SOX9 transcriptional activity is differentially regulated in healthy and osteoarthritic chondrocytes

Purpose: SOX9 is the master transcription factor for the cartilage development and homeostasis and its dysfunction is often detected in osteoarthritis (OA). Although much is known about the signals regulating SOX9 mRNA expression, little is known about the factors regulating SOX9 protein activity. We have previously applied Fluorescence Recovery After Photobleaching (FRAP) to measure SOX9-mGFP transcriptional activity in a chondrocyte cell line. To further understand the role of SOX9 in OA pathophysiology, we study the differences in SOX9 transcriptional activity between healthy and OA chondrocytes in response to extracellular stimuli.

Methods: Healthy human primary chondrocytes (Healthy hPCs) were purchased from Articular Engineering, USA. Human osteoarthritic chondrocytes (OA-hPCs) were isolated from patients undergoing total knee replacement therapy. hPCs were grown on glass coverslips and transfected with SOX9-mGFP. Recombinant proteins, WNT3A, BMP7, DKK1, FRZB, IL1β (R&D systems) and IL1-Ra (Sigma) were added 24 h post-transfection at 10 or 100 ng/ml. hPCs were incubated with the recombinant proteins for 30 min. FRAP was performed in at least 40 cells per condition in 3 donors each for healthy and OA. In FRAP, a small region of the nucleus is bleached and the recovery of the fluorescence in the bleach spot is recorded. Results were analyzed using Matlab™ and statistics were performed using Mann-Whitney U tests. To correlate protein mobility with DNA binding, Chromatin immunoprecipitation (ChIP) assays were performed.

Results: We observed that without any treatment, the SOX9-mGFP immobile fraction (IF, measured as the fraction that showed no fluorescence recovery in the bleach spot) was significantly (19%) lower in OA-hPCs as compared to healthy chondrocytes. IL1β and WNT3A treatment significantly decreased the SOX9 activity in healthy hPCs, but not in OA-hPCs. The iNOS inhibitor 1400W partially reversed IL1β induced effects in healthy hPCs, but not in the OA-hPCs. In contrast, BMP7 significantly increased SOX9 activity in both healthy and OA-hPCs. IL1 inhibitor IL1-Ra, and WNT antagonists DKK1 and FRZB restored SOX9 activity in the healthy and OA-hPCs (Figure 1). The values for the immobile fraction, protein diffusion rate and ratio of free diffusing population of SOX9 were calculated and showed significant differences. ChIP assays showed that WNT3A treatment decreased SOX9 binding to the ACAN and COL2A1 promotor sites.

Conclusions: Here we show that the SOX9 transcriptional activity is differentially regulated in healthy and OA-hPCs. We observed that the basal SOX9 activity was lower in OA-hPCs and that the activity was not significantly changed in the presence of IL1β or WNT3A. Healthy hPCs were more responsive to catabolic (IL1 and WNT) and anabolic (BMP7) signals and SOX9 activity was restored by IL1 and WNT inhibitors. These data indicate that the response of cells is highly dependent on the disease state. This is relevant for the treatments of OA joints where heterogeneous cell populations with varying OA grades are present. In implicates that treatment strategies solely based on restoring SOX9 mRNA/protein expression will not result in increased SOX9 transcriptional activity.