Liver progenitor cells may construct cysts having heterogeneous gene expression of liver-enriched transcription factors in mice with conditional knockout of the Hhex gene.

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb-Cre/HhexloxP/loxP) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases.

Analysis of regulatory mechanisms of an insulin-inducible SHARP-2 gene by (S)-Equol.

Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between -4687 and -4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus.

(-)-Epigallocatechin-3-gallate enhances the expression of an insulin-inducible transcription factor gene via a phosphoinositide 3-kinase/atypical protein kinase C lambda pathway.

The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is an insulin-inducible transcriptional repressor. In this study, we examined issues of whether (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, regulates the expression of the rat SHARP-1 gene and which signaling pathway mediates the regulation. When H4IIE cells were treated with EGCG, SHARP-1 mRNA levels rapidly increased. Pretreatments with inhibitors for either phosphoinositide 3-kinase (PI 3-K) or protein kinase C partially blocked EGCG induction. Atypical protein kinase C lambda (aPKCλ) is known as a downstream target of PI 3-K in the liver. When a dominant-negative form of aPKCλ was expressed, the EGCG-induced SHARP-1 mRNAs was inhibited. Finally, Western blot analysis revealed that EGCG rapidly and temporarily stimulates aPKCλ phosphorylation. Thus, we conclude that EGCG induces SHARP-1 gene expression via a PI 3-K/aPKCλ signaling pathway.

Identification of cis-regulatory elements and trans-acting proteins of the rat carbohydrate response element binding protein gene.

Carbohydrate response element binding protein (ChREBP) is a transcription factor that activates liver glycolytic and lipogenetic enzyme genes in response to high carbohydrate diet. Here we report the transcriptional regulatory mechanisms for the rat ChREBP gene. Firstly, we determined the transcription initiation site and the nucleotide sequences of the rat ChREBP promoter region encompassing approximately 900bp from the ATG initiation codon. Reporter gene assays demonstrated that the major positive regulatory region exists in the nucleotide sequence between -163 and -32 of the ChREBP gene. This region contains a cluster of putative transcription factor binding elements that consist of two specificity protein 1 (Sp1) binding sites (-66 to -50 and -93 to -78), a sterol regulatory element (-101 to -110), and two nuclear factor-Y (NF-Y) binding sites (-23 to -19 and -131 to -127). Mutations introduced into these sites caused marked reduction of ChREBP promoter activities. Functional synergisms were observed between Sp1/NF-Y and Sp1/sterol regulatory element-binding protein. Additionally, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that these factors bound to these elements. Thus, we conclude that functional synergisms between these transcription factors are critical for ChREBP gene transcription.

Molecular cloning, expression and chromosomal localization of mouse MM-1.

The protooncogene product Myc associates with many proteins. The isolation of the mouse MM-1; c-Myc binding protein (Myc-Modulator 1) cDNA is described. The cDNA contains a 462 bp open reading frame that encodes a polypeptide of 154 amino acid residues. The deduced amino acid sequence indicates that mouse MM-1 has a 99% identity with the sequence of human MM-1. The expression of mouse MM-1 mRNA was detected in the fetal liver, but its level was 3-fold higher than that in the normal adult liver, and was slightly increased after a partial hepatectomy. It is expressed widely in a variety of adult mouse tissues. Thus, MM-1 may play a role in liver development and growth. A bioinformatics analysis indicates that mouse MM-1 gene consists of 6 exons. Furthermore, the chromosomal location of the mouse MM-1 gene was on the F2-F3 band of chromosome 15, as determined by fluorescence in situ hybridization.

Nuclear factor 1 family members interact with hepatocyte nuclear factor 1alpha to synergistically activate L-type pyruvate kinase gene transcription.

Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cis-regulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1alpha binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5'-flanking region up to -189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1alpha, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1alpha-binding site, raising the possibility that NF1 proteins function as HNF1alpha co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1alpha bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1alpha via their DNA-binding domains. This interaction enhanced HNF1alpha binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1alpha plays an important role in hepatic L-PK transcription.

Hex stimulates the hepatocyte nuclear factor 1alpha-mediated activation of transcription.

The homeodomain protein Hex can function either as a transcriptional repressor or activator in animals. Recent reports have indicated that Hex is involved in liver development. However, its target genes and interacting proteins are largely unknown. We found that Hex functionally interacted with hepatocyte nuclear factor (HNF) 1alpha to further stimulate its activity using reporter gene containing multiple copies of HNF1alpha-binding site of the L-type pyruvate kinase (L-PK) gene promoter or natural L-PK promoter. This stimulation required the homeodomain and the acidic carboxyl-terminal region of Hex. Over-expression of Hex in primary cultured hepatocytes resulted in stimulation of the L-PK gene expression. Glutathione S-transferase pull-down assay and co-immunoprecipitation revealed that Hex physically interacted with HNF1alpha in mammalian cells through the homeodomain of Hex and POU-homeodomain of HNF1alpha. Since HNF1alpha is an important liver-enriched transcription factor involved in liver differentiation, Hex may contribute to liver differentiation through interaction with HNF1alpha.

Identification of the transactivating region of the homeodomain protein, hex.

The homeodomain-containing protein Hex acts as an activator as well as a repressor of transcription in animals. While its repression domain has been mapped to the amino-terminal region, the activation domain has never been identified. Here, we show that the homeodomain and the acidic carboxyl-terminal region are necessary for full activation of the sodium-dependent bile acid cotransporter gene promoter in a cell type-independent manner, suggesting that the carboxyl-terminal region comprising residues 197 to 271 functions as the activation domain. In addition, we observed that a Hex mutant without this activation domain acts as a dominant-negative mutant as to the transactivating function of Hex.

Identification and characterization of the hematopoietic cell-specific enhancer-like element of the mouse hex gene.

Hex is one of the homeobox genes suggested to be important for hematopoietic cell differentiation. However, its biological function and mechanism of transcriptional regulation in hematopoietic cells remain elusive. We have identified the regulatory region necessary for transcription of the mouse Hex gene in K562 leukemia cells through transient reporter assays involving various deletion mutants. This region, comprising +775 to +1177 in the first intron, had enhancer-like properties and showed high activity in other hematopoietic cell lines such as U937, HEL, and RAW264.7, but little activity in other Hex-expressing cell lines such as MH(1)C(1) and H4IIE hepatoma cells, suggesting that this region functions as a hematopoietic cell-specific enhancer-like element. Binding site mutation of hematopoietic transcription factors, such as GATAs and c-Myb present in the enhancer-like element, significantly decreased the luciferase reporter gene expression in K562 cells. Electrophoretic mobility shift assays showed that GATA-1, GATA-2, or c-Myb actually binds to three of these putative binding sites, and also suggested that several unidentified factors might interact with the enhancer-like element. Overexpression of GATA-1, GATA-2, or c-Myb stimulated the enhancer-like activity via these three binding sites. Thus, we conclude that Hex expression in hematopoietic cells is mainly regulated by GATA-1, GATA-2, and c-Myb via this intronic enhancer-like element.

Interaction between hex and GATA transcription factors in vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial growth factor signaling.

Recent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells. The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors. Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells. The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells. Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures. In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5'-untranslated region GATA motif. Finally, in RNase protection assays, transforming growth factor beta1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells. Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor beta-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes.

Sequence analysis of mouse muscle-type phosphofructokinase gene introns 10 and 11 with special reference to the exon skipping.

To elucidate physiological relevance of the skipping of exon 11 in muscle-type phosphofructokinase (PFK-M) transcripts, we partially cloned and analyzed the cDNA and the genomic fragment of mouse PFK-M. In RT-PCR analysis using a pair of primers which carry the region corresponding to human exon 11 in between, any minor transcript without exon 11 was not detected. Partial sequencing analysis of mouse PFK-M gene revealed that the junctions of intron 10 of human gene were both less identical to the consensus sequences than those of mouse gene, but that there was no appreciable difference in the junctions in intron 11 between mouse and human. These results suggest that the skipping of exon 11 in PFK-M gene transcripts would be found mainly in human. Although further investigation would be required to understand the mechanisms and physiological significance of exon-skipping, the exon-skipping in PFK-M transcripts would be unlikely to have the physiological significance.

Insulin stimulates expression of the pyruvate kinase M gene in 3T3-L1 adipocytes.

M2-type pyruvate kinase (M2-PK) mRNA is produced from the PKM gene by an alternative RNA splicing in adipocytes. We found that insulin increased the level of M2-PK mRNA in 3T3-L1 adipocytes in both time- and dose-dependent manners. This induction did not require the presence of glucose or glucosamine in the medium. The insulin effect was blocked by pharmacological inhibitors of insulin signaling pathways such as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase. A stable reporter expression assay showed that the promoter activity of an about 2.2-kb 5'-flanking region of the rat PKM gene was stimulated by insulin, but the extents of these stimulations were lower than those of the mRNA stimulation. Thus, we suggest that insulin increases the level of M2-PK mRNA in adipocytes by acting at transcriptional and post-transcriptional levels through signaling pathways involving both PI3K and MAPK kinase.

Insulin induces the expression of the SHARP-2/Stra13/DEC1 gene via a phosphoinositide 3-kinase pathway.

Transcription of the rat fatty acid synthase (FAS) gene in the rat liver can be regulated by feeding a high carbohydrate diet. A carbohydrate response element (ChoRE) located on the rat FAS gene promoter has been identified. Using multiple copies of the ChoRE as the bait in a yeast one-hybrid system, a rat liver cDNA library was screened, and the cDNA of ChoRE-binding proteins was cloned. A positive clone that encodes a basic helix-loop-helix protein, enhancer of split- and hairy-related protein-2 (SHARP-2), was obtained. Northern blot analysis revealed that the levels of SHARP-2 mRNA increase when a high carbohydrate diet is fed to normal rats or when insulin is administered to diabetic rats. In primary cultured rat hepatocytes, insulin rapidly induced an accumulation of SHARP-2 mRNA even in the absence of glucose. A time course for the increase in SHARP-2 mRNA levels indicated that it followed by those of FAS and L-type pyruvate kinase mRNAs and that the initial time course of SHARP-2 mRNA was similar to changes in the levels of glucokinase mRNA and phosphoenolpyruvate carboxykinase mRNA. Although wortmannin, LY294002, and actinomycin D blocked the increase in SHARP-2 mRNA levels by insulin, rapamycin, staurosporine, PD98059, okadaic acid, and 8-bromocyclic AMP had no effect. In addition, nuclear run-on assay revealed that transcription of the rat SHARP-2 gene was induced by insulin. Thus, we conclude that insulin induces the transcription of the rat SHARP-2 gene via a phosphoinositide 3-kinase pathway.