Functional Analysis Of67kD Laminin Receptor And The Pathways It Regulates In Cell Growth And Larval Development Of Clam Meretrix Meretrix

Laminin receptor (LR) is a transmembrane glycoprotein expressed on the surfaceof the cell, which mediated interactions between cell and cell, cell and extracellularmatrix. The combination of LR and its ligand laminin (LN) conduct the signalingtransduction pathways between cell and extracellular matrix. It plays great roles inearly embryonic development, organogenesis and maintaining normal physiologicalactivities of organisms. At present, studies on LR and the signaling pathways itregulates mainly focused on the animal model, few studies on marine invertebrateswas reported.The clam Meretrix meretrix is a representative species of marine bivalve, whichis an important commercial bivalve in China. In the present study, we cloned andcharacterized a67kD LR gene from M. meretrix and named it MmeLR. On this basis,further studies at the cellular level, larval and adult stages of the regulatorymechanisms of MmeLR and the pathways it regulates on the growth and developmentof M. meretrix were carried out.A recombinant MmeLR (rMmeLR) protein was obtained by prokaryoticexpression. Its tissue expression characteristics were examined on both thetranscriptional and translational levels. The MmeLR mRNA and protein detected byrealtime PCR and western blots were primarily distributed in muscle tissues.Far-western analysis showed a specific interaction between rMmeLR and LN.We successfully cultured primary cells from the different tissues of M. meretrixfor at least2weeks by a modified culture condition. The results of the binding assaysuggested a role of LR in cell adhesion and apoptosis in cultured primary cells ofmantle tissues from M. meretrix. The bcl-2mRNA expression levels in primary cells cultured in matrigel (mainly laminin) coated plates was significantly higher than incells cultured in non-coated plates at48h of culture, while the p53mRNA expressionpattern was inversely related to that of bcl-2, suggesting that MmeLR is involved inp53-dependent apoptosis, and the binding between MmeLR and laminin inhibitsapoptosis during primary cell culture.Double strand RNA (dsRNA) and siRNA (small interference RNA) of MmeLRwere prepared to develop the method of RNAi in the primary cells of M. meretrix. Thesilencing efficiency was about60%. LR-pEGFP-N1and LR-pEGFP-ie1plasmidswere construted. Transfection experiments in HeLa and SF9cell lines were performedand EGFP positive signals can be stably detected. In M. meretrix primary cells, thetransfection conditions were explored, including electroporation, liposometransfection methods.Realtime PCR, Western blot and immunofluorescence were used to analyze theexpression profile of MmeLR in different larval stages of M. meretrix. RNAi wasproformed on M. meretrix larvae using siRNA, and the downstream pathways wereanalyzed. The MmeLR downstream protein calmodulin (MmeCAM), calmodulinkinase IV (MmeCAMKIV) were cloned and analyzed. The CAM inhibitortrifluoperazine (TFP) was used to discover roles of MmeCaM in larval developmentof M. meretrix.This study may increase the understanding of the role of LR in cell adhesion andapoptosis and help to improve the culture of primary cells of marine invertebrates.