Regulation of mRNA Expression of 3/?-Hydroxy-5-Ene Steroid Dehydrogenase in Porcine Granulosa Cells in Culture: A Role for the Protein Kinase-C Pathway P. Jorge Chedrese, David Zhang, Van Luu The, Fernand Labrie, Augusto V. Juorio, and Bruce D. Murphy Reproductive Biology Research Unit (P.J.C., D.Z., B.D.M.) Neuropsychiatric Research Unit (A.V.J.) University of Saskatchewan Saskatoon, Saskatchewan, Canada S7N OXO Department of Molecular Endocrinology CHUL Research Centre (V.L.T., F.L.) Quebec, Quebec, Canada G1V 4G2 We studied the effect of the tumor-promoting phor- bol ester phorbol 12-myristate 13-acetate (PMA), which activates protein kinase-C, on porcine granu- losa cells in culture. PMA as well as cholera toxin, forskolin, and hCG increased cAMP accumulation. PMA further augmented the elevation in cAMP ac- cumulation induced by cholera toxin, forskolin, and hCG. In the same cell culture model, hCG induced a time-dependent increase in the 3/?-hydroxy-5-ene steroid dehydrogenase (3/JHSD) mRNA levels with a maximal 3-fold stimulation obtained at 8-16 h of incubation with 1 IU hCG/ml. PMA inhibited the in- crease in 30HSD mRNA levels induced by hCG in a dose-dependent manner. The phorbol ester also in- hibited the increase in 30HSD mRNA levels stimu- lated by LH as well as cholera toxin and forskolin and the cAMP analogs (Bu) 2 cAMP and 8-bromo- cAMP. Activation of protein kinase-C by mezerein similarly inhibited hCG stimulation of 30HSD mRNA levels. The present data indicate that activation of the protein kinase-C pathway induces generation of cAMP, but causes a near-complete inhibition of the stimulatory effects of hCG, LH, forskolin, cholera toxin, and cAMP analogs on 30HSD mRNA levels in porcine granulosa cells in culture. (Molecular Endo- crinology 4: 1532-1538,1990) INTRODUCTION Stimulation of the corpus luteum by LH initiates a cascade of reactions, which include the formation of 0888-8809/90/1532-1538$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society the intracellular second messenger cAMP (1). The cAMP activates cytoplasmic protein kinase-A (PKA), inducing a wide range of effects in ovarian cells, includ- ing the initiation and maintenance of steroidogenesis (2). An alternative or concurrent system mediating sur- face ligand signals in luteal tissue is the phosphatidyli- nositol pathway (3). In this pathway hormone-receptor interaction is followed by an increase in the membrane inositol breakdown and consequent activation of the intracellular protein kinase-C (PKC) by diacylglycerol (4). In mammalian cells PKC can be activated by tumor- promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA) (5). Treatment of granulosa and luteal cells from different species with PMA has revealed both stimulatory and inhibitory effects of PKC activation on steroidogenesis. PMA stimulates steroidogenesis with- out altering cAMP generation in bovine luteal cells (6). In cultured human granulosa cells, PMA increases the accumulation of cAMP and elevates progesterone syn- thesis (7). In contrast, PMA inhibition of LH-induced steroidogenesis has been demonstrated in rat granu- losa and Leydig cells (8) and in pig granulosa cells (9). Accordingly, activation of PKC produces an acute dose- dependent inhibition of progesterone production in un- stimulated large and LH-stimulated small ovine luteal cells (10). Recent studies have demonstrated that PKC activa- tion by PMA induces cAMP production in porcine gran- ulosa (11) and luteal cells (12), suggesting an interaction between the two pathways. Despite controversy about the overall stimulatory and inhibitory effects of the PKC pathway on luteal cell steroidogenesis, there is a gen- eral agreement that PMA produces some interference in the hormone response cascade beyond cAMP for- mation. 1532 Downloaded from https://academic.oup.com/mend/article/4/10/1532/2714020 by guest on 30 December 2022