The orphan nuclear receptor Nur77 (NR4A1) is a member of the nuclear receptor superfamily and plays an important role in the regulation of genes involved 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 the testis. Moreover, LH treatment induced a transient increase in Nur77 mRNA, and this induction was LH dose dependent in mouse Leydig tumor cell line, K28. Western blot analysis showed that LH transiently induced Nur77 protein. The protein kinase inhibitor H-89, bisindolymaleimide I, and wortmannin strongly inhibited this inductive effect of LH on Nur77 gene expression. Transient transfection assay demonstrated that LH significantly increased the Nur77 promoter-driven luciferase reporter activity in a dose-dependent manner, and LH also increased the activity of a luciferase reporter gene driven by a promoter containing multi copies of a Nur77-responsive element. Moreover, EMSA showed that Nur77 DNA-binding activity was increased in response to LH. Finally, overexpression of dominant negative Nur77 reduced LH-mediated progesterone biosynthesis. Taken together, these results demonstrate that LH induces Nur77 gene expression, and Nur77 may play an important role in the LH-mediated steroidogenesis in Leydig cells.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is an important hypophysiotrophic factor as well as a regulator for immune, reproductive, and neural tissues. We recently found that TTF-1, a homeodomain-containing transcription factor essential for the development of the fetal diencephalon, is postnatally expressed in the hypothalamic area and plays a transcription regulatory role for certain neurohormones. Based on the similarity of synthesis sites between PACAP and TTF-1 and, moreover, on the presence of conserved core TTF-1 binding motifs in the 5-flanking region of the PACAP gene, we sought to uncover a regulatory role of TTF-1 in PACAP gene transcription. The TTF-1 homeodomain binds to six of the seven putative binding domains observed in the 5-flanking region of the PACAP gene. In the C6 glioma cell-line, TTF-1 activates the PACAP promoter in a dose-dependent manner. This transactivation of PACAP by TTF-1 was totally removed when the core TTF-1 binding motif at ؊369 was deleted. RNase protection assays showed that TTF-1 and PACAP mRNAs have daily fluctuations in the rat hypothalamus. They both were at low levels during the day and high levels during the night. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased the PACAP mRNA level as well as TTF-1 protein content in the rat hypothalamus, suggesting that TTF-1 also regulates PACAP transcription in vivo. Moreover, the TTF-1 promoter was inhibited by molecular oscillators of CLOCK and BMAL-1. Taken together, these data suggest that TTF-1 plays an important regulatory role in the gene transcription for PACAP, which may be important for the generation of a daily rhythm of hypothalamic PACAP gene expression.Pituitary adenylate cyclase-activating polypeptide (PACAP) 1 was originally isolated from the ovine hypothalamus (1, 2) and was classified as a member of the secretin-glucagon-vasoactive intestinal peptide family (1). PACAP plays the role of a hypophysiotropic factor and regulates the synthesis and secretion of pituitary hormones through the hypothalamo-hypophysial portal system (3). Although PACAP immunoreactivity is found in nearly all brain regions, its highest concentration is in the hypothalamus of both rat and human (3, 4). In the hypothalamus, PACAP immunoreactivity is primarily localized to the cells of the paraventricular and supraoptic nuclei (3, 4). PACAP is found both in neurons terminating on hypophysial blood vessels of the median eminence as well as in neurons projecting to the posterior pituitary. Therefore, PACAP may act not only as a hypophysiotropic factor regulating anterior pituitary hormone release but as a posterior pituitary hormone as well. It is also an important factor in the regulation of peripheral tissues such as ovary and testis and of the immune system.Recently, the 5Ј-flanking region of PACAP gene was characterized and shown to have several sequence motifs homologous to the cAMP response element (CRE), the TPA response element (TRE), and the growth hormone factor...

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