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

Tumor necrosis factor-alpha (TNFalpha), a cytokine secreted by activated macrophages, has been shown to modulate Leydig cell steroidogenesis. The present study examined the regulation of mouse Leydig cell function by TNFalpha at the molecular level. The effects of TNFalpha on both basal and 8-Br-cAMP-stimulated testosterone production, as well as cholesterol side-chain cleavage enzyme (P450scc) and 17alpha-hydroxylase/C-17, C-20 lyase (P450c17), were investigated. Treatment of Leydig cells with 0.1, 1.0, and 10.0 ng/ml TNFalpha inhibited basal testosterone secretion by 20 +/- 5.0%, 61.1 +/- 6.6%, and 60.7 +/- 5.8% of control, respectively, but had no effects on basal P450scc messenger RNA (mRNA) or protein levels. Treatment of Leydig cells with 8-Br-cAMP caused a 150.7 +/-32.9-fold increase in testosterone production and marked stimulation of P450scc and P450c17 mRNA and protein accumulation. TNFalpha caused a significant and dose-dependent inhibition of 8-Br-cAMP-stimulated 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. TNFalpha 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 TNFalpha 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 observed with 0.1, 1.0, and 10.0 ng/ml TNFalpha, respectively. The inhibitory effect of TNFalpha on 8-Br-cAMP-induced P450c17 mRNA expression was reversible. Within 48 h after the removal of TNFalpha from culture, P450c17 mRNA was restored to 80.6 +/- 3.1% of the level in cultures treated 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 TNFalpha-treated cells. These data demonstrate that TNFalpha inhibits both basal and 8-Br-cAMP-stimulated testosterone secretion from Leydig cells in a dose-dependent manner. The inhibition of 8-Br-cAMP-stimulated testosterone production was parallel to the TNFalpha-mediated repression of P450scc and P450c17 mRNA and protein levels. The inhibitory effects of TNFalpha were not due to cytotoxicity to Leydig cells.