Effects Of MKP5 On The Interaction Between Islet Cells And Macrophages Upon Glycolipidtoxicity

Type 2 diabetes mellitus(T2DM)is a serious disease to harm human health and is characterized by insulin resistance and disfunction of islet cells.Obesity is one of the risk factors of T2 DM.In obese pancreatic tissue,chronic low-grade inflammation occurs frequently,wherein amount of macrophages infiltrate and release inflammatory cytokines,which further impairing islet cells.Therefore,crosstalk of macrophage and islet cell plays crucial role in T2 DM progression.Mitogen-activated protein kinase(MAPK)pathway is mainly composed of Extracellular-signal-regulated Protein Kinase(ERK),C-jun N-terminal Kinase(JNK)and P38 protein kinase,which vitally functioned in cell differentiation,proliferation,Carcinogenesis,apoptosis and inflammation.Recently,studies found that mitogen activated MAPK phosphatase 5(MKP5)can negative regulate MAPK pathway to alleviate the dysfunction and apoptosis of islet cells upon obesity.Besides,MKP5 also involve in GP induced inflammation in macrophage of adipose tissue.However,it is unclear that whether MKP5 is associated with macrophage-islet cell interaction under glucolipotoxicity.In this study,we constructed MKP5 overexpressed macrophage and islet ? cell line and investigated the role of MKP5 in the interaction of islet ? cell and macrophage upon glucolipotoxicity.Furthermore,the molecular mechanism was also discussed.Method:1.Human MKP5 DNA fragment was cloned into pcDNA3.1 vector and transfected into RAW264.7 and MIN6 cells.The stable MKP5 overexpression cell lines were obtained after screened by G418,which referred asc RAW-MKP5 and MIN6-MKP5,respectively.The cells transfected with pcDNA3.1 plasmid were controls and named as RAW-PC and MIN6-PC respectively.2.To investigate the effects of MKP5 on apoptosis,dysfunction,inflammation,endoplasmic reticulum stress,oxidative stress and MAPK activation in macrophages.RAW-PC or RAW-MKP5 cells were co-cultured with MIN6 cells in Transwell chamber for 12 hours,and GP was treated for 12 hours.Apoptosis-related markers including cleaved Caspase-9,cleaved Caspase-3,PARP and PDX1,GCK,IL-1,IL-6,MCP-1,XBP-1s,GRP-94,CHOP,BIP were analyzed by western blot or Real-time PCR.The expression and activation of HIF-1,PHD3,P38,JNK and ERK were also determined by western blot.The insulin secretion of MIN6 cells induced by GP was detected by GSIS,and the reactive oxygen species(ROS)in MIN6 cells were detected by ROS.Otherwise,RAW-PC and RAW-MKP5 cells were treated with GP for 12 hours,then collected supernatant combined with GP to stimulate MIN6 cells for 16 hours,similar tests were performed as described above.3.The effect of MKP5 overexpression in islet ? cell to macrophages: MIN6-PC or MIN6-MKP5 cells were co-cultured with RAW264.7 cells for 12 h and treated with GP for 12 h.Expression of IL-1?,IL-6 and MCP-1 in Raw264.7 cells were determined by Real-time PCR.Results:1.Construction of MKP5 stable overexpression cell lineThe expression of MKP5 in RAW-MKP5 and MIN6-MKP5 cells was significantly increased compared with the control groups,which indicated that the stable overexpression of MKP5 cell lines were successfully constructed.2.MKP5 overexpression in RAW264.7 cells affects apoptosis and function of MIN6 cellsThe results showed that RAW-PC cells enhanced the activation of Caspase-9,Caspase-3 and the decline of PARP expression in islet cells which induced by GP.However,RAW-MKP5 cells could significantly alleviate the changes described above.In addition,RAW-PC cells aggravated the dysfunction of MIN6 cells induced by GP and decreased the level of insulin secretion of MIN6 cells,but RAW-MKP5 cells significantly alleviated these phenomena.Therefore,overexpression of MKP5 in macrophages could alleviate GP induced apoptosis and dysfunction of islet cells underthe interaction condition.3.Effects of MKP5 overexpression in RAW264.7 cells on endoplasmic reticulum stress,oxidative stress and inflammatory response of MIN6 cellsGP significantly increased the expression of IL-1,IL-6,MCP-1,CHOP,BIP,XBP-1s and GRP-94 in MIN6 cells compared with control group.Co-cultured with RAW-PC cells further increased the expression of these markers.However,co-cultured with RAW-MKP5 cells could significantly alleviate the highly expression of these markers in MIN6 cells.In addition,co-culture with macrophages increased GP induced ROS production in MIN6 cells,and MKP5 overexpression in macrophages significantly inhibited ROS production.The expression of HIF-1 and PHD3 was detected by Real-time PCR.The results showed that macrophages could further enhance the oxidative stress response activated by GP by inhibiting the expression of PHD3 and promoting the expression of HIF-1,however,overexpression of MKP5 in macrophages inhibited HIF-1 expression in MIN6 cells,and decreased the expression of PHD3,which alleviated the oxidative stress induced by macrophage coculture.Therefore,over-expression of MKP5 in macrophages could alleviate the inflammatory response,endoplasmic reticulum stress and oxidative stress pathway of islet cells,thus indirectly affect the apoptosis and dysfunction of islet cells.4.Effects of MKP5 overexpression in RAW264.7 cells on MAPK pathway in MIN6 cellsThe results showed that GP could activate P38,JNK and ERK pathway in MIN6 cells.Overexpression of MKP5 in macrophages and co-culture with islet cells showed a significant decrease in JNK and P38 activation,but the activation of ERK pathway was not affected by MKP5.It is suggested that MKP5 may regulate JNK and P38 signaling pathway upon macrophage and islet cell interaction.5.Effect of overexpression of MKP5 in MIN6 cells on inflammatory response of RAW264.7 cells upon co-cultureThe expressions of IL-1?,IL-6 and MCP-1 in RAW264.7 cells were significantly increased under the stimulation of GP.After co-cultured with islet cells,the expression of these pro-inflammatory cytokines was further increased.Compared withthe MIN6-PC cells co-cultured group,the highly expression of these cytokines were significantly inhibited in MIN6-MKP5 co-cultured group.These results showed that overexpression of MKP5 in islet cells could alleviate the inflammatory response of macrophages which induced by GP.Conclusion:1.MKP5 relieves the dysfunction and apoptosis of islet cells in the process of interaction between islet cells and macrophages by inhibiting endoplasmic reticulum stress,oxidative stress and inflammatory response,it also inhibited the activation of P38 and JNK-MAPK pathway in islet cells.2.MKP5 in islet cells can alleviate the inflammation of macrophages during the interaction between islet cells and macrophages.Innovativeness:MKP5 is involved in the regulation of obesity-associated cinteraction between macrophages and islet cells.