Liver-Enriched Transcription Factors

HNF4α在胆汁酸合成和结合调控中的作用。在HNF4pha肝缺失小鼠(HNF4pha-deltaL)的许多有趣的表型中，血清胆汁酸升高。HNF4Alpha-deltaL小鼠的血清胆汁酸(BAs)水平显著高于对照小鼠(HNF4Alpha-F/F)。由于胆汁酸是由肝脏中的胆固醇产生的，而且参与胆汁酸生物合成的许多酶在肝脏中优先表达，因此我们研究了HNF4pha在BA产生中的作用。这在一定程度上是由于编码氧化甾醇7α-羟基酶(CYP7A1)、固醇12α-羟基酶(CYP8B1)和固醇载体蛋白x的基因下调所致。只有在HNF4alphadeltaL小鼠的暗周期中，CYP7A1的mRNA和蛋白表达减少，而在光周期中，HNF4alphadeltaL和HNF4pha-F/F小鼠的表达没有差异。无论是光周期还是暗周期，细胞色素P8B1mRNA和酶活性均降低。在小鼠Cyp8b1启动子中发现了一个HNF4pha结合位点，它能够指导HNF4pha依赖的转录。令人惊讶的是，由于CYP8B1活性而产生的胆酸衍生的bas，仍然在这些小鼠的血清和胆囊中观察到。这些研究表明，HNF4α通过调节BA生物合成的相关基因，包括体内胆固醇的羟化和侧链β-氧化，在BA的动态平衡中发挥核心作用。HNF4pha-deltaL小鼠还表现出超长链酰辅酶A合成酶相关基因(VLACSR)和胆汁酸辅酶A：氨基酸N-酰基转移酶(BAT)的表达减少。这与HNF4α-deltaL小鼠的胆汁中未结合胆汁酸和甘氨酸结合胆汁酸水平显著升高有关。与体内的发现一致，HNF4？还发现可以直接与小鼠的VLACSR和BAT基因启动子结合，在体内通过直接调节VLACSR和BAT的表达，启动子的活性依赖于HNF4α结合部位和HNF4α的表达。这些研究表明，HNF4α在胆汁酸的合成和结合中发挥着核心作用，并解释了为什么HNF4α-deltaL小鼠的胆汁酸稳态异常。HNF4α在许多肝脏特异性功能的维持中起着重要作用。肝脏特异性HNF4pha缺失小鼠被用来确定肝脏HNF4pha在体内是否调节凝血。这些小鼠表现出肝脏凝血因子V、IX、XI、XII和XIIIB的表达减少，活化的部分凝血活酶时间延长，但没有凝血酶原时间。对小鼠FXII和FXIIIB基因进行启动子分析，以确定HNF4α是否直接调节编码这些凝血因子的基因。序列分析显示，在小鼠FXII和FXIIIB基因中分别存在一个和两个HNF4pha结合位点。对小鼠FXII和FXIIIB启动子的瞬时转染和凝胶迁移率分析表明，高水平的启动子活性依赖于HNF4pha结合部位和HNF4pha的表达。综上所述，HNF4α通过指导FXII和XIIIB基因的转录在凝血动态平衡中起关键作用。最近的研究发现，转录因子相互作用通过禁食对基因的代谢进行调节。辅活化子PGC-1α和HNF4α通过位于其近端启动子的保守的HNF4反应元件，直接调节构成雄烷受体(CAR)基因的表达。PGC-1α在细胞中的表达增加了CAR的表达和非配体依赖的CAR活性。对HNF4pha缺失小鼠的研究表明，肝脏表达HNF4pha是产生禁食诱导的CAR表达和活性所必需的。禁食通过涉及肝脏中cAMP、PGC-1α、HNF4α、CAR和CAR靶基因的相互作用网络，增加编码关键代谢酶和摄取转运蛋白的基因的表达

Role of HNF4alpha in control of bile acid synthesis and conjugation. Among the many interesting phenotypes in the HNF4alpha liver null mice (HNF4alpha-deltaL) is an elevation in serum bile acids. HNF4alpha-deltaL mice have markedly increased levels of serum bile acids (BAs) compared with control floxed mice (HNF4alpha-F/F). Because bile acids are produced from cholesterol in liver and many enzymes involved in their biosynthesis are preferentially expressed in liver, the role of HNF4alpha in BA production was examined. This is due in part to the downregulation of genes encoding oxysterol 7alpha-hydroxylase (CYP7A1), sterol 12alpha-hydroxylase (CYP8B1), and sterol carrier protein x. CYP7A1 mRNA and protein were diminished only during the dark cycle in HNF4alphadeltaL mice, whereas expression in the light cycle was not different between HNF4alphadeltaL and HNF4alpha-F/F mice. CYP8B1 mRNA and enzyme activity was reduced regardless of light or dark cycle. An HNF4alpha binding site was found in the mouse Cyp8b1 promoter that was able to direct HNF4alpha-dependent transcription. Surprisingly, cholic acid-derived BAs, produced as a result of CYP8B1 activity, were still observed in the serum and gallbladder of these mice. These studies reveal that HNF4alpha plays a central role in BA homeostasis by regulation of genes involved in BA biosynthesis, including hydroxylation and side chain beta-oxidation of cholesterol in vivo. HNF4alpha-deltaL mice also exhibited decreased expression of the very long chain acyl-CoA synthase-related gene (VLACSR), also called bile acid-CoA ligase, and bile acid-CoA:amino acid N-acyltransferase (BAT). This was associated with markedly elevated levels of unconjugated and glycine-conjugated bile acids in gallbladder of the HNF4alpha-deltaL mice. In agreement with this in vivo finding, HNF4? was also found to bind directly to the mouse VLACSR and BAT gene promoters, and the promoter activities were dependent on HNF4alpha-binding sites and HNF4alpha expression by direct regulation of VLACSR and BAT in vivo. These studies indicate HNF4alpha plays a central role in bile acid synthesis and conjugation and explain why the HNF4alpha-deltaL mice have abnormal bile acid homeostasis. HNF4alpha plays an important role in the maintenance of many liver-specific functions. Liver-specific HNF4alpha-null mice were used to determine whether hepatic HNF4alpha regulates blood coagulation in vivo. These mice exhibited reduced expression of hepatic coagulation factors V, IX, XI, XII, and XIIIB and a prolonged activated partial thromboplastin time but not prothrombin time. Promoter analysis of the mouse FXII and FXIIIB genes was performed to determine whether HNF4alpha directly regulates the genes encoding these coagulation factors. Sequence analysis revealed the presence of one and two HNF4alpha binding sites in the mouse FXII and FXIIIB genes, respectively. Using transient transfection and electrophoretic mobility shift analyses with the mouse FXII and FXIIIB promoters, it was established that the high levels of promoter activity were dependent on HNF4alpha binding sites and the expression of HNF4alpha. In conclusion, HNF4alpha has a critical role in blood coagulation homeostasis by directing transcription of the FXII and XIIIB genes Recent studies have uncovered transcription factor interactions in the metabolic regulation of genes by fasting. The coactivator PGC-1alpha along with HNF4alpha directly regulates expression of the constitutive androstane receptor (CAR) gene expression through a conserved HNF4-response element located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Studies in HNF4alpha-null mice revealed that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver