The Mechanism Of Osteoblastic CXCL2 In Regulation Of Bone Formation And Osteoblastic Differentiation

1.BackgroundBone formation is closely related to osteoblasts.Osteoblastic differentiation can be regulated by mass of factors and signaling pathways in human body.The activation of ERK1/2 pathway,the downstream of MAPK signaling pathway can promote the expression of RUNX2 in osteoblasts and play a regulatory role in promoting the differentiation of osteoblasts.CXCL2,a member of CXC Chemokine,can be expressed and secreted in kinds of cell types and organs in human body,especially in liver and bone.By specific binding with its membrane receptors CXCR1 and CXCR2,CXCL2 activate downstream signal transduction,including ERK1/2 pathway.The past researches focused on functions of chemokines in immune related reactions,but ecent studies have found that they can participate in non-immune-related reactions in humans.However,whether CXCL2 can affect bone formation is still unknown2.ObjectiveIn order to broaden the understanding of chemokines and osteoporosis,the author of this paper explored the correlation between CXCL2 and osteoporosis,investigated the effect of CXCL2 on bone formation,and studied the mechanism of ERK1/2 regulating the process of CXCL2 affects osteoblastic differentiation.3.Materials and MethodsA total of 20 peripheral blood samples and 16 bone marrow blood samples were collected after conditional screening.The results of two-photon bone mineral density examination after hospitalization were recorded.Osteoporosis was diagnosed if the T value less than-2.5,and the value greater than-2.5 was regarded as normal control.The concentration of CXCL2 in blood samples was detected by ELISA.The difference of CXCL2 between peripheral blood and medullary blood in normal control group and osteoporosis group were compared.The correlation between CXCL2 and bone mineral density in peripheral blood and medullary blood was analyzed.In vivo,Ovariectomy(OVX)osteoporosis model in mice was established,and both Sham and OVX group contains 10 mice.BMD of mice was analyzed by micro-CT.The author performed double-labelling immunofluorescence staining to mark CXCL2 and Osteocalcin(OCN).The expression of CXCL2 in osteoblasts was observed,the ratio of CXCL2 + OCN + cells to total osteoblasts was calculated.OVX mice and senile mice were underwent medullary cavity injection with CXCL2 neutralizing antibody.After 3 months,BMD parameters of each group were compared by micro-CT scanning.OCN was labeled with immunohistochemical staining,and the ratio of osteoblasts number to trabecular area between control and antibody injection group was compared.In vitro,CXCL2 was knocked down or overexpressed by RNA interference or plasmid transfection.The OD values to evaluate proliferation were detected by CCK8 assay.Flow cytometry was applied to detect the apoptotic rate.Then,the expression levels of RUNX2 and OCN were detected by Real-time PCR and Western blot,and ALP and alizarin red stains were performed on the 7th and 21st day after osteogenesis induction respectively.At last,cells were co-treated with SCH772984 or C6 ceramide to perform a rescue experiment.4.ResultsThe expression of CXCL2 in medullary blood samples was significantly higher than in peripheral blood,and it was also negatively correlated with BMD.The expression level of CXCL2 in osteoporosis group was alos higher than in control group.In vivo,the expression of CXCL2 in osteoblasts and the ratio of CXCL2+osteoblasts to total osteoblasts of OVX was significantly higher than sham.The number of osteoblasts on the trabecular bone surface in the antibody injection group was higher than control group,and the antibody injection group also had a larger ratio of osteoblast number to trabecular area.The result of micro-CT scanning indicated that neutralization of CXCL2 in medullary cavity could achieve a effectively remission to osteoporosis.The result of in vitro experiment shows,CCK8 assay observed a smoother curve while CXCL2 was over-expressed.The expression of RUNX2 and OCN wereinhibited.ALP expression and mineralized nodules were significantly reduced.Opposite results were found while CXCL2 was knocked down.Functional changes of the transfected cells were reversed after co-treated with SCH772984 or C6 ceramide.5.ConclusionsCXCL2 plays an essential role in osteoporosis and shows an enrichment trend into bone marrow.Osteoblasts secreted increased CXCL2 in osteoporotic mice.Neutralizing CXCL2 in bone marrow can effectively alleviated osteoporosis in mice.CXCL2 inhibits osteoblastic differentiation and bone formation by inhibiting the activity of ERK1/2 pathway upstream of RUNX2.And CXCL2 maybe a new expected target to treat with osteoporosis in future.