Soo-Hee Park

GROWTH HORMONE ACTIVATION OF STAT 1, STAT 3, AND STAT 5 IN RAT LIVER : DIFFERENTIAL KINETICS OF HORMONE DESENSITIZATION AND GROWTH HORMONE STIMULATION OF BOTH TYROSINE PHOSPHORYLATION AND SERINE/THREONINE PHOSPHORYLATION

Intermittent plasma growth hormone (GH) pulses, which occur in male but not female rats, activate liver Stat 5 by a mechanism that involves tyrosine phosphorylation and nuclear translocation of this latent cytoplasmic transcription factor (Waxman, D. J., Ram, P. A., Park, S. H., and Choi, H. K.(1995) J. Biol. Chem. 270, 13262-13270). We demonstrate that physiological levels of GH can also activate Stat 1 and Stat 3 in liver tissue, but with a dependence on the dose of GH and its temporal plasma profile that is distinct from Stat 5 and with a striking desensitization following a single hormone pulse that is not observed with liver Stat 5. GH activation of the two groups of Stats leads to their selective binding to DNA response elements upstream of the c-fos gene (c-sis-inducible enhancer element; Stat 1 and Stat 3 binding) and the β-casein gene (mammary gland factor element; liver Stat 5 binding). In addition to tyrosine phosphorylation, GH is shown to stimulate phosphorylation of these Stats on serine or threonine in a manner that either enhances (Stat 1 and Stat 3) or substantially alters (liver Stat 5) the binding of each Stat to its cognate DNA response element. These findings establish the occurrence of multiple, Stat-dependent GH signaling pathways in liver cells that can target distinct genes and thereby contribute to the diverse effects that GH and its sexually dimorphic plasma profile have on liver gene expression.

Distinctive Roles of STAT5a and STAT5b in Sexual Dimorphism of Hepatic P450 Gene Expression IMPACT OF Stat5a GENE DISRUPTION

Stat5b gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239–7244). In the present study, disruption of the mouse Stat5a gene, whose coding sequence is ∼90% identical to the Stat5b gene, resulted in no loss of expression in male mice of several sex-dependent, GH-regulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5b feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16α-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6β-hydroxylase). Since STAT5a is thus nonessential for these male GH responses, STAT5b homodimers, but not STAT5a-STAT5b heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either Stat5a or Stat5bled to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. Stat5a or Stat5b gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5a, which is much less abundant in liver than STAT5b, and STAT5b are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.

STAT5b-deficient Mice Are Growth Hormone Pulse-resistant ROLE OF STAT5b IN SEX-SPECIFIC LIVER P450 EXPRESSION

The signal transducer and transcriptional activator STAT5b is required to maintain the adult male pattern of liver gene expression and whole body pubertal growth rates, as demonstrated by the loss of these growth hormone (GH) pulse-dependent responses in mice with a targeted disruption of the STAT5b gene. The present study investigates whether these phenotypes of STAT5b-deficient mice result from impaired intracellular GH signaling associated with a loss of GH pulse responsiveness, as contrasted with a feminization of the pituitary GH secretory profile leading to the observed feminization of body growth and liver gene expression. Pulsatile GH replacement in hypophysectomized mice stimulated body weight gain in wild-type but not in STAT5b-deficient mice. Expression of the male-specific liver P450 enzyme CYP2D9, which is reduced to female levels in hypophysectomized male mice, was restored to male levels by GH pulse replacement in wild-type but not in STAT5b-deficient mice. Similarly, a female-specific liver CYP2B P450 enzyme that was up-regulated to female levels following hypophysectomy of males was suppressed to normal basal male levels by GH pulses only in wild-type hypophysectomized mice. Finally, urinary excretion of the male-specific, GH pulse-induced major urinary protein was restored to normal male levels following pulsatile GH treatment only in the case of wild-type hypophysectomized mice. STAT5b-deficient mice are thus GH pulse-resistant, supporting the proposed role of STAT5b as a key intracellular mediator of the stimulatory effects of plasma GH pulses on the male pattern of liver gene expression.

Synergistic Action of Hepatocyte Nuclear Factors 3 and 6 onCYP2C12 Gene Expression and Suppression by Growth Hormone-activated STAT5b PROPOSED MODEL FOR FEMALE-SPECIFIC EXPRESSION OFCYP2C12 IN ADULT RAT LIVER

Growth hormone (GH) exerts sexually dimorphic effects on liver gene transcription through its sex-dependent temporal pattern of pituitary hormone secretion. CYP2C12 encodes a female-specific rat liver P450 steroid hydroxylase whose expression is activated by continuous GH stimulation of hepatocytes. Presently, we investigated the role of liver-enriched and GH-regulated transcription factors in the activation of CYP2C12 gene expression in GH-stimulated liver cells. Transcription of a CYP2C12 promoter-luciferase reporter gene in transfected HepG2 cells was activated 15–40-fold by the liver-enriched hepatocyte nuclear factor (HNF) 3α, HNF3β, and HNF6. Synergistic interactions leading to an ∼300-fold activation of the promoter by HNF3β in combination with HNF6 were observed. 5′-Deletion analysis localized the HNF6 response to a single 5′-proximal 96-nucleotide segment. By contrast, the stimulatory effects of HNF3α and HNF3β were attributable to five distinct regions within the 1.6-kilobase CYP2C12 proximal promoter. GH activation of the signal transducer and transcriptional activator STAT5b, which proceeds efficiently in male but not female rat liver, inhibited CYP2C12 promoter activation by HNF3β and HNF6, despite the absence of a classical STAT5-binding site. The female-specific pattern of CYP2C12 expression is thus proposed to reflect the positive synergistic action in female liver of liver-enriched and GH-regulated transcription factors, such as HNF3β and HNF6, coupled with a dominant inhibitory effect of GH-activated STAT5b that is manifest in males.

Inhibitory Cross-talk between STAT5b and Liver Nuclear Factor HNF3β IMPACT ON THE REGULATION OF GROWTH HORMONE PULSE-STIMULATED, MALE-SPECIFIC LIVER CYTOCHROME P-450 GENE EXPRESSION

STAT5b is repeatedly activated in rodent liver by the male pattern of intermittent plasma growth hormone (GH) stimulation and is required to maintain the GH pulse-regulated, male-specific pattern of liver gene expression. We presently investigate the interactions between STAT5b and hepatocyte-enriched nuclear factors (HNFs) that contribute to regulation of GH pulse-inducible, male-specific liver cytochrome P-450 (CYP) genes. STAT5 binding sites were identified in the 5′-flank of the adult male-expressed genesCYP2A2 (nucleotides −2255 to −2247), CYP4A2(nucleotides −1872 to −1864), and CYP2C11 (nucleotides −1150 to −1142). STAT5-DNA complexes were formed by eachCYP sequence with nuclear extract from GH pulse-activated male, but not female, rat liver. TheCYP2C11 STAT5 site, which is flanked by HNF3 consensus sequences, conferred STAT5b-inducible reporter gene activity in GH-treated HepG2 cells. trans-Activation of the intactCYP2C11 promoter (1.8-kilobase 5′-flank) was strongly induced by the liver nuclear factors HNF1α and HNF3β but, unexpectedly, was inhibited by GH-activated STAT5b. This STAT5b inhibitory effect could be reversed by HNF1α and reflects a functional antagonism between STAT5b and HNF3β, as evidenced by the inhibition of HNF3β DNA binding and transcriptional activity by STAT5b. HNF3β, in turn, inhibited STAT5b by a novel mechanism that leads to suppression of GH-inducible STAT5b tyrosine phosphorylation, DNA binding activity, and transcriptional activity. The potential for GH-activated STAT5b to stimulate male-specific liver CYPexpression can thus be modulated by HNF3β, highlighting the complex interrelationship between STAT5b and liver transcription factors controlling expression of GH-regulated CYPgenes.

Signalling cross-talk between hepatocyte nuclear factor 4α and growth- hormone-activated STAT5b

In the present study, we have characterized signalling cross-talk between STAT5b (signal transducer and activator of transcription 5b) and HNF4alpha (hepatocyte nuclear factor 4alpha), two major regulators of sex-dependent gene expression in the liver. In a HepG2 liver cell model, HNF4alpha strongly inhibited beta-casein and ntcp (Na+/taurocholate cotransporting polypeptide) promoter activity stimulated by GH (growth hormone)-activated STAT5b, but had no effect on interferon-gamma-stimulated STAT1 transcriptional activity. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4alpha towards the ApoCIII (apolipoprotein CIII) promoter. The inhibitory effect of HNF4alpha on STAT5b transcription was associated with the inhibition of GH-stimulated STAT5b tyrosine phosphorylation and nuclear translocation. The short-chain fatty acid, butyrate, reversed STAT5b transcriptional inhibition by HNF4alpha, but did not reverse the inhibition of STAT5b tyrosine phosphorylation. HNF4alpha inhibition of STAT5b tyrosine phosphorylation was not reversed by pervanadate or by dominant-negative phosphotyrosine phosphatase 1B, suggesting that it does not result from an increase in STAT5b dephosphorylation. Rather, HNF4alpha blocked GH-stimulated tyrosine phosphorylation of JAK2 (Janus kinase 2), a STAT5b tyrosine kinase. Thus STAT5b and HNF4alpha exhibit bi-directional cross-talk that may augment HNF4alpha-dependent gene transcription while inhibiting STAT5b transcriptional activity via the inhibitory effects of HNF4alpha on JAK2 phosphorylation, which leads to inhibition of STAT5b signalling initiated by the GH receptor at the cell surface.