EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.     

Mutation of Human Connexin43 Amino Acids S279/S282 Increases Protein Stability Upon Treatment with Epidermal Growth Factor

Connexins are the structural units of gap junctions, structures allowing interchanging of information between the adjacent cells. Connexin43 (Cx43) is the most abundant gap junction protein. Cx43 can be degraded by lysosome- and proteasome-mediated processes upon internalisation of the entire structure. Only little is known about the role of phosphorylation during the gap junction degradation. In Cx43, a protein containing 14 amino acids susceptible to be phosphorylated, amino acids S279 and S282 are phosphorylated upon epidermal growth factor (EGF) treatment by erk1/2 MAP kinases. Here, we show that the wild-type Cx43 protein, as well as HeLa cells expressing the mutated Cx43 proteins S279A, S282A, and S279A/S282A, is mainly located at the plasma membrane. However, the protein stability is not altered in the isolated single mutants, whereas the double mutant S279A/S282A is strongly degradation impaired upon EGF treatment. This effect is not due to the decreased Cx43 internalisation, but seems to be related to a reduced ubiquitination.     

TCF7 is highly expressed in immune cells on the atherosclerotic plaques,and regulating inflammatory signaling via NFκB/AKT/STAT1 signaling

Atherosclerosis, which is the fundamental basis for cardiovascular diseases in the global world, is driven by multiple roles of the immune system in the circulation and vascular plaque. Recent studies demonstrated that T-cell infiltrates into aorta plaque and plays an important role in recruiting macrophages to the vascular wall. Here, using single-cell sequencing, we found T cells in patients’ plaques and differentially expressed genes (DEGs) of T cells in atherosclerosis mice. T cells and macrophages were continuously activated in atherosclerotic plaque in patients. Besides, other immune cells also take part in atherogenesis, such as natural killer (NK) cells, granulocytes. Interferon (IFN)/NFκB signaling, the AKT signaling pathway was highly activated in mouse (in vivo) and cell line (in vitro). TCF7 and XCL1 were regulated by AKT and NFκB, respectively through protein–protein network analysis. Therefore, we attempt to clarify and discover potential genes and new mechanisms associated with atherosclerosis for drug development.