GJB2 Positively Regulates B Cell Differentiation By Upregulating BCR Signals And Actin Recombination

Background:Gap junction protein beta-2(GJB2),a member of connexin family,also known as Connexin 26(Cx26),which is expressed in almost all cell types,mainly in epithelial cells.The GJB2 protein monomer can assemble itself or with other members of the gap junction protein family to form a hexamer on the endoplasmic reticulum or Golgi body to form a half channel,and then a gap junction channel is formed end to end with a half channel of another cell.These channels allow ions,small molecules,nucleotides and peptides to be transported from cell to cell.In addition to its role as a channel,GJB2 involves in many biological activities including cell adhesion and migration,differentiation and development,neuronal activity and immune response.Studies have shown that other gap junction protein family members involved in the T-cell dependent antigen stimulation of B cell synthesis immunoglobulin Ig M and the differentiation or function of lymphocytes in the immune response.Whether GJB2 protein is involved in the development,differentiation and function of B cells has not been studied yet.Objectives:1.To explore the important role of GJB2 in the development and differentiation of B cells.2.To explore whether GJB2 is involved in the regulation of BCR signal and actin recombination?3.To explore the irreplaceable important role of GJB2 in B cell function.Methods:a mouse model of specific Gjb2 gene knockout in B-cells was constructed.Flow cytometry to detect the number and proportion of B cell subsets in bone marrow,spleen and abdominal cavity,and spleen B cell proliferation and apoptosis of WT and KO mice.Western blot is used to detect the changes of BCR signaling molecules,actin molecules in spleen B cells of WT and KO mice.BCR signal molecules and the co-localization of these signal molecules with BCR in B cells from spleens of WT and KO mice are detected by confocal.TIRFM is used to detect the early activation events of B cells,including the formation of BCR clusters,the expansion of B cells,and the expression of BCR signaling molecules.Finally,flow cytometry is used to detect the cell proportion of positive B cell subsets capable of producing various types of antibodies after spleen purified B cells of two groups of mice stimμlated by different stimμlants,so as to evaluate the changes in the isotype switching ability of B cells.Resμlts:1.There was no significant change in the number and proportion of bone marrow B cell subsets after GJB2 deficiency.2.GJB2 deficiency resulted in a decreased proportion of B cells in the marginal region of the spleen(MZ B)and down-regulated expression of CD21.3.GJB2 deficiency resulted in decreased activation of both positive and negative proximal signals of BCR.4.GJB2 deficiency leads to decreased activation of actin regulatory molecules and actin aggregation.5.GJB2 deficiency leads to reduced B cell expansion,BCR cluster formation and weakened signal transduction,which affects the early activation of B cells.6.There was no significant change in the isotype switching ability of B cells after GJB2 deficiency.Conclusion: GJB2 is not necessary for the development of B cells,but GJB2 participates in the early activation of B cells and thus regulates the differentiation process of B cells by upregμlating BCR signal and actin molecules and regulating actin recombination.