Sex differences in the expression of hepatic drug metabolizing enzymes.

Sex differences in pharmacokinetics and pharmacodynamics characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Sex-based differences in drug metabolism are the primary cause of sex-dependent pharmacokinetics and reflect underlying sex differences in the expression of hepatic enzymes active in the metabolism of drugs, steroids, fatty acids and environmental chemicals, including cytochromes P450 (P450s), sulfotransferases, glutathione transferases, and UDP-glucuronosyltransferases. Studies in the rat and mouse liver models have identified more than 1000 genes whose expression is sex-dependent; together, these genes impart substantial sexual dimorphism to liver metabolic function and pathophysiology. Sex differences in drug metabolism and pharmacokinetics also occur in humans and are due in part to the female-predominant expression of CYP3A4, the most important P450 catalyst of drug metabolism in human liver. The sexually dimorphic expression of P450s and other liver-expressed genes is regulated by the temporal pattern of plasma growth hormone (GH) release by the pituitary gland, which shows significant sex differences. These differences are most pronounced in rats and mice, where plasma GH profiles are highly pulsatile (intermittent) in male animals versus more frequent (nearly continuous) in female animals. This review discusses key features of the cell signaling and molecular regulatory mechanisms by which these sex-dependent plasma GH patterns impart sex specificity to the liver. Moreover, the essential role proposed for the GH-activated transcription factor signal transducer and activator of transcription (STAT) 5b, and for hepatic nuclear factor (HNF) 4alpha, as mediators of the sex-dependent effects of GH on the liver, is evaluated. Together, these studies of the cellular, molecular, and gene regulatory mechanisms that underlie sex-based differences in liver gene expression have provided novel insights into the physiological regulation of both xenobiotic and endobiotic metabolism.

Liver-specific hepatocyte nuclear factor-4alpha deficiency: greater impact on gene expression in male than in female mouse liver.

Hepatocyte nuclear factor (HNF)-4alpha is a liver-enriched transcription factor that regulates numerous liver-expressed genes including several sex-specific cytochrome P450 genes. Presently, a liver-specific HNF4alpha-deficient mouse model was used to characterize the impact of liver HNF4alpha deficiency on a global scale using 41,174 feature microarrays. A total of 4994 HNF4alpha-dependent genes were identified, of which about 1000 fewer genes responded to the loss of HNF4alpha in female liver as compared with male liver. Sex differences in the impact of liver HNF4alpha deficiency were even more dramatic when genes showing sex-specific expression were examined. Thus, 372 of the 646 sex-specific genes characterized by a dependence on HNF4alpha responded to the loss of HNF4alpha in males only, as compared with only 61 genes that responded in females only. Moreover, in male liver, 78% of 508 male-specific genes were down-regulated and 42% of 356 female-specific genes were up-regulated in response to the loss of HNF4alpha, with sex specificity lost for 90% of sex-specific genes. This response to HNF4alpha deficiency is similar to the response of male mice deficient in the GH-activated transcription factor signal transducer and activator of transcription 5b (STAT5b), where 90% of male-specific genes were down-regulated and 61% of female-specific genes were up-regulated, suggesting these two factors cooperatively regulate liver sex specificity by mechanisms that are primarily active in males. Finally, 203 of 648 genes previously shown to bind HNF4alpha near the transcription start site in mouse hepatocytes were affected by HNF4alpha deficiency in mouse liver, with the HNF4alpha-bound gene set showing a 5-fold enrichment for genes positively regulated by HNF4alpha. Thus, a substantial fraction of the HNF4alpha-dependent genes reported here are likely to be direct targets of HNF4alpha.

Role of STAT5a in regulation of sex-specific gene expression in female but not male mouse liver revealed by microarray analysis.

Sexual dimorphism in mammalian liver impacts genes affecting hepatic physiology, including inflammatory responses, diseased states, and the metabolism of steroids and foreign compounds. Liver sex specificity is dictated by sex differences in pituitary growth hormone (GH) secretion, with the transcription factor signal transducer and activator of transcription (STAT)5b required for intracellular signaling initiated by the pulsatile male plasma GH profile. STAT5a, a minor liver STAT5 form >90% identical to STAT5b, also responds to sexually dimorphic plasma GH stimulation but is unable to compensate for the loss of STAT5b and the associated loss of sex-specific liver gene expression. A large-scale gene expression study was conducted using 23,574-feature oligonucleotide microarrays and livers of male and female mice, both wild-type and Stat5a-inactivated mice, to elucidate any dependence of liver gene expression on STAT5a. Significant sex differences in expression were found for 2,482 mouse genes, 1,045 showing higher expression in males and 1,437 showing higher expression in females. In contrast to the widespread effects of the loss of STAT5b, STAT5a deficiency had a limited but well-defined impact on liver sex specificity, with 219 of 1,437 female-predominant genes (15%) specifically decreased in expression in STAT5a-deficient female liver. Analysis of liver RNAs from wild-type mice representing three mixed or outbred strains identified 1,028 sexually dimorphic genes across the strains, including 393 female-predominant genes, of which 89 (23%) required STAT5a for normal expression in female liver. These findings highlight the importance of STAT5a for regulation of sex-specific gene expression specifically in female liver, in striking contrast to STAT5b, whose major effects are restricted to male liver.

Characterization of three growth hormone-responsive transcription factors preferentially expressed in adult female liver.

Plasma GH profiles regulate the sexually dimorphic expression of cytochromes P450 and many other genes in rat and mouse liver; however, the proximal transcriptional regulators of these genes are unknown. Presently, we characterize three liver transcription factors that are expressed in adult female rat and mouse liver at levels up to 16-fold [thymus high-mobility group box protein (Tox)], 73-fold [tripartite motif-containing 24 (Trim24)/transcription initiation factor-1alpha (TIF1alpha)], and 125-fold [cut-like 2 (Cutl2)/cut homeobox 2 (Cux2)] higher than in adult males, depending on the strain and species, with Tox expression only detected in mice. In rats, these sex differences first emerged at puberty, when the high prepubertal expression of Cutl2 and Trim24 was extinguished in males but was further increased in females. Rat hepatic expression of Cutl2 and Trim24 was abolished by hypophysectomy and, in the case of Cutl2, was restored to near-female levels by continuous GH replacement. Cutl2 and Trim24 were increased to female-like levels in livers of intact male rats and mice treated with GH continuously (female GH pattern), whereas Tox expression reached only about 40% of adult female levels. Expression of all three genes was also elevated to normal female levels or higher in male mice whose plasma GH profile was feminized secondary to somatostatin gene disruption. Cutl2 and Trim24 both responded to GH infusion in mice within 10-24 h and Tox within 4 d, as compared with at least 4-7 d required for the induced expression of several continuous GH-regulated cytochromes P450 and other female-specific hepatic genes. Cutl2, Trim24, and Tox were substantially up-regulated in livers of male mice deficient in either of two transcription factors implicated in GH regulation of liver sex specificity, namely, signal transducer and activator of transcription 5b (STAT5b) and hepatocyte nuclear factor 4alpha (HNF4alpha), with sex-specific expression being substantially reduced or lost in mice deficient in either nuclear factor. Cutl2 and Trim24 both display transcriptional repressor activity and could thus contribute to the loss of GH-regulated, male-specific liver gene expression seen in male mice deficient in STAT5b or HNF4alpha. Binding sites for Cutl1, whose DNA-binding specificity is close to that of Cutl2, were statistically overrepresented in STAT5b-dependent male-specific mouse genes, lending support to this hypothesis.

Loss of sexually dimorphic liver gene expression upon hepatocyte-specific deletion of Stat5a-Stat5b locus.

Hepatocyte-specific, albumin-Cre recombinase-mediated deletion of the entire mouse Stat5a-Stat5b locus was carried out to evaluate the role of signal transducer and activator of transcription 5a and 5b (STAT5ab) in the sex-dependent transcriptional actions of GH in the liver. The resultant hepatocyte STAT5ab-deficient mice were fertile, and unlike global STAT5b-deficient male mice, postnatal body weight gain was normal, despite a 50% decrease in serum IGF-I. Whole-liver STAT5ab RNA decreased by approximately 65-85%, and residual STAT5 immunostaining was observed in a minority of the hepatocytes, indicating incomplete excision by Cre-recombinase. Quantitative PCR analysis of 20 sexually dimorphic, liver-expressed genes revealed significant down-regulation of 10 of 11 male-specific genes in livers of male hepatocyte STAT5ab-deficient mice. Class I female-specific liver genes were markedly up-regulated (de-repressed), whereas the expression of class II female genes, belonging to the Cyp3a subfamily, was unaffected by the loss of hepatocyte STAT5ab. STAT5ab is thus required in the liver for positive regulation of male-specific genes and for negative regulation of a subset of female-specific genes. Continuous GH infusion strongly induced (>500-fold) the class II female gene Cyp3a16 in both wild-type and hepatocyte STAT5ab-deficient male mice, indicating sex-specific transcriptional regulation by GH that is STAT5ab independent. In contrast, hepatocyte STAT5ab deficiency abolished the strong suppression of the male-specific Cyp2d9 by continuous GH seen in control mouse liver. Analysis of global STAT5a-deficient mice indicated no essential requirement of STAT5a for expression of these sex-specific liver Cyp genes. Thus, the major loss of liver sexual dimorphism in hepatocyte STAT5ab-deficient mice can primarily be attributed to the loss of STAT5b.

Sex-dependent liver gene expression is extensive and largely dependent upon signal transducer and activator of transcription 5b (STAT5b): STAT5b-dependent activation of male genes and repression of female genes revealed by microarray analysis.

Sexual dimorphism in mammalian liver contributes to sex differences in physiology, homeostasis, and steroid and foreign compound metabolism. Many sex-dependent liver genes are regulated by sex differences in pituitary GH secretion, with the transcription factor, signal transducer and activator of transcription (STAT5b), proposed to mediate signaling by the pulsatile, male plasma GH profile. Presently, a large-scale gene expression study was conducted using male and female mice, wild type and Stat5b inactivated, to characterize sex differences in liver gene expression and their dependence on STAT5b. The relative abundance of individual liver RNAs was determined for each sex-genotype combination by competitive hybridization to 23,574-feature oligonucleotide microarrays. Significant sex differences in hepatic expression were seen for 1603 mouse genes. Of 850 genes showing higher expression in males, 767 (90%) were down-regulated in STAT5b-deficient males. Moreover, of 753 genes showing female-predominant expression, 461 (61%) were up-regulated in STAT5b-deficient males. In contrast, approximately 90% of the sex-dependent genes were unaffected by STAT5b deficiency in females. Thus: 1) STAT5b is essential for sex-dependent liver gene expression, a characteristic of approximately 1600 mouse genes (4% of the genome); 2) male-predominant liver gene expression requires STAT5b, or STAT5b-dependent factors, which act in a positive manner; and 3) many female-predominant liver genes are repressed in males in a STAT5b-dependent manner. Several of the STAT5b-dependent male genes encode transcriptional repressors; these may include direct STAT5b targets that repress female-predominant genes in male liver. Several female-predominant repressors are elevated in STAT5b-deficient males; these may contribute to the major loss of male gene expression seen in the absence of STAT5b.

Codependence of growth hormone-responsive, sexually dimorphic hepatic gene expression on signal transducer and activator of transcription 5b and hepatic nuclear factor 4alpha.

Targeted disruption of the signal transducer and activator of transcription 5b gene (STAT5b) leads to decreased expression in male mouse liver of a male-predominant cytochrome (Cyp) 2d protein, whereas female-predominant Cyp2b proteins are increased. Presently, we characterize the effects of STAT5b deficiency on 15 specific, individual Cyp RNAs and other sexually dimorphic liver gene products. All seven male-specific RNAs investigated were decreased to normal female levels in STAT5b-deficient male liver, whereas five of eight female-specific RNAs, designated class I female genes, were increased in expression up to 200-fold or more. STAT5b deficiency had a much more modest effect on the expression of these genes in females. Hypophysectomy and GH replacement studies demonstrated positive GH pulse regulation of all seven male RNAs and negative GH pulse regulation of class I, but not class II, female RNAs in wild-type, but not in STAT5b-deficient, male mice. A majority of the sex-specific genes responded in parallel to the loss of STAT5b and the loss of hepatocyte nuclear factor 4alpha, indicating that both transcription factors are essential and suggesting they may coregulate sexually dimorphic liver gene expression. Continuous GH treatment of intact male mice, which overrides the endogenous male, pulsatile plasma GH pattern, down-regulated all seven male RNAs and induced expression of the five class I female RNAs within 4-7 d; however, induction of class II female RNAs was delayed until d 7-14. Given the slow responses of all 15 genes to changes in plasma GH status, GH regulation of sex-specific Cyp expression is proposed to be indirect and mediated by STAT5b- and hepatocyte nuclear factor 4alpha-dependent factors that may include repressors of female-specific Cyps and other targets of GH action.