Research Progress In Receptor Of Hepassocin

Hepassocin(HPS), also known as hepatocytes derived fibrinogen-related protein 1 or Fibrinogen-Like 1(FGL1), a liver-specific of mitogenic factor and partial hepatectomy, carbon tetrachloride(CCl4), D-galactosamine(D-gal N) induced hepatocyte damage repair plays an important role. Our previous studies have shown that HPS though epidermal growth factor receptor(EGFR) activation of MAPK signaling pathwaydependent manner and mediates promotes normal liver cell proliferation. However, if there exist HPS specific receptors and how does it work is still unclear. In this study, we used CHO cell expression system to get rh HPS and established a biological activity assay method in vitro, that confirmed to obtain high activity rh HPS. Further immunofluorescence experiments and receptor binding experiments confirmed the presence of liver-derived cell specific receptor HPS. Finally, We found ANXA2 receptor molecules HPS candidate molecule and confirmed ANXA2 molecules involved in membrane transfer HPS signal, indicating that at least one receptor molecule ANXA2 component HPS variety of ways. These findings for the study of signaling pathways mediated HPS and severe an envidence in liver disease.We successfully constructed CHO-HPS-expressing cell lines, and got multiple batches HPS sample, this study verified the physical and chemical properties and putity of recombinant human HPS, which confirmed to obtain high purity HPS. Further, we optimized experimental conditions and established Ed U method and LDH method to measure the acticity of HPS. Experimental results show that the recombinant HPS dose-dependent manner to promote hepatocyte proliferation, and inhibition of CCl4-induced LDH release, shows that we obtain recombinant HPS with high purity and high activity, which can be used for HPS and HPS receptor and its signaling pathway function study. Both of these methods are simple, reproducible, better reflects the HPS in vivo by promoting liver cell proliferation, inhibition of liver cell necrosis treatment of acute liver injury in mice activity and can be used to evaluate the in vitro biological activity of recombinant HPS. So we build a quality control and research functions of HPS. To confirm the existence of HPS specific receptors on the cell membrane, we first use FITC fluorescence labeled HPS, and HPS was observed by immunofluorescence whether the cell membrane and liver found FITC-labeled specific binding may HPS in liver cell membrane portion of L02. It remind us that HPS specific binding receptor in L02 presents. To verify this result, we used the classic method of 125I-HPS, in vitro biological activity test showed 125 IHPS maintained promoting liver cell proliferation activity, suggesting that 125I- without breaking the spatial structure of HPS. Receptor binding experiments found that HPS and L02 human hepatocytes, binding Hep G2 human hepatoma cells and mouse primary hepatocytes showed typical way binding kinetics of receptor and ligand, the saturation concentration of L02 cells 1546.32ng/m L, the saturation concentration of Hep G2 cells was 2753.84ng/m L, the saturation concentration of mouse hepatocytes 2242.76ng/m L. Competition experiments showed that the concentration of non-standard high power HPS can effectively compete with cell binding 125I-HPS, in line with competition curves between receptor ligands. These results indicate the presence of HPS said liver-derived cell specific membrane receptors.Scatchard analysis showed that three cells there is only one source of liver receptor HPS. HPS and L02 cells, Hep G2 cells, mouse primary hepatocytes binding constants were 6.71pmol/L, 7.7nmol/L and 2.17nmol/L. L02 cells, Hep G2 cells, mouse primary hepatocytes HPS surface receptor number was 3.1×105sites/cell, 1.8×105sites/cell and 4.8×105sites/cell. Mouse primary hepatocytes have larger number of receptors, which may be related to the relatively large size. Detecting 125 IHPS and non-liver-derived cells A549, Hela and EA926 cells found in combination with HPS have liver-specific receptors. High concentrations of EGF, HGF binding HPS can not compete with the liver cells, indicating that different from the EGF receptor HPS and HGF receptor. Furthermore, we use the ligand to the receptor crosslinking experiments also confirmed the presence of HPS specific receptor. Autoradiography indicated that 125I-HPS and receptor complex formed was about the size of the 110 k D, the molecular weight of the 34 k D HPS, dimerization for activity,HPS receptor presumed molecular weight of about 42 k D.To confirm HPS receptor molecules, we used proteomic techniques identified after HPS stimulate cell membrane protein phosphorylation changes found HPS stimulate L02 cells significantly induced tyrosine phosphorylation of the protein band of about 40 k D, this one with mass spectrometry identified four candidate proteins obtained by the candidate protein tissue distribution, cellular localization, functional analysis, comprehensive judgment, we may speculate ANXA2 as HPS receptor. Further, we can verify the HPS induce ANXA2 tyrosine phosphorylation, and HPS can be copositioned with ANXA2 in the cell membrane and interact; ANXA2 expression using RNA interference reduced, significantly reduced binding to 125I-HPS to L02 cells; down-regulated expression ANXA2, that inhibits HPS activity EGFR phosphorylation and MAPK pathway and inhibits cell proliferation and HPS promote L02 reduce CCl4 induced liver injury activity.These experimental results suggest ANXA2 is a key molecule HPS signaling pathways involved in the binding process HPS liver derived cells, the receptor is HPS HPS or at least part of the receptor.We also established a method for homology modeling three-dimensional structure of HPS, through the model to optimize the eventual establishment of a high-quality three-dimensional structural model of HPS. This model ANXA2 be rigid docking, combined for multiple configurations optimized to obtain the best combination of HPS and ANXA2 configuration. ANXA2 with HPS interacting residues, PHE72, GLN69, MET11.Corresponding HPS role residues, Glu59, Leu55, Lys65.To confirm HPS receptor is important to elucidate the various aspects of HPS signal pathway, function and role in severe liver disease, etc., at the same time, HPS receptors may also become a target for liver disease, for the development of new drugs. While the preliminary experiments confirm ANXA2 participation HPS and cell binding process, but the lack of strict ANXA2 liver tissue specificity, can not be explained in terms of tissue distribution of HPS specific mechanisms to promote hepatocyte proliferation, in addition, our previous studies have shown that stimulation of HPS hepatocyte proliferation is dependent on the activation of EGFR, ANXA2 has been reported to interact with the EGFR occurred and regulate its activation, therefore, the next step we need to investigate the role of HPS-ANXA2- EGFR-MAPK signaling pathway in the HPS and biologic function, particularly liver-specific selection mechanism.