Cytokine regulation of the expression of estrogenic biosynthetic enzymes in cultured rat granulosa cells.

Both P450 aromatase (P450arom) and 17beta-hydroxysteroid dehydrogenase (17HSD) type 1 are key enzymes in the ovarian E(2) biosynthesis. Cytokines have been suggested to be mediators between the immune and the reproductive systems, and they may play a role as paracrine or autocrine ovarian regulatory factors. Interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha) have been shown to modulate the FSH-induced E(2) production in immature rat granulosa cells. The aim of the present study was to investigate the effects of these cytokines on the activity and expression of the 17HSD type 1 enzyme in cultured undifferentiated granulosa cells. Furthermore, the expression of P450arom was also analyzed. The granulosa cells obtained from the ovaries of immature DES-treated rats were initially cultured for 48 h with no other treatment and then incubated with or without the test reagents for an additional 48 h. The treatment of the granulosa cells with cytokines alone did not affect the activity of 17HSD type 1 as assessed by the conversion of tritiated substrate. However, both TNFalpha and IL-1beta caused a dose-dependent inhibition of the recombinant FSH-induced enzyme activity and the Forskoline-induced expression of 17HSD type 1 and P450arom mRNAs. The cytokines only slightly inhibited the 8-Br-cAMP-induced P450arom expression. In contrast, the inhibitory cytokine effects on 17HSD type 1 expression and activity were not abolished by the presence of 8-Br-cAMP. Despite the presence of inhibitors of protein kinase C (staurosporine) or tyrosine kinases (genistein), the inhibitory effects of TNFalpha and IL-1beta on the Forskoline-induced expression of 17HSD type 1 and P450arom and the Forskoline-induced 17HSD activity were not blocked. The data show a dose dependent inhibitory effect of TNFalpha and IL-1beta on gonadotropin action, opposite to the follicular development by down-regulating the expressions of estrogen biosynthetic enzymes. The cytokine effects on P450arom expression are mainly derived from a decrease in gonadotropin-induced cAMP production, while the inhibitory mechanisms on 17HSD type 1 expression involve distal sites from cAMP generation. The protein kinase C and tyrosine kinase pathways are likely not to be involved in the latter mechanisms.