Regulation Of Raf-MEK1/2 Signaling Pathway By HCV E2 Protein Via DC-SIGN

Hepatitis C virus (HCV) is an enveloped and positive-stranded RNA virus belonging to the Flaviviridae family. HCV infection causes chronic hepatitis and is linked to the development of hepatocellular carcinoma. The HCV pathogenesis remains to be clarified based on the regulation of cellular signaling pathways by HCV proteins.Recently, culture systems that support HCV replication and viral particle formation in vitro have been developed. Albeit this limitation, capture molecules involved in low-affinity interaction for the initial contact of HCV with target cells and potential high-affinity receptor candidates mediated HCV trafficking and fusion have been described. HCV like particles (HCV-LPs), solubilized and chimeric HCV glycoprotein E1E2, soluble E2 protein, and pseudoparticles (HCVpp) were proposed to be robust model systems for the study of HCV entry. Some cell surface molecules have thereby been identified as HCV receptors including human tetraspanin CD81, low-density lipoprotein receptor (LDLR), scavenger receptor B type I, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin, CD209), and L-SIGN (liver and lymph node-specific, CD209L). Curiously, these molecules are naturally expressed on a number of cell lines of hepatic and non-hepatic origin. Several groups have shown that these receptors are necessary but not sufficient for HCV entry into target cells. DC-SIGN and L-SIGN are known to be capture receptors for HCV. Moreover, capture of HCV by SIGN molecules depends on the presence of CRD, indicating that recognition of high mannose oligosaccharides in the viral envelope glycoproteins is critical for HCV binding.MAPK signaling pathways regulate many cellular functions such as cell growth, transformation, and apoptosis. Raf and MEK1/2, serine/threonine kinase, are essential components of the MAPK pathways. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK, MAPK) which phosphorylates various cytoplasmic and nuclear proteins. Regulation of Raf and MEK1/2 is crucial in the maintenance of cell growth as oncogenic mutations in the genes lead to high transforming activity. The regulation of Raf is complex and involves the integration ofother signaling pathways as well as phosphorylation, dephosphorylation, and protein-protein interaction. HCV core and nonstructural proteins were suggested to modulate various intracellular signaling pathways, which was involved in the promotion of target cell proliferation and maintenance of HCV-infected cell survival.Previously, we demonstrated that the soluble HCV E2 protein expressed in CHO cells up-regulated MAPK signaling pathways via CD81 and LDLR. It is known that the interaction of HCV E2 protein with cellular receptors is a major step in determining the replication and the outcome of infection. The response of cells to extracellular signals is mediated by surface receptors and intracellular signal molecules. Here, we showed that HCV E2 protein activated Raf-MEKl/2 pathway through DC-SIGN on the surface of cells. Studies on early signaling events triggered by HCV E2 protein will be fit for further understanding pathogenesis of HCV and opening an approach for new therapies for HCV.Regulation of Raf-MEK1/2 signaling pathway by HCV E2 protein via DC-SIGNIn this study, NIH3T3/MX-DC-SIGN and NIH3T3/MX-L-SIGN cells stably expressing DC-SIGN and L-SIGN were used to be cell models. FACS analysis showed that DC-SIGN and L-SIGN were expressed at a high level on the surface of cells. Binding of HCV E2 protein to NIH3T3/MX-DC-SIGN and NIH3T3/MX-L-SIGN cells was detected. The E2 binding was inhibited by DC-SIGNmAb or L-SIGNmAb in a dose-dependent manner. In contrast, expression of DC-SIGN and L-SIGN as well as E2 binding was undetectable in parental NIH3T3 cells.Moreover, the cellular distribution pattern of DC-SIGN and binding of HCV E2 protein were also revealed by confocal microscopy. DC-SIGN expression (green fluorescence) was found to localize on the surface of NIH3T3/MX-DC-SIGN cells. E2 protein (red fluorescence) distributed both on the cell membrane and in the cytoplasmic regions. The areas of overlap (yellow fluorescence) indicated the interplay between E2 protein and DC-SIGN. At the same time, no fluorescence was observed in NIH3T3 cells, which was consistant with the FACS result.NIH3T3/MX-DC-SIGN cells were treated with HCV E2 protein for different time lengths, and the cell lysates were then collected for detection of Raf and MEK1/2 by Western blotting. Rabbit antibodies against Phospho-Raf and Phospho-MEKl/2 respectively detect Raf andMEK1/2 when catalytically activated by phosphorylation at Ser 338 of Raf and Ser 217/221 of MEK1/2. Levels of Raf and MEK1/2 phosphorylation were increased in the cells treated with E2 protein as compared to the untreated cells. Stimulation of the cells with 2 jug/ml E2 protein for 30 min resulted in significant phosphorylation of Raf and MEK1/2. The constant amounts of total Raf and MEK1/2 were confirmed. The levels of Raf and MEK1/2 phosphorylation induced by E2 protein were reduced by blockage of DC-SIGN with DC-SIGNmAb as well as L-SIGNmAb. In addition, MEK1/2 inhibitor U0126 pretreatment prevented E2-induced MEK1/2 phosphorylation. In response to E2 protein, DC-SIGNmAb or L-SIGNmAb, there was no obvious difference in Raf and MEK1/2 phosphorylation in parental NIH3T3 cells. These results suggested that HCV E2 protein activated Raf-MEKl/2 signaling pathway via DC-SIGN.Construction of DC-SIGN and L-SIGN expressing plasmidsTo investigate regulation of Raf-MEKl/2 pathway by HCV E2 protein in transient transfected cells, pEGFP-Nl-DC-SIGN and pEGFP-Nl-L-SIGN expressing plasmids were successfully constructed. These plasmids were then transfected into HEK293T and HeLa cells. Kinetic fluorescence changes were monitored using fluorescence microscope. Green fluorescence was detectable in the two cells transfected with pEGFP-Nl-DC-SIGN or pEGFP-Nl-L-SIGN plasmid, while strong fluorescence was observed in the HEK293T cells. Up to 48 h, the expression of DC-SIGN protein was confirmed by Western blot analysis. The result showed that DC-SIGN was expressed at a high level in the HEK293T cells but at a low level in the HeLa cells. Therefore, the HEK293T cells effectively expressing DC-SIGN are suitable for the investigation of Raf-MEKl/2 signaling pathway triggered by HCV E2 protein.