Dual Actions of Phorbol Ester on Cytochrome P450 Cholesterol Side-Chain Cleavage Messenger Ribonucleic Acid Accumulation in Porcine Granulosa Cells1

Abstract

In earlier studies in cultures of porcine granulosa cells prepared from small antral follicles, steroidogenesis-related loci were inhibited by treatment for 48 h with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC). In the present investigation, cells were incubated in serum-free medium for 48 h, with various agents present during the last 2-24 h. With TPA at 30 ng/ml, the FSH-stimulated cAMP accumulation was markedly enhanced at all time points. FSH increased the concentration of cytochrome P450 cholesterol side-chain cleavage (P450scc) mRNA throughout the 24-h incubation. At 4 and 8 h, TPA increased the accumulation of P450scc mRNA, having an additive effect with FSH. However, at 24 h, TPA markedly suppressed the FSH-induced increased in P450scc mRNA. Pretreatment of cells with FSH did not shorten the time required for TPA to become inhibitory. The stimulatory effect of 8-bromo-cAMP on P450scc mRNA also was augmented by TPA at 4 h, but significant inhibition was not observed at 24 h. The concentration of glyceraldehyde-3-phosphate dehydrogenase mRNA, intended to be used for correction of gel loading, was stably increased by both cAMP and TPA. These effects of TPA suggest multiple actions of PKC(s) on the regulation of P450scc expression and other endpoints in ovarian granulosa cells.

Regulation of Granulosa Cell-Derived Insulin-Like Growth Factor Binding Proteins (IGFBPs): Role for Protein Kinase-C in the Pre- and Posttranslational Modulation of IGFBP-4 and IGFBP-51

“…However, attempts at normalizing the data with the above ''housekeeping'' genes failed due to significant changes in the relative expression of CHOB and ␤-actin transcripts in PMA-treated cells (data not shown). Similar effects of PMA were noted for other housekeeping genes (i.e., cyclophilin and GADPH), in keeping with previous reports [46]. Due to the inability to identify PMA-independent transcripts, strict quantitative normalization of Northern blots was not possible.…”

NH2terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element

“…The inhibition of steroidogenesis by TNF-␣ was mediated through the IGFRE (24). Finally, there is evidence that the protein kinase C pathway can also regulate steroidogenesis by effects on FSH-stimulated steroidogenesis, indicating that this important cellular pathway may influence P450scc expression through the IGFRE (10,15). Therefore, understanding the mechanisms and interactions of PSF and Sp1 on this important 30-bp response element can result in an increased understanding of P450scc gene expression by multiple cellular pathways.…”

Calcium Ions Positively Modulate Follicle-Stimulating Hormone- and Exogenous Cyclic 3',5'-Adenosine Monophosphate-Driven Transcription of the P450scc Gene in Porcine Granulosa Cells

“…CYP17, CYP19, CYP11A1, CYP11B2) control steroidogenic gene expression (52, 58 -61). Although FSH is not known to stimulate PKC activity in granulosa cells (7)(8)(9)11), effectors of the PKC pathway can significantly enhance (at 4 -6 h) and then down-regulate (at 18 -48 h) FSH-stimulated P450 scc gene expression in ovarian cells (62,63). Thus, the ability of Ca 2ϩ to selectively potentiate FSH/cAMP's stimulation of P450 scc gene transcription in granulosa cells differs from PKC-associated bidirectional regulation of this and other steroidogenic genes.…”