Studies Of Dlx5 Participating In Regulation Of The Specific Expression Of Mouse Type X Collagen Gene

Background The specific expression of type X collagen gene(COL10A1)in hypertrophic chondrocytes is an important part of endochondral ossification.Mutation or abnormal expression of human COL10A1 is often accompanied by impaired chondrocyte maturation and seen in Schmid metaphyseal dysplasia or various other bone diseases.Mice depletion of Col10a1 also shows abnormalities of bone growth plates.Therefore,elucidating the regulation of type X collagen gene is essential for understanding bone development and disease.Our laboratory has previously succeeded in localization of the Col10a1 cis enhancer to the 150 bp sequence of its distal promoter,and demonstrated that Runx2 is able to bind this enhancer and is an indispensable transcriptional regulator that mediates the specific expression of Col10a1 in hypertrophic chondrocytes.At the same time,multiple transcription regulators including Dlx5,Nrf2,Hoxa3 etc.,that may bind to the 150 bp Col10a1 enhancer have been identified by the bioinformatics prediction software TRAP.It is worth noting that the binding site of Dlx5(Distal-Less Homeobox 5)and Col10a1 enhancer is adjacent to the Runx2 site,suggesting that Dlx5 may be involved in regulation of murine Col10a1 gene expression.PurposesThis study intends to explore the regulation and mechanism of Dlx5 involving in mouse Col10a1 gene expression and its potential effect on the differentiation and maturation of chondrocytes.Methods(1)Using the bioinformatics software JASPAR to verify whether the transcription factors such as Dlx5,Nrf2,Hoxa3,etc.bind the 150 bp Col10a1 cis-enhancer.(2)Detecting the expression of Dlx5 and Col10a1 in proliferative and hypertrophic MCT cells,the immortalized mouse chondrocyte respectively by q RT-PCR and Western blot.(3)Detecting the effect of Dlx5 on Col10a1 gene expression by q RT-PCR and Western blot after using lipo3000 to transiently transfect pcDNA3.1/Dlx5 and Dlx5 si RNA-A in MCT cells.(4)Determining the effect of Dlx5 on the activity of the Col10a1 enhancer by dual luciferase reporter gene assay.(5)Exploring the effect of Dlx5 on the expression of chondrocyte maturation-related genes by q RT-PCR and Western blot after interfering with the expression of Dlx5 in MCT cells.(6)Detecting the effect of Dlx5 on the proliferation of MCT cells by Ed U after interfering with the expression of Dlx5 in MCT cells.Results(1)JASPAR confirmed that the binding site of Dlx5 and the 150 bp Col10a1 enhancer is consistent with the prediction results of TRAP software.(2)The results of q RT-PCR and Western blot showed that the m RNA levels of Col10a1 and Dlx5 were higher in hypertrophic than in proliferative MCT cells(p<0.05),their protein levels in hypertrophic MCT cells also rised compared with proliferative MCT cells.(3)The results of q RT-PCR and Western blot indicated that the m RNA levels of Dlx5 and Col10a1 in MCT cells transfected with pcDNA3.1/Dlx5 were significantly increased compared with the control group(p<0.05),and the protein levels also obviously increased;In comparison with the control group,the m RNA levels of Dlx5 and Col10a1 in MCT cells interfering with si RNA-A were significantly decreased(p<0.05),and the protein levels were also greatly decreased.(4)The results of dual luciferase reporter gene assay declared that overexpression of Dlx5 enhanced the activity of the 150 bp Col10a1 enhancer compared with the control group(p<0.05).(5)The results demonstrated that compared with the control group,the m RNA level of Runx2 was higher in MCT cells transfected with pcDNA3.1/Dlx5(p<0.05)and its protein level was significantly increased.However,there were no obvious differences in expression of Sox9 and Mmp13.In comparison with the control group,the m RNA level of Runx2 was obviously decreased in MCT cells transfected with si RNA-A(p<0.05)and its protein level also declined.However,there was no significant effect on Sox9 gene expression after the intervention of Dlx5 si RNA-A in MCT cells.The protein level of Mmp13 was decreased in MCT cells transfected with si RNA-A compared with control group and there was no obvious change in Mmp12 m RNA.(6)Results of Ed U cell proliferation assay suggest that overexpression of Dlx5 inhibited chondrocyte proliferation compared with the control group.While interfering with Dlx5 had no significant effect on MCT cell proliferation.Conclusions(1)Dlx5 can increase the activity of Col10a1 enhancer and promote the expression of Col10a1 in hypertrophic chondrocytes.Dlx5 is a positive regulator of Col10a1 gene expression.(2)Dlx5 promotes Runx2 expression in chondrocytes and it may cooperate with Runx2 to regulate Col10a1 gene expression and chondrocyte differentiation and maturation.