Regulation Mechanism Of CaSR-PTHrP In Abnormal Proliferation And Differentiation Of Superficial Chondrocytes In TMJ Condyle

BackgroundThe relationship between temporomandibular joint and occlusion is very close.The latest research results of our research group showed that bilateral anterior elevation(BAE)can promote the proliferation of condylar chondrocytes and cartilage thickening in mice.Parathyroid hormone-related protein(PTHr P)is a macromolecular protein secreted by superficial condylar chondrocytes,which plays an important role in promoting cell proliferation.Calcium sensing receptor(Ca SR)is a receptor protein widely expressed on cell membrane,it is very sensitive to calcium signal.Ca SR can activate a series of downstream signals and play important biological functions.The purpose of this study is to explore the activation of Ca SR and PTHr P signals of condylar superficial chondrocytes stimulated by BAE by using molecular biology research methods including specific knockout of Ca SR or PTHr P gene in superficial condylar chondrocytes and to demonstrate the response law of condylar superficial chondrocytes to BAE stimulation and the regulation mechanism of Ca SR-PTHr P signal combined with in vitro loading experiment.Materials and methods1.36 6-week-old female C57 BL / 6J mice were randomly divided into 3-week,7-week,and 11-week groups.At each time point,they were divided into control group(CONT)and BAE group(n = 6).All BAE groups’ mice were given BAE stimulation at the age of 6 weeks.in control group mice,except that mice were not given BAE stimulation,other operations were the same as those in BAE group.The samples were taken at the end of 3 weeks,7 weeks,and 11 weeks respectively,and the morphological changes of mouse condylar cartilage were further detected.2.The condyle superficial chondrocytes of 3-week SD rats were isolated under a microscope and cultured in vitro.Si RNA and agonists were used to inhibit or activate the expression of Ca SR and PTHr P in cultured cells in vitro,and the cells were stimulated by fluid flow shear stress(FFSs)with an intensity of 16 dyn / cm2 for 2 h.The cells were collected and the expression of molecules related to cell proliferation and differentiation was detected by Western blotting(WB).3.We crossed Ca SRflox/flox mice with Prg4-Cre ERT2 mice to obtain Ca SRflox/flox;Prg4-Cre ERT2 mice,which Ca SR gene can be specifically knocked out in superficial chondrocytes expressing Prg4 in condyle induced by tamoxifen(TAM).Transgenic mice(PTHr Pflox/flox;Prg4-Cre ERT2)with specific knockout of PTHr P gene were obtained by the same method.Tam was injected intraperitoneally at the age of 6 weeks,continuously for 5 days,BAE stimulation was given the next day,and Ca SR agonist(cinacalcet HCl,Cina)or PTHr P agonist(PTHr P87-139 fragment)was injected around the joint at the age of 9 weeks.At the end of the 13 th week,the morphological changes of superficial condylar cartilage and the expression of molecules related to cell proliferation and differentiation were observed by chemical staining and immunohistochemical staining.Main results1.BAE stimulation on wild-type mice showed that the superficial condylar chondrocytes of mice increased significantly and the cartilage thickened.Compared with the control group,the rate of Ca SR positive cells and PTHr P positive cells in the superficial condylar cartilage of BAE group increased significantly from the third week(all P<0.05).2.The results in vitro showed that FFSS could induce the proliferation and early differentiation of superficial condylar chondrocytes,which was manifested by the increased expression levels of Ca SR,PTHr P,Ki67,and Indian hedgehog(IHH)protein(all P<0.05).Cinacalcet or PTHr P 87-139 fragment will further enhance the proliferation and early differentiation of FFSS on superficial condylar chondrocytes(all P<0.05),while si RNA transfection of Ca SR or PTHr P will inhibit the proliferation and early differentiation of FFSS on superficial condylar chondrocytes(all P>0.05).More importantly,activation and inhibition of Ca SR can promote and inhibit the expression of PTHr P,respectively(all P<0.05),while activation or inhibition of PTHr P has no significant effect on the expression of Ca SR(all P>0.05).3.The results of transgenic mice in vivo showed that knockout of Ca SR or PTHr P gene could prevent the proliferation and early differentiation of superficial condylar chondrocytes induced by BAE(all P>0.05).4.In vivo experiments showed that when PTHr P gene was knocked out and Ca SR agonist cinacalcet was injected around the joint,it could also prevent the proliferation and early differentiation of superficial condylar chondrocytes induced by BAE(all P>0.05).However,when Ca SR gene was knocked out and PTHr P87-139 fragment was injected around the joint,BAE induced proliferation and early differentiation of superficial condylar chondrocytes would not be prevented(all P<0.05).Conclusion1.In condylar superficial chondrocytes,Ca SR can be used as the upstream molecule of PTHr P to promote the proliferation and early differentiation of condylar superficial chondrocytes.2.Ca SR-PTHr P signaling pathway plays an important role in the regulation of BAE and FFSS promoting the proliferation and early differentiation of superficial condylar chondrocytes.