The Experimental Research On Enhancement Of Osteoblast Survival By CCN2 And JAK2 Signaling Pathway Through Autophagy In Inflammatory Environment

Background:Bone homeostasis is maintained by the dynamic balance of bone resorption and bone formation.Immunocytes regulate bone homeostasis in inflammation and produce cytokines to affect the balance between bone resorption and bone formation.TNF-?,one of the canonical pro-inflammatory factors,promote osteoclastogensis and inhibit survival and function of osteoblasts.Long-term inflammation such as osteoporosis reduces bone mass and increases bone fragility.CCN2 and JAK2 signaling pathway taking part in virous biological and pathological processes are involved in inflammation.The differentiation of osteoblasts can be affected by its survival ability.Studies have reported that CCN2 and JAK2 signaling pathway can protect tumor cells and increase the resistance to apoptosis.It is meaningful for inflammation control and bone homeostasis maintaining to explore whether the protective effects of CCN2 and JAK2 signaling pathway work on osteoblasts and the relationship between CCN2 and JAK2 signaling pathway.Purposes:Gain-and lost-of function method was performed to explore the effect of CCN2 and JAK2 signaling pathway on TNF-?-induced apoptosis in osteoblasts and the mechanism in the process.The relationship between CCN2 and JAK2 pathway was also discussed.Materials and Methods:1.TNF-?-induced-apoptosis and autophagy in MC3T3-E1 was tested by flow cytometry,Western Blot and immunofluorescence technique.The expression of CCN2 and p-JAK2 were also detected.2.CCN2-overexpression MC3T3-E1 was established with lentivirus transfection.Western Blot was performed to examine the effect of CCN2 on TNF-?-inducedapoptosis and autophagy.CQ was used to explore whether CCN2 promote osteoblasts survival through autophagy.Akt and Erk pathways were also detected with Western Blot whether participated in the process.The effect of CCN2 on differentiation of MC3T3-E1 in inflammation was detected by ALP activity test,Alizarin S Red staining and Western Blot.3.The effect of JAK2 pathway inhibition by AG490 on apoptosis and autophagy was detected by flow cytometry and Western Blot.Rapamycin and CQ were used to explore whether JAK2 pathway affect cell apoptosis through autophagy.P-STAT3,pAkt and p-Erk were detected with Western Blot to examine the effect of JAK2 inhibition on other pathways.4.Si RNA was used to knockdown the expression of CCN2 in MC3T3-E1.Western Blot was used to examine the expression of p-JAK2 to explore the effect of CCN2 on JAK2 pathway.Proteins related to apoptosis and autophagy after inhibition of JAK2 pathway by AG490 were detected by Western Blot to further probe the relationship between CCN2 and JAK2 pathway.Results:1.TNF-? could increase percentage of apoptotic cells in MC3T3-E1.The level of apoptotic markers increased with the concentration and stimulation time of TNF-?.The ratio of LC3II/LC3 I didn't increase until 12 h.The fluorescence intensity of LC3 B was higher with TNF-? treatment for 72 h than control group.The expression of CCN2 was downregulated,but the expression of p-JAK2 was upregulated.2.The expression level of CCN2 was higher in Lv CCN2 group than Lv NC group.This result suggested CCN2-overexpression system was established.Overexpression of CCN2 attenuated TNF-?-induced apoptotic marker upregulation and increased the ratio of LC3II/LC3 I.CQ inhibited autophagy resulting in increasing apoptotic markers in Lv CCN2 group.Akt and Erk pathways were activated with TNF-? treatment.The level of p-Akt and p-Erk were higher in Lv CCN2 group than Lv NC group.Inhibition of Akt and Erk pathways attenuated the effect of CCN2 on apoptosis and autophagy.TNF-? decreased ALP activity,mineralized nodules and the expression of Runx2 and Osx.Overexpression of CCN2 upregulated ALP activity,mineralized nodules and the expression of Runx2 and Osx.3.Inhibition of JAK2 pathway by AG490 increased Cl-PARP and p62,decreased the ration of LC3II/LC3 I.Enhancement of autophagy by rapamycin inhibited the effect of AG490 on apoptosis but CQ promoted the effect.AG490 also decreased p-STAT3,p-Akt and p-Erk in MC3T3-E1.4.CCN2-overexpression increased TNF-?-induced p-JAK2 expression and downregulation of CCN2 decrease p-JAK2 in MC3T3-E1.The effect of CCN2 on cell apoptosis and autophagy could be attenuated by JAK2 pathway in inflammation.Conclusion:1.TNF-? could induce apoptosis and autophagy in MC3T3-E1.CCN2 and JAK2 pathway might participate in these processes.2.Overexpression of CCN2 attenuated TNF-?-induced apoptosis through enhancing autophagy.Akt and Erk pathways were involved in the process.Overexpression of CCN2 could also reverse the inhibition of TNF-? on MC3T3-E1 differentiation in inflammation.3.Inhibition of JAK2 pathway by AG490 promoted TNF-?-induced apoptosis through inhibiting autophagy.P-STAT3,p-Akt and p-Erk were affected with JAK2 pathway inhibition.4.JAK2 pathway was downstream pathway of CCN2 in TNF-?-treated MC3T3-E1.