TGF-β1/WISP1/Integrin-α interaction mediates human chondrocytes dedifferentiation

OBJECTIVE: To clarify the interaction between TGF-beta 1 and WISP1, and the effect of Integrin alpha 5/V subunits on the WISP1 caused chondrocyte (CH) dedifferentiated phenotype. PATIENTS AND METHODS: The knee joint cartilage from the trauma and osteoarthritis (OA) patients were collected. The patients of trauma group were confirmed to have no OA history. The protein level of WISP1. Integrin-alpha 5/V, and type II/I collagen were analyzed by Western blotting. Besides, we isolated the CHs from the cartilage without OA and treated CHs with exogenic TGF-beta 1 and WISP1 protein. In addition to this, to regulate the alpha 5 and alpha V subunits expression of CHs, we silenced two genes by siRNA transfection and upregulated them by exogenic protein supplement. Then, the CHs with different alpha 5 and alpha V expression were treated with WISP1. To value the chondrogenic gene expression, we determined the type II collagen and SOX9 gene expression by immunofluorescence (IF) and RT-PCR, respectively. Meanwhile, the dedifferentiation markers of CH, type I collagen, and Runx2 expression was also analyzed. Cell proliferation was measured by MIT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. RESULTS: The OA cartilage contains a higher level of type I collagen, WISP1. Integrin alpha 5, and Integrin alpha V, but low type II collagen. The upregulation of TGF-beta 1 caused the increase of WISP1, as well as the high level of Integrin alpha 5/V, and de-differentiated gene. Besides, the upregulation of WISP1 also contributed to the TGF-beta 1 expression and CHs dedifferentiation. Apart from this, the silencing of the alpha 5 subunit of Integrin aggravated the WISP1 induced CHs dedifferentiation, which was reversed by alpha 5 upregulation. However, the aV subunit played an opposite role that mediated the WISP1-induced CHs dedifferentiation. Additionally, the interaction between TGF-beta 1 and WISP1 promoted the CHs proliferation, which was not affected by the Integrin-alpha 5/V expression. CONCLUSIONS: TGF-beta 1 and WISP1 interact to induce CHs dedifferentiation, which was mainly by the mediation of the Integrin-alpha V subunit. On the contrary, Integrin-alpha 5 shows a protective effect during the WISP1 caused CHs dedifferentiation.