The effects of Connexin-43 on osteoblasts cells proliferation and osteogenic differentiation / 中华骨科杂志

ABSTRACT

Objective:To investigate the effects of Connexin-43 (Cx43) on osteoblasts proliferation and osteogenic differentiation and its regulatory mechanism.Methods:Osteoblasts were isolated and cultured in vitro. The osteogenic activity of osteoblasts was detected by alizarin red staining and alkaline phosphatase (ALP) staining after dexamethasone treatment. The expression of Cx43, Runt-related transcription factor 2 (Runx2), ALP, collagen I type (COL-I) and proliferation-related proteins PCNA and CDK4 in osteoblasts were detected by Western-blot. The expressions of osteoblast proteins were detected by immunofluorescence staining. The proliferation of osteoblasts was detected by CCK8 assay. The lentivirus-mediated Cx43 gene overexpression plasmid (Lv-Cx43) was constructed and transfected into osteoblasts. The osteogenic activity and proliferation ability of osteoblasts were further detected by the above methods. Cx43 in osteoblasts was overexpressed by pretreating PD98059. The osteogenic activity and proliferation of Cx43 in overexpressed osteoblasts was detected by CCK8 and alizarin red staining.Results:The isolated osteoblasts have osteogenic differentiation ability. Compared with the control group, 1×10 -6 mol/L dexamethasone treatment could reduce the formation of calcium nodules in osteoblasts. With the increase of dexamethasone treatment duration, the protein expression of Cx43, Runx2, ALP and COL-I in osteoblasts decreased gradually, while the expression of PCNA, CDK4 and p-ERK1/2 decreased. The OD values of normal osteoblasts at 0, 1, 2, 3 and 4 d were 0.316±0.043, 0.891±0.623, 1.683±0.154, 2.315±0.721 and 2.891±0.323, respectively. However, The OD values of osteoblasts treated with dexamethasone were 0.376±0.021, 0.657±0.121, 1.124±0.285, 1.521±0.272, 1.987±0.584, respectively. OD values of dexamethasone treated osteoblasts were lower than those of normal group at 2, 3 and 4 days ( P<0.05). The relative expression levels of Cx43 mRNA in control group, Lv-NC group and Lv-Cx43 group were 0.541±0.086, 0.598±0.018 and 1.000±0.082, respectively. The mRNA expression level of Cx43 in Lv-Cx43 group was higher than that in control group and Lv-NC group ( P<0.05). The ratio of Cx43 protein band to the gray value of GAPDH band in control group, Lv-NC group and Lv-Cx43 group were 0.816±0.737, 0.738±0.643 and 1.145±1.101, respectively. The expression level of Lv-Cx43 was higher than that in control group and Lv-NC group ( P<0.05). The expressions of Runx2, ALP, COL-I mRNA and related marker proteins in Lv-Cx43 group were higher than those in control group and Lv-NC group ( P<0.05). The number of calcium nodules in the Lv-Cx43 group was significantly higher than that in the control group and Lv-NC group. The OD value of osteoblasts and the number of calcium nodules in Lv-Cx43+PD98059 group were significantly lower than those in Lv-Cx43 group ( P<0.05). Conclusion:The proliferation and differentiation ability of osteoblasts is significantly decreased after the treatment of dexamethasone with decreased expression of Cx43. Overexpression of Cx43 can promote the proliferation and osteogenic differentiation of osteoblasts, which may be regulated through the ERK1/2 pathway.