Interaction of a Novel Sex-dependent, Growth Hormone-regulated Liver Nuclear Factor with CYP2C12Promoter*

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 CYP2C12gene 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 CYP2C12in 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 CYP2C11and other male-expressed liver genes.