CTGF regulates mineralization in human mature chondrocyte by controlling Pit-1 and modulating ANK via the BMP/Smad signaling

Objective: Connective tissue growth factor (CTGF) has a potent proliferative, differentiated, and mineral effect, and is thought to be involved in cartilage mineralization in Osteoarthritis (OA). However, little is known about the mechanism of chondrocytic mineralization that is induced by Connective tissue growth factor (CTGF). As a crucial mediator in regulating mineral responses, the type III phosphate transporter 1 (Pit-1) has been implicated in the pathogenesis of articular mineralization. The aim of this study was, therefore, to determine whether Connective tissue growth factor regulates the formation of mature chondrocyte mineralization and the mechanisms underlying such mineralization. Methods: The effect of Connective tissue growth factor (CTGF) was analyzed with human C-28/I2 chondrocyte. The chondrocytes were treated with Connective tissue growth factor (CTGF) or related inhibitor, and transfected with Overexpression and siRNA transfection of the type III phosphate transporter 1(Pit-1). The chondrocytes were subjected to Alizarin red S staining, PiPer phosphate assay kit, Alkaline Phosphatase Diethanolamine Activity Kit, ELISA, RT-PCR or Western blot analysis. Results: Stimulation with Connective tissue growth factor (CTGF) markedly upregulated expression of the type III phosphate transporter 1(Pit-1) and levels of mineralization in the chondrocyte through activation of α5β1 integrin and BMP/Samd1/5/8 signaling pathways. Moreover, treatment of overexpressed Pit-1 could significantly upregulate expression of multipass transmembrane ankylosis (ANK) transporter in the cells. Blockade of CTGF receptor using α5β1 Integrin blocking antibody significantly suppressed the expression of Pit-1 and ANK transporter, and the events of chondrocyte mineralization. Conclusions: Our data indicate that Connective tissue growth factor (CTGF) playing a critical role in the chondrocyte mineralization is dependent on the expression of the type III phosphate transporter 1(Pit-1) and multipass transmembrane ankylosis (ANK) transporter, while inhibition of CTGF activity might serve as a novel therapeutic approach in the management of OA.