Transcription factors in liver development, differentiation, and regeneration

SCIENCE FRONTIER Transcription Factors in Liver Development, Differentiation, and Regeneration Robert H. Costa,1 Vladimir V. Kalinichenko,1 Ai-Xuan L. Holterman,1,2 and Xinhe Wang1

Regulation of Hepatocyte-Specific Gene Transcription Binding of Multiple Hepatocyte Nuclear Factors to DNA Regulatory Regions Is Required for Synergistic Transcriptional Activation. The liver performs essential functions in the body by uniquely expressing hepatocyte-specific genes encoding plasma proteins, clotting factors and enzymes involved in detoxification, gluconeogenesis, glycogen synthesis, and glucose, fat, and cholesterol metabolism.1 Functional analysis of numerous hepatocyte-specific DNA regulatory regions (promoter and enhancer regions) reveals that they are composed of multiple cis-acting DNA sequences that bind different families of liver-enriched transcription factors named hepatocyte nuclear factors (HNF). The analysis involved transient transfections of hepatoma cells lines utilizing DNA regulatory regions containing targeted mutations in each of these transcription factor binding sites fused to a reporter gene, whose expression levels were used to monitor promoter activity.2-11 The results of these transfection studies suggested that hepatocyte-specific gene transcription requires simultaneous binding of multiple, distinct Abbreviations: HNF, hepatocyte nuclear factor; TTR, transthyretin; Fox, Forkhead box; C/EBP, CCAAT/enhancer binding protein; bZIP, basic region leucine zipper; FTF, fetoprotein transcription factor; TBP, TATA binding protein; TAF, TBP associated factors; CBP, Creb binding protein; Stat, signal transducer and activator of transcription; Pepck, phosphoenolpyruvate carboxykinase; PPAR-␥, peroxisome proliferator-activated receptor ␥; PAH, phenylalanine hydroxylase; AFP, ␣-fetoprotein; dpc, days post coitum; IHBD, intrahepatic bile ducts; NF-␬B, nuclear factor ␬B; TNF, tumor necrosis factor; AFPp-Cre, ␣-fetoprotein enhancer albumin promoter-enhancer driven Cre recombinase transgene; Cdk, cyclin dependent kinase; TFN, transferrin; Alb-Cre, albumin promoter and enhancer driven Cre recombinase; TG, transgenic; WT, wild type; PHx, partial hepatectomy; TGF-␣, tumor growth factor ␣; IL-6, interleukin 6; CCl4, carbon tetrachloride. From the 1Department of Biochemistry and Molecular Genetics and 2Department of Surgery, Division of Pediatric Surgery, University of Illinois at Chicago, College of Medicine, Chicago, IL. The work in the laboratory is supported by US Public Health Service Grants RO1 DK 54687-05 from NIDDK (R.H.C.), R01 GM43241-14 from the NIGMS (R.H.C.), and RO1 AG 21842-01 from NIA (R.H.C.). Received August 4, 2003; accepted September 14, 2003. Address reprint requests to: Robert H. Costa, Ph.D., Department of Biochemistry and Molecular Genetics (M/C 669), University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave., Room 2220 MBRB, Chicago, IL 60607-7170. E-mail: [email protected]; fax: 312-355-4010. Copyright © 2003 by the American Association for the Study of Liver Diseases. 0270-9139/03/3806-0002$30.00/0 doi:10.1016/j.hep.2003.09.034

HNF transcription factors to the gene regulatory region providing synergistic transcriptional activation (Fig. 1). The requirement for combinatorial protein interactions between multiple HNF factors to achieve synergistic transcriptional stimulation of hepatocyte-specific genes thus plays an important role in maintaining tissue-specific gene expression. Furthermore, maintenance of hepatocyte-specific expression of any of the HNF transcription factors (Fig. 1), in turn, involves cross-regulation by other HNF transcription factors.10,12-21 In addition, these transfection studies showed that specific strong-affinity promoter binding sites for HNF transcription factors are critical for expression of distinct hepatocyte-specific target genes. For example, disruption of a single proximal strong-affinity HNF3 (Foxa)/HNF6 site in the transthyretin (TTR) proximal promoter region (Fig. 1) abolished expression of the TTR regulatory region6,10 and mutation of the proximal strong-affinity HNF1 site inhibited transcriptional activity of the albumin promoter/enhancer region.11 Analysis of liver gene expression profiles in HNFdeficient (knockout) mice shows that HNF deficiency caused distinct liver phenotypes because each HNF transcription factor is critical for high transcriptional levels of distinct sets of hepatocyte-specific genes (see below).

Liver Transcription Factor Structure and Function Liver Transcription Factor DNA-Binding Domains. Isolation of the cDNA clones encoding the HNF transcription factors facilitated identification of their DNA-binding and transcriptional activation domains. The DNA-binding domain of a transcription factor is composed of amino acid sequences that conform to a structural motif, which mediates DNA sequence-specific recognition. The DNA-binding domain provides specificity to the transcription factor by recognizing DNA sites located in either the proximal promoter or distal enhancer sequences (regulatory regions) of hepatocyte-specific genes. The specificity between the transcription factor DNA binding domain and its promoter/enhancer DNA recognition site defines the hepatocyte-specific genes that are regulated by a particular transcription factor (Fig. 1). Depending on the transcription factor, the DNA-binding domain may recognize these DNA regulatory sequences 1331

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Fig. 1. Transcription factors that regulate expression of the TTR and FoxA2 genes and their binding sites. Schematically shown are the FoxA2 (HNF3␤) and TTR promoter constructs and their corresponding transcription factors. Members of 4 different liver-enriched transcription factors HNF1, Foxa, HNF4, HNF6, and C/EBP10,188,189 and the growth factor inducible AP-1 protein190 bind the TTR regulatory regions. The strong affinity FoxA binding site (⫺106 to ⫺96 bp) overlaps with the HNF6 binding site in the TTR promoter.10 The TTR enhancer is also recognized by an uncharacterized ubiquitous factor (UF) and contains one FoxA binding site, which is selectively recognized by the FoxA2 isoform.189 Four families of liver transcription factors regulate the FoxA2 promoter: FoxA, HNF6, C/EBP, and FTF/Nkx 2.8,18,34,46 the latter of which has binding specificity similar to that of Nkx-2.8.45 Two additional binding sites are recognized by generally expressed factors: UF1-H3␤9 and the interferon (IFN) response factor-1 (IRF-1), which is activated in response to IFN␦.20,191

as either a monomer or a dimer. Protein dimers consist of either homodimers between the same transcription factors or heterodimers between distinct, related proteins from the same family of transcription factors. On the basis of homology within DNA-binding domains, the liver-enriched transcription factors were grouped into related protein families (Fig. 2). The HNF3␣, HNF3␤, and HNF3␥ proteins (renamed as Forkhead box a1 [Foxa1], Foxa2, and Foxa3 proteins, respectively)5,22,23 bind to DNA as a monomer using the winged helix DNA-binding domain,24,25 which also contains sequences essential for nuclear localization26 and transcriptional activation.27 The Foxa1, Foxa2, and Foxa3 winged helix domains share greater than 90% homology in their amino acid sequence (Fig. 2A) and therefore bind to similar DNA target sequences within hepatocyte-specific regulatory regions and exhibit functional redundancy in hepatocytes.10,23,28 The mesodermspecific Foxf1 (previously called HFH8 or Freac1) and the proliferation-specific FoxM1B (previously called HFH11B or Trident) transcription factors share 54% and 39% homology with the Foxa1 winged helix sequence, respectively (Fig. 2A). Both the Foxf1 and FoxM1B proteins bind to DNA as monomers and contain potent transcriptional activation domains that reside in the Cterminal region of the protein.29-31 The HNF6 or ONECUT-1 (OC-1) transcription factors define a new class of transcription factors (Fig. 2B) containing a single cut domain and a divergent homeodomain motif.32 The HNF6 protein binds to its DNA recognition site as a monomer through the cut-homeodomain or the “ONECUT” DNA-binding domain,10,33,34

which contains sequences that mediate nuclear localization and transcriptional activation.27,35 Interestingly, a second member of the ONECUT family, called OC-2, is expressed in the liver and shares DNA-binding site specificity and amino acid sequence homology with HNF6 (OC-1) protein and may, therefore, provide functional redundancy with HNF6 in hepatocytes.32 The CCAAT/enhancer binding proteins (C/EBP) utilize an amino-terminal basic region leucine zipper (bZIP) bipartite DNA-binding domain (Fig. 2C) consisting of a dimerization interface composed of heptad-repeated leucine residues termed the “leucine zipper” and a DNAbinding interface consisting of basic amino acids.36 The C/EBP␣ and C/EBP␤ proteins are coexpressed in hepatocytes and are able to form either homodimers or heterodimers for DNA sequence-specific binding through the bZIP protein motif.37,38 The HNF1␣ uses a POU-homeodomain sequence and a myosin-like dimerization domain located at the amino terminus of the protein (Fig. 2D) to bind its DNA recognition sequence as a dimer.39,40 HNF1␣ dimers are stabilized through association with dimerization cofactor of HNF1␣ (DcoH) protein, which is identical to the aromatic amino acid metabolizing enzyme 4␣ carbinolamine dehydratase.41 In the liver, HNF1␣ is coexpressed with the isoform HNF1␤ (previously called vHNF1) and forms heterodimers with the HNF1␤-related family member.42 The orphan nuclear receptor HNF4␣ protein utilizes the zinc finger DNA-binding domain to recognize DNA while both the DNA- and ligand-binding domain (Fig. 2E) are used to form homodimers or heterodimers with retinoic X receptor ␣.43

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Fig. 2. Functional protein domains in liver transcription factors. (A) Functional protein domains of the Fox transcription factors. Schematically shown is the Forkhead/winged helix DNA-binding domain (magenta, percent amino acid homology) of the 5 liver Fox transcription factors, which contains sequences required for nuclear localization.26 Indicated on the HNF3 proteins are their conserved N-terminal (purple) and C-terminal (conserved region II [red] and region III [orange]) transcriptional activation domains.26,56 Also shown are the transcriptional activation domains (TAD; various colors) for Foxf1, and FoxM1B proteins.29-31 (B) Functional protein domains of the HNF6 transcription factor. The HNF6 protein binds to DNA as a monomer through the cut-homeodomain DNA-binding domain and defines a new class of transcription factors called the “ONECUT” (OC) family.10,32-34 The cuthomeodomain contains sequences that mediate nuclear localization and transcriptional activation and an N-terminal STP box sequence is also required for transcriptional activity.27,35 (C) Functional protein domains of the C/EBP␣ transcription factor. The C/EBPs utilize a basic leucine zipper (bZIP) bipartite DNA-binding domain consisting of a dimerization interface composed of heptad-repeated leucine residues termed the “leucine zipper” and a DNA-binding interface consisting of basic amino acids.36-38 The C/EBP␣ protein contains two regions (AD1 and AD2) required for transcriptional activation.108 (D) Functional protein domains of the HNF-1␣ transcription factor. The HNF1␣ binds to its DNA recognition sequence as a dimer using a POU-homeodomain DNA-binding domain through a myosin-like dimerization domain located at the amino terminus of the protein.39,40 The HNF1␣ transcriptional activation domain is located at the C-terminus of the protein.192 (E) Functional protein domains of the HNF4␣ transcription factor. The orphan receptor family member HNF4␣ protein utilizes both the zinc finger DNA-binding and ligand-binding domain to recognize DNA either as a homodimer or a heterodimer with retinoic X receptor ␣.43 The location of the HNF4␣ transcriptional activation domains (AF-1 and AF-2) and proline rich region was obtained from Sladek and Seidel, 2001.193

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Additional liver-enriched transcription factors have been identified through the analysis of DNA regulatory regions of liver genes. These include ␣-fetoprotein promoter-linked coupling element factor,44 the Nkx-2.8 homeodomain family member45 and the fetoprotein transcription factor (FTF), the latter of which is a member of the orphan nuclear receptor Drosophila FTZ-F1 family.18,46 Although the Nkx-2.8 and FTF proteins are members of different families of transcription factors, they exhibit similar DNA-binding specificities and target promoter regulation.47 Liver Transcription Factor Activation Domains. The transcription factor activation domain contains amino acid sequences that mediate increased transcription or synthesis of an RNA copy of the gene, which is processed into mature mRNA and transported into the cytoplasm for translation of encoded protein. Initiation of gene transcription involves the formation of a transcriptional initiation complex that assembles at the TATA box sequences located 25 to 35 nucleotides proximal to the start site of gene transcription. This transcriptional initiation complex consists of the TATA box binding protein (TBP), which forms a complex with a large number of distinct TBP-associated factors (TAF) and basal transcription factors that are involved in recruiting RNA polymerase II to the transcriptional initiation site of a gene.48 A subset of the transcriptional activation domains interacts with the TAFs to stabilize this transcriptional initiation complex, thus stimulating the rate of gene transcriptional initiation.49 Transcription factor activation domains also recruit coactivator proteins involved in either chromatin remodeling such as the SWI/SNF family of ATP-dependent chromatin remodeling enzymes50 or chromatin modifications such as the p300/CREB binding protein (CBP) family of histone acetyltransferases.49,51-54 The p300/CBP coactivator proteins neutralize the positively charged residues of histone proteins, diminishing their association with DNA, and thus provide accessibility of the DNA regulatory regions to bind other transcription factors.49 The transcriptional activation domains of nuclear receptors recruit distinct complexes of coregulator proteins that either stimulate or repress transcription depending on ligand occupancy.55 The FoxA (HNF3) proteins possess homology in the N-terminal and C-terminal transcriptional activation domains,26,56 the latter of which contain the functionally important region II and III sequences (Fig. 2A) that are conserved in the Fox protein family.57 FoxA proteins are involved in organizing the nucleosome architecture of the ⫺10-kb albumin enhancer sequences,58-60 and they exhibit more stable binding to the nucleosome-assembled

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DNA templates containing the albumin enhancer sequences.61 The HNF6 protein possesses an N-terminal transcriptional activation domain within an STP Box sequence (Fig. 2).35 The HNF6 cut-homeodomain sequences were shown to interact with CBP in vitro and in vivo through the LXXLL motif within the cut domain and methionine (M) and phenylalanine (F) residues within the homeodomain.27,35 Moreover, expression of the HNF6 transcription factor is increased in response to growth hormone through a signal transducer and activator of transcription 5 (Stat5) binding site in the HNF6 promoter region and through preventing C/EBP␣-mediated reduction in transcription of the Hnf6 gene.62-64 The HNF6 cut-homeodomain associated with the FoxA2 winged helix motif and synergistically stimulated FoxA2 transcriptional activity in cotransfection assays through recruitment of the p300/C/EBP coactivator proteins.27 Interestingly, using the HNF6-regulated Glut2 promoter, we observed that FoxA2 association with HNF6 prevented HNF6 from binding to its DNA recognition sequence, thereby significantly diminishing HNF6 transcriptional activation.27 These data suggest that at a FoxA-specific site, HNF6 serves as a coactivator protein to recruit the p300/CBP protein to enhance FoxA2 transcription, thus providing a mechanism for synergistic transcriptional activation among liver transcription factors (Fig 3). In contrast, at an HNF6-specific site, FoxA2 represses HNF6 transcription by inhibiting HNF6 DNA– binding activity (Fig. 3). Consistent with an important role of the p300/CBP coactivators in regulating transcription of hepatocyte-specific genes, transcriptional activity of the HNF4␣,65,66 HNF1␣,67,68 HNF6,35 and C/EBP69 proteins involves recruitment of the p300/CBP coactivator proteins. The p300/CBP proteins acetylate a lysine residue within the HNF4␣ nuclear localization sequence and stimulate HNF4␣ nuclear retention, DNA-binding activity, and transcriptional activity via enhanced recruitment of the CBP protein.70 Transcriptional activity of HNF4␣ has been reported to be modulated through binding of the endogenous ligand fatty acyl CoA thioesters (long chain) and through protein phosphorylation.71-73 More recent studies have shown that Hnf4␣ is essential for transcription of the phosphoenolpyruvate carboxykinase (Pepck) and glucose-6 phosphatase genes through recruitment of the peroxisome proliferator-activated receptor ␥ (PPAR␥) coactivator-1␣ (PGC-1␣) protein.74 Transfection studies utilizing the albumin promoter containing an HNF1␣-binding site mutation abrogated transcriptional activity of the albumin promoter and enhancer region, suggesting that HNF1␣ is of critical

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Fig. 3. FoxA2 and HNF6 interactions either synergistically stimulate transcription at FoxA sites or repress transcription at HNF6 sites. We showed that association between these liver transcription factors requires the HNF6 cut-homeodomain and FoxA2 winged helix DNA-binding domains. On a FoxA-dependent promoter, HNF6 and FoxA2 protein interaction synergistically stimulates transcription by HNF6-mediated recruitment of the p300/CBP histone acetyltransferase proteins.27 The p300/CBP proteins possess histone acetyltransferase activity that enhances transcription by acetylating the lysine residues of histone proteins, thereby diminishing their association with DNA.49,51-54 On an HNF6-specific site, the association between the HNF6 and FoxA2 proteins causes inhibition of HNF6 DNA binding activity, thereby causing reduced HNF6 transcriptional activity.

importance in coordinating activity between the transcription factors binding to the proximal and distal regulatory regions.11 Furthermore, disruption of the Hnf1␣ transcription factor causes methylation of the phenylalanine hydroxylase (PAH) promoter region, blocks hepatic chromatin remodeling of the PAH locus, and results in undetectable transcription levels of the PAH gene.75 Using human hepatoma cell lines that express fetoprotein (AFP) gene, antisense inhibition of Nkx2.8 homeodomain mRNA translation selectively reduced expression of endogenous human AFP gene, suggesting that Nkx2.8 protein is critical for transcriptional activation of the AFP gene.47

Transcription Factors and Liver Development During mouse embryonic development, the liver bud begins to form at 9 days post coitum (dpc) as a proliferative outgrowth from the ventral foregut endoderm. Primitive hepatic cells derived from the cranial part of the liver primordium proliferate, delaminate from the foregut endoderm, and invade the septum transversum mesenchyme.76,77 Hepatic specification involves inductive and growth factor signaling originating from both the septum transversum and cardiac mesenchyme to give rise to hepatic cords and establish the final hepatic architecture.76,77 At later stages of liver development, bipotential

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Table 1. Transcription Factors in Development of Liver, Bile Ducts, and Gallbladder Transcription Factor and DBD

References

Hex ⫺/⫺ mice homeodomain

81, 82

Hlx ⫺/⫺ mice divergent homeodomain

83

c-Jun ⫺/⫺ mice bZIP RelA ⫺/⫺ or IKK-␤ ⫺/⫺ or IKK-␥ ⫺/⫺ mice

84,85

Rel A ⫺/⫺ Tnfr-1 ⫺/⫺ double knockout mice XBP-1 ⫺/⫺ mice bZIP Jumonji ⫺/⫺ mice

92

89-91

93 94

Foxf1 ⫹/⫺ mice winged helix

100

Hnf6 ⫺/⫺ mice cut-homeodomain (ONECUT)

96,97,187

AFPp Cre Hnf1␤ ⫺/⫺ Mice divergent Pou-homeodomain

98

Description of Embryonic or Postnatal Liver Phenotype in Knockout Mice

Hex ⫺/⫺ embryos displayed defective liver development, but normal formation of the mouse liver bud and expression of hepatocyte transcription factors. Hepatic progenitor cells fail to migrate into the septum transversum mesenchyme and undergo liver morphogenesis. Embryonic lethality at 10 dpc. Hlx is expressed in visceral mesenchyme of the developing liver, gallbladder, and gut. Hlx ⫺/⫺ embryos exhibit normal formation of the liver diverticulum and differentiation of hepatocytes, but liver failed to proliferate and undergo morphogenesis (3% of normal size). Severe decrease in proliferative expansion of the intestine also observed. c-Jun ⫺/⫺ embryos exhibit lethality at 13.5 dpc resulting from impaired hepatogenesis and increased apoptosis of hepatoblasts in developing liver. Disrupted activation of NF-␬B transcription factor by mutation of genes critical for the NF-␬B signaling pathway (RelA ⫺/⫺, IKK-␤ ⫺/⫺, or IKK-␥ ⫺/⫺) causes embryonic lethality from extensive hepatoblast apoptosis in developing liver. Rel A is p65 subunit of the NF-␬B transcription factor, whereas IKK-␤ and IKK-␥ are involved in activating NF-␬B via phosphorylating inhibitor I␬B. Tnfr-1 ⫺/⫺ NF-␬B p65 RelA ⫺/⫺ double knockout embryonic livers exhibit no apoptosis and normal liver development, suggesting TNFR-1 signaling is causing apoptosis in the absence of NF-␬B activation. XBP-1 is a CREB/ATF family transcription factor highly expressed in hepatocellular carcinomas. Impaired development of XBP-1 ⫺/⫺ livers with diminished proliferation and increased apoptosis of hepatoblasts. Jumonji ⫺/⫺ livers display defects in hepatoblast differentiation, increased apoptosis of hepatoblasts at the periphery of the liver, and abnormal development of thymus and spleen. Foxf1 is expressed in gut (visceral) mesoderm and septum transversum mesoderm during mouse development. Foxf1 ⫹/⫺ mice exhibit severe defects in development of gallbladder—diminished mesoderm expression of vascular cell adhesion molecule-1 (VCAM-1), ␣5 integrin, platelet-derived growth factor receptor ␣ and hepatocyte growth factor (HGF). Hnf6 ⫺/⫺ embryos fail to develop a gallbladder and displayed severe defects in formation of extrahepatic and intrahepatic bile ducts. Hnf6 ⫺/⫺ mice are diabetic with severe defects in pancreatic islets. Hnf6 is required for transcription of the liver glucokinase gene. AFPp-Cre mediates deletion of Hnf1␤ LoxP/LoxP (fl/fl) targeted allele in developing liver. HNF1␤ deficiency causes severe jaundice caused by abnormalities of the gallbladder and intrahepatic bile ducts (IHBD). Diminished hepatic expression of Oatp1 bile acid transporter and fatty acid dehydrogenase VLCAD.

Abbreviation: DBD, DNA-binding domain.

hepatoblasts selectively differentiate into either hepatocytes or bile duct epithelial cells.76-78 Beginning at approximately 10 dpc of mouse embryogenesis, the caudal part of the liver primordium gives rise to the extrahepatic bile ducts, the cystic duct and the gallbladder, which remain in continuity with the foregut and connect the liver hilum with the digestive tract.78,79 At 15.5 dpc, periportal hepatoblasts coalesce around the portal mesenchyme and undergo a complex morphogenesis process culminating in the formation of intrahepatic bile ducts (IHBD) at 17 dpc.78 In the adult liver, bile is synthesized in hepatocytes, is secreted into the bile canaliculi, and flows through the IHBD into the extrahepatic bile ducts to the gallbladder where it is released into the digestive tract to emulsify lipids.78,80 Liver Development Requires Retention of Numerous Proliferation-Specific Transcription Factors and Mesoderm-Epithelial Cell Cross Talk. Targeted disruption of the homeodomain Hex (⫺/⫺) gene, which is expressed in the anterior visceral and definitive endoderm and the hepatic diverticulum, allows normal development of the mouse liver bud, but these cells fail to migrate into the septum transversum and undergo neither liver growth nor morphogenesis.81,82 The homeodomain Hlx gene is

expressed in the gut (visceral) mesoderm that regulates expression of mesenchymal genes, which are critical for the induction of organ morphogenesis from the gut endoderm.83 Consistent with this mesoderm-inductive signaling, Hlx ⫺/⫺ embryos exhibit severe defects in liver and intestine development.83 Hlx ⫺/⫺ embryos exhibit normal liver bud formation and hepatocyte differentiation, but failure of the hepatoblasts to proliferate led to an embryonic lethal phenotype with diminutive embryonic livers that are 3% of their normal size.83 These studies show that transcription factors expressed in both the gut endoderm and mesoderm play essential roles in liver development. A number of proliferation-specific transcription factors play important roles in liver development and survival (see Table 1). Disruption of the bZIP c-Jun gene causes extensive apoptosis of both hematopoeitic cells and hepatoblasts in 13 dpc mouse fetal livers, resulting in embryonic lethality.84,85 The nuclear factor ␬B (NF-␬B) transcription factor is negatively regulated and sequestered in the cytoplasm through association with its inhibitor I␬B protein. Growth factor or cytokine signaling results in activation of I␬B kinases (IKK) which phosphorylates the I␬B protein, causing its dissociation from

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NF-␬B protein and allowing NF-␬B nuclear translocation and transcriptional activation of its target genes.86-88 Liver degeneration and hepatic apoptosis were found in mouse embryos where the signaling pathway that activates the NF-␬B transcription factor89-91 such as the IKK-␤ ⫺/⫺ or IKK-␦ ⫺/⫺ mice or the RelA (NF-␬B p65) ⫺/⫺ mice, which encodes the p65 subunit of the NF-␬B transcription factor was disrupted. Interestingly, the tumor necrosis factor receptor-1 ⫺/⫺ (TNFR-1) RelA (NF-␬B p65) ⫺/⫺ double knockout embryos exhibit no liver apoptosis and normal development suggesting that apoptosis in RelA (NF-␬B p65) ⫺/⫺ mice is mediated by TNFR-1 signaling.92 XBP-1 ⫺/⫺ embryos die in utero from a severe liver phenotype resulting from significantly reduced growth of the liver and prominent apoptosis of hepatoblasts.93 Jumonji ⫺/⫺ livers displayed defects in hepatoblast differentiation, increased apoptosis of hepatoblasts at the periphery of the liver and abnormal development of thymus and spleen.94 These studies identified transcription factors mediating the proliferative expansion and survival of hepatoblasts, which are essential for normal liver development. Transcription Factors Involved in Development of the Gallbladder and Intrahepatic Bile Ducts. In the developing mouse liver, HNF6 is expressed in hepatocytes and in the epithelial cells of the intrahepatic and extrahepatic bile ducts.34,95 Hnf6 ⫺/⫺ mouse embryos also fail to develop a gallbladder and exhibited severe abnormalities in both extrahepatic and intrahepatic bile ducts, which was associated with diminished expression of the Hnf1␤ transcription factor.96,97 Interestingly, use of the ␣-fetoprotein enhancer albumin promoter-enhancer– driven Cre recombinase transgene (AFPp-Cre) to delete the Hnf1␤ LoxP (Floxed) targeted allele in hepatoblasts caused developmental abnormalities in the gallbladder and IHBD similar to, but less severe than those found in the Hnf6 ⫺/⫺ mouse embryos.98 The defects in AFPpCre Hnf1␤ ⫺/⫺ IHBD also caused developmental abnormalities in the intralobular arteries, suggesting an interaction between development of periportal hepatic bile ducts and arteries.98 These studies show that HNF6 is essential for regulating expression of Hnf1␤ that plays an important role in development of the gallbladder and IHBD. The Foxf1 transcription factor is expressed in embryonic gut mesoderm and septum transversum mesoderm, suggesting that Foxf1 participates in mesenchymal-epithelial cell inductive signaling of the internal organs. Consistent with this hypothesis, Foxf1 ⫹/⫺ mice displayed a variety of developmental abnormalities in the lung, gallbladder, esophagus, and trachea.99-102 Foxf1 ⫹/⫺ gallbladders exhibited malformation of the external smooth

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muscle cell layer, reduction in mesenchymal cell number, and in some cases, lack of a discernible biliary epithelial cell layer.100 However, Foxf1 ⫹/⫺ mice displayed normal liver function and development of IHBD.100 Abnormal Foxf1 ⫹/⫺ gallbladders fail to express genes critical for mesenchymal-epithelial cell induction of gallbladder morphogenesis such as vascular cell adhesion molecule-1, ␣5 integrin, platelet-derived growth factor receptor ␣ and hepatocyte growth factor genes.100

Transcription Factors and Hepatocyte Differentiation C/EBP␣ Regulates Transcription of Genes Involved in Hepatic Glycogen Synthesis, Gluconeogenesis, and Lipid Homeostasis. Expression of C/EBP␣ in developing liver and intestine is observed in 13-dpc mouse embryos and in adult mice, C/EBP␣ is expressed in differentiated hepatocytes, adipocytes, keratinocytes, and myeoloid cells as well as epithelial cells of the lung, intestine, adrenal gland, and placenta.103 C/EBP␣ ⫺/⫺ mice die from hypoglycemia within 8 hours postpartum due to a complete absence of hepatic glycogen storage and a failure to store lipid in hepatocytes and adipocytes.104 The hepatic glycogen storage defect is due to diminished postnatal expression of glycogen synthase and gluconeogenic enzymes Pepck and glucose-6 phosphatase (Table 2). Consistent with the antiproliferative activity of C/EBP␣ protein, C/EBP␣ ⫺/⫺ hepatocytes show increased proliferation and disruption of the normal liver and lung architecture.105,106 Further characterization of this aberrant proliferation phenotype in C/EBP␣ ⫺/⫺ hepatocytes shows that C/EBP␣ inhibits cyclin-dependent kinase 2 (Cdk2) by cooperating with the Cdk inhibitor p21Cip1 protein.107,108 These studies show that C/EBP␣ regulates expression of genes involved in hepatic glucose and lipid homeostasis as well as negatively regulating hepatocyte proliferation. Hnf1␣ and Hnf4␣ Regulate Transcription of Genes Essential for the Hepatocytic Cell Lineage. In the adult mouse, the Pou-homeodomain HNF1␣40,109 and steroid hormone receptor HNF4␣110-112 transcription factors are expressed in hepatocytes and in epithelial cells of the pancreas (islets), intestine, stomach, and kidney. The Hnf1␣ and Hnf4␣ genes are also mutated in pedigrees of human families suffering from maturity onset diabetes of the young (MODY), which is manifested by non–insulin-dependent diabetes.113,114 The fact that mutation of Hnf1␣ and Hnf4␣ transcription factors causes non–insulin-dependent diabetes suggests that they regulate expression of genes critical for glucose homeostasis. Consistent with this hypothesis, Hnf1␣ ⫺/⫺ mice

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Table 2. Transcription Factors Required for Hepatocyte Differentiation Transcription Factor and DBD

References

C/EBP␣ ⫺/⫺ mice basic domain leucine zipper (bZIP)

104,105

Hnf1␣ ⫺/⫺ mice divergent Pouhomeodomain

109,117

Hnf4␣ ⫺/⫺ mice (tetraploid rescue) zinc finger orphan receptor AFPp-Cre Hnf4␣ ⫺/⫺ mice zinc finger orphan receptor Alb-Cre Hnf4␣ ⫺/⫺ mice zinc finger orphan receptor Foxa3 (Hnf3␦) ⫺/⫺ mice winged helix TTR-FoxA2 (HNF3␤) TG mice winged helix

16

119

120

129,130

131,133

Description of Embryonic or Postnatal Liver Phenotype in Transgenic or Knockout Mice

C/EBP␣ ⫺/⫺ hepatocytes fail to store glycogen—hepatocytes and adipocytes do not store lipid. Diminished postnatal hepatic expression of glycogen synthase, gluconeogenic enzymes PEPCK, and glucose-6phosphatase. Increased proliferation levels of C/EBP␣ ⫺/⫺ hepatocytes. C/EBP␣ ⫺/⫺ pups die from hypoglycemia within 8 hours after birth. Hnf1␣ ⫺/⫺ livers display undetectable postnatal hepatic expression of phenylalanine hydroxylase leading to phenylketonuria. Hnf1␣ ⫺/⫺ livers exhibit diminished expression of albumin, ␣1-antitrypsin, and ␤fibrinogen, and compensatory increase in hepatic levels of Hnf1␤. Defective pancreatic secretion of insulin and severe kidney defect. Tetraploid rescued Hnf4␣ ⫺/⫺ fetal livers exhibit impaired hepatocyte differentiation—diminished expression of pregnane-X-receptor (PXR); Hnf1␣; albumin (Alb); ␣-fetoprotein (AFP); transferrin (TFN); apolipoprotein (Apo) A1, A4, B, C3, and C2; phenylalanine hydroxylase (PAH); L-type fatty acid binding protein; erythropoietin and retinol binding protein (RBP). AFPp-Cre Hnf4␣ ⫺/⫺ embryonic hepatocytes (18.5 dpc) failed to store glycogen—decreased expression of glycogen synthase (Gys2), phosphoenolpyruvate carboxykinase (Pepck; Pck1), and glucose-6-phosphatase (G6pc). Disruption in hepatocyte expression of cell adhesion/junction molecules E-cadherin, ZO-1, CEACAM1, connexins 32 (Gjb1) and 26 (Gjb2), and severely affected livers have disrupted architecture. Use of Alb-Cre for postnatal hepatocyte-specific deletion of Hnf4␣fl/fl targeted allele. Alb-Cre Hnf4␣ ⫺/⫺ hepatocytes exhibited accumulation of lipid, reduced serum cholesterol and triglyceride levels, and increased serum bile acid concentrations. Diminished hepatic expression of Apo B, microsomal triglyceride transfer protein, liver fatty acid binding protein, and bile acid transport proteins (Ntcp and Oatp1). Foxa3 ⫺/⫺ livers display 50% reduction in tyrosine aminotransferase, Pepck, transferrin (TFN) expression. Compensatory increase in expression of Foxa1 and Foxa2. Foxa3 is required for maintenance of glucose homeostasis during a prolonged fast with diminished hepatic expression of glucose transporter 2 (Glut-2). TTR-FoxA2 transgenic hepatocytes do not store glycogen and display transient increase in lipid accumulation, which is associated with increased synthesis of lipid and lipid ␤-oxidation resulting in mitochondrial damage. TTR-FoxA2 livers exhibited 50% decrease in expression of Pepck, glycogen synthase, Glut-2, C/ EBP␣, and HNF4 and significant reduction in endogenous levels of Foxa1, Foxa2, Foxa3, and Hnf6. Increase serum levels of bile acid (diminished expression of Ntcp) and bilirubin (conjugating enzyme).

Abbreviation: DBD, DNA-binding domain.

exhibit defective glycolytic signaling of pancreatic ␤-cells resulting in diminished insulin secretion in response to a glucose challenge.115-117 Hnf1␣ ⫺/⫺ mice died at the time of weaning due to a severe wasting syndrome109 with massive glucosuria, phosphaturia, and aminoaciduria from renal tubular dysfunction (Table 2). Hepatic expression of PAH is also completely extinguished in the Hnf1␣ ⫺/⫺ mice, causing a phenotype seen in the human disease phenylketonuria.109,116 Hnf1␣ ⫺/⫺ mice also exhibited a partial reduction in hepatic expression of albumin, ␣1-antitrypsin, and fibrinogen and probably because of a compensatory increase in vHNF1 (HNF1␤) levels.109,116 Thus, Hnf1␣ plays an important role in the transcriptional activation of differentiated hepatocyte-specific genes critical for liver function but is not required for specification of the hepatocytic cell lineage. In mouse development, Hnf4␣ is expressed in the primary and extraembryonic visceral endoderm prior to gastrulation and in epithelial cells at the onset of liver, pancreas, and intestine formation.110 Consistent with this early embryonic expression pattern, Hnf4␣ ⫺/⫺ embryos exhibited a severe visceral endoderm defect preventing gastrulation and causing a failure to develop past 6.5 dpc.118 Tetraploid rescue of the visceral endoderm defect

in Hnf4␣ ⫺/⫺ embryos restores gastrulation and allowed formation of the liver and other organs to proceed.13 Analysis of the tetraploid rescued 12.5-dpc mouse Hnf4␣ ⫺/⫺ liver for altered expression of genes showed that Hnf4␣ is critical for transcriptional regulation of the orphan receptor pregnane-X-receptor (PXR) and for cross regulation of the Hnf1␣ transcription factor, which is required for PAH expression.16 Diminished expression of albumin, AFP, transferrin (TFN), several distinct apolipoproteins, L-type fatty acid binding protein, erythropoietin, and retinol binding protein was also found in the 12.5 dpc Hnf4␣ ⫺/⫺ liver (see Table 2). Consistent with a role of Hnf4␣ in glucose homeostasis, AFPp-Cre Hnf4␣ ⫺/⫺ embryonic (18.5 dpc) hepatocytes failed to store glycogen (Table 2), which was associated with decreased expression of glycogen synthase (Gys2), Pepck (Pck1), and glucose-6-phosphatase (G6pc) genes.119 Disruption in hepatocyte expression of cell adhesion/junction molecules E-cadherin, ZO-1, CEACAM1, and connexins 32 (Gjb1) and 26 (Gjb2) was found in Hnf4␣-deficient hepatocytes and in the severely affected AFPp-Cre Hnf4␣ ⫺/⫺ embryos, in which the liver architecture was markedly disrupted with loss of organized hepatic cords and sinusoids.119 Moreover, infec-

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tion of NIH 3T3 fibroblasts with Hnf4␣-expressing retrovirus causes conversion to epithelial cell morphology as evidenced by induced expression of E-cadherin and ZO-1.119 These studies showed that Hnf4␣ is critical for regulating transcription of genes involved in gluconeogenesis and glycogen synthesis and in cell adhesion required for epithelial cell morphology. Postnatal hepatocyte-specific deletion of Hnf4␣ fl/fl targeted allele with the albumin promoter and enhancer driven Cre recombinase (Alb-Cre) showed that Hnf4␣ ⫺/⫺ hepatocytes displayed aberrant accumulation of lipid, reduced serum cholesterol and triglyceride levels, and increased serum bile acid concentrations.120 Alb-Cre Hnf4␣ ⫺/⫺ liver exhibited diminished hepatic expression of apolipoprotein B, microsomal triglyceride transfer protein, liver fatty acid binding protein, and bile acid transport proteins sodium taurocholate cotransporter protein (Ntcp) and organic anion transporter protein 1 (Oatp1).120 These studies showed that Hnf4␣ is critical for regulating transcription of genes involved in postnatal lipid, cholesterol, and bile acid homeostasis. The Three Homologous FoxA Proteins Display Functional Redundancy in the Developing and Adult Liver. Foxa2 ⫺/⫺ embryos died in utero prior to liver formation because of severe defects in gastrulation of the embryo resulting in abnormalities of the node, notochord, gut endoderm, and visceral endoderm.121,122 During organogenesis, Foxa2 and Foxa1 genes are expressed in epithelial cells of the developing liver, esophagus, trachea, salivary gland, lung, pancreas, intestine, and stomach.123 Furthermore, Foxa1 is expressed exclusively in epithelial cells of the renal pelvis, prostate gland, bladder, and urinary tract,17,124 whereas Foxa3 (Hnf3␥) expression is restricted to the liver, pancreas, stomach, gut, testes, and ovaries.125 Because of the functional redundancy of the 3 Foxa proteins in the liver, use of the Alb-Cre transgene protein to conditionally delete the Foxa2 LoxP/LoxP (fl/fl) targeted allele in adult hepatocytes had no apparent effect on liver gene expression.126 No liver phenotype was noted in the Foxa1 ⫺/⫺ postnatal mice, but they were hypoglycemic because of reduced pancreatic islet expression and secretion of glucagon.127,128 Foxa3 (Hnf3␦) ⫺/⫺ livers (Table 2) displayed a 50% reduction in hepatic expression of tyrosine aminotransferase, Pepck, and TFN with compensatory increases in levels of Foxa1 and Foxa2 genes, suggesting that disruption of the Foxa3 gene is not sufficient to cause severe defects in hepatic function.129 Moreover, Foxa3 ⫺/⫺ mice display reduced hepatic expression of glucose transporter-2 (Glut-2) and diminished serum levels of glucose during a prolonged fast,130 suggesting

HEPATOLOGY, December 2003

that Foxa3 plays a unique role in mediating hepatic glucose homeostasis during fasting. Increased Hepatocyte Foxa2 Levels Cause Depletion of Glycogen, Increased Hepatocyte Lipid Accumulation, and Mitochondrial Damage. The early lethality of Foxa2 ⫺/⫺ embryos along with the hepatic functional redundancy of the 3 hepatic Foxa proteins has precluded examination of Foxa2 in vivo hepatocyte target genes. To circumvent the functional redundancy of Foxa proteins, we developed the T-77 transgenic (TG) mouse line in which the ⫺3-kb TTR promoter was used to increase hepatocyte expression of the FoxA2 (HNF3␤) cDNA.131 Adult TTR-FoxA2 TG mice exhibited reduced hepatocyte glycogen, but maintained normal serum levels of glucose, insulin, and glucagon and displayed increased serum levels of bile acid and bilirubin (Table 2). This phenotype was associated with a 50% reduction in the hepatic expression of Pepck, glycogen synthase, and Glut-2 genes and bile acid sinusoidal transporter sodium taurocholate cotransporter protein (Ntcp) and bilirubin conjugating enzyme UDP glucuronosyltransferase.131,132 Significantly reduced levels of the endogenous Foxa1, Foxa2, Foxa3, and Hnf6 transcription factors as well as a 50% reduction in C/EBP␣ and Hnf4␣ expression was found in TTRFoxA2 TG livers.131,132 Postnatal TTR-FoxA2 TG mice displayed significant reduction in hepatocyte glycogen storage and serum glucose levels without increased serum levels of ketone bodies and free fatty acid, suggesting that they are not undergoing a starvation response. We demonstrate that TG liver developed a substantial transient accumulation of lipid (steatosis), which reached a maximum at postnatal day 5 and is associated with increased expression of hepatic genes involved in fatty acid and triglyceride synthesis, lipid ␤-oxidation, and amino acid biosynthesis.133 Furthermore, transmission electron microscopy analysis of postnatal TG liver revealed extensive mitochondrial membrane damage, which is likely due to reactive oxygen species generated from lipid ␤-oxidation. HNF6 Mediates Transcriptional Synergy With FoxA2. Because the TTR-FoxA2 TG liver phenotype was associated with an increase in FoxA2 levels and significantly reduced expression of HNF6, we used adenovirus-mediated delivery to increase hepatic levels of both Foxa2 and HNF6. Equivalent increases in hepatic expression of FoxA2 and HNF6 restored hepatic glycogen and Glut-2 mRNA to levels found in wild-type (WT) liver,134 suggesting that FoxA2 and HNF6 interacted synergistically to influence hepatocyte-specific gene transcription. Consistent with this hypothesis, cotransfection studies showed that HNF6 synergizes with FoxA2 to potentiate FoxA2 transcriptional activity by recruiting the p300/C/ EBP coactivator proteins.27

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Transcription Factors and Liver Regeneration Cell Cycle Regulatory Proteins Tightly Control Cellular Proliferation. Cellular proliferation involves stimulation of the mitogen-activated protein kinase (MAPK) pathway consisting of the Ras/Raf1/MEK/MAPK cascade135,136 and activation of the phosphoinositide 3-kinase (PI3K) pathway consisting of the PI3K/phosphoinositide-dependent kinase-1 (PDK1)/Akt cascade.137 However, cell division is tightly regulated at the G1/S (DNA replication) and G2/M (mitosis) transitions of the cell cycle by temporal activation of multiple cyclindependent kinases (Cdk) complexed with their corresponding cyclin regulatory subunits. Stimulation of Cdk2-cyclinE/A or Cdk1-cyclin B complex kinase activity is essential for progression through the cell cycle G1/S and G2/M transitions, respectively. Furthermore, Cdk activity is inhibited by phosphorylation of Thr 14 and Tyr 15 by the dual specific Myt1 kinase,138-141 and these residues are dephosphorylated by Cdc25A phosphatase (G1/S phase), Cdc25B (late S-phase), and Cdc25C phosphatases (G2/M phase) to stimulate Cdk activity.142-144 It is well established that Cdk2 activity in complex with either cyclin E or cyclin A cooperates with cyclin D-Cdk4/6 to phosphorylate the retinoblastoma protein, which releases bound E2F transcription factor and allows it to stimulate expression of genes required for S-phase.145,146 The Cdk1-cyclin B complexes drive Mphase progression through phosphorylation of protein substrates that are essential for chromosome segregation, breakdown of the nuclear envelope, and cytokinesis.147-152 Hepatocyte Proliferation During Liver Regeneration Requires Activation of Immediate Early Transcription Factors. The mammalian liver is one of the few adult organs capable of completely regenerating its original size in response to cellular injury from toxins, viral hepatitis, or tissue resection.153-155 Liver regeneration induced by two-thirds partial hepatectomy (PHx) results in synchronous induction of hepatocyte DNA replication (S-phase) and mitosis. This proliferative response is initiated by the release of the growth factors tumor growth factor ␣ (TGF-␣), hepatocyte growth factor, and the cytokines tumor necrosis factor ␣ (TNF-␣) and interleukin 6 (IL-6), which mediate reentry of terminally differentiated hepatocytes into the cell cycle.155-158 This hepatocyte proliferative response during liver regeneration is coincident with a potent activation of immediate early transcription factors including c-Jun, c-Fos, c-Myc, NF-␬B, Stat3, and the C/EBP␤ proteins. Mouse genetic studies showed that the cytokine IL-6 plays an important role in establishing responsiveness of hepatocytes to growth fac-

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tors, which are released following liver injury.159,160 Following PHx, IL-6 ⫺/⫺ or TNF receptor type 1 (Tnfr-I) ⫺/⫺ mice exhibited a 70% reduction in hepatocyte DNA replication, which was reversed by an intraperitoneal injection of IL-6 prior to surgery.159-161 This decrease in proliferation of regenerating hepatocytes was accompanied by failed induction of immediate early transcription factors including Stat3, c-Jun and c-Fos (AP-1), NF-␬B, and c-Myc. Genetic studies using transgenic and knockout mice have implicated several growth-regulated transcription factors in mediating hepatocyte proliferation during liver regeneration. Liver regeneration studies using either the H-2K promoter or albumin promoter to drive premature expression of the c-Myc transgene resulted in a 10-hour earlier onset of hepatocyte proliferation, which correlated with premature expression of cyclin A and cdc2 (Cdk1) genes.162,163 Regenerating liver exhibits decreased levels of the anti-proliferative C/EBP␣ protein, while significant increases in expression of the C/EBP␤ and C/EBP␦ isoform proteins were found.158,164,165 Consistent with this induction, the C/EBP␤ ⫺/⫺ mice displayed a 75% reduction in replicating hepatocytes following PHx with coincident decreases in expression of immediate early EGR-1 transcription factor and in cyclin B and E expression levels.166 Furthermore, TGF-␣ activation of p90Rsk kinase mediates site-specific phosphorylation of C/EBP␤ protein, which is critical for C/EBP␤ transcriptional activity required to mediate both hepatocyte proliferation and survival during liver regeneration.167-169 Regenerating cAMP responsive promoter element modulator (Crem) ⫺/⫺ livers exhibited delayed hepatic proliferation with a 50% reduction in hepatocyte DNA replication.170 This is associated with diminished expression of c-Fos as well as the cell cycle regulators cyclin A, B, D, and E and cdc2 (Cdk1). Liver regeneration studies with AFPp-Cre c-Jun ⫺/⫺ mice showed that c-Jun deficiency caused significant reduction in hepatocyte DNA replication with an aberrant increase in hepatocyte apoptosis and lipid accumulation, suggesting that c-jun induction was critical for hepatocyte cell survival during liver regeneration.171 The Stat proteins are activated by tyrosine phosphorylation, which mediates dimerization through the Src homology 2 (SH2) domain and their nuclear translocation to activate Stat target genes.172 Regenerating Alb-Cre Stat3 ⫺/⫺ liver displayed 66% reduction in hepatocyte DNA replication with diminished expression of S-phase promoting cyclin D1 and cyclin E genes with compensatory increases in Stat1 protein levels.173 These studies show that the induction of immediate early transcription factors is essential for hepatocyte DNA replication following PHx.

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Premature Expression of the FoxM1B Transcription Factor Stimulates Earlier Expression of Cell Cycle Genes During Liver Regeneration. The FoxM1B transcription factor (previously known as HFH11B, Trident, Win, or MPP2) is expressed in every proliferating cell examined, but its expression is extinguished in cells undergoing terminal differentiation.31,174-176 More recent studies have shown that activation of FoxM1B protein is dependent on cell cycle signaling because FoxM1B transcriptional activity requires both Cdk-cyclin phosphorylation-dependent recruitment of p300/CBP and binding of activated Cdk-cyclin complexes to the activation domain.177 In regenerating liver, increased hepatic expression of FoxM1B occurs at the G1/S transition of the cell cycle, and its levels remain elevated throughout the period of proliferation.31 FoxM1B thus displays the expression kinetics of a delayed early transcription factor. To examine the role of FoxM1B in hepatocyte proliferation, we developed TG mice in which the ⫺3-kb TTR promoter was used to prematurely express the human FoxM1B (HFH11B) cDNA in regenerating hepatocytes.178,179 Premature expression of FoxM1B caused an 8-hour acceleration of hepatocyte entry into S-phase and mitosis. This was associated with earlier hepatic expression of S-phase promoting cyclin D1 and C/EBP␤, diminished induction of cdk inhibitor p21Cip1 (p21), as well as earlier expression of M-phase promoting cyclin B1, cyclin B2, cdc-2 (Cdk1), and cdc25B phosphatase.178,179 Increased Levels of FoxM1B Restore Hepatocyte Proliferation in Regenerating Livers of Old-Aged Mice. Liver regeneration experiments showed that diminished expression of FoxM1B and cell cycle regulatory genes is associated with reduced proliferation in regenerating livers of 12-month-old (old-aged) CD-1 mice compared with 2-month-old (young) WT mice.180 Interestingly, maintaining hepatic expression of FoxM1B alone in old-aged TTR FoxM1B TG mice is sufficient to restore hepatocyte DNA synthesis and mitosis to levels similar to those found in young regenerating mouse liver.180 Furthermore, FoxM1B-mediated stimulation of hepatocyte DNA replication was associated with increased expression of S-phase promoting cyclin D1 and cyclin A2 and diminished levels of p21 protein180 (Fig. 4). Increase in hepatocyte mitosis was associated with stimulated expression of cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55Cdc (Fig. 4). Cotransfection assays show that FoxM1B can directly activate the transcription of cyclin B1 and cyclin D1 promoters.180 Furthermore, adenovirus-mediated delivery of the FoxM1B cDNA (AdFoxM1B) in regenerating liver of old-aged Balb/c mice caused a significant increase in FoxM1B expression, hepatocyte DNA replication, and mitosis.181 This stimu-

HEPATOLOGY, December 2003

Fig. 4. Summary of cell cycle promotion genes stimulated by restoring FoxM1B in old-aged regenerating liver. Liver regeneration experiments demonstrated that diminished expression of FoxM1B and cell cycle regulatory genes is associated with reduced proliferation in regenerating livers of 12-month-old (old-aged) CD-1 mice compared with 2-month-old (young) WT mice.180 Maintaining hepatic expression of FoxM1B alone in old-aged TTR-FoxM1B TG mice is sufficient to restore hepatocyte DNA synthesis and mitosis and expression of cell cycle genes to levels similar to those found in young regenerating mouse liver.180 Schematically shown are the different phases of the cell cycle (arrows) and corresponding cell cycle promotion genes whose expression is stimulated (green, ⴙ) by elevated FoxM1B levels in regenerating liver of old-aged TTR-FoxM1B TG mice.180 We find that increased FoxM1B levels cause diminished protein levels of p21Cip1 (unpublished) or p27Kip1 (red, ⴚ) in regenerating liver.179,181 In addition, Foxm1b ⫺/⫺ regenerating hepatocytes displayed increased nuclear expression of p21Cip1 protein.

lation in regenerating hepatocyte DNA replication was associated with diminished expression of Cdk inhibitor p27Kip1 (p27) protein, leading to stimulation of Cdk2cyclinE/A complexes (Fig. 4). Similar to regenerating TG liver, AdFoxM1B-infected regenerating livers displayed elevated levels of M-phase promoting genes.181 Taken together, our studies show that FoxM1B controls the transcriptional network of genes that regulate cell cycle progression and that maintaining FoxM1B levels will prevent age-mediated decrease in cellular proliferation. The FoxM1B Transcription Factor Regulates Cell Cycle Genes Required for Hepatocyte Proliferation During Liver Regeneration. Recent studies with regenerating Alb-Cre Foxm1b ⫺/⫺ livers showed that Foxm1b deficiency caused a significant reduction in hepatocyte DNA replication and mitosis.182 Reduced hepatocyte DNA replication was associated with increased levels of Cdk inhibitor p21Cip1 (p21) protein and diminished expression of Cdc25A phosphatase, leading to decreased Cdk2 activation and progression into S-phase (Fig. 5). Activation of Cdk1 involves removal of inhibitory phosphate at residues Thr 14 and Tyr 15 by the Cdc25B phosphatase during late S-phase and thus Cdc25B activity is required to mediate the transition from S-phase to G2 phase of the cell cycle.142-144 Regenerating Alb-Cre

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Fig. 5. Diagram depicting FoxM1B regulation of cell cycle genes. Regenerating Alb-Cre Foxm1b ⫺/⫺ livers exhibited a significant reduction in hepatocyte DNA replication and mitosis.182 Regenerating Alb-Cre Foxm1b ⫺/⫺ hepatocytes display increased nuclear protein levels of the Cdk inhibitor p21Cip1 ( p21), suggesting that FoxM1B controls the expression of a gene that regulates p21 protein stability and diminishes Cdc25A phosphatase levels. Blue arrows represent positive regulation and black lines represent negative regulation. Because regenerating p21 ⫺/⫺ liver displayed increased Cdc25A expression and earlier nuclear localization of Cdc25A,194 we propose that increased p21 levels may influence Cdc25A phosphatase levels. Furthermore, the significant reduction in hepatocyte mitosis is associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase, which is required for Cdk1 activation and entry into mitosis.142-144 Cotransfection assays showed that FoxM1B transcriptionally activates the Cdc25B promoter region.182

Foxm1b ⫺/⫺ livers displayed undetectable expression of the Cdc25B phosphatase and delayed accumulation of cyclin B1, both of which are essential for progression into mitosis through activation of the Cdk1-cyclinB complex. Cotransfection assays showed that FoxM1B is capable of directly activating transcription of the ⫺200-bp Cdc25B promoter region.182 Diminished hepatocyte proliferation in regenerating Alb-Cre Foxm1b ⫺/⫺ liver was thus associated with altered expression of proteins that limit Cdk1 and Cdk2 activity required for normal cell cycle progression into DNA replication and mitosis. Foxf1 ⴙ/ⴚ Mice Exhibit Defective Stellate Cell Activation and Abnormal Liver Regeneration Following CCl4 Injury. We showed that the Foxf1 transcription factor is expressed in septum transversum mesenchyme during mouse embryonic development and that its expression continues in the hepatic stellate cells in developing and adult liver.183 We used carbon tetrachloride (CCl4) liver injury because this liver regeneration process requires a transient differentiation of stellate cells into myofibroblasts, which secrete type I collagen into the extracellular matrix.184 We found that regenerating Foxf1 ⫹/⫺ liver exhibited defective stellate cell activation and increased pericentral hepatocyte apoptosis following CCl4 liver injury.183 Defective stellate cell activation in regenerating liver was associated with diminished induc-

tion of type I collagen, ␣-smooth muscle actin, and Notch-2 protein and decreased levels of interferon-inducible protein-10 and monocyte chemoattractant protein-1 (Table 3). Collectively, Foxf1 ⫹/⫺ mice exhibited abnormal liver repair, and diminished activation of hepatic stellate cells, implicating the Foxf1 transcription factor in regulating stellate cell function. Summary and Future Directions. The liver-enriched transcription factors (C/EBP, HNF1, HNF3, HNF4, and HNF6) bind to multiple promoter/enhancer sites and synergistically interact with each other to stimulate hepatocyte-specific gene transcription. Liver development requires retention of numerous proliferationspecific transcription factors that required proliferation, migration, and survival of hepatic progenitor cells. The HNF6 and HNF1␤ proteins are critical for gallbladder and bile duct development, while the mesodermal Foxf1 transcription factor is essential for gallbladder development. The HNF1␣, HNF4␣, Foxa2, and Foxa3 transcription factors regulate expression of genes critical for hepatocyte differentiation during embryonic and postnatal liver development. The nuclear orphan receptors LXR, FXR, and PPAR are also known to play critical roles in regulating expression of hepatic genes involved in lipid, cholesterol, and bile acid metabolism. These nuclear receptor knockout mouse studies have been summarized in

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HEPATOLOGY, December 2003

Table 3. Transcription Factors Involved in Hepatocyte Proliferation During Liver Regeneration Transcription Factor and DBD

AFPp-Cre c-Jun ⫺/⫺ mice bZIP H-2K or Alb c-myc TG mice C/EBP␤ ⫺/⫺ mice bZIP

References

171 162,163 166

Crem ⫺/⫺ mice bZIP

170

Alb-Cre Stat3 ⫺/⫺ mice Phospho-Tyr SH2mediated dimerization

173

TTR-FoxM1B (HFH11B) TG mice winged helix

178,179

TTR-FoxM1B 12-month-old (old-aged) TG mice winged helix

180,181

Alb-Cre Foxm1b ⫺/⫺ mice winged helix

182

Foxf1 ⫹/⫺ mice winged helix

183

Function of Transcription Factor in Adult Liver Regeneration

Conditional hepatocyte-specific deletion of c-Jun LoxP/LoxP (fl/fl) targeted allele using AFPp-Cre recombinase transgene. Regenerating AFPp Cre c-Jun ⫺/⫺ liver displayed reduced hepatocyte DNA replication and aberrant increase in hepatocyte apoptosis and lipid accumulation. c-Myc transgenic mice that overexpress c-Myc protein exhibited a 10-hour earlier onset of hepatocyte proliferation, which correlated with premature expression of cyclin A and cdc2 (Cdk1) genes. Regenerating C/EBP␤ ⫺/⫺ livers display a 75% reduction in replicating hepatocytes with coincident decreases in expression of immediate early EGR-1 transcription factor and in cyclin B and E expression levels. Regenerating Crem ⫺/⫺ livers exhibit a 50% reduction in hepatocyte DNA replication, which is delayed by 10 hours following PHx and paralleled by diminished expression of c-Fos as well as the cell cycle regulators cyclin A, B, D, and E and cdc2 (Cdk1). Alb-Cre recombinase transgene was used to delete Stat3 fl/fl targeted allele during liver regeneration. Regenerating Alb-Cre Stat3 ⫺/⫺ liver displays 66% reduction in hepatocyte DNA replication with diminished expression of cyclin D1 and cyclin E, which mediate progression into S-phase. Compensatory induction of Stat1 protein, which is not found in WT regenerating liver. Premature expression of FoxM1B in regenerating TTR-FoxM1B TG liver accelerated onset of hepatocyte DNA replication and mitosis and earlier expression of cell cycle regulatory genes. These include earlier expression of S-phase promoting cyclin D1 and C/EBP␤ and diminished induction of cdk inhibitor p21Cip1 (p21) and M-phase promoting cyclin B1, cyclin B2, cdc-2 (Cdk1), and cdc25B phosphatase. Maintaining FoxM1B expression in old-aged regenerating liver increases hepatocyte DNA replication and mitosis to levels found in young regenerating liver. This is associated with increased expression of S-phase promoting cyclin D1 and cyclin A2 and diminished levels of Cdk inhibitor proteins p21Cip1 and p27Kip1 and stimulates expression of M-phase promoting cyclin F, cyclin B1, cyclin B2, Cdc25B, and p55Cdc. Conditional hepatocyte-specific deletion of Foxm1b fl/fl targeted allele using Alb-Cre recombinase transgene. Diminished regenerating hepatocyte DNA replication and mitosis. Increased protein levels of cyclin dependent kinase (Cdk) inhibitor p21Cip1 and diminished expression of Cdc25A and Cdc25B phosphatases (required for activation of the Cdk-cyclin complexes). Foxf1 ⫹/⫺ mice display abnormal liver regeneration and increased apoptosis following CCl4 liver injury. Defective activation of stellate cells as evidenced by lack of induced expression of collagen and ␣-smooth muscle actin proteins. Reduced hepatic expression of interferon-inducible protein-10 and Notch-2 receptor after liver injury.

Abbreviation: DBD, DNA-binding domain.

several recent excellent reviews.55,185,186 Liver regeneration is initiated by the release of hepatic growth factors and cytokines, which mediate reentry of terminally differentiated hepatocytes into the cell cycle. This hepatocyte proliferative response during liver regeneration is coincident with a potent activation of immediate early transcription factors including c-Jun, c-Fos, c-Myc, NF-␬B, Stat3, and C/EBP␤ proteins. Furthermore, the delayed early Foxm1b transcription factor regulates expression of cell cycle regulatory proteins mediating hepatocyte progression into both S-phase (DNA replication) and mitosis. Future studies identifying the complex network of interaction between hepatic transcription factors in the regulation of liver development, differentiation, and homeostatic function may facilitate development of novel methods of therapeutic intervention in human liver diseases. Acknowledgment: The authors thank D. Hughes, M. Major, and F. Rausa for critically reviewing the manuscript.

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2. Cereghini S, Raymondjean M, Carranca AG, Herbomel P, Yaniv M. Factors involved in control of tissue-specific expression of albumin gene. Cell 1987;50:627-638. 3. Courtois G, Morgan JG, Campbell LA, Fourel G, Crabtree GR. Interaction of a liver specific nuclear factor with the fibrinogen and alpha 1-antitrypsin promoters. Science 1987;238:688-692. 4. Courtois G, Baumhueter S, Crabtree GR. Purified hepatocyte nuclear factor 1 interacts with a family of hepatocyte-specific promoters. Proc Natl Acad Sci U S A 1988;85:7937-7941. 5. Costa RH, Grayson DR, Darnell JE, Jr. Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and ␣1-antitrypsin genes. Mol Cell Biol 1989;9:1415 1425. 6. Costa RH, Grayson DR. Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region. Nucleic Acids Res 1991;19:4139-4145. 7. DiPersio CM, Jackson DA, Zaret KS. The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology. Mol Cell Biol 1991;11:4405-4414. 8. Monaci P, Nicosia A, Cortese R. Two different liver-specific factors stimulate in vitro transcription from the human ␣1-antitrypsin promoter. EMBO J 1988;7:2075-2087. 9. Pani L, Qian XB, Clevidence D, Costa RH. The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3␤ involves a cell-specific factor and positive autoactivation. Mol Cell Biol 1992;12:552-562. 10. Samadani U, Costa RH. The transcriptional activator hepatocyte nuclear factor six regulates liver gene expression. Mol Cell Biol 1996;16:62736284. 11. Vorachek WR, Steppan CM, Lima M, Black H, Bhattacharya R, Wen P, Kajiyama Y, et al. Distant enhancers stimulate the albumin promoter

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