CTGF promotes cementogenesis and cementum repair via Cx43/β-catenin axis

Abstract Background: Orthodontic tooth movement always induces cementum resorption inevitably, which is an urgent problem for orthodontists to confront. Human Periodontal ligament stem cells (hPDLSCs) possess multidirectional differentiation ability. Connective tissue growth factor (CTGF) is an important extracellular matrix proteins for bone homeostasis and cell differentiation. The purpose is to explore the role of CTGF in cementum repair and cementogenesis, and to elucidate its underlying mechanism.Methods: The cementum defect model was established by the gravity tooth movement, and the cementum repair effect of CTGF was observed by Micro CT, HE staining and immunohistochemical staining. RT-qPCR, Western bolt (WB), alizarin red and ALP activity experiments verified the mineralization ability of hPDLSCs stimulated by CTGF. In vivo and in vitro experiments, the expression of Cx43 in periodontal ligament cells was detected after CTGF stimulation by WB and immunofluorescence (IF) experiments. Subsequently, the mineralization ability of hPDLSCs were observed after the application of small interfering RNA Si-Cx43 and CTGF. Additionally, the co-intervention of small interfering RNA Si-CTGF and Cx43 agonist ARAT on hPDLSCs was applied to deepen the mechanism. Next, WB, IF experiments and co-immunoprecipitation were applied to confirm whether CTGF triggered Cx43/β-catenin axis to regulate cementoblast differentiation of hPDLSCs.Results: Local oral injection of CTGF to the cementum defects in vivo facilitated cementum repair. CTGF facilitated the cementogenesis of hPDLSCs in concentration-dependent manners. Cx43 acted as a CTGF downstream effector protein to regulate cementoblast differentiation. Si-Cx43 diluted CTGF-induced cementoblast differentiation. Cx43 agonists ATRA restored the low differentiation capacity induced by Si-CTGF. Further mechanistic studies showed that CTGF triggered the activation of β-catenin in a dose-dependent manner. Besides, colocalization analysis by IF and co-immunoprecipitation demonstrated that Cx43 interacted with β-catenin at the cell-cell connection. Si-Cx43 attenuated the enormous expression of β-catenin induced by CTGF. Cx43 agonists reversed the Si-CTGF-induced inhibition of β-catenin. IF demonstrated that the nuclear importation of β-catenin is related to the immense expression of Cx43 at cell-cell junctions. Conclusions: Taken together, these data demonstrated that CTGF promoted cementum repair and cementogenesis through activation of the Cx43/β-catenin signaling axis.