The Roles Of Hepatitis B Small Surface Antigen In Regulating The Functions Of Cellular Protein LEF-1 And CyPA And Their Implications

The roles of hepatitis B small surface antigen in regulating the functions of cellular protein LEF-1 and CyPA and their implicationsHepatitis B virus(HBV),one of the most widely spread viruses,is the prototype of hepadnavirus family.It is estimated that about 350 million people are infected chronically and become carriers of this virus worldwide.Persistent HBV infection leads to chronic hepatitis,and is closely associated with the development of liver cirrhosis and hepatocellular carcinoma(HCC).Three forms of viral particles can be detected in the serum of HBV infected patients,namely,42 nm diameter mature virion particles,22 nm diameter spherical particles and 22 nm diameter filamentary particles. Uniquely,22 nm subviral particles,which are composed of the hepatitis B surface antigen(HBsAg) and do not contain viral DNA,usually outnumber the virions in patient serum by a factor of 1000-fold or more.Serum HBsAg is an important serological marker in chronic hepatitis B.Patients who are positive for HBsAg for at least 6 months are diagnosed as chronic infections.It is difficult to achieve the endpoint of HBsAg clearance and serum turnover in treatment.HBsAg plays a very important role in the pathogenesis of HBV infection.In the first part of this study,the overall effects of hepatitis B small surface antigen(SHBs) on the expression of cellular genes were examined by microarray assays to study the effects of persistent expression of HBsAg on host cell functions.A head-to-head comparison was made between two cell lines that were cultured under the same conditions.One was an SHBs-secreting stable cell line(HepG2-S-G2), which was transfected with a plasmid harboring the small S gene;the other was the corresponding control cell line(HepG2-Neo-F4),transfected with the vector only. Cellular genes involved in cell metabolism,growth and death,signal-transduction pathways,cytoskeleton and extracellular matrix formation showed altered transcription in HepG2-S-G2 cells.Among these genes,lymphoid enhancer-binding factor 1(LEF-1) was up-regulated consistently and markedly in cells expressing SHBs and HepG2.2.15 cells(a HepG2-derived cell line which produces HBV virion particles).In addition,the phenotype changes of HepG2-S-G2 cells were studied according to the microarray data.Secretion of cyclophilin A(CyPA),the major target for the immunosuppressive drug cyclosporin A(CsA),was found to be induced by the expression and secretion of SHBs.In the second part of this study,the molecular mechanism of SHBs induced LEF-1 up-regulation was investigated.LEF-1 is a crucial downstream mediator of the Wnt signal transduction pathway.Activation of LEF-1 promotes the expression of dozens of downstream effector proteins,which subsequently regulate the progress of cell proliferation and cell cycle.Marked up-regulation of LEF-1 was found in SHBs transient expressing cells and was confirmed by interference experiments with small interfering RNA.By immunofluorescence staining,cellular distribution of LEF-1 protein was found to be altered by SHBs expression.A higher concentration of LEF-1 protein was detected in the cytoplasm of HepG2-S-G2 cells,whilst in HepG2-Neo-F4 cells LEF-1 was located mainly in the nucleus.In addition,38 kDa LEF-1 truncated isoform rather than 55 kDa full-length LEF-1 was induced by the expression of SHBs. These results indicated that SHBs expression resulted in altered expression and distribution patterns of LEF-1 protein in cell compartments and up-regulation of LEF-1 isoforms seemed to suppress,rather than enhance the Wnt pathway.In the third part of this study,the molecular mechanism and its clinical implications of the interaction between SHBs and CyPA were investigated.CyPA secretion was increased in SHBs transfected Huh7 cells.Similar phenomenon was observed in HBsAg positive transgenic mice and HBV infected patients.Serum CyPA levels were markedly increased in HBsAg positive mice and patients,compared to those of normal controls.Increased CyPA secretion was found in association with infiltration of inflammatory cells surrounding HBsAg-expressing hepatocytes in SHBs DNA hydrodymanic injected mice.It indicated that secreted CyPA played an important role in SHBs-induced stress and inflammatory responses.Furthermore, direct and specific protein-protein interaction was observed between CyPA and SHBs. As an intracellular chaperon molecule,the direct binding of CyPA to SHBs might facilitate the folding and production of HBsAg.To address this issue,cyclosporine A (CsA) was added to cell cultures and the expression and secretion of HBsAg in cell cultures were suppressed.In summary,effects of persistent SHBs expression on the gene expression profiles of host cells were studied in a global view.The molecular mechanisms of interactions between SHBs and cellular proteins(LEF-1 and CyPA) were revealed. Their clinical implications and patho-physiological significance were discussed. These findings revealed the pathogenic roles of HBsAg in HBV infections and provided important imformation to the development of new drugs targeting HBsAg.