BackgroundActivation of the Wnt/β-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/β-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC.Methodology/Principal FindingsUsing real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of β-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/β-catenin signaling pathway, as shown by the TCF/β-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice.Conclusions/SignificanceOur findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/β-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.
DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.
The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler.
The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. The Hoxb3 gene is expressed in the vagal neural crest and in the enteric ganglia of the developing gut during embryogenesis. We have identified a cis-acting enhancer element b3IIIa in the Hoxb3 gene locus. In this study, by transgenic mice analysis, we examined the tissue specificity of the b3IIIa enhancer element using the lacZ reporter gene, with emphasis on the vagal neural crest cells and their derivatives in the developing gut. We found that the b3IIIa-lacZ transgene marks only the vagal region and not the trunk or sacral region. Using cellular markers, we showed that the b3IIIa-lacZ transgene was expressed in a subset of enteric neuroblasts during early development of the gut, and the expression was maintained in differentiated neurons of the myenteric plexus at later stages. The specificity of the b3IIIa enhancer in directing gene expression in the developing ENS was further supported by genetic analysis using the Dom mutant, a spontaneous mouse model of Hirschsprung's disease characterized by the absence of enteric ganglia in the distal gut. The colonization of lacZ-expressing cells in the large intestine was incomplete in all the Dom/b3IIIa-lacZ hybrid mutants we examined. To our knowledge, this is the only vagal neural crest-specific genetic regulatory element identified to date. This element could be used for a variety of genetic manipulations and in establishing transgenic mouse models for studying the development of the ENS. Developmental Dynamics 233:473-483, 2005.
Although the bactericidal effects of gastric juice are well established, little is known of the role of components other than hydrochloric acid. Indeed, it has been suggested that gastric acid is the only antibacterial factor in gastric juice. The aim of the study therefore was to compare the effects of human gastric juice, acid and pepsin on bacterial survival. 15 gastric juices obtained from patients undergoing routine gastroscopy or gastric drainage and solutions of HCI at p H values of 1.5-6.0 with or without pig pepsin A were used for 'killing' experiments. E. coli C690 and H.pylori E5 were selected as the test organisms. Suspensions of bacteria (1x106 E coli and1xlO8 H pylori) were pre-incubated with test solutions at 37°C. Samples were taken at 0,5, 10,20 then every 20 mins up to Zhrs, neutralised and the viable colonies counted after culturing at 37"C, 15hrs for E coli and 3 days for H pylori.Growth of bacteria was inhibited at pHs3.5, whereas killing required pH<2.5. 14 of the 15 gastric juices had bactericidal activity at pH3.5. The rate of killing was juice dependent with complete death of E.coli occurring between 5-40mins pre-incubation. In two juices 'killing' was observed at pH6.0. Preincubation with pig pepsin 0.5,l.O and 2.0mg/ml in separate experiments at pH's 2.5, 3.0 and 3.5 showed little bactericidal effect on E.coli C690. However, with H.pylori, the viable colonies were reduced to 50% of the control after 20mins incubation in lmg/ml pepsin at p H 3.5and pH3.0. In conclusion, killing of E.coli can occur in gastric juice at pH's 3.5-6.0 inferring component(s) other than acid and the proteolytic activity of pepsin cause this effect. Whereas, pepsin can facilitate killing of H.pylori.The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. Abnormal development of the vagal neural crest causes aganglionosis of the distal colon, leads to Hirschsprung's disease in humans and megacolon in mice. Several genes have been found to be involved in ENS development. The mouse megacolon mutant Dom was recently found to be due to a mutation in the Sox10 gene. We have in our laboratory a transgenic mouse line carrying the lac2 gene driven by a vagal neural crest-specific enhancer element, b3-IIIa, which was previously identified from the Hoxb-3 gene locus. To assess the tissue-specificity of this element, we examined the expression of lac2 in the transgenic mouse line during the course of ENS development. Our results showed that in the b3-IIIa mouse line, lac2 was expressed in the neural tube at the vagal region at 9.5 dpc. From 10.5 to 14.5 dpc, the IacZ-marked cells appeared as a migrating wave from the vagal region down to the developing gut. The lac2-marked cells first appeared in the foregut at 10.5 dpc, then at the mid-and hindgut by 14.5 dpc. Histological sections of 12.5 to 16.5 dpc gut samples showed that the lac2-marked cells appeared as a concentric ring in the mesenchyme. Using an antibody for neurofilamen...
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