Stimulation of [3H]serine-labeled A431 cells with tumor necrosis factor-alpha (TNFalpha) or bacterial sphingomyelinase (SMase) resulted in a rapid decrease (approximately 50% by 15 min) in cellular [3H]sphingomyelin content and generation of the lipid moiety [3H]ceramide, which remained elevated 60 min later. Sphingomyelin hydrolysis in response to TNFalpha or bacterial SMase resulted in a time-dependent decrease in the phosphorylation state of c-Jun protein, an effect that was also observed in cells treated with the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-ceramide). The rapid dephosphorylation of the c-Jun gene product in response to TNFalpha, SMase, or C6-ceramide was not observed in A431 cells treated with the serine-threonine phosphatase inhibitor okadaic acid. After the initial steps of previously described methods for the purification of a ceramide-activated protein phosphatase termed CAPP (Dobrowsky, R. T., Kamibayashi, C., Mumby, M. C., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 15523-15530), we obtained a cytosolic fraction from A431 cells that specifically dephosphorylated 32Pi-labeled c-Jun protein used as substrate in an immunocomplex phosphatase assay. Phosphatase activity in vitro was apparent only in the presence of ceramide (5 micro) and was specifically abrogated when okadaic acid (1 n) was included in the immunocomplex phosphatase assay. These results provide strong evidence for c-Jun as a downstream target for CAPP activated in response to post-TNF signaling in A431 cells.

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0.01-1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3 beta-hydroxysteroid dehydrogenase/delta 5-4 isomerase (3 beta-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0.1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 microgram/ml), forskolin (15 mumol/l) or 1-methyl-3-isobutyl-xanthine (0.1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0.1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3 beta-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

In cultured granulosa cells, interleukin-1 beta (IL-1 beta) induced a time-dependent (16-72 h) and dose-related (0.3-30 ng/ml) stimulation of nitric oxide (NO) synthase (NOS) activity, as determined by the catalytic conversion of [3H]arginine to [3H]citrulline and NO2- accumulation in the culture medium. Although FSH alone failed to stimulate NOS activity, concomitant treatment with the gonadotropin (200 ng/ ml) or the cell-permeant cAMP analog (Bu)2cAMP (0.5 mM) markedly enhanced IL-1 beta-induced NO generation in cultured granulosa cells. The effect of IL-1 beta on citrulline biosynthesis and NO2- accumulation was abrogated by the NOS inhibitor NG-methyl-L-arginine or the IL-1-receptor antagonist protein. In contrast bacterial endotoxin (lipopolysaccharide), interferon-gamma, or tumor necrosis factor-alpha, which are well known inducers of inducible NOS (iNOS) in a variety of immunocompetent and nonimmunocompetent cell types, failed to increase [3H]citrulline formation or NO2- accumulation in untreated or FSH-stimulated cells. As demonstrated by reverse transcriptase-PCR analysis, IL-1 beta-stimulated NO generation was accompanied by a time-dependent increase in messenger RNA levels for iNOS and GTP-cyclohydrolase (GTPCH), the rate-limiting step for de novo tetrahydrobiopterin (BH4) biosynthesis. Treatment with FSH augmented only GTPCH messenger RNA expression, and a more than additive GTPCH signal was observed when cells were simultaneously challenged with IL-1 beta and FSH. Treatment with the GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine prevented IL-1 beta-induced NOS activity in untreated or FSH-stimulated cells, and this inhibition was completely reversed by sepiapterin, a substrate for BH4 biosynthesis, via an alternative pterin salvage pathway present in many cell types. As BH4 is an essential cofactor for NOS catalytic activity, these observations strongly suggest that FSH-induced biosynthesis of endogenous BH4 is essential for full iNOS biosynthetic capacity in IL-1 beta-stimulated granulosa cells.

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