Hepatitis B Virus Proteins Induce Activation Of Hfgl2 Gene Transcription Through C-Ets-2 Transcription Factor

Fibrinogen-like protein 2 (fgl2) /fibroleukin also known as fgl2 prothrombinase was identified to belong to the fibrinogen protein superfamily. Fgl2 was expressed mainly in activated macrophages and endothelial cells. Fgl2 gene encodes for two different types of proteins which are membrane bound fgl2 and soluble fgl2. Fgl2 prothrombinase has been shown to have the attributes of a serine protease capable of directly cleaving prothrombin to thrombin leading to fibrin deposition and act as blood coagulation factor X. Previous work has demonstrated that fgl2 participated in the pathogenesis of fulminant hepatitis or human severe acute on chronic (AOC) hepatitis B, fetal loss, xenotransplant rejection and so on. Previous work also found mouse fgl2 (mfgl2) expressed strongly in susceptible mouse post murine hepatitis virus-3 (MHV-3) infection. The nucleocapsid protein of MHV-3 induced transcription of mfgl2 and invoked the hepatic nuclear factor 4αas a key transcription factor participating in the regulation of mfgl2 gene expression.In Asia, hepatitis B virus (HBV) is still one of the most frequent causes of liver failure. Fgl2/fibroleukin gene plays a pivotal role in the pathogenesis of both experimental and human severe form of viral hepatitis. However the viral and host factors involved in the transcription of hfgl2 gene have not been defined. The aim of this study was to define the viral and host factors involved in the transcription of human fgl2 (hfgl2). HBc, HBs or HBx expression plasmids were cotransfected with a hfgl2 luciferase report construct into Chinese Hamster Ovary (CHO) cells and HepG2 cells respectively. Luciferase assay showed that HBc or HBx protein, but not HBs protein significantly enhanced hfgl2 transcription activity in both CHO cells and HepG2 cells. Expression of these plasmids in eukaryotic cells was detected by immunohistochemistry and Western blottingting. The transcriptional activity which HBV proteins induced hfgl2 gene was determined by the activity of luciferase andβ-galactosidase served as internal control. The results showed that CHO cells transfected with eukaryotic expressing plasmids of HBV proteins could express HBV encoding proteins transiently. Relative luciferase activity in CHO cells transfected with pcDNA-HBc or pcDNA-HBx was at an average of 5.4-fold and 6-fold elevation when compared with the control group, and 8.7-fold and 11-fold respectively in HepG2 cells. These results indicate that HBc and HBx protein but not HBs activate the transcription of hfgl2 gene.A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hfgl2 gene transcription in response to HBc or HBx proteins expressed by respective expressional plasmids. By site-directed mutagenesis, the overlapping cis-elements LEF/c-Ets in the region of -712/-568 was demonstrated to play an important role in hfgl2 gene transcription in response to HBc protein, while two cis-elements LEF/c-Ets and HSTF in the same domain were found to account for hfgl2 transcription in response to HBx protein. EMSA assays using nuclear extracts from THP-1 cells showed that an Ets family member c-Ets-2 bound to the cognate cis-element in hfgl2 promoter which was shown to be responsible for hfgl2 gene transcription in response to viral proteins. ChiP assay using c-Ets-2 antibody found that the DNA fragment, which bound to transcription factor c-Ets-2, was within the sequence of hfgl2 promoter. It was evidenced c-Ets-2 partially translocated into the nucleus of THP-1cells in response to both HBc and HBx by confocal immunofluorescence study and Western blotting. shRNA interference of c-Ets-2 expression was able to decrease the relative luciferase activity of hfgl2 promoter by 64.8%.In human, c-Ets-2 protein was highly expressed in PBMC isolated from patients with severe AOC hepatitis B when compared with health controls. Increased activation of P-ERK, P-JNK and P-p38 MAPK were found in PBMC isolated from patients with severe AOC hepatitis B when compared with health controls. Treatment with ERK inhibitor PD098059 but not P-p38 MAPK inhibitor SB203580 or JNK inhibitor SP600125 abolished the upregulated expression of c-Ets-2 in response to HBc protein, while JNK inhibitor SP600125 but not ERK inhibitor PD098059 or P-p38 MAPK inhibitor SB203580 abolished the upregulated expression of c-Ets-2 in response to HBx protein, suggesting ERK and JNK MAPK signal pathways were involved in the expression of c-Ets-2 in response to HBc and HBx proteins respectively.In conclusion, these studies have demonstrated that HBc and HBx initiated the transcription of hfgl2 gene through c-Ets-2 transcription factor, which was dependent on the activation of ERK and JNK signal pathway in corresponding to either HBc or HBx proteins. This work provides new insights in the interaction between HBV virus and host gene hfgl2 expression. The transcription factor c-Ets-2 could be a new therapeutic target for diseases intervention such as fulminant or severe AOC hepatitis B.