The role of tumor necrosis factor-alpha in the regulation of mouse Leydig cell steroidogenesis.

Abstract

Tumor necrosis factor-alpha (TNF alpha), 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 TNF alpha at the molecular level. The effects of TNF alpha on both basal and 8-Br-cAMP-stimulated testosterone production, as well as cholesterol side-chain cleavage enzyme (P450scc) and 17 alpha-hydroxylase/C17,20 lyase (P450c17), were investigated. Treatment of Leydig cells with 0.1, 1.0, and 10.0 ng/ml TNF alpha 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. TNF alpha 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 TNF alpha, respectively. TNF alpha also caused a decrease in P450scc and P450c17 mRNA and protein. Treatment with 0.1, 1.0, and 10.0 ng/ml TNF alpha 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 TNF alpha 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 TNF alpha, respectively. The inhibitory effect of TNF alpha on 8-Br-cAMP-induced P450c17 mRNA expression was reversible. Within 48 h after the removal of TNF alpha 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. TNF alpha-mediated inhibition of 8-Br-cAMP-stimulated testosterone secretion from Leydig cells was also reversible. In addition, no significant cell mortality was noted in TNF alpha-treated cells. These data demonstrate that TNF alpha inhibits both basal and 8-Br-cAMP-stimulated testosterone secretion from Leydig cells in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)