Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells

Abstract

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-α (TNF-α) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 ± 3.1 ng/10 6 cells/h (mean ± SE). TNF-α in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-α almost completely inhibited testosterone formation ( p < 0.001). TNF-α (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-α (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 ± 9 ng/10 6 cells/h to 4.9 ng/10 6 cells/h. This indicates that the major effect of TNF-α is at $teps beyond LH receptor site. To further evaluate the site(s) of action of TNF-α, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-α on hCG-in- duced testosterone formation. TNF-α had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-α is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-α had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1β. In the present study, we demonstrated that both murine TNF-α and human IL-1β were potent inhibitors of hCG-induced testosterone formation. IL-1β alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-α (10 ng/ml), IL-1β in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-α on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-α and IL-1β inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-α on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-α inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-α is a potent inhibitor of Leydig cell function. TNF-α inhibited both P450scc and IGF-I mRNA gene expression.