The Mechanism By Which ClC-3 Chloride Channel Mediates Estrogen Regulation Of Osteoblast In The Bone Formation

Objective:This study explores the role of ClC-3 chloride channel in estrogen regulation of osteoblast in the bone formation,and further analyzes the downstream signal transduction pathway and mechanism after estrogen activation of chlorine channel.Hence,it provides theoretical basis for development of new ion channel type anti-osteoporosis drugs.Methods:Construction and screening of mouse osteoblast cell line with stable low expression of CLC-3 chloride channel were established by lentivirus（MC3T3-E1/CLC-3 KO）.We used confocal laser microscopy immunofluorescence technique to investigate the effect of E₂on the bone formation function of osteoblasts before and after ClC-3knockout.N-（Ethoxycarbonylmethyl）-6-methoxyquinolinium bromidethe chloride ion fluorescence probe（MQAE）technique was used to detect the chloride channel activity of ClC-3 osteoblast cell lines with different expression（MC3T3-E1）,and to explore the effect of estrogen on the opening of chloride channel in osteoblasts.Alkaline phosphatase staining and alizarin red staining were used to detect alkaline phosphatase and calcified nodules formation in osteoblasts before and after ClC-3knockout.The qPCR assay was used to investigate the effect of estrogen on the expression of ClC-3 gene in osteoblasts.Proteomic analysis was performed to evaluate the changes in downstream proteomic signals of osteoblasts before and after ClC-3 knockout.Results:The immunofluorescence of ClC-3 in the estrogen group was significantly higher than that in the blank group,and the immunofluorescence of estrogen receptor was significantly higher,suggesting that the expression of ClC-3 gene could be enhanced after estrogen treatment of osteoblasts.In the chloride ion fluorescence probe test,the intensity of the chloride ion fluorescence probe was increased in the estrogen group,suggesting that estrogen increases the opening of chloride ion channels in the membrane of osteoblasts,and the chloride ion efflux and the chloride ion concentration in the cytoplasm is decreased.Staining osteoblasts by alkaline phosphatase and alizarin showed that Estrogen could promote the maturation of osteoblasts,and the alkaline phosphatase staining was increased.The calcified nodules were more obvious in the estrogen treated group than in the blank group.The expression of ClC-3 gene in osteoblasts was detected by RT-qPCR.The increase of estrogen treatment time,increased the expression of ClC-3 gene.Then,proteomic analysis presented that 304 differential proteins were changed.Among them,P26187,Q61107 and Q64282 were significantly up-regulated,while P04918,P3804 and Q923B6 were significantly down-regulated.Conclusion:Estrogen can promote the opening of chloride channels in osteoblasts.It also promotes the formation of alkaline phosphatase and calcified nodules in osteoblasts,increase the expression of ClC-3 gene,and participate in bone formation.Proteomics and Go/KEGG enrichment analysis found that down-regulated differential proteins were mainly concentrated in the endoplasmic reticulum protein synthesis,protein digestion and absorption,and Relaxin signaling pathways.The up-regulated proteins were mainly concentrated in ribosomal biosynthesis,proteasome and NOD-like receptor signaling pathways.