Abstract
CYP2C12 is a steroid hydroxylase cytochrome P450 whose female-specific expression in adult rat liver is transcriptionally activated by the continuous plasma growth hormone (GH) profile characteristic of adult female rats. DNase I footprinting and gel mobility shift analysis of the 5'-flank of the CYP2C12 gene were carried out to identify cis-acting elements and trans-acting factors that may contribute to the GH-regulated, sex-dependent transcription of this P450 gene. DNase I footprinting analysis revealed sex- and GH-regulated DNase I hypersensitivity sites at the boundaries of several protein binding sites detected along a 1560-nucleotide upstream segment of CYP2C12. Five distinct sites bound a novel continuous GH-regulated nuclear factor, GHNF, which is enriched in adult female and continuous GH-treated male liver nuclear extracts compared to untreated male liver nuclear extracts. Two other footprinted sites correspond to binding sites for the liver transcription factors C/EBP and albumin D element-binding protein and a third to an HNF1 binding site. A specific binding site for GHNF was also found in the 5'-proximal promoter of CYP2C11, an adult male-specific liver P450 gene, suggesting that GHNF may contribute to the down-regulation of that gene by continuous GH. GHNF was distinguished from the nuclear factors that bind to a GH response element upstream of the rat Spi 2.1 gene and is also distinct from the GH-activatable latent cytoplasmic transcription factors STAT 1, STAT 3, and STAT 5. These findings support the hypothesis that continuous GH-activated transcription of CYP2C12 in adult female rat liver (a) involves the activation of a novel GH-regulated nuclear factor which binds to multiple sites along the 5'-flank of this cytochrome P450 gene, and (b) proceeds via a signaling pathway distinct from the GH pulse-activated STAT5 pathway proposed to induce CYP2C11 and other male-expressed liver genes.
Highlights
IntroductionDiscrimination by the hepatocyte between male and female plasma growth hormone (GH) profiles is likely to occur at the cell surface and may involve the activation of distinct intracellular signaling pathways by a chronic (female) as compared to an intermittent (male) pattern of plasma GH stimulation
These DNase I footprinting patterns were compared to those of liver nuclear extracts prepared from male rats given a continuous growth hormone (GH) infusion for 7 days, since this treatment feminizes the pattern of liver gene expression and leads to transcriptional activation and high level expression of the CYP2C12 gene in adult male rats (24, 25)
In view of the possibility that liver STAT 5 may serve as an important intracellular mediator of the effects of plasma GH pulses on 2C11 expression (20), and given its ability to enhance expression of a GH-regulated hamster liver cytochrome P450 (CYP) gene (39), we examined whether a STAT-related protein might play a corresponding role in the stimulation of 2C12 gene expression by continuous GH
Summary
Discrimination by the hepatocyte between male and female plasma GH profiles is likely to occur at the cell surface and may involve the activation of distinct intracellular signaling pathways by a chronic (female) as compared to an intermittent (male) pattern of plasma GH stimulation This hypothesis is supported by the recent demonstration that intermittent, but not continuous, plasma GH activates the latent cytoplasmic transcription factor liver STAT 5 by a mechanism that involves both Jak kinase-catalyzed tyrosine phosphorylation and serine or threonine phosphorylation followed by nuclear translocation of the STAT protein (20, 21). GH pulse frequency is the most critical determinant for intermittent GH stimulation of male liver P450 expression, which is characterized by a requirement for a well-defined minimum recovery period that is not met in the case of female rat hepatocytes exposed to GH continuously (22) This recovery period may serve to reset the Jak2/STAT 5 intracellular sig-. In contrast to the GH pulse-responsive STAT proteins (20, 21), GHNF is shown to be activated by continuous plasma GH, establishing the occurrence in hepatocytes of multiple GH plasma pattern-responsive nuclear signaling factors
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