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.
The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-alpha (TNF-alpha) 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-alpha in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-alpha almost completely inhibited testosterone formation (p < 0.001). TNF-alpha (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-alpha (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-alpha is at steps beyond LH receptor site. To further evaluate the site(s) of action of TNF-alpha, 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-alpha on hCG-induced testosterone formation. TNF-alpha had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-alpha is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-alpha had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1beta. In the present study, we demonstrated that both murine TNF-alpha and human IL-1beta were potent inhibitors of hCG-induced testosterone formation. IL-1beta 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-alpha (10 ng/ml), IL-1beta in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-alpha 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-alpha and IL-1beta inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-alpha 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-alpha 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-alpha is a potent inhibitor of Leydig cell function. TNF-alpha inhibited both P450scc and IGF-I mRNA gene expression.
Lin
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WJB Dorn Veterans Hospital and Department of Medicine, University of South Carolina School of Medicine, Columbia 29201, USA.
Wang
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Nagpal
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Chang
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D000818:Animals; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D007985:Leydig Cells / Q000378:metabolism*; D008297:Male; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007334:Insulin-Like Growth Factor I; D002786:Cholesterol Side-Chain Cleavage Enzyme