ABSTRACT
BACKGROUND: Maturation of enterocytes along the small intestinal crypt-villus axis is associated with significant changes in gene expression profiles. fls485 coding a putative chaperone protein has been recently suggested as a gene involved in this process. The aim of the present study was to analyze fls485 expression in human small intestinal mucosa. METHODS: fls485 expression in purified normal or intestinal mucosa affected with celiac disease was investigated with a molecular approach including qRT-PCR, Western blotting, and expression strategies. Molecular data were corroborated with several in situ techniques and usage of newly synthesized mouse monoclonal antibodies. RESULTS: fls485 mRNA expression was preferentially found in enterocytes and chromaffine cells of human intestinal mucosa as well as in several cell lines including Rko, Lovo, and CaCo2 cells. Western blot analysis with our new anti-fls485 antibodies revealed at least two fls485 proteins. In a functional CaCo2 model, an increase in fls485 expression was paralleled by cellular maturation stage. Immunohistochemistry demonstrated fls485 as a cytosolic protein with a slightly increasing expression gradient along the crypt-villus axis which was impaired in celiac disease Marsh IIIa-c. CONCLUSIONS: Expression and synthesis of fls485 are found in surface lining epithelia of normal human intestinal mucosa and deriving epithelial cell lines. An interdependence of enterocyte differentiation along the crypt-villus axis and fls485 chaperone activity might be possible.
Subject(s)
Celiac Disease/genetics , Intestinal Mucosa/metabolism , Intestine, Small/metabolism , Molecular Chaperones/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Celiac Disease/metabolism , Cell Line , Child , Chromaffin System/metabolism , Enterocytes/metabolism , Female , Gene Expression Regulation , Humans , Male , Mice , Middle Aged , Open Reading Frames , RNA, Messenger/analysis , Reference Values , Young AdultABSTRACT
BACKGROUND: Chronic infection with hepatitis B virus (HBV) is a major factor associated with the development of hepatocellular carcinoma, but the mechanism by which this occurs is unknown. Treatment of chronic hepatitis B with lamivudine results in virological suppression and histological improvement; however, the role of lamivudine in preventing the development of hepatocellular carcinoma is less well defined. We recently reported that replication of HBV in a cell-culture system was associated with the upregulation of pERK, pAkt, pc-Myc, nuclear cyclin B1, p21(cip1) and p53 together with G2 cell cycle arrest. METHODS: In order to determine whether lamivudine is able to reverse the HBV-induced changes on signal transduction and cell cycle, we infected Huh7 cells with a recombinant adeno-HBV virus in the presence of 0-50 microM of lamivudine. Signal transduction and cell cycle regulatory proteins were analysed by western immunoblot. RESULTS: Although lamivudine was able to inhibit HBV replication, it failed to reverse the changes on ERK and Akt phosphorylation. Correspondingly, levels of phospho-GSK3beta and p21(cp1/waf1) were increased, as were cyclin D1, cyclin B1, p53 and pc-Myc. CONCLUSIONS: Lamivudine was ineffective in reversing the HBV-induced changes in signal transduction pathways and cell cycle regulatory proteins, indicating that the HBV-infected cells remained primed for oncogenic transformation despite viral suppression.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/pathogenicity , Lamivudine/pharmacology , Signal Transduction/drug effects , Cell Cycle Proteins/drug effects , Cell Cycle Proteins/metabolism , Cell Line , Extracellular Signal-Regulated MAP Kinases/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Hepatitis B virus/enzymology , Humans , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
BACKGROUND/AIMS: Chronic infection with the hepatitis B virus (HBV) is strongly associated with the development of hepatocellular carcinoma but the mechanism by which this occurs is unknown. Numerous studies have focused on the HBV X protein showing that it activates signal transduction pathways while few have investigated these changes in HBV-replicating hepatocytes. METHODS: We utilized the recombinant adenovirus system to deliver a replication competent HBV genome into Huh7 and primary marmoset hepatocytes (PMH) to examine the effects of active viral replication on the regulation of Ras-ERK signal transduction and related pathways. RESULTS: Huh7 cells and PMHs replicating HBV demonstrated significant upregulation in phosphorylated ERK, Akt, c-myc together with increased p53, cyclin B1 and p21(cip1) expression and cell cycle progression to G2 phase in the absence of increased cell proliferation. Phosphorylation of the key cell survival kinase, Akt, was significantly increased, resulting in increased serine phosphorylation of the downstream target, GSK3-beta. CONCLUSIONS: These results demonstrated simultaneous activation of the MAP Kinase and Akt pathways in HBV-replicating hepatocytes that resulted in dysregulation in the control of cell cycle progression and which help explain the early pathogenic mechanisms that underlie malignant transformation associated with chronic hepatitis B infection.
