulation of PKC (w~th PMA for 5 h) followed by a 2 h incubation with dbcAMP (0 001 to 1 mM) there was an increase m basal-, submax~mal- and maximal-stimulated steroldogenes~s (unpubhshed observations) In our prewous studies[I] we found that phorbol esters desensmzed LH-st~mulated cAMP production In the above studies no effect of PMA or arach~domc acid on LHstimulated cAMP was found This lack of effect on cAMP production was due to the h~gh concentrations (1%) of albumin used In the presence of lower concentrations (0 1%), lnhlbmon of LH-st~mulated cAMP production by arach~domc acid (unpubhshed observations) and desensmzatlon of LH-medmted cAMP productlon by PMA does occur ACTIVATION AND DESENSITIZATION OF THE LH RECEPTOR (LHR)

Continued exposure of the Leydlg cells to LH leads to desensmzatlon[1, 13] Previously we demonstrated that desensmzaUon of the rat Ley&g cell involves an uncouphng of the LHR from ~ts transducing system i e a G-protein coupled to adenylate cyclase [1] This process could be numlcked by sUmulatlon of PKC but not cAMP-dependent protein kmase (PKA) The LH- or phorbol ester-Induced desensltlza-

tlon led to a 50% decrease m cAMP production In mouse Ley&g cells, stimulation of both PKA and PKC leads to complete desensmzaUon with regard to cAMP and steroid producUon [14, 15] Thus, there are marked species differences Although the ewdence suggests that the substrate for PKC/PKA-medlated phosphorylatlon may be the LHR Itself[I, 16], this has not been &rectly demonstrated under phystologlcal condmons The results could equally be explained by phosphorylatlon of a protein(s) that interacts with the LHR DOWN-REGULATION OF THE LHR

It was previously shown that down-regulaUon l e loss of the LHR, only occurred after a considerable time period followmg exposure to LH/hCG i e 12-24 h [17, 18] We have recently shown that there is a rapid proteolytlc cleavage of the LHR during culture of rat and mouse Ley&g cells, which results m the release of the LHR extracellular domain [15] This process is continuous m the freshly ~solated rat Ley&g cells (up to 4 h), whereas m the mouse Ley&g cell ~t is a cAMP-dependent process Actwatlon of PKC is ineffective m causmg truncation [15] Paradoxically, inhibition of LHR truncaUon with protease lnhlbltors, also inhibits cAMP production in the mouse (but not the rat) Leydlg cell [19], indicating that proteolysls of the LHR ~s reqmred for activation of adenylate cyclase to occur The depleted receptors m the mouse cells are replemshed by a cAMP-dependent process It ~s possible therefore, that loss of receptor in the Ley&g cell occurs at two levels acute, revolving truncation, and chronic, mvolwng mternahzatlon and degradation and also a decrease of LHR mRNAs [20, 21] THE STRUCTURE OF THE LHR

The LHR has been cloned from human and rat ovaries and porcine Leydlg cells Its sequence indicates that ~t ~s a member of the 7 transmembrane hehx-G protein coupled receptor family [22, 23] The LH, (and FSH and TSH receptors) &ffer from other members of this receptor family in having a very large extracellular domain (50-60 kDa), which is the site of the hormone binding [22-27] There is also very httle homology w~th the other receptors m the transmembrane domains and the C-terminal sequences which are the sites of binding of the other hormones and the G-proteins [28] There

Ley&g cell steroldogenesls

IS approx 85% homology between the cloned LHRs. A region spanning the 2nd and 3rd transmembrane domains is highly conserved among the LH, FSH and TSH receptors However, anUbo&es raised against the whole LHR show species and cell specificity [29] and the hLHR is unusual in that [125I]hCGbinding is not displaced by equine, rat or ovlne LH [26] THE ANTI-SENSE INHIBITORY STRATEGY

This methodology involves incubating cells with anti-sense ohgodeoxynucleotides (AS oligos) to the gene or mRNA coding for the N-terminal regions of the specific proteins The AS ohgos are taken up by the cells and brad to the DNA/mRNA, preventing transcription/ translation and thus completely blocking the synthesis of specific proteins This methodology has been used successfully In many lnvestlgaUons e g to study the effects o f c - m y c [30] and the prohferatmg cell nuclear antigen (cychn) on cell proliferation and cell differentiation [31] It has also been successfully used to determine the role of type Ilfl regulatory subunlt mRNA of PK m the cAMP-reduced differentiation of HL60 leukaemia cells [32] We have shown that this strategy will also inhibit LHR synthesis [33] In MA10 cells depleted of their functional LHR by treatment w~th trypsin, complete resynthesls occurs during subsequent incubation for 48 h [34] When the incubation of the MA10 cells was camed out m the presence of an AS ohgo &rected against a LHR N-terminal sequence no resynthesls occurred and there was a complete loss of LH-lnduced cAMP production and steroldogenesis We have adapted the strategy in an attempt to produce truncated LHRs with deleted Cterminal phosphorylatlon sites [33] We used AS ohgos coding for 7 amino acids m the putative 3rd extracellular loop and to different sequences of the mtracellular C-terminal loop of the rat ovarian LHR The presence of the AS ollgos 2 or 3 was found to have no effect on the replenIshment of the LH receptors, as measured by [125]IhCG binding, or on the LH stlmulaUon of cAMP or steroldogenesls However, they completely prevented the LH- and phorbol ester-induced desensitlzaUon of adenylate cyclase by these newly-synthesized (and presumably C-terminal truncated) receptors cAMP- and phorbol ester-reduced desensltlZaUon of steroldogenesls was also prevented However, no effect was found on LH-lnduced

447

desensitization of steroidogenesis, thus indicating that LH reduces additional desensitization processes which are independent of PKA and PKC The losses of surface LHRs caused by LH and cAMP were also investigated It was found that pretreatment of the MA10 cells with all the C-terminal AS ohgos including the sequence coding for the end C-terminal peptlde, prevented the 50% loss of binding sites detected in the controls The mechanisms of the AS ohgos against the N-terminus sequence in preventing the synthesis of the LHR can be understood However, those involved m altering the function of the Cterminus are unknown It is possible that they act as a stop codon on the mRNA so that the LHR IS synthesized up to the position where the AS ohgo is bound and is then released Alternatwely, mRNAs coding for the LHR lacking the cytoplasmic C-terminus may exist, addition of the AS ohgos may completely prevent the synthesis of the full length LHR and thus allow the translation of the mRNAs coding for the truncated receptors Multiple mRNAs for LHR do exist CONCLUSIONS

Because of the marked species differences in the mechamsms of desensitization and downregulation found in the mouse and rat Leydig cells, ~t ~s apparent that these mechanisms cannot be studied in only one species It is well established however, that all Ley&g cells do contain spare LHRs [18], full cAMP and steroid production can be mamtained by low occupancy of the receptors In addition, the antisense expenments in our studies demonstrate that almost complete depletion of the LHR has to occur before cAMP or steroidogenesls is decreased The acute decreases in the ability of Leydig cells after exposure to LH and other hgands to form cAMP is therefore not due to a loss m the numbers of LHRs but to a loss m the ablhty of the LHR to couple to its transducing systems In the rat Leydlg cells, acute exposure m vttro to LH causes desensitization This desensitization can be mduced by PKC activators but not by cAMP Prolonged exposure is required before down-regulation of the LHR However, continuous proteolytlc cleavage of the LHR does occur dunng the first 4 h of incubation in vttro This results m the excretion of the extracellular bmdmg domain of the LHR into the

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mcubatton medmm It is possible that this proteolysls ~s controlled m v t v o via regulatmn w~th protease mh~b~tors, perhaps from adjacent cells e g semlnfferous tubular myo~d cells In the mouse Ley&g cells, desenslt~zaUon occurs after exposure to LH, cAMP and phorbol esters Th~s results m very low cAMP and steroid product~on Proteolysls of the LHR occurs m w t r o If lnternahzatlon ~s mhtb~ted This process ~s sUmulated by cAMP but not phorbol esters Curiously, mh~blUon of proteolysls mh~blts cAMP producUon, th~s does not necessarily ~mply that the proteolys~s of the LHR extracellular domam is involved, other changes m the LHR structure or assocmted proteins may Occur

The anU-sense strategy ~s obviously a powerful techmque for mvesUgatmg structure--actwlty relatlonsh~ps of regulator proteins m homologous expression systems The results of its apphcat~on to the LHR ~mply that C-terminal residues are revolved m the processes of desensmzatmn and proteolyt~c cleavage of the extracellular domam Further work ~s reqmred to identify the proposed truncated protems and the mechanisms of mhlbmon of translation/transcription Acknowledgements--We are grateful to the following for financml s,,pport The Spanish Government, The Science and Enm'aeenng Council, The Me&cal Research Courted, The Pete1 Samual Royal Free Fund

REFERENCES

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29 Dlrarm G , Poulter L W and Cooke B A Separation and charactenzatmn of Leydlg cells and macrophages from rat testes J Endocr 130 (1991) 357-365 30 Helkklla R , Schwab G , Wiekstrom E , Loke S L , Lpuzmk D H , Watt R and Neckers L M A c-muc antlsense oligodeoxynucleotlde inhibits entry into S phase but not progress from Go to Gl Nature 328 (1987) 445~,49 31 Jaskulskl D , Klm deRlel J , Calabretta B and Baserga R Inhlbluon of cellular proliferation by antlsens¢ ohgodeoxynucleotldes to PCNA cychn Scwnce 240 (1988) 1544-1546 32 Tortora G , Clair T and Cho-Chung Y S An antisense ollgodeoxynucleotide targeted against the type IIb regulatory subtype mRNA of protein klnas¢ inhibits cAMP-mduc~d &fferentlatlon m HL-60 leukemia cells without affecting phorbol ester effects Proc Natn Acad Set U S .4 87 (1990) 705-708 33 West A P and Cooke B A A novel method to modulate desensmzatmn and truncation of LH receptors using antlsense ohodeoxynucleotldes Molec Cell Endocr 79 (1991) R9-R14 34 W e s t A P , R o s e M P and CookeB A Induction of lutropm receptors by lutropm and Y5'-cychc AMP m cultured mouse tumour (MA10) Leydlg cells Bwchem J 270 (1990) 499-503