CCN4/ WISP-1 is a positive Regulator of TGF-β3 Induced Chondrogenic Differentiation.

Objectives: The goal of this study was to clarify the role and mechanism of action of CCN4 in chondrogenic differentiation.
Methods: In vitro gain-and-loss of function analysis using human bone marrow stromal cells (hBMSCs) were performed with adenovirus expressing CCN4 (adCCN4), and small interfering RNA of CCN4 (siCCN4) to investigate the influence of CCN4 to chondrogenic differentiation and the influence of CCN4 to TGF-β3 signaling that is well known as the cascade of chondrogenic differentiation. To understand how CCN4 controls TGF-β3 signaling, immunoprecipitation, western blotting and cell protein binding assay were performed. To investigate the function of CCN4 in vivo, we generated Ccn4 conventional knock-out mice (Ccn4 KO mice), and collected RNA from articular cartilage and compared the mRNA expression levels of chondrocyte markers with wild type mice (WT mice) by real-time RT-PCR. Moreover, full thickness articular cartilage defects in knee joints of Ccn4 KO mice and WT mice were created and further analyzed histologically 4 weeks after surgery to confirm the CCN4 function of healing the defect.
Results: Gain of CCN4 function by transduction with adCCN4 significantly enhanced TGF-β3 induced chondrogenic differentiation in vitro by enhancing activation of TGF-β3 downstream signaling Smad2/3. On the other hand, loss of CCN4 function by transduction with siCCN4 significantly inhibited the Smad signaling and subsequent TGF-β3 induced chondrogenic differentiation. Immunoprecipitation and western blotting analysis showed that CCN4 bound to TGF-β3, and cell protein binding assay analysis showed that CCN4 increased TGF-β3 binding to the surface of hBMSCs. In vivo data showed that the mRNA expression levels of chondrocyte markers in articular cartilage was lower in Ccn4 KO mice and the articular cartilage regeneration was repressed in Ccn4 KO mice.
Conclusions: CCN4 has a positive influence on chondrogenic differentiation by modulating the effects of TGF-β3 and is important for cartilage repair in vivo.