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Molecular Endocrinology, doi:10.1210/me.2004-0129
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Molecular Endocrinology 18 (8): 1975-1987
Copyright © 2004 by The Endocrine Society

Sexually Dimorphic P450 Gene Expression in Liver-Specific Hepatocyte Nuclear Factor 4{alpha}-Deficient Mice

Christopher A. Wiwi, Minita Gupte and David J. Waxman

Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: David J. Waxman, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: djw{at}bu.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hepatocyte nuclear factor (HNF) 4{alpha} is a liverenriched nuclear receptor that plays a critical role in regulating the expression of numerous hepatic genes, including members of the cytochrome P450 (CYP) superfamily, several of which are expressed in a sex-dependent manner. Presently, we use a liver-specific Hnf4{alpha}-deficient mouse model to investigate the role of HNF4{alpha} in regulating liverenriched transcription factors and sexually dimorphic Cyps in liver in vivo. Real-time PCR analysis of RNA isolated from livers of wild-type and Hnf4{alpha}-deficient mice revealed the following: 1) HNF4{alpha} exerts both positive regulation (Hnf{alpha}, C/ebp{alpha}, and C/ebpß) and negative regulation (Hnf3{alpha} and the HNF4{alpha} coactivator Pgc-1{alpha}) on liver transcription factor expression; 2) a strong dependence on HNF4{alpha} characterizes several male-predominant Cyps (2d9 and 8b1), female-predominant Cyps (2b10, 2b13, 3a41, and 3a44) and Cyps, whose expression is sex independent (3a11, 3a25); 3) HNF4{alpha} confers a unique, positive regulation of two male-expressed genes (Cyp4a12 and GST{pi}) and a negative regulation of several female-predominant genes (Cyp2a4, Cyp2b9, Hnf3ß, and Hnf6), both of which are manifest in male but not female mouse liver. These trends were confirmed at the protein level by Western blot analysis using antibodies raised to Cyp2a, Cyp2b, and Cyp3a family members. Thus, HNF4{alpha} is an essential player in the complex regulatory network of liver-enriched transcription factors and the sexually dimorphic mouse Cyp genes that they regulate. HNF4{alpha} is proposed to contribute to the sex specificity of liver gene expression by positively regulating a subset of male-specific Cyp genes while concomitantly inhibiting the expression of certain female-specific Cyps and liver transcription factors, by mechanisms that are operative in male, but not female, mouse liver.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CYTOCHROME P450s (CYPs) are heme-containing membrane-bound enzymes that play an important role in the detoxification of endogenous steroids and fatty acids, as well as foreign compounds, including many drugs and environmental chemicals. In rodent liver models, CYP expression differs markedly between the sexes, in part reflecting gender differences in the requirements for site-specific steroid hormone hydroxylation (1). Well-characterized examples of male-specific or male-predominant liver CYPs include CYPs 2C11, 2A2, 3A2, and 4A2 in the rat (2, 3) and Cyps 2d9 and 4a12 in the mouse (4, 5). Female-specific liver CYPs include rat CYP2C12 (6) and mouse Cyps 2a4, 2b9, 3a41, and 3a44 (4, 7, 8, 9). Sex-dependent liver CYP gene expression is not a direct response to gonadal steroids acting on the liver; rather, it reflects the action of the gonadal hormone-regulated, sexual dimorphic pattern of pituitary GH secretion (10). In male rats, GH is secreted by the pituitary gland in a manner that is essential for transcriptional activation of CYP2C11, a male-specific steroid 16{alpha}- and 2{alpha}-hydroxylase, whereas in female rats, GH is secreted by the pituitary in a near-continuous fashion, and stimulates transcription of CYP2C12, a female-specific steroid sulfate 15ß-hydroxylase (3, 11). Similarly, in male mice, GH, via its sexually dimorphic plasma profile, activates the male-specific steroid 16{alpha}-hydroxylase Cyp2d9, and suppresses the female-specific steroid 15{alpha}-hydroxylase Cyp2a4 (4).

The transcription factor signal transducer and activator of transcription 5b (STAT5b) is uniquely responsive to the male pulsatile GH pattern and is proposed to be a key mediator of the sexually dimorphic response of liver CYPs to GH (12). The importance of STAT5b in GH pulse-stimulated, sex-specific liver gene expression is consistent with the GH pulse-induced, intermittent high levels of active STAT5b present in adult male but not female rat liver (13, 14, 15) and is strongly supported by the loss of sexually dimorphic Cyp gene expression in STAT5b-null male mice (16, 17, 18, 19). Elevated STAT5b activity has also been seen in male compared with female mouse liver (20). Although STAT5b is clearly required for the sexually dimorphic expression of liver CYPs, STAT5b by itself is not sufficient to induce the adult male pattern of liver CYP expression (14), suggesting a requirement for additional liver factors. CYP promoter sequences contain consensus binding sites for several hepatocyte-enriched nuclear factors (HNFs) (21), which may act in concert with STAT5b to regulate expression of sexually dimorphic CYP genes. These liver transcription factors are characterized by structurally diverse DNA binding domains, and include the variant homeodomain containing protein HNF1{alpha}, CCAAT/enhancer binding proteins (C/EBPs), HNF3 winged helix factors, the one-cut homeoprotein HNF6, and the orphan nuclear receptor HNF4{alpha} (22).

HNF4{alpha} (nuclear receptor NR2A1) is highly expressed in liver, where it regulates the expression of genes involved in fatty acid, cholesterol and glucose metabolism, urea biosynthesis, apolipoprotein synthesis, and liver development (23, 24, 25, 26). HNF4{alpha} binds DNA as a homodimer and can activate gene transcription in the absence of exogenous ligand (27). Fatty acyl-coenzyme A derivatives and protein kinase A-mediated phosphorylation may modulate the ability of HNF4{alpha} to bind to certain DNA response elements (28, 29). Other studies show that HNF4{alpha} can cooperate with other liver transcription factors to regulate liver-specific genes, acting in part through the influence of GH (30). For example, the sex-dependent expression of HNF6 is positively regulated by GH through a mechanism involving HNF4{alpha} and GH-activated STAT5b (31). Moreover, HNF4{alpha} can induce the re-expression of several liver-specific genes in dedifferentiated hepatoma cells (32).

HNF4{alpha} may also play a key role in the regulation of hepatic CYP genes, as suggested by in vitro analysis carried out with human CYPs (33, 34, 35), members of the rabbit and rat CYP2C (36, 37) and CYP3A families (38), and the mouse Cyp2a, Cyp2d, and Cyp3a subfamilies (39, 40, 41). However, little is known about the role that HNF4{alpha} plays in vivo in regulating the expression of liver CYPs, in particular, sexually dimorphic CYPs. Investigation of the function of HNF4{alpha} in liver in vivo has been hampered by the fact that targeted disruption of HNF4{alpha} is embryonic lethal (42). Presently, we use a liver-specific Hnf4{alpha}-deficient mouse model (24) to investigate the role of HNF4{alpha} in the regulation of liver-enriched transcription factors and the sexually dimorphic Cyps that they regulate. Liver-specific disruption of Hnf4{alpha} is shown to markedly alter the expression of several sexually dimorphic Cyp genes and liver transcription factors. The data presented suggest a model whereby HNF4{alpha} contributes to sex-dependent liver Cyp expression in male liver by positively regulating a subset of male-specific Cyp genes while concomitantly inhibiting the expression of certain female-specific Cyps.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HNF4{alpha} mRNA Is Ablated in Liver-Specific HNF4{alpha}-Deficient Mouse Liver
We first established that Hnf4{alpha} gene expression is, in fact, abolished in the HNF4{alpha}-targeted mouse livers used in this study. Quantitative real-time PCR (QPCR) using primers specific to the deleted exons 4 and 5 of the mouse Hnf4{alpha} gene were used to quantify total liver HNF4{alpha} RNA levels in 48-d-old HNF4{alpha}-flox and HNF4{alpha}-deficient mice. Data were normalized to 18S rRNA levels, which were highly consistent between individuals within each group, and were unchanged between HNF4{alpha}-flox and HNF4{alpha}-deficient males and females (Table 1Go, last line). Liver STAT5b RNA and glyceraldehyde phosphate dehydrogenase RNA were also unaffected by Hnf4{alpha} deletion (Table 1Go), indicating that the loss of Hnf4{alpha} expression does not have a global effect on the liver RNA profile. Liver HNF4{alpha} RNA was expressed at a more than 50-fold higher level in HNF4{alpha}-flox (control) liver compared with HNF4{alpha}deficient liver, consistent with the near-complete ablation of Hnf4{alpha} expression reported earlier in HNF4{alpha}-deficient mice (24). HNF4{alpha} showed no difference in expression between the sexes in control mice (Table 1Go).


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  		Table 1. Liver Transcription Factor Expression in Liver-Specific HNF4{alpha}-Deficient Male and Female Mice

 
Impact of Liver-Specific HNF4{alpha} Gene Disruption on Expression of Other Liver Transcription Factors
Analysis of CYP promoter sequences indicates the presence of consensus binding sites for several hepatocyte-enriched nuclear factors (HNFs), several of which may regulate liver gene expression, including sexually dimorphic liver CYP expression, through a complex transcriptional control hierarchy involving HNF4{alpha} (43). We therefore investigated the effect of HNF4{alpha} loss on the expression of seven individual HNFs (HNF1{alpha}, HNF3{alpha}, HNF3ß, HNF3{gamma}, HNF6, C/EBP{alpha}, and C/EBPß) and the HNF4{alpha} coactivators PGC-1{alpha} (peroxisome proliferator-activated receptor (PPAR) {gamma} coactivator-1] and PGC-1ß. These studies were carried out in both male and female mice, enabling us to identify sex differences in mouse Hnf gene expression. Positive regulation by HNF4{alpha} was evident for C/EBP{alpha}, C/EBPß, and HNF1{alpha} RNA, which were down-regulated in livers of HNF4{alpha}-deficient mice of both sexes. In contrast, HNF3{alpha} and PGC-1{alpha} RNA levels were both significantly higher in HNF4{alpha}-deficient liver compared with HNF4{alpha}-flox liver, both in males and in females, indicating negative regulation. No significant sex difference in hepatic expression of Hnf1{alpha}, Hnf3{alpha}, Hnf3{gamma}, C/ebp{alpha}, C/ebpß, Pgc-1{alpha}, and Pgc-1ß was apparent in either HNF4{alpha}-flox or HNF4{alpha}-deficient mice (Table 1Go). Liver RNA levels of HNF3ß and HNF6 were significantly elevated in female as compared with male HNF4{alpha}-flox mice, consistent with the sex dependence seen in rat liver (44), where HNF3ß and HNF6 are proposed to act in a synergistic manner to positively regulate certain female-specific CYP genes (45). Interestingly, Hnf4{alpha} disruption significantly increased the expression of HNF3ß and HNF6 RNA in male mouse liver, by 1.8- and 3.2-fold, respectively, but had no effect in females. Thus, HNF4{alpha} exerts an inhibitory effect on liver Hnf3ß and Hnf6 expression that is restricted to male mice.

HNF4{alpha} Positively Regulates Liver Expression of Apolipoprotein CIII (ApoCIII), Cyp3a11, and Cyp3a25
Twelve liver-expressed mouse genes, including 10 Cyp family members, were analyzed using the web-based program Cluster Buster (46) and the TransFac database (47) to identify clustered DNA-binding motifs present in 5'-flanking DNA sequences. Analysis of the clustered DNA-binding elements revealed multiple consensus DNA-binding sites for HNF4{alpha} within 5 kb of the transcriptional start site of each gene (Table 2Go). This suggests that HNF4{alpha} may be an important regulator of these Cyp genes, several of which are expressed in a sex-dependent manner. To test this hypothesis, we investigated the effect of Hnf4{alpha} disruption on expression of the 12 putative target genes in both male and female mouse liver. Our initial studies investigated liver RNAs encoded by three genes whose expression does not show strong sex dependence: ApoCIII, Cyp3a11, and Cyp3a25. Expression of all three genes was decreased significantly in both male and female HNF4{alpha}-deficient mice (Fig. 1Go).


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  		Table 2. Potential HNF4{alpha}-Binding Sites Located in the 5'-Flank of Mouse ApoCIII, GST{pi}, and CypGenes

 


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  		Fig. 1. HNF4{alpha} Is Required for Full Expression of ApoCIII, Cyp3a11, and Cyp3a25

Control (HNF4{alpha}-flox) and HNF4{alpha}-deficient male and female mouse livers were assayed for expression of ApoCIII, Cyp3a11, and Cyp3a25 RNAs by QPCR as described in Table 1Go. Data shown represent mean ± SE values for eight individual livers per group after normalization to 18S rRNA, with the highest level of expression in each panel set to 100 (relative expression). Statistical differences (t test) are reported as follows: HNF4{alpha}-flox male vs. HNF4{alpha}-flox female ({Delta}, P < 0.05); HNF4{alpha}-flox vs. HNF4{alpha}-deficient; **, P < 0.01. ApoCIII mRNA was expressed at an approximately 1.4-fold higher level in HNF4{alpha}-flox female compared with HNF4{alpha}-flox male liver. This moderate sex difference was significant at P < 0.05.

 
Positive Regulation of Male-Specific and Male-Predominant Liver Genes by HNF4{alpha}
We next determined the impact of Hnf4{alpha} disruption on the expression of Cyp and other genes whose expression is substantially higher in male than female mouse liver. QPCR analysis of HNF4{alpha}-flox mice revealed a substantial male specificity for Cyp2d9 (male:female expression ratio = 12.7), Cyp8b1 (2.8-fold sex difference), Cyp4a12 (7.8-fold) and glutathione-S-transferase {pi} RNA (GST{pi}; 8.6-fold) (Fig. 2Go). Cyp2d9 and Cyp8b1 mRNA levels were decreased 50- to 60-fold in HNF4{alpha}-deficient male livers compared with the corresponding HNF4{alpha}-flox controls (Fig. 2Go), indicating strong positive regulation by HNF4{alpha}. A substantial decrease in expression was also seen for both genes in HNF4{alpha}-deficient female mice (Fig. 2Go, A and B, right side of each panel). The near complete loss of gene expression in both sexes suggests that the HNF4{alpha} dependence of these genes is mechanistically unrelated to their sex-dependent expression. Furthermore, the strong HNF4{alpha} dependence of mouse Cyp8b1 in vivo supports previous in vitro studies indicating a direct role of HNF4{alpha} in the regulation of the human CYP8B1 gene (34). Interestingly, the other two male-specific RNAs examined, Cyp4a12 and GST{pi},1 were down-regulated, by approximately 50%, in livers of male but not female HNF4{alpha}-deficient mice (Fig. 2Go, C and D). Thus, the positive regulation of these genes by HNF4{alpha} is exclusively associated with males. Liver GST{pi} RNA expression was increased somewhat in HNF4{alpha}-deficient females, effectively abolishing the sex difference in liver GST{pi} expression in the HNF4{alpha}-deficient mice. This increase could reflect the loss of a female-specific repressor protein in the HNF4{alpha}-deficient females.



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  		Fig. 2. Positive Regulation of Male-Specific and Male-Predominant Liver Genes by HNF4{alpha}

Liver RNA was analyzed for Cyp2d9, Cyp8b1, Cyp4a12, and GST{pi} expression by QPCR as described in Table 1Go. Data shown represent mean ± SE values for eight individual livers per group after normalization to 18S rRNA, with the highest level of expression in each panel set to 100 (relative expression). Statistical differences (t test) are reported as follows: HNF4{alpha}-flox male vs. HNF4{alpha}-flox female ({Delta}{Delta}, P < 0.01); and HNF4{alpha}-flox vs. HNF4{alpha}-deficient compared separately for each sex (**, P < 0.01).

 
Regulation of Female-Specific and Female-Predominant Liver Cyp Genes by HNF4{alpha}
The effects of Hnf4{alpha} disruption were next determined for six Cyp genes whose expression was found to be substantially higher in female than in male mouse liver: Cyp2b10 (female:male expression ratio = 3.6), Cyp2b13 (>1000-fold sex difference), Cyp3a41 (17-fold), Cyp3a44 (15-fold), Cyp2b9 (77-fold), and Cyp2a4 (3-fold) (Figs. 3Go and 4Go). Cyp2b10, Cyp2b13, Cyp3a41, and Cyp3a44 RNAs were down-regulated, by as much as 94%, in HNF4{alpha}-deficient female livers (Fig. 3Go), demonstrating that HNF4{alpha} plays a dominant, positive role in the regulation of these female-specific Cyps. The striking HNF4{alpha} dependence of these Cyp genes differed markedly from that of two other female-predominant genes, Cyp2b9 and Cyp2a4, whose hepatic expression was unaffected by the loss of HNF4{alpha} in female mice. Remarkably, however, the latter two genes were strongly up-regulated in HNF4{alpha}-deficient male mice (Fig. 4Go). This finding is strikingly similar to the selective up-regulation of the female-predominant liver transcription factors HNF3ß and HNF6 seen in HNF4{alpha}-deficient males (Table 1Go) and demonstrates the ability of HNF4{alpha} to negatively regulate gene expression in a sex-dependent manner.



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  		Fig. 3. Positive Regulation of Female-Specific Liver Genes by HNF4{alpha}

Liver RNA was analyzed for Cyp2b10, Cyp2b13, Cyp3a41, and Cyp3a44 expression by QPCR as described in Table 1Go. Data shown represent mean ± SE values for eight individual livers per group after normalization to 18S rRNA, with the highest level of expression in each panel set to 100 (relative expression). Statistical differences (t test) are reported as follows: HNF4{alpha}-flox male vs. HNF4{alpha}-flox female ({Delta}, P < 0.05; {Delta}{Delta}, P < 0.01); and HNF4{alpha}-flox vs. HNF4{alpha}-deficient compared separately for each sex, (*, P < 0.05; **, P < 0.01).

 


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  		Fig. 4. Female-Specific Cyp2b9 and Female-Predominant Cyp2a4 Are Negatively Regulated by HNF4{alpha} in Male But Not Female Mouse Liver

QPCR analysis of liver RNA was carried out as described in Table 1Go. Data shown represent mean ± SE values for eight individual livers per group after normalization to 18S rRNA, with the highest level of expression in each panel set to 100 (relative expression). Statistical differences (t test) are reported as follows: HNF4{alpha}-flox male vs. HNF4{alpha}-flox female ({Delta}, P < 0.05; {Delta}{Delta}, P < 0.01); and HNF4{alpha}-flox vs. HNF4{alpha}-deficient compared separately for each sex (*, P < 0.05; **, P < 0.01).

 
Western Blot Analysis of Hepatic Cyp Genes in Hnf4{alpha}-Deficient Mouse Liver
Western blot analysis was carried out using whole liver extracts prepared from HNF4{alpha}-flox and HNF4{alpha}-deficient livers (n = 5 livers/group), to confirm and extend the overall patterns of liver Cyp expression seen in the above QPCR analyses. Figure 5Go (bottom panel) shows that the higher level of Cyp3a expression in female compared with male liver (c.f. Cyp3a41 and Cyp3a44 RNAs; Fig. 3Go) was confirmed at the protein level. Moreover, there was an overall decrease in Cyp3a protein in HNF4{alpha}-deficient livers of both sexes (c.f. Cyps 3a11, 3a25, 3a41, and 3a44 RNAs; Figs. 1Go and 3Go). Likewise, the negative regulation of Cyp2b9 by HNF4{alpha} in male mice (Fig. 4AGo) was confirmed at the protein level by the striking up-regulation of the female-specific Cyp2b immunoreactive band c in HNF4{alpha}-deficient male mouse liver (Fig. 5Go). Cyp2b bands a and d, which were present at somewhat higher levels in HNF4{alpha}-flox females than males, were down-regulated in HNF4{alpha}-deficient males and females in a pattern similar to that of Cyp2b10 RNA (c.f. Fig. 3AGo). Cyp2b band b was present at similar levels in HNF4{alpha}-flox and HNF4{alpha}-deficient liver, in animals of both sexes, indicating the presence Cyp2b family members whose expression is independent of HNF4{alpha}. Finally, the negative regulation of Cyp2a4 RNA by HNF4{alpha} seen in male mice (Fig. 4AGo) was confirmed at the protein level by the moderate increase in Cyp2a immunoreactive band b in HNF4{alpha}-deficient male liver (Fig. 5Go, top panel). Cyp2a band a, which was present at similar levels in HNF4{alpha}-flox males and females, was significantly decreased in HNF4{alpha}-deficient mice of both sexes indicating the presence of additional Cyp2a family members whose sex-independent expression is dependent on HNF4{alpha}. Overall, these results support our conclusion that HNF4{alpha} confers a unique, negative regulation on certain female-specific (Cyp2b9) and female-predominant (Cyp2a4) genes that is restricted to male mouse liver.



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  		Fig. 5. Western Blot Analysis of Sex-Dependent Cyp2a, Cyp2b, and Cyp3a Proteins in HNF4{alpha}-Deficient Mice

Whole liver homogenates prepared from individual male and female HNF4{alpha}-flox and HNF4{alpha}-deficient mice (40 µg protein/lane) were analyzed on Western blots probed with polyclonal antibodies raised to CYP2A, CYP2B, or CYP3A proteins. Two immune cross-reactive CYP2A bands are seen (bands a and b). CYP2A band b is female predominant, and band a is sex independent. Four immune cross-reactive CYP2B bands were deleted (bands a, b, c, and d, with band d corresponding to a doublet). CYP2B band c is female specific, whereas bands a and d are somewhat more intense in female compared with male liver samples. The single CYP3A band is likely to be comprised of multiple CYP3A proteins of similar electrophoretic mobility. Liver homogenates were prepared from the same individual livers analyzed in Figs. 1–4GoGoGoGo and in Table 1Go.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In rodents, hepatic expression of several CYP family members differs markedly between the sexes in response to sexually dimorphic plasma GH profiles. The sexually dimorphic response of liver CYPs to GH is regulated at the level of gene transcription and requires the GH pulse-activated transcription factor STAT5b (48). Experiments in immature rats demonstrate, however, that the activation of STAT5b is not sufficient to confer the adult pattern of GH-regulated liver CYP gene expression (14), suggesting a requirement for additional liver factors. The regulation of many liver-specific genes, including GH-regulated CYP genes, reflects the coordinated action of multiple hepatocyte-enriched nuclear factors that are interlinked in a complex transcriptional hierarchy. HNF4{alpha} plays a key role in this transcriptional cascade, as illustrated by its ability to positively regulate HNF1{alpha} expression through HNF4 binding sites located in 5'-regulatory sequences (49). HNF4{alpha} itself is subject to positive regulation by HNF6 (50) and negative regulation by HNF1{alpha} (51). A detailed examination of the role of HNF4{alpha} in the regulation of sexually dimorphic Cyps in liver in vivo had been precluded by the fact that targeted disruption of the Hnf4{alpha} gene is embryonic lethal (42). The recent development of a liver-specific HNF4{alpha}-deficient mouse model enabled us to address this issue, and to elucidate the role of HNF4{alpha} in the sexually dimorphic expression of Hnfs and Cyps.

Liver-specific Hnf4{alpha}-disruption altered the expression of liver transcription factors through both positive regulation (HNF1{alpha}, C/EBP{alpha}, and C/EBPß) and negative regulation (HNF3{alpha}, HNF3ß, HNF6, and PGC-1{alpha}) (Table 3Go). Cre-mediated recombination at the Hnf4{alpha} locus results in an approximately 50% reduction in intact HNF4{alpha} mRNA by 4 wk of age. By 6 wk of age, HNF4{alpha} mRNA is reduced at least 90% (24) and by 7 wk by 98% (Table 1Go), suggesting that the differences in liver gene expression observed here do not reflect an early developmental role of HNF4{alpha} in hepatocyte differentiation. Although the involvement of HNF4{alpha} in regulation of HNF1{alpha} and HNF6 has been demonstrated in vitro, HNF4{alpha} was not previously implicated in the regulation of C/ebp{alpha}, C/ebpß, Hnf3{alpha}, or Hnf3ß. Given the complex hierarchical network of HNF regulation noted above, it is likely that the changes in liver transcription factor profiles seen in HNF4{alpha}-deficient mice are the result of direct effects, as well as indirect effects of HNF4{alpha} deficiency.


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  		Table 3. Gene Expression in HNF4{alpha}-Null Mice

 
Three of the transcription factors examined, STAT5b, PGC-1ß, and HNF3{gamma}, were not altered in their expression in HNF4{alpha}-deficient mice, indicating that they are not targets of HNF4{alpha} (Table 3Go). STAT5b undergoes repeated cycles of tyrosine phosphorylation, nuclear translocation and DNA-binding in direct response to the regular plasma GH pulses found in male rodents (13, 14) and is essential for maintaining the normal male pattern of liver CYP expression (48). Although STAT5b RNA levels were similar in control and HNF4{alpha}-deficient mouse liver, HNF4{alpha} deficiency may nevertheless affect the sexdependent, GH-regulated activation of STAT5b and thereby contribute to the altered expression of certain liver CYPs seen in HNF4{alpha}-deficient mice. The HNF4{alpha} coactivator Pgc-1{alpha} was increased in HNF4{alpha}-deficient mouse liver, consistent with the increase in Pgc-1{alpha} expression in liver-specific HNF4{alpha}-null hepatocytes, and supporting the conclusion that HNF4{alpha} is a critical component of PGC-1{alpha}-mediated gluconeogenesis (52). However, the related coactivator PGC-1ß was not up-regulated in HNF4{alpha}-deficient mouse liver, despite its ability to trans-activate many of the same target genes as PGC-1{alpha} (53).

Liver RNA levels of HNF3ß and HNF6 were found to be higher in female as compared with male mice, in accord with the female-predominant expression of these factors seen in rat liver (Ref. 44 ; and Wiwi, C. A., and D. J. Waxman, unpublished experiments). Our discovery that Hnf4{alpha} disruption increases hepatic HNF3ß and HNF6 RNA levels in males, but has no effect in females, indicates that HNF4{alpha} confers negative regulation to these genes in a male-specific manner. The up-regulation of HNF3ß and HNF6 RNAs to normal female liver levels seen in HNF4{alpha}-deficient males could contribute to the associated up-regulation of the female-predominant Cyp genes 2b9 and 2a4 (Table 3Go), in view of the finding that HNF3ß and HNF6 synergistically trans-activate another female-specific liver CYP gene, rat CYP2C12 (45). Conceivably, the sex-dependent effects of HNF4{alpha} on HNF3ß and HNF6 expression revealed by the present study could involve the action of GH and its sex-dependent plasma hormone profile. Notably, the expression of HNF6 mRNA is GH dependent, as revealed by the loss of HNF6 expression in rat liver after hypophysectomy and by its restoration to normal female levels in response to continuous GH replacement (44). The increase in HNF6 expression is due, in part, to a GH-stimulated increase in HNF4{alpha} and STAT5b binding to their respective sites in the HNF6 promoter (31). Further investigation is required to elucidate the mechanism whereby HNF4{alpha} exerts an apparent inhibitory effect on HNF3ß and HNF6 expression in male liver, and the role of GH in this response.

Multiple HNF4{alpha} consensus DNA binding sites were localized to clusters of liver transcription factor binding sites in the 5'-regulatory regions of genes belonging to several mouse Cyp subfamilies (Table 2Go). The implication of this finding, that HNF4{alpha} plays an important role in regulating hepatic Cyp expression, is given direct support by the substantial decreases in expression of many Cyp genes seen in HNF4{alpha}-deficient mice. These findings are consistent with the demonstration that HNF4{alpha} binding motifs are important for activation of mouse Cyp2a4 (40) and Cyp2d9 (39). Similarly, the substantial down-regulation of Cyp8b1 and ApoCIII in livers of male and female HNF4{alpha}-deficient mice (Table 3Go) supports earlier transfection studies showing that these genes are regulated in a positive manner by HNF4{alpha} (34, 54, 55, 56). This regulation may involve cooperative interactions between HNF4{alpha} and other nuclear receptors. For example, in the case of CYP3A4, binding of HNF4{alpha} to its promoter site is essential for pregnane X receptor and constitutive androstane receptor to trans-activate the gene (57). By contrast, in the case of ApoCIII, farnesoid X receptor suppresses gene expression by displacement of HNF4{alpha} from a shared binding site on the promoter (58). The loss of expression of certain hepatic genes seen in HNF4{alpha}-deficient mice may thus involve indirect factors and mechanisms, such as increased bile acid levels (24) leading to increased farnesoid X receptor inhibition of target genes, as exemplified by ApoCIII.

Liver-specific HNF4{alpha}-deficient mice are characterized by an accumulation of lipid in the liver and greatly reduced serum cholesterol and triglyceride levels (24). The expression of several nuclear receptors involved in lipid homeostasis, including retinoid X receptor {alpha}, pregnane X receptor, and farnesoid X receptor is unchanged in HNF4{alpha}-deficient mice. PPAR{alpha} RNA levels are reduced in HNF4{alpha}-deficient liver; nevertheless, several known PPAR{alpha} target genes are elevated in expression (24). The up-regulation of certain Cyp genes seen in HNF4{alpha}-deficient mice (Table 3Go) could therefore result from changes in the levels of lipids, bile acids, or other endogenous ligands for nuclear receptors involved in their regulation. However, this indirect mechanism would not explain the exclusive up-regulation of Cyps 2a4 and 2b9 in male liver. It also would not explain the loss of expression of Cyp4a12 seen in males, or the lack of Cyp4a12 induction in HNF4{alpha}-deficient females, given that Cyp4a12 expression is highly responsive to lipid and other activators of PPAR{alpha}, which strongly induces its expression in female mouse liver (5).

Liver-specific expression of GH-responsive, sexually dimorphic CYPs is likely to be achieved through the combined action of multiple HNFs acting in concert. In rats, the male-specific CYP2C11 is induced by HNF1{alpha} and HNF3ß (59), whereas the female-specific CYP2C12 is activated through a robust synergy between HNF3ß and HNF6 (45). Based on the present study, Cyp2d9 and Cyp8b1, both male predominant in their expression, exhibit a strong dependence on HNF4{alpha} in vivo (Table 3Go), extending previous in vitro studies of these genes (34, 39, 60). However, the HNF4{alpha} dependence of these male-specific Cyps appears to be mechanistically unrelated to their sex-dependent expression, given the decrease in expression that is seen in both male and female HNF4{alpha}-deficient mice. In contrast, the positive regulation by HNF4{alpha} of two other male-specific mouse genes, Cyp4a12 and GST{pi},1 was specifically associated with males. Correspondingly, expression of the female-predominant Cyp2b10 and the female-specific Cyps 2b13, 3a41, and 3a44 was down-regulated, by as much as 94%, in HNF4{alpha}-deficient females, indicating that HNF4{alpha} can positively regulate the female-specific expression of certain Cyps. Collectively, the HNF4{alpha} dependence of Cyp4a12 and GST{pi}, in males, and of Cyps 2b10, 2b13, 3a41, and 3a44, in females, highlights the critical role that HNF4{alpha} plays in the sex-dependent expression of a wide range of GH-regulated mouse Cyp genes. Moreover, HNF4{alpha} was found to confer a unique, sex-dependent negative regulation on two other female-specific Cyp genes, 2b9 and 2a4, whose expression was unchanged in HNF4{alpha}-deficient females but was strongly up-regulated in HNF4{alpha}deficient male mice as compared with control mice (Table 3Go). This finding parallels the selective up-regulation in male liver of the female-predominant genes Hnf3ß and Hnf6, discussed above, and establishes that HNF4{alpha} can negatively regulate HNFs as well as CYPs in a sex-dependent manner.

Recently, a pair of Krüppel-associated box zinc finger genes, named Rsl1 and Rsl2, has been implicated in the negative regulation of certain mouse liver genes that are expressed in a sexually dimorphic, GHdependent manner. Notably, Rsl1 and Rsl2 repress male-specific Slp and Cyp2d9 (Rsl1) and Mup (Rsl2) gene expression in adult male and female mouse liver before puberty (61, 62 ; for review, see Ref. 63). This repression is selectively relieved in males at puberty, apparently in response to pulsatile GH signaling. Conceivably, HNF4{alpha}-dependent expression of Rsl1 and Rsl2 could contribute to the moderate up-regulation of GST{pi} mRNA seen in HNF4{alpha}-deficient female mouse liver. Moreover, HNF4{alpha} regulation of other, as of yet unidentified Krüppel-associated box zinc finger repressor proteins that may repress female-specific genes in male mouse liver could help explain the negative regulation of several female-predominant genes (Cyp2a4, Cyp2b9, Hnf3ß, and Hnf6) seen in male but not female HNF4{alpha}-deficient mouse liver.

Thus, although many of the HNF4{alpha}-regulated actions identified in the present study were manifest in males and females (positive HNF4{alpha}-regulation of ApoCIII, Cyp3a11, Cyp3a25, Cyp2d9, and Cyp8b1), several examples of positive regulation by HNF4{alpha} (Cyp4a12 and GST{pi}) and negative regulation by HNF4{alpha} (Cyp2a4, Cyp2b9, Hnf3ß, and Hnf6) were restricted to male liver. HNF4{alpha} thus plays a unique role, and presumably is subject to a unique hormonedependent regulation that enables it to regulate a subset of the sexually dimorphic genes expressed in male liver. The selective up-regulation of Cyp2b9 in HNF4{alpha}-deficient male liver demonstrated here is reminiscent of the increased expression of certain female-specific Cyp2b family members seen in STAT5b-deficient male mouse liver (19). Given the involvement of GH-activated STAT5b in the hierarchy of liver transcription expression and activity, noted above, HNF4{alpha} and STAT5b may conceivably act in a coordinate or a cooperative manner to regulate the expression of GH-dependent, sexually dimorphic Cyps.

In conclusion, the present findings establish a critical role for HNF4{alpha} in controlling the expression of liver-enriched transcription factors and their sexually dimorphic mouse Cyp target genes. These findings in the mouse model take on added significance in view of recent evidence that sex is a major determinant in the expression CYP3A4 in human liver (64), and that this sex dependence is in part determined by sexually dimorphic plasma GH profiles (65). HNF4{alpha} is proposed to contribute to sex-dependent liver Cyp expression through the positive regulation of a subset of male-specific Cyp genes and via the concomitant repression of female-specific Cyps. Further study will be required to elucidate the hormonal factors that dictate these sex-dependent actions of HNF4{alpha}, and the molecular mechanisms that enable HNF4{alpha} to exert gender-specific and gene-selective effects on sexually dimorphic liver gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Liver-specific HNF4{alpha}-deficient mice were generated by albumin promoter-regulated Cre-loxP-mediated deletion of exons 4 and 5 of the HNF4{alpha} gene (24). Liver tissue from 48-d-old liver-specific HNF4{alpha}-deficient and HNF4{alpha}-flox (control) male and female mice (n =8 for each group), killed between 1000 and 1500 h, obtained from Y. Inoue and F. J. Gonzalez (National Cancer Institute, Bethesda, MD), were used in this study. Liver tissues were snap frozen in liquid nitrogen at the time of collection and stored at –80 C until use.

QPCR
QPCR primers specific to each of the mouse mRNAs to be investigated were designed using Primer Express software (Applied Biosystems, Foster City, CA) (Table 4Go). To ensure specificity among multigene Cyp subfamily members, genes belonging to each Cyp subfamily were aligned with each other and QPCR primers were designed based on the regions with the greatest sequence divergence. In general, PCR primers were chosen to introduce a minimum of a two nucleotide mismatch with all related Cyp subfamily members to ensure primer specificity. For example, the Cyp2b9 forward primer (oligo 1135, Table 4Go) contains 4, 3, and 3 mismatches with the corresponding sequences of Cyps 2b10, 2b13, and 2b19, respectively, whereas the 2b9 reverse primer (oligo 1136) contains 2, 3, and 4 mismatches with the same corresponding three Cyps. Gene specificity was further verified using the National Center for Biotechnology Information (NCBI) Blast program. QPCR results presented for GST{pi} represent the combined contribution of GST{pi}1 and GST{pi}2 RNA because the two GST{pi} forms are approximately 99% identical at the DNA level (66) and could not be distinguished by QPCR.


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  		Table 4. Mouse QPCR Primer Sets and GenBank Accession Numbers

 
Total RNA from mouse tissue was isolated using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions, then treated for 60 min at 37 C with 1 unit of RQ1 ribonuclease-free deoxyribonuclease (Promega, Madison, WI) followed by heating for 5 min at 75 C. cDNA was generated by reverse transcription of 1 µg of total rat liver RNA using random hexamers and murine leukemia virus reverse transcriptase (Applied Biosystems) in a total reaction volume of 20 µl. A typical QPCR mixture (16 µl) included 4 µl of cDNA template diluted 1:100, 1 µl each PCR primer (312 nM, final concentration each primer), 8 µl SYBR Green I PCR master mix (Applied Biosystems) and 3 µl deionized water. Triplicate samples (5 µl each) were then pipetted into separate wells of a 384-well plate and run through 40 cycles on an ABS 7900HT QPCR sequence detection system (Applied Biosystems). Relative quantitation of each HNF, CYP, or other target RNA was calculated as described in the manufacturer’s bulletin. Dissociation curves were generated after each QPCR run to ensure that a single, specific product was amplified. QPCR data for each gene were graphed as fold-activation values, normalized to the 18S rRNA content of each cDNA. Statistical differences were calculated by t test analysis using GraphPad Prism version 3.0 for Windows software (San Diego, CA) for each of the following comparisons: 1) HNF4{alpha}-flox male vs. HNF4{alpha}-flox female; 2) HNF4{alpha}-flox male vs. HNF4{alpha}-deficient male; and 3) HNF4{alpha}-flox female vs. HNF4{alpha}-deficient female. P < 0.05 was deemed significant.

Western Blot Analysis
Rabbit polyclonal antibodies raised to mouse Cyp2a4 (anti-C-P45015{alpha}) (67), rat CYP2B1 (68), and rat CYP3A (69) were used for Western blot analysis. Antimouse Cyp2a4 antibody was kindly provided by Dr. M. Negishi (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC). Whole liver homogenates, prepared from frozen individual mouse livers (70), were electrophoresed for 5 h on a 7.5% nondenaturing polyacrylamide sodium dodecyl sulfate gel (40 µg protein/lane) and transferred overnight onto nitrocellulose membranes. Membranes were blocked for 1 h at 37 C in TST buffer [10 mM Tris-HCl (pH 7.5), 0.1% Tween 20, 0.1 M NaCl] containing 1% BSA and 5% nonfat dry milk (CYP2B1 antibody) or 1% BSA and 3% nonfat dry milk (Cyp2a4 and CYP3A antibodies). Membranes were incubated at 4 C overnight with anti-CYP2B1 antisera (diluted 1:4000 in blocking solution) or for 1 h at room temperature with anti-CYP3A or anti-Cyp2a4 antisera (diluted 1:4000 in blocking solution), washed and probed for 1 h with goat antirabbit secondary antibody conjugated to alkaline phosphatase (1:20,000 dilution) (Pierce Biotechnology Inc., Rockford, IL). Antibody binding was visualized on x-ray film by enhanced chemiluminescence using the ECL kit from Amersham Pharmacia Biotech (Piscataway, NJ). X-ray films were scanned using a Microtek Scanmaker V6USL (Hauppauge, NY) scanner and ScanWizard 5 version 5.12 scanning software (Microtek, Inc.).

Promoter Analysis and Identification of HNF4 Binding Sites
The web-based program PromoSer (http://biowulf.bu.edu/zlab/promoser) (71) was used to retrieve DNA sequence information, based on the October 2003 mouse genome update, for the proximal promoter regions of 12 liver-expressed mouse genes, including 5 kb of sequence upstream of the transcriptional start site. GenBank accession numbers, used to identify genes of interest by PromoSer, are shown in Table 2Go (see Results). The promoter sequences retrieved were evaluated for correct genomic location and orientation using the NCBI Blast program. Proximal promoters were then analyzed using the web-based program Cluster Buster (http://zlab.bu.edu/cluster-buster) (46) to identify clustered DNA-binding motifs in the 5'-flanking DNA for a set of 26 liver-expressed transcription factors based on consensus sequences for these factors defined by the TransFac database (47). HNF4{alpha} DNA-binding motifs found within the clusters and having scores greater than 3.5 are reported in Table 2Go.


   FOOTNOTES
 
This work was supported by National Institutes of Health Grant DK33765 (to D.J.W.).

Abbreviations: ApoCIII, Apolipoprotein CIII; C/EBP, CCAAT/enhancer binding protein; CYP, cytochrome P450; GST, glutathione-S-transferase; HNF, hepatocyte nuclear factor; PGC-1, PPAR {gamma} coactivator-1; PPAR, peroxisome proliferator activated receptor; STAT5b, signal transducer and activator of transcription 5b; QPCR, quantitative real-time PCR.

1 The QPCR primers used to measure GST{pi} do not distinguish between GST{pi}1 and GST{pi}2 RNA, which are approximately 99% identical (66 ). Consequently, the partial (~50%) HNF4{alpha} dependence of GST{pi} RNA seen in male liver may indicate that one, but not both, GST{pi} genes is HNF4{alpha} regulated. Back

Received for publication March 29, 2004. Accepted for publication May 12, 2004.


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 ABSTRACT
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