Effect Of CKIP-1 On C3H/10T1/2 Cell Osteogenic Differentiation And Immunomodulatory Function

Mesenchymal stem cells (MSCs) are present in almost all tissues and are self-renewing pluripotent stem cells. Their potential for osteogenic differentiation,immunomodulation and treatment of multiple inflammatory-related diseases has recently been widely concerned, however, its regulatory mechanism is still unclear.CKIP-1 is a bone-forming negative regulator that interacts with a variety of proteins and proinflammatory cytokine mTNF, what's more, CKIP-1 expression in THP-1 monocyte model is significantly up-regulated under LPS-stimulated. It is unclear how MSCs, which have osteogenic differentiation and immune function, are regulated by CKIP-1. To study the effect of CKIP-1 on MSCs osteogenic differentiation and immunoregulation is of great significance to its clinical application.Objective: To investigate the effect of CKIP-1 on C3H/10T1/2 cells Osteogenic differentiation and Immunomodulatory function.Method:Part one: CKIP-lregulates the Osteogenic Differentiation of C3H/10T1/2 Cells.Osteogenic differentiation of C3H1/10T1/2 cells was identified by ALP and RT-qPCR.miR-20a mimics and CKIP-1 siRNA were transfected into C3H/10T1/2 cells with lipo3000. The expression of osteoblast marker genes, miR-20a and CKIP-1 were quantitatively assessed by RT-qPCR.Part two: CKIP-lregulates the immune capacity of C3H/10T1/2 Cells.1. C3H/10T1/2 cells were stimulated with increasing concentration LPS and the expression profile of CKIP-1 mRNA and protein were analyzed by RT-qPCR and Western blot.2. Lentiviruses expressing CKIP-1 shRNA cells (sh-CKIP-1) were generated to knockdown CKIP-1 in C3H/10T1/2. The transfection efficiency and the change of surface molecules and cell cycle of CKIP-1-knockdown cells were analyzed by flow cytometry. The CFSE fluorescence intensity of T cells proliferation was performed by flow cytometry and the expression of TNF-a, IFN-y, IL-10 and p38 MAPK phosphorylation was tested by RT-qPCR and Western blot, respectively.Result:Part one: CKIP-lregulates the Osteogenic Differentiation of C3H/10T1/2 Cells.Ckip-1 is down-regulated, while miR-20a is up-regulated during osteogenic differentiation of C3H/10T1/2 cells. Knockdown of CKIP-1 promotes osteogenic differentiation. Furthermore, overexpression of miR-20a promotes osteogenic differentiation and inhibites the expression of bone formation negative regulator CKIP-1.Part two: CKIP-lregulates the immune capacity of C3H/10T1/2 Cells.CKIP-1 expression was induced in murine MSC cell line C3H/10T1/2 cells stimulated with LPS. Knockdown of CKIP-1 did not bring much difference on the cell cycle and immune phenotype of MSCs. However, MSCs with CKIP-1 knockdown showed enhanced immunosuppressive capacity. RT-qPCR and western blot data exhibited that, compared with the control, CKIP-1-knockdown C3H/10T1/2 cells showed higher IL-10 mRNA production and p38 MAPK activation after treated with LPS. Interestingly, the expression of CKIP-1 was decreased in C3H/10T1/2 cells with high glucose treatment, while IL-10 level was increased. C3H/10T1/2 cells became more suppressive after treated with high glucose, showing as inhibiting T cell proliferation at lower ratio (C3H/10T1/2 to T).Conclusion:CKIP-1 played an important role in modulating the osteogenic differentiation and immunoregulatory function of C3H/10T1/2 cells, CKIP-1 knockdown enhanced the immunosuppressive capacity of MSCs, which may through the activation of the p38 MAPK pathway and lead to decreased TNF-a, IFN-y secretion as well as the increased of IL-10 production.