THE ROLE OF TUMOR-NECROSIS-FACTOR-ALPHA IN THE REGULATION OF MOUSE LEYDIG-CELL STEROIDOGENESIS

Tumor necrosis factor-alpha (TNFalpha), a cytokine secreted by activat ed macrophages, has been shown to modulate Leydig cell steroidogenesis . The present study examined the regulation of mouse Leydig cell funct ion by TNFalpha at the molecular level. The effects of TNFalpha on bot h basal and 8-Br-cAMP-stimulated testosterone production, as well as c holesterol side-chain cleavage enzyme (P450scc) and 17alpha-hydroxylas e/C-17, C-20 lyase (P450c17), were investigated. Treatment of Leydig c ells with 0.1, 1.0, and 10.0 ng/ml TNFalpha inhibited basal testostero ne secretion by 20 +/- 5.0%, 61.1 +/- 6.6%, and 60.7 +/- 5.8% of contr ol, respectively, but had no effects on basal P450scc messenger RNA (m RNA) or protein levels. Treatment of Leydig cells with 8-Br-cAMP cause d a 150.7 +/-32.9-fold increase in testosterone production and marked stimulation of P450scc and P450c17 mRNA and protein accumulation. TNFa lpha caused a significant and dose-dependent inhibition of 8-Br-cAMP-s timulated testosterone secretion by 35.9 +/- 9.9%, 90.9 +/- 1.7%, and 96.9 +/- 1.4% with 0.1, 1.0, and 10.0 ng/ml TNFalpha, respectively. TN Falpha also caused a decrease in P450scc and P450c17 mRNA and protein. Treatment with 0.1, 1.0, and 10.0 ng/ml TNFalpha decreased 8-Br-cAMP- stimulated P450scc mRNA by 11.5 +/- 6.9%, 29.3 +/- 2.7%, and 59.2 +/-8 .7%, and decreased 8-Br-cAMP-induced P450c17 mRNA 41.9 +/-13.5%, 95.7 +/- 2.3%, and 98.5 +/- 1.2%, respectively. The inhibitory effects of T NFalpha on 8-Br-cAMP-stimulated P450 enzyme protein accumulation were also dose dependent, 35.6 +/- 11.4%, 52.9 +/- 14.1%, and 56.0 +/- 7.9% inhibition of P450scc protein levels, and 65.8 +/- 9.4%, 95.5 +/- 1.9 %, and 96.9 +/- 2.1% suppression on P450c17 protein levels were observ ed with 0.1, 1.0, and 10.0 ng/ml TNFalpha, respectively. The inhibitor y effect of TNFalpha on 8-Br-cAMP-induced P450c17 mRNA expression was reversible. Within 48 h after the removal of TNFalpha from culture, P4 50c17 mRNA was restored to 80.6 +/- 3.1% of the level in cultures trea ted with 8-Br-cAMP alone for 4 days. TNFalpha-mediated inhibition of 8 -Br-cAMP-stimulated testosterone secretion from Leydig cells was also reversible. In addition, no significant cell mortality was noted in TN Falpha-treated cells. These data demonstrate that TNFalpha inhibits bo th basal and 8-Br-cAMP-stimulated testosterone secretion from Leydig c ells in a dose-dependent manner. The inhibition of 8-Br-cAMP-stimulate d testosterone production was parallel to the TNFalpha-mediated repres sion of P450scc and P450c17 mRNA and protein levels. The inhibitory ef fects of TNFalpha were not due to cytotoxicity to Leydig cells.