3β-hydroxy-5-ene Steroid dehydrogenase gene expression regulation in porcine granulosa cells

Abstract

The objective of this study was to investigate the effect of the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) on FSH- and LH-induced 3β-HSD-gene expression in cultured porcine granulosa cells. FSH and LH induced a dose dependent increase in the accumulation of 3β-HSD mRNA, measured by Northern blot. A 1.6- to 1.8-fold increase (p<0.01) was observed with 10 ng/mL of FSH or LH. Maximal levels of 2.5- to 2.9-fold increases, relative to control, were reached at 30 and 100 ng/mL of the gonadotropins. When granulosa cells were treated with PMA (100 nM) just before the addition of FSH, the 3β-HSD rnRNA levels induced by 10 or 30 ng/mL of FSH were inhibited or partially inhibited, respectively. PMA did not inhibit elevated levels of 3β-HSD mRNA induced by FSH at concentrations of 100, 300, and 1000 ng/mL. Alternatively, PMA added just before LH, inhibited LH-stimulated 3β-HSD mRNA levels at all doses of LH tested (10, 30, 100, 300, and 1000 ng/mL). The protein kinase A-stimulators, dibutyryl-cAMP (cAMP) (0.5 mM) and forskolin (10 nM), also elevated the 3β-HSD-gene transcription, 3.5- and 4.0-fold respectively. PMA prevented the stimulation of the 3β-HSD-gene transcription when it was added just before cAMP or forskolin. We concluded that stimulation of PKC by PMA appears to have inhibited the gonadotropin-induced increase in 3β-HSD mRNA levels by preventing cAMP-activated 3β-HSD-gene transcription. The data also suggest that the effect of PMA appears to be more specific for regulation of LH-stimulated intracellular signals than those of FSH. This effect may indicate a site of differential regulation of FSH and LH on the stimulation of 3β-HSD-gene transcription.