Molecular Mechanism Of Estrogen Regulating TRPV5 Expression In Osteoclasts

Objective:1.Determination of the transcriptional activity region of TRPV5 gene promoter in osteoclasts activated by estrogen(E2).2.To explore the role of transcription binding factors AP-1,SP1 and NF-kappa B in regulating the transcriptional activation of TRPV5 gene in osteoclasts activated by estrogen.Methods:1.TRPV5 gene which contains 5' UTR and its promoter region specific size sub fragments by PCR,their length were 2000 bp,1000bp,500 bp,150bp,respectively.Connect it to the p GL3-basic plasmid,then we get luciferase reporter gene plasmid Trpv5-2k-luci,Trpv5-1k-luci,Trpv5-500-luci,Trpv5-150-luci,which was driven by mouse TRPV5 gene promoter.They were transfected to Raw264.7 cells by liposome transfection.We detected the expression of luciferase before and after the stimulus of estrogen,and found out the region of the largest contribution of estrogen induced TRPV5 transcription activation.2.RNAi models of AP-1,SP1 and NF-?B was constructed,which co-transfected to Raw264.7 cells.After stimulation with E2,the transcription and expression of TRPV5 gene was detected.(1)Real-time PCR was used to detect the transcription and expression of TRPV5 in the models above.(2)Western-blot was used to detect the transcription and expression of TRPV5 in the models above.(3)Ch IP was used to detect the transcription and expression of TRPV5 gene in the models above.Results :1.Statistical analysis of the dual luciferase assay results showed obvious luciferase expression when the length of TRPV5 promoter are 2000bp(p=0.011<0.05),1000bp(p=0.005<0.05),500bp(p=0.002<0.05)and 150bp(P=0.415>0.05)compared with the control group.It indicates that TRPV5 gene promoter has complete activity with the length in the range of 500 bp to 2000 bp and 500 bp length may be the necessary section of gene promoter transcription activation.2.In the models which TRPV5-500-luci transfected the RAW264.7 cells,RNAi were used to silence the AP-1,SP1 and NF-?B.(1)The results of three groups of independent experiments showed that the double luciferase assay showing a significant decrease in AP-1 silenced group(p<0.008),SP1 silenced group(p<0.006)and NF-?B silenced group(p<0.001)compared with the control group.AP-1,SP1 and NF-?B are involved in the regulation of luciferase reporter gene driven by the promoter of TRPV5 gene.Inter group analysis showed thatthe activity of thedouble luciferase decreased most significantly when silenced NF-?B.The results showed that AP-1,SP1,NF-?Bwere all involved in the expression of osteoclast TRPV5 stimulated by E2.NF-?B may account for the most important role in the expression of TRPV5.(2)Real-time PCR was used to detect TRPV5 expression which showed that when silenced the NF-k B,AP-1 or SP1 repectively.(3)Western blot was used to detect TRPV5 expression which showed that when silenced the NF-?B,TRPV5 protein expression decreased compared to mock group.The protein electrophoresis bands of TRPV5 were narrow than NC group,wider than mock group,which indicate that other transcriptional regulators are still present in TRPV5 gene.(4)Ch IP was used to detect TRPV5 expression which showed that when silenced the NF-?B,TRPV5 protein expression decreased compared to mock group.The protein electrophoresis bands of TRPV5 were narrow than NC group,wider than mock group,which indicate that other transcriptional regulators are still present in TRPV5 gene.Conclusions :1.TRPV5 gene promoter has better activity in the range of 500 bp to 2000 bp,and 500 bp length may have the most of the transcriptional binding sites that are required for gene transcription and expression in the 500 bp range of gene promoter.2.AP-1,SP1 and NF-?B play an important role in the transcriptional activation of estrogen stimulated TRPV5,in which NF-kappa B may play a major role.