LH induces orphan nuclear receptor Nur77 gene expression in testicular Leydig cells

The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear recept or superfamily and plays an important role in the regulation of genes invol ved in steroidogenesis and cell death. Northern blot analysis revealed that the expression of Nur77 mRNA was increased after puberty in mouse testis, and-hCG treatment of peripubertal animals induced this gene expression in t he testis. Moreover, LH treatment induced a transient increase in Nur77 mRN A, and this induction was LH dose dependent in mouse Leydig tumor cell line , K28. Western blot analysis showed that LH transiently induced Nur77 prote in. The protein kinase inhibitor H-89, bisindolymaleimide I, and wortmannin strongly inhibited this inductive effect of LH on Nur77 gene expression. T ransient transfection assay demonstrated that LH significantly increased th e Nur77 promoter-driven luciferase reporter activity in a dose-dependent ma nner, and LH also increased the activity of a luciferase reporter gene driv en by a promoter containing multi copies of a Nur77-responsive element. Mor eover, EMSA showed that Nur77 DNA-binding activity was increased in respons e to LH. Finally, overexpression of dominant negative Nur77 reduced LH-medi ated progesterone biosynthesis. Taken together, these results demonstrate t hat LH induces Nur77 gene expression, and Nur77 may play an important role in the LH-mediated steroidogenesis in Leydig cells.