Structure Based Mutational Analysis Of Homophilic Interaction Of HAb18G/CD147and Its Function In Invasion Of Hepatocellular Carcinoma

Metastasis is the most frequent cause of death in cancer patients. Matrixmetalloproteinases (MMPs) play an important role in cancer progression. MMPscan be expressed and secreted by tumor cells and by most of the cells present inthe microenvironment of the tumor, such as fibroblasts, macrophages andendothelial cells. MMPs are important for degradation numerous substrate,including ECM proteins, growth factors, cell-cell adhesion molecules andMMPs themselves~[1,2]. The most well-known function of MMPs is matrixremodeling. Matrix remodeling increases invasion by removing a physicalmatrix barrier. CD147was initially reported that interstitial collagenase (MMP-1)production was induced during the co-culturing of tumor cells and fibroblasts~[3,4]. Further studies revealed that CD147is capable of inducing the expression ofseveral MMPs other than MMP-1, including MMP-2, MMP-3, MMP-9andMMP-11, it was also determined that either CD147expressing tumor cells orconditioned media from the same cells were equally capable of inducing MMPproduction in co-cultured fibroblast cells~[5,6,7].HAb18G is a novel hepatoma-associated antigen cloned by HCC monoclonal antibody HAb18screening from human HCC cDNA library. It isabundantly expressed in human HCC tissues and on the cell surface of severalhighly metastatic HCC cell lines~[8]. The amino acid sequence of HAb18G isidentical to that of CD147~[9]. CD147expressed on tumor cells plays a key rolein inducing the production of MMPs by surrounding stromal cells and tumorcells themselves~[10]. Homophilic interaction is an important model of action formany cell surface proteins and transmembrane proteins, such as the integrinsuperfamily, the cadherin superfamily and the Ig superfamily. Homophilicinteraction of HAb18G/CD147has been revealed in previous studies, however,the exact relationship between dimerization and function of HAb18G/CD147still remains to be elucidated. This study is expected to provide insight into thefunction of dimerizaition of HAb18G/CD147in hepatoma invasion.To determine what amino acid residues are required for dimerization ofHAb18G/CD147, we have created mutant types of HAb18G/CD147(MTs)coding for proteins in which residues on the dimer interface were altered,recombinant expressed and purified the extracellular region of mutant protein(rMT), and tested them for their ability to form dimers in solution. Immunoblotanalysis of the purified fusion protein resolved by Native-PAGE revealed thatrWT, rM2, rM5, rM6, rM12could form dimers in solution, and the othermutants were effective mutants which could not form dimers in solution. Alsowe checked their dimerization state in living cells by BiFC assay. We concludedthat M7and M14have significantly reduced but not eliminated homophilicinteraction in living cells. It was demonstrated that targeted mutagenesis coulddecrease dimerization of HAb18G/CD147.The evidence that rWT maintaining a native conformation can activate the MAP kinase signaling pathway and stimulate MMPs expression in fibroblasts[11,12]suggested that dimerization might be involved in these functions. To test this,We chose four representative MTs, namely, MT4, MT5, MT7, MT14, toparticipate the subsequent functional experiments. We observed that rWT, rMT4and rMT5could activate MAP kinase signaling pathway in SMMC-7721cells,HeLa cells and a SMMC-7721–MRC-5coculture system and stimulate MMP2expression in the coculture system. In sharp contrast, rMT7and rMT14lostthese functions. Furthermore, MAP kinase phosphorylation level andinvasiveness was significantly increased in SMMC-7721cells overexpressingMT4, MT5and WT compared to those overexpressing MT7, MT14and GFP. Ina coculture system where the stably transfected cells above were cocultrued withMRC-5we observed the similar effect.In conclusion, the data presented herein conform that the non-glycosylatedextracellular region of HAb18G/CD147can form dimers and activate MAPkinase signaling pathway leading to increased expression of MMP2. For the firsttime, we demonstrate that dimerization of HAb18G/CD147can be significantlydecreased by structure-based targeted mutagenesis and identify the key aminoacid residues on the dimer interface. This study also demonstrates thathemophilic interaction of HAb18G/CD147is involved in activation of MAPkinase signaling pathway, induction of MMP2production and hepatomainvasion. Thus this study puts forward a novel mechanism of malignancy ofhepatoma, in that the increased expression of HAb18G/CD147progresses tumorinvasion along with the activation of MAP kinase signaling pathway and theincreased expression of MMP-2via hemophilic interaction of HAb18G/CD147.Finally, considering the pathological significance of HAb18G/CD147in hepatoma invasion, these findings may provide a promising strategy fordeveloping competent antibodies and small molecule antagonists againstdimerization of HAb18G/CD147for treatment of hepatoma.