Elongation of very long chain fatty acids-3 (Elovl3) is activated by ZHX2 and is a regulator of cell cycle progression.

Zinc fingers and homeoboxes 2 (Zhx2) are transcriptional regulators of liver gene expression with key functions in embryonic development as well as tissue regeneration in response to damage and disease, presumably through its control of target genes. Previous microarray data suggested that elongation of very long chain fatty acids-3 (Elovl3), a member of the ELOVL family of enzymes that synthesize very long chain fatty acids (VLCFAs), is a putative Zhx2 target gene. VLCFAs are core component of ceramides and other bioactive sphingolipids that are often dysregulated in diseases and regulate key cellular processes including proliferation. Since several previously identified Zhx2 targets become dysregulated in liver damage, we investigated the relationship between Zhx2 and Elovl3 in liver development, damage, and regeneration. Here, using mouse and cell models, we demonstrate that Zhx2 positively regulates Elovl3 expression in the liver and that male-biased hepatic Elovl3 expression is established between 4 and 8 wk of age in mice. Elovl3 is dramatically repressed in mouse models of liver regeneration, and the reduced Elovl3 levels in the regenerating liver are associated with changes in hepatic VLCFAs. Human hepatoma cell lines with forced Elovl3 expression have lower rates of cell growth; analysis of synchronized cells indicates that this reduced proliferation correlates with cells stalling in S-phase and lower mRNA levels of cell cyclins. Taken together, these data indicate that Elovl3 expression helps regulate cellular proliferation during liver development and regeneration, possibly through control of VLCFAs.NEW & NOTEWORTHY Numerous targets of the transcription factor Zhx2 are dysregulated in liver disease. We show that the elongase Elovl3 is a novel Zhx2 target. Elovl3 and Zhx2 expression change during liver regeneration, which is associated with changes in very long chain fatty acids. Forced Elovl3 expression reduces cell growth and blocks cell cycle progression. This suggests that Elovl3 may account, at least in part, for the relationship between Zhx2 and proliferation during liver development and disease.

The prorenin receptor and its soluble form contribute to lipid homeostasis.

Obesity is associated with alterations in hepatic lipid metabolism. We previously identified the prorenin receptor (PRR) as a potential contributor to liver steatosis. Therefore, we aimed to determine the relative contribution of PRR and its soluble form, sPRR, to lipid homeostasis. PRR-floxed male mice were treated with an adeno-associated virus with thyroxine-binding globulin promoter-driven Cre to delete PRR in the liver [liver PRR knockout (KO) mice]. Hepatic PRR deletion did not change the body weight but increased liver weights. The deletion of PRR in the liver decreased peroxisome proliferator-activated receptor gamma (PPARγ) and triglyceride levels, but liver PRR KO mice exhibited higher plasma cholesterol levels and lower hepatic low-density lipoprotein receptor (LDLR) and Sortilin 1 (SORT1) proteins than control (CTL) mice. Surprisingly, hepatic PRR deletion elevated hepatic cholesterol, and up-regulated hepatic sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-R) genes. In addition, the plasma levels of sPRR were significantly higher in liver PRR KO mice than in controls. In vitro studies in HepG2 cells demonstrated that sPRR treatment upregulated SREBP2, suggesting that sPRR could contribute to hepatic cholesterol biosynthesis. Interestingly, PRR, total cleaved and noncleaved sPRR contents, furin, and Site-1 protease (S1P) were elevated in the adipose tissue of liver PRR KO mice, suggesting that adipose tissue could contribute to the circulating pool of sPRR. Overall, this work supports previous works and opens a new area of investigation concerning the function of sPRR in lipid metabolism and adipose tissue-liver cross talk.NEW & NOTEWORTHY Hepatic PRR and its soluble form, sPRR, contribute to triglyceride and cholesterol homeostasis and hepatic inflammation. Deletion of hepatic PRR decreased triglyceride levels through a PRR-PPARγ-dependent mechanism but increased hepatic cholesterol synthesis through sPRR-medicated upregulation of SREBP-2. Our study highlighted a new paradigm of cross talk between the liver and the adipose tissue involving cholesterol and sPRR.

Liver size and lipid content differences between BALB/c and BALB/cJ mice on a high-fat diet are due, in part, to Zhx2.

BALB/cJ mice exhibit considerable phenotypic differences with other BALB/c substrains. Some of these traits involve the liver, including persistent postnatal expression of genes that are normally expressed only in the fetal liver and reduced expression of major urinary proteins. These traits are due to a mutation that dramatically reduces expression of the gene encoding the transcription factor Zinc fingers and homeoboxes 2 (Zhx2). BALB/cJ mice also exhibit reduced serum lipid levels and resistance to atherosclerosis compared to other mouse strains when placed on a high-fat diet. This trait is also due, at least in part, to the Zhx2 mutation. Microarray analysis identified many genes affecting lipid homeostasis, including Lipoprotein lipase, that are dysregulated in BALB/cJ liver. This led us to investigate whether hepatic lipid levels would be different between BALB/cJ and BALB/c mice when placed on a normal chow or a high-fat chow diet. On the high-fat chow, BALB/cJ mice had increased weight gain, increased liver:body weight ratio, elevated hepatic lipid accumulation and markers of liver damage when compared to BALB/c mice. These traits in BALB/cJ mice were only partially reversed by a hepatocyte-specific Zhx2 transgene. These data indicate that Zhx2 reduces liver lipid levels and is hepatoprotective in mice on a high-fat diet, but the partial rescue by the Zhx2 transgene suggests a contribution by both parenchymal and non-parenchymal cells. A model to account for the cardiovascular and liver phenotype in mice with reduced Zhx2 levels is provided.

Zhx2 (zinc fingers and homeoboxes 2) regulates major urinary protein gene expression in the mouse liver.

The mouse major urinary proteins (Mups) are encoded by a large family of highly related genes clustered on chromosome 4. Mups, synthesized primarily and abundantly in the liver and secreted through the kidneys, exhibit male-biased expression. Mups bind a variety of volatile ligands; these ligands, and Mup proteins themselves, influence numerous behavioral traits. Although urinary Mup protein levels vary between inbred mouse strains, this difference is most pronounced in BALB/cJ mice, which have dramatically low urinary Mup levels; this BALB/cJ trait had been mapped to a locus on chromosome 15. We previously identified Zhx2 (zinc fingers and homeoboxes 2) as a regulator of numerous liver-enriched genes. Zhx2 is located on chromosome 15, and a natural hypomorphic mutation in the BALB/cJ Zhx2 allele dramatically reduces Zhx2 expression. Based on these data, we hypothesized that reduced Zhx2 levels are responsible for lower Mup expression in BALB/cJ mice. Using both transgenic and knock-out mice along with in vitro assays, our data show that Zhx2 binds Mup promoters and is required for high levels of Mup expression in the adult liver. In contrast to previously identified Zhx2 targets that appear to be repressed by Zhx2, Mup genes are positively regulated by Zhx2. These data identify Zhx2 as a novel regulator of Mup expression and indicate that Zhx2 activates as well as represses expression of target genes.

HBV suppresses ZHX2 expression to promote proliferation of HCC through miR-155 activation.

Initiation of hepatocellular carcinoma (HCC) by chronic hepatitis B virus (HBV) infection is a complex process that includes both oncogene activation and tumor suppressor inhibition. The HBV X (HBx) protein has an important and complex role in processes leading to HCC. We previously identified the mammalian Zinc fingers and homeoboxes 2 (ZHX2) gene as an HCC-associated tumor suppressor gene. In the present study, we investigated whether the oncogenic properties of HBV and, more specifically, HBx, involved ZHX2 silencing. Our data indicates that ZHX2 expression is significantly decreased in tumor tissues from HBV-positive HCC patients and livers from HBV transgenic mice. In vitro and in vivo studies confirmed that HBV-encoded proteins, particularly HBx, inhibits both the expression and tumor suppression properties of ZHX2. Further analyses identified miR-155, a well-known oncomiR in various cancers, as an important link between HBx and ZHX2 inhibition. Increased miR-155 levels were found in HBV-positive tumors, livers of HBV transgenic mice and HBx-overexpressing hepatoma cell lines. MiR-155 overexpression reduced ZHX2 levels via miR-155 seed sites in the ZHX2 3'UTR, whereas blocking miR-155 levels led to increased ZHX2 levels. Taken together, our data indicate that HCC-promoting properties of HBV may include ZHX2 silencing via a miR-155 dependent pathway and suggests a novel therapy for HBV-related HCC.

Zinc Fingers and Homeoboxes 2 (Zhx2) Regulates Sexually Dimorphic Cyp Gene Expression in the Adult Mouse Liver.

The mammalian cytochrome P450 (Cyp) gene family encodes a large number of structurally related enzymes that catalyze a variety of metabolic and detoxification reactions. The liver is the primary site of Cyp expression in terms of expression levels and number of expressed genes, consistent with this organ's essential role in metabolism of endogenous and xenobiotic compounds. Many Cyp genes exhibit sexually dimorphic expression. For example, Cyp2a4 is expressed significantly higher in the adult liver of female mice compared to male mice. An exception to this pattern is seen in BALB/cJ mice, where male hepatic Cyp2a4 mRNA levels are substantially elevated compared to male mice of other strains. The Zinc fingers and homeoboxes 2 (Zhx2) protein governs the silencing of several genes in the postnatal liver, including α-fetoprotein, H19, and glypican 3. Zhx2 also regulates numerous hepatic genes that govern lipid homeostasis. We previously showed that the Zhx2 gene is mutated in BALB/cJ mice, which led us to consider whether elevated male hepatic Cyp2a4 levels in this strain are due to this Zhx2 mutation. Using mice with a conditional Zhx2 deletion, we show here that the absence of Zhx2 in hepatocytes results in increased Cyp2a4 expression in adult male liver. We extend this finding to show that additional Cyp genes are disregulated in the absence of Zhx2. We also show that mRNA levels of Cyp2a4 and several other female-biased Cyp genes are increased, and male-biased Cyp4a12 is decreased in mouse liver tumors. These data indicate that Zhx2 is a novel regulator of sex-biased Cyp gene expression in the normal and diseased liver.

Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of Cyclins A and E.

BACKGROUND & AIMS: Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in the development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells. METHODS: We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry. RESULTS: ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of Cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes Cyclin E) and inhibited their transcription. Knockdown of Cyclin A or Cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter, <5 cm), compared with adjacent nontumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation. CONCLUSIONS: ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.

Pericentral activity of alpha-fetoprotein enhancer 3 and glutamine synthetase upstream enhancer in the adult liver are regulated by ß-catenin in mice.

UNLABELLED: We previously showed that mouse alpha-fetoprotein (AFP) enhancer 3 activity is highly restricted to pericentral hepatocytes in the adult liver. Here, using transgenic mice, we show that the upstream enhancer of the rat glutamine synthetase gene is also active, specifically in pericentral regions. Activity of both enhancers is lost in the absence of ß-catenin, a key regulator of zonal gene expression in the adult liver. Both enhancers contain a single, highly conserved T-cell factor/lymphoid enhancer factor binding site that is required for responsiveness to ß-catenin. We also show that endogenous AFP messenger RNA levels in the perinatal liver are lower when ß-catenin is reduced. CONCLUSION: These data identify the first distinct zonally active regulatory regions required for ß-catenin responsiveness in the adult liver, and suggest that postnatal AFP repression and the establishment of zonal regulation are controlled, at least in part, by the same factors.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

BACKGROUND: Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). MATERIALS AND METHODS: Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. RESULTS: Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 µM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 µM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). CONCLUSIONS: LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation.

Zhx2 and Zbtb20: novel regulators of postnatal alpha-fetoprotein repression and their potential role in gene reactivation during liver cancer.

The mouse alpha-fetoprotein (AFP) gene is abundantly expressed in the fetal liver, normally silent in the adult liver but is frequently reactivated in hepatocellular carcinoma. The basis for AFP expression in the fetal liver has been studied extensively. However, the basis for AFP reactivation during hepatocarcinogenesis is not well understood. Two novel factors that control postnatal AFP repression, Zhx2 and Zbtb20, were recently identified. Here, we review the transcription factors that regulate AFP in the fetal liver, as well as Zhx2 and Zbtb20, and raise the possibility that the loss of these postnatal repressors may be involved in AFP reactivation in liver cancer.

Zinc fingers and homeoboxes 2 is required for diethylnitrosamine-induced liver tumor formation in C57BL/6 mice.

Liver cancer, comprised primarily of hepatocellular carcinoma (HCC), is the third leading cause of cancer deaths worldwide and increasing in Western countries. We previously identified the transcription factor zinc fingers and homeoboxes 2 (Zhx2) as a regulator of hepatic gene expression, and many Zhx2 target genes are dysregulated in HCC. Here, we investigate HCC in Zhx2-deficient mice using the diethylnitrosamine (DEN)-induced liver tumor model. Our study using whole-body Zhx2 knockout (Zhx2KO ) mice revealed the complete absence of liver tumors 9 and 10 months after DEN exposure. Analysis soon after DEN treatment showed no differences in expression of the DEN bioactivating enzyme cytochrome P450 2E1 (CYP2E1) and DNA polymerase delta 2, or in the numbers of phosphorylated histone variant H2AX foci between Zhx2KO and wild-type (Zhx2wt ) mice. The absence of Zhx2, therefore, did not alter DEN bioactivation or DNA damage. Zhx2KO livers showed fewer positive foci for Ki67 staining and reduced interleukin-6 and AKT serine/threonine kinase 2 expression compared with Zhx2wt livers, suggesting that Zhx2 loss reduces liver cell proliferation and may account for reduced tumor formation. Tumors were reduced but not absent in DEN-treated liver-specific Zhx2 knockout mice, suggesting that Zhx2 acts in both hepatocytes and nonparenchymal cells to inhibit tumor formation. Analysis of data from the Cancer Genome Atlas and Clinical Proteomic Tumor Consortium indicated that ZHX2 messenger RNA and protein levels were significantly higher in patients with HCC and associated with clinical pathological parameters. Conclusion: In contrast to previous studies in human hepatoma cell lines and other HCC mouse models showing that Zhx2 acts as a tumor suppressor, our data indicate that Zhx2 acts as an oncogene in the DEN-induced HCC model and is consistent with the higher ZHX2 expression in patients with HCC.

Highly homologous mouse Cyp2a4 and Cyp2a5 genes are differentially expressed in the liver and both express long non-coding antisense RNAs.

The mammalian Cytochrome P450 (Cyp) gene superfamily encodes enzymes involved in numerous metabolic pathways and are frequently expressed in the liver. Despite the remarkably high sequence similarity of Cyp2a4 and Cyp2a5 genes and their surrounding genomic regions, they exhibit differences in expression in the adult mouse liver. For example, Cyp2a4 is highly female-biased whereas Cyp2a5 is only moderately female-biased and Cyp2a4, but not Cyp2a5, is activated in liver cancer. We hypothesized that the limited sequence differences may help us identify the basis for this differential expression. An antisense expressed sequence tag had been uniquely annotated to the Cyp2a4 gene which led us to investigate this transcript as a possible regulator of this gene. We characterized the full-length antisense transcript and also discovered a similar transcript in the Cyp2a5 gene. These transcripts are nuclear long noncoding RNAs that are expressed similarly to their sense mRNA counterparts. This includes the sex-biased and liver tumor differences seen between the Cyp2a4 and Cyp2a5 genes, but we also find that these two genes and their antisense transcripts are expressed within different zones of the liver structure. Interestingly, while the differences in sex-biased expression of the mRNAs are established 1-2 months after birth, the antisense transcripts exhibit these expression differences earlier, at 3-4 weeks after birth. By analyzing published genomic data, we have identified candidate transcription factor binding sites that could account for differences in Cyp2a4/Cyp2a5 expression. Taken together, these studies characterize the first antisense RNAs within the Cyp supergene family and identify potential transcriptional and post-transcriptional mechanisms governing different Cyp2a4 and Cyp2a5 expression patterns in mouse liver.

The alpha-fetoprotein enhancer region activates the albumin and alpha-fetoprotein promoters during liver development.

The four members of the albumin gene family encode the serum transport proteins albumin, alpha-fetoprotein, alpha-albumin, and vitamin D-binding protein. These genes are transcribed primarily in the liver with each having a different pattern of developmental expression. The tight linkage of these genes, particularly that of albumin, alpha-fetoprotein and alpha-albumin, and their liver-specific expression, has led to the suggestion that these genes share common regulatory elements. To directly examine whether the alpha-fetoprotein enhancer region could regulate the albumin gene family, expression of these genes was monitored in mice in which this region was deleted by homologous recombination. Our data indicate that this enhancer region is required for alpha-fetoprotein and albumin activation early in liver development and alpha-fetoprotein reactivation during liver regeneration, but that albumin, alpha-albumin, and vitamin D-binding protein expression later in hepatic development is not affected by the absence of these enhancers. We also demonstrate that RNA polymerase II loading on the alpha-fetoprotein and albumin promoters is reduced in the absence of this enhancer region, indicating a direct role for these enhancers in the assembly of the RNA Polymerase II complex during liver development.

Early growth response genes regulate B cell development, proliferation, and immune response.

Egr-1 (early growth response gene-1) is an immediate early gene encoding a zinc finger motif-containing transcription factor. Upon cross-linking of BCR, mature B cells undergo proliferation with an increase in Egr-1 message. Immature B lymphoma cells that express Egr-1 message and protein constitutively are growth inhibited when Egr-1 is down-regulated by negative signals from BCR or by antisense oligonucleotides. To test the hypothesis that Egr-1 is important for B cell development, we examined B cells from primary and secondary lymphoid organs in Egr-1(-/-) mice. Marginal zone B cell development was arrested in these mice, whereas the B cells in all other compartments were increased. To test the hypothesis that Egr-1 function may be partially compensated by other Egr family members, we developed transgenic mice expressing a dominant negative form of Egr-1, which lacks the trans activation domain but retains the DNA-binding domain, in a B cell-specific manner. There was a decrease in B lymphopoiesis in the bone marrow accompanied by a reduction in splenic immature and mature B cells as well as marginal zone B cells in the transgenic mice. Moreover, transgenic mice respond poorly to BCR cross-linking in vitro and T-independent and T-dependent Ags in vivo.

Effects of cigarette smoke on the activation of oxidative stress-related transcription factors in female A/J mouse lung.

Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine whether cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 d (6 hr/d, 5 d/wk). Cigarette smoke did not increase NF-kappaB activation at any of these times, but NF-kappaB DNA binding activity was lower after 15 d and 56 d of smoke exposure. The DNA binding activity of AP-1 was lower after 10 d and 56 d but was not changed after 42 d of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 d of smoke exposure but decreased after 56 d. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 d of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-kappaB, AP-1, and HIF are largely unaffected or reduced.

Evolutionary Analysis of the Zinc Finger and Homeoboxes Family of Proteins Identifies Multiple Conserved Domains and a Common Early Chordate Ancestor.

The Zinc Fingers and Homeoboxes (Zhx) proteins, Zhx1, Zhx2, and Zhx3, comprise a small family of proteins containing two amino-terminal C2-H2 zinc fingers and four or five carboxy-terminal homeodomains. These multiple homeodomains make Zhx proteins unusual because the majority of homeodomain-containing proteins contain a single homeodomain. Studies in cultured cells and mice suggest that Zhx proteins can function as positive or negative transcriptional regulators. Zhx2 regulates numerous hepatic genes, and all three Zhx proteins have been implicated in different cancers. Because Zhx proteins contain multiple predicted homeodomains, are associated with interesting physiological traits, and seem to be only present in the vertebrate lineage, we investigated the evolutionary history of this small family by comparing Zhx homologs from a wide range of chordates. This analysis indicates that the zinc finger motifs and homeodomains are highly similar among all Zhx proteins and also identifies additional Zhx-specific conserved regions, including a 13 amino acid amino-terminal motif that is nearly identical among all gnathostome Zhx proteins. We found single Zhx proteins in the sea lamprey (Petromyzon marinus) and in the nonvertebrate chordates sea squirt (Ciona intestinalis) and lancelet (Branchiostoma floridae); these Zhx proteins are most similar to gnathostome Zhx3. Based on our analyses, we propose that a duplication of the primordial Zhx gene gave rise to Zhx3 and the precursor to Zhx1 and Zhx2. A subsequent tandem duplication of this precursor generated Zhx1 and Zhx2 found in gnathostomes.

An Underlying Mechanism of Dual Wnt Inhibition and AMPK Activation: Mitochondrial Uncouplers Masquerading as Wnt Inhibitors.

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target ß-catenin in this pathway. We now report that several "Wnt inhibitors" that allegedly target ß-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known "Wnt inhibitor", FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M), was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

The oncofetal gene glypican 3 is regulated in the postnatal liver by zinc fingers and homeoboxes 2 and in the regenerating liver by alpha-fetoprotein regulator 2.

UNLABELLED: The Glypican 3 (Gpc3) gene is expressed abundantly in the fetal liver, is inactive in the normal adult liver, and is frequently reactivated in hepatocellular carcinoma (HCC). This reactivation in HCC has led to considerable interest in Gpc3 as a diagnostic tumor marker and its possible role in tumorigenesis. Despite this interest, the basis for Gpc3 regulation is poorly understood. On the basis of the similarities between Gpc3 and alpha-fetoprotein expression in the liver, we reasoned that common factors might regulate these 2 genes. Here we identify zinc fingers and homeoboxes 2 (Zhx2) as a regulator of Gpc3. Mouse strain-specific differences in adult liver Gpc3 messenger RNA levels and transgenic mouse studies indicate that Zhx2 represses Gpc3 expression in the adult liver. We also demonstrate that Gpc3 is activated in the regenerating liver following a carbon tetrachloride treatment and that the level of Gpc3 induction is controlled by alpha-fetoprotein regulator 2 (Afr2). CONCLUSION: We show that Zhx2 acts as a repressor of Gpc3 in the adult liver, and this raises the interesting possibility that Zhx2 might also be involved in Gpc3 reactivation in HCC. We also show that Gpc3 is activated in the regenerating liver in an Afr2-dependent manner. Zhx2 and Afr2 represent the first known regulators of Gpc3.

Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-kappa B in mice.

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-kappaB on the hepatic tumor promoting activity of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50-/- male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 micromol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P=0.09) increased the tumor incidence in wild-type and p50-/- mice. PCB-153 increased the total tumor volume in both wild-type and p50-/- mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50-/- mice administered PCB-153 compared to vehicle controls, and inhibited in p50-/- mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50-/- mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50-/- mice; this increase was inhibited in p50-/- mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P=0.10) increased apoptosis in p50-/- but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-kappaB p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.

Effect of dietary selenium on the promotion of hepatocarcinogenesis by 3,3', 4,4'-tetrachlorobiphenyl and 2,2', 4,4', 5,5'-hexachlorobiphenyl.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that have promoting activity in the liver. PCBs induce oxidative stress, which may influence carcinogenesis. Epidemiological studies strongly suggest an inverse relationship between dietary selenium (Se) and cancer. Despite evidence linking Se deficiency to hepatocellular carcinoma and liver necrosis, the underlying mechanisms for Se cancer protection in the liver remain to be determined. We examined the effect of dietary Se on the tumor promoting activities of two PCBs congeners, 3,3', 4,4'-tetrachlorobiphenyl (PCB-77) and 2,2', 4,4', 5,5'-hexachlorobiphenyl (PCB-153) using a 2-stage carcinogenesis model. An AIN-93 torula yeast-based purified diet containing 0.02 (deficient), 0.2 (adequate), or 2.0 mg (supplemental) selenium/kg diet was fed to Sprague-Dawley female rats starting ten days after administering a single dose of diethylnitrosamine (150 mg/kg). After being fed the selenium diets for 3 weeks, rats received four i.p. injections of either PCB-77 or PCB-153 (150 micromol/kg) administered every 14 days. The number of placental glutathione S-transferase (PGST)-positive foci per cm(3) and per liver among the PCB-77-treated rats was increased as the Se dietary level increased. Unlike PCB-77, rats receiving PCB-153 did not show the same Se dose-response effect; nevertheless, Se supplementation did not confer protection against foci development. However, the 2.0 ppm Se diet reduced the mean focal volume, indicating a possible protective effect by inhibiting progression of preneoplastic lesions into larger foci. Cell proliferation was not inhibited by Se in the liver of the PCB-treated groups. Se did not prevent the PCB-77-induced decrease of hepatic Se and associated reduction in glutathione peroxidase (GPx) activity. In contrast, thioredoxin reductase (TrxR) activity was not affected by the PCBs treatment or by Se supplementation. These findings indicate that Se does not inhibit the number of PGST-positive foci induced during promotion by PCBs, but that the size of the lesions may be inhibited. The effects of Se on altered hepatic foci do not correlate with its effects on GPx and TrxR.