The Molecular Mechanism Of Scaffold Protein GIT2in Modulating Immune Liver Injury

GIT2(G protein-coupled receptor kinase interacting proteins2) is a kind ofmultidomain signaling scaffold protein, and belongs to the Arf GAPs family (ARFGTPase-activating protein). The Git2-/-mice display multiple symptoms ofimmunodeficiency, such as extramedullary hematopoiesis, splenomegaly, marrowneutrophils increased, pulmonary infiltration of lymphocytes increased, more sensitivityto infecting the pathogen. Many researches show that GIT2palys a key role in immunesystem. The deficiency of GIT2could lead to the damage of direction response aboutchemotaxis of neutrophils and excessive production of superoxide. In the thymus ofTCR genetically modified Git2-/-mice, the damage in the development process fromCD4+CD8+T cells to CD4+CD8-T cells can result in the reducing of CD4+CD8-T cells.These all mentions that GIT2is critical in the system of natural immunity and acquiredimmune system. And other evidence also indicates that GIT2could form a tri-moleculecomplex with αPIX, PAK1to regulating the TCR alternative path and activating PAK1indepent with LAT、SLP-76、Nck and Rac. Our former laboratory studies found thatGIT2-deficent mice could avoid Con A(Concanavalin A) induced immunological liverinjury. So we speculated GIT2may be involved in regulating the development ofimmunoligical liver injury.We isolated the spleen mononuclear cells from GIT2knocked-out mice and pickedout CD3+and CD4+T cells to be stimulated in vitro. And then check proliferation,cytokine secretion. Due to Con A mainly function on T cell receptor, the study werefoucused on the effect on the activation of TCR pathway, including phosphorylation of signaling molecules, changes of cytoskeletal remodeling, formation of immune synapseand small GTPase activity of Rac/Cdc42. The results showed that the deletion of GIT2could decrease the proliferation and cytokine production of T lymphocytes. Althoughthis cannot affect the activation of classic TCR pathway, it will impact on the formationof GIT2-αPIX-PAK1trimer and damage the accumulation of F-actin at the site ofimmunological synapse and formation of cap structure. In addition, we also found thatdeletion of GIT2reduced the stability of αPIX and led to its ubiquitination, PAK1inhibitor, IPA3, can inhibit T lymphocyte proliferation and activation in wild-type mice.Thus, we infer that the lack of GIT2lead to the down-regulation of αPIX protein leveland resulting GIT2-αPIX-PAK1mediated unclassic TCR pathway was damaged. PAK1can not be effectively phosphorylated and can not futher weaken the activation of T cellin immune liver injury and cytokine secretion to play a protective role.Further, since GIT2was highly expressed in damaged parenchymal liver cell, sowe studied the expression change of critical molecules in damaged liver of Git2+/+andGit2-/-mice induced by Con A, also incude the differences of signal path, target geneexpression. The results suggest that the NF-κB was activated, phosphorylation ofSmad3and ERK were inhibited. Previous study found GIT2can raise A20to affectNEMO ubiquitination and play negative role in the regulation of NF-κB. Therefore, weuse the Real-Time PCR technique to study the transcription level of NF-κB downstreamtarget genes affected by GIT2in damaged liver tissue induced by Con A, and the levelincreased. From two-hybrid yeast screening, we found that Smad3is a candidateinteracting molecule of GIT2. And evidences supported that Smad3deficient micecould be away with immune liver damage induced by ConA. Therefore, we used co-IPto verify the interaction between GIT2and Smad3and overexpression of GIT2increased Smad3-dependent transcription induced by TGF-β1. These findings raise theintriguing possibility that GIT2is a new regulator of Smad3and involved in regulatingthe hepatocytes injury． In this study, we reserch the different mechanisms of how GIT2worked in theprocess of liver immune damagement on T lymphocytes and hepatocytes. In the Tlymphocytes, GIT2form a trimer together with PIX and PAK1to take part in mediatingunclassic TCR pathway and activating PAK1and eventually leading to the activation ofT cells in liver and the cytokine secretion. Cytokine could cause the damagement tohepatocytes. In hepatocytes, GIT2were involved in the inhibition of NF-κB activationand then inhibited the transcription of downsteam anti-apoptotic gene leading to thefinal apoptosis of hepatocytes. In addition, GIT2can interact with Smad3regulateing itstranscriptional activity and possibly participate in regulating the process of immunizingdamagement of hepatocytes. But the detailed functional mechanism need to be furtherresearched.