The Role And Possible Mechanism Of Connexin 43 (Cx43) In The Occurrence And Development Of Multiple Myeloma

Gap junction is a membrane channel in all cells of the human body,which is very important for the physiological function of cells.Gap junctions are formed by connexins,which are responsible for the transfer of bioactive molecules,metabolites and salts between adjacent cells or cells and their extracellular environment.Connexin 43(Cx43)is the most highly expressed gap junction substance in human body.It can be expressed in a variety of cells,including hematopoietic stem cells and stromal cells,and participate in the regulation of hematopoiesis.The abnormal expression of connexin 43(Cx43)is related to cancer recurrence,metastasis and poor survival.The permeability of Cx43 channels to small and large molecules makes it an attractive target for the direct delivery of drugs to the cytoplasm.Because Cx43 can be used as both tumor suppressor and oncogene,it is necessary to analyze the biological characteristics and better understand how Cx43 mediates cancer phenotype in order to develop clinically feasible Cx43-based therapy.GJIC plays an important role in hematopoietic regulation,especially in the initial stage of blood cells development and hematopoietic reconstitution after chemotherapy.Detecting the expression of Cx43 in bone marrow stromal cells in hematological diseases,especially in the hematopoietic microenvironment of myeloma,may provide a new opportunity for the diagnosis and treatment of myeloma.At present,most studies on the failure of hematopoietic stem cell transplantation in the treatment of hematological diseases,some solid tumors and genetic metabolic diseases focus on the source and number of stem cells,but lack of attention to the structural and functional integrity of the recipient hematopoietic microenvironment.What is the effect of pretreatment regimens or a large number of preventive GVHD drugs on Cx43 expression and gap junction function of bone marrow stromal cells in recipients?Is it possible to promote implantation and hematopoietic recovery by changing the functional state of Cx43-mediated GJIc?These problems need to be further studied.In this study,Cx43-/-mouse model was used to observe the effect of Cx43 gene knockout on bone marrow hematopoietic reconstitution at the level of chemotherapy and transplantation,respectively.Functional gap junction communication(GJIC)plays an important role in maintaining normal hematopoiesis.Multiple myeloma(multiplemyeloma,MM)is a common malignant tumor in the hematological system,but little is known about the effect of Cx43 molecules on the biological behavior of MM cells.In this study,we further investigated the effects of bone marrow stromal cells(bonemarrowstromalcells,BMSC)from normal and MM patients on the biological characteristics of MM cells under the condition of direct co-culture.in addition,To further study the effect of GJIC on MM and stromal cells.We constructed CX43-NT overexpressed cells to determine whether cx43C-terminal mediated signal transduction contributes to MM cell survival and migration,and whether bone marrow stromal cell-mediated GJIC is involved in MM clonal proliferation.In addition,we further explored the role of AD in the development and progression of mm in the bone marrow microenvironment of MM.Part 1 Role of connexin43 in regulation and reconstruction of hematopoiesisAim:To establish a conditional connexin 43 gene knock-out mouse model and to explore its role in proliferation and differentiation of developing haematopoietic cells in these mice.Methods:Two pairs of transgenic mice,Cx43 loxP/-_Lyz-Cre/+which are brought from Jackson lab,USA and Lyz-Cre/+brought from Center of Experimental Animal,Jingus,were inbreeded to produce hybrid mice F1.The Cx43 loxP/-_LYZ-Cre/+female F1 mice were selected to mate with Cx431oxP/loxP male mice to finally produce the Cx43-/in hematopoietic system.The gene knock-out in these mice was confirmed by PCR.We detected the expression of Cx43 in different organ of these mice by PCR.Both Cx43-/-and Cx43+/+ mice were all received intravenous injection of 125 mg/kg of 5-FU or 7.5 Gy irradiation with 60Co-y ray equipment and bone marrow cells transfusion,the recovery of their hematpoietic tissues was assessed with different assays at different time interval.In order to discover the change of phenotype of immunologic cells,it was determined by flow cytometry by using different fluorochrome conjugated,such as CD4、CD45R、TCRαβ,Gr-1、Mac-1,CD8a、Sca-1,TER119 and CD117.Results:The Cx43 gene knock-out mouse model was successfully established by mating those 2 pairs of transgenic mice.No Cx43 expression was been observed on blood cell either from peripheral blood or bone marrow.The expression of Cx43 on the liver and spleen cells of these Cx43 gene conditional knock-out mice was decreased significantly as compared with wild type mice(P<0.01),and no effect on the heart and renal cells to express Cx43 was found.The bone marrow cellularity and peripheral blood counts of Cx43-/-mice were markedly reduced as analyzed on day+15 post-5-FU treatment,while the wild type mice recovered to normal(P<0.01).Moreover,hematopoiesis recovery after 5-FU treatment was severely impaired as demonstrated by decreasing proliferation of hematopoietic progenitor content,in which granulomacrophagic colony forming units(CFU-GM)and erythroid forming units(BFU-E)in BM of Cx43 deficient mice were greatly decreased(P<0.01).However,the content of Lin-/c-Kit+/Sca-1+ in Cx43-/-BM was maintained as compared with Cx43+/+BM.Cx43 deficiency mice not only delay the hematopoiesis reconstruction after chemotherapy or bone marrow transplantation,Compared with Cx43/mice,bone marrow cells in bone marrow microenvironment decreased and adipocytes increased.The B and T lineage cell developments were abnormal in Cx43-/-mice.And CD4 and CD8 cells increased significantly(P<0.05),while the CD4+T cells markedly reduced in comparison with Cx43-/-,especially the decrease of TCRαβsubpopulation(P<0.01).Moreover,the CD45R+IgM-subgroup comparison with its counterpart,the CD45R+IgM-subgroup in Cx43 deficiency mice was markedly reduced.Conclusion:It is critical for developing hematopoietic stem cells for the expression of Cx43 on bone marrow microenvironment,especially in response to hematopoietic stress.The hematopoietic regeneration and lymphoid cell development are blocked in Cx43-deficient mice.Part 2 Impact of connexin 43 coupling on survival and migration of multiple myeloma cellsIntroduction:Gap junction(GJS),which represents the most famous intercellular communication(IC)system,is a kind of transmembrane channel that facilitates intercellular communication by allowing small signal molecules to be transferred between cells.In this study,we constructed the amino terminus of human Cx43(CX43nt-GFP),verified cx43-NT overexpression in human umbilical vein endothelial cells(HUVEC),and investigated the role of gap junction(GJS)in multiple myeloma(MM).Material and methods:The cellular expression membrane of Cx43 transmembrane region was constructed,and its half-channel activity was observed.On this basis,the phosphorylation level of the carboxyl terminal(S368)of the full-length Cx43 molecule and the changes of related molecules such as ERK1/2,JNK1/2,p38 and NF κ B in the MAPK signal pathway of mitogen-activated protein kinase pathway were analyzed.The changes of Cx43mRNA and protein expression in cell lines and bone marrow mesenchymal stem cells(MSCs)were detected.Dye transfer was used to analyze the existence of functional GJIC between MM,MSCs or MM and HUVECCx43-NT.Results:It was confirmed that the migration activity of MM cells to MSCs or HUVECCx43-N was channel dependent,which was stronger than that of spontaneous migration,and could protect MM cells from apoptosis mediated by dexamethasone plate by secreting cytokines.At the same time,it was also found that the up-regulated expression of p38 and JNK signal pathway at the carboxyl terminal of Cx43 was involved in the migration of MM cells to MSCs or HUVECCx43-NT.Conclusion:The GJIC between MM and MSCs is one of the important factors of tumor cell proliferation and drug sensitivity,and is related to the pathogenesis of MM.Part 3 Bone Marrow Adipocytes Promote Multiple Myeloma Cells Survival and Up-Regulate Its ChemoresistanceIntroduction:To investigate the role of adipocytes in the bone marrow microenvironment of patients with multiple myeloma(MM)on pathogenesis MM.Material and methods:Bone marrow adipocyte(BMA)in normal and newly diagnosed MM(ND-MM)bone marrow smears was evaluated with oil red O staining.The mesenchymal stem cell(MSC)from normal and ND-MM patients were isolated and cultured in vitro.In vitro co-culture assay was used to explore the effects MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells.The expression of adipogenic/osteogenic differentiation associated genes PPRA-γ,DLK1,DGAT1,FABP4,FASN and ALP both in MSC and MSC derived adipocytes was determined with real time PCR.The western blot was employed to detect the levels of IL-6,IL-10,SDF-1α,TNF-α and IGF-1 in the supernatant with or without PPRA-y inhibitor.Results:The oil red O staining of bone marrow smears confirmed that BMA increased significantly in patients of ND-MM and the bone marrow adiposity was related to the disease state.The number of BMA decreased in those who are response to the chemotherapy.Our results showed that MM cells upregulated MSC adipogenic differentiation associated genes PPRA-γ,DLK1 DGAT1,FABP4 and FASN expression,but the osteogenic differentiation related gene ALP was significantly down regulated.This means that the direct consequence of the interaction between MM and MSC in the bone marrow microenvironment is to push MSC differentiation into adipocytes at the expense of osteoblasts.At the same time,we demonstrated that cytokines detected in supernatant changed.PPRA-γ inhibitor G3335 could partially reverse the release of cytokines by BMA.Those results confirmed that increased BMA regulated its cytokine release via PPAR-γsignal,which in turn could protect the MM cells from drug-induced apoptosis,and that PPRA-γ inhibitor G3335 could distort PPRA-γ mediated BMA maturation and cytokine release.BMA could protect the MM cells from drug-induced apoptosis.Conclusion:Our finding suggested that increased in BMA and its associated cytokines acted as a promotion factor in MM cells survival,proliferation and migration.BMA can protect MM cells from drug-induced apoptosis and plays an important contribution to MM treatment failure and disease progression.