RNAseq of ATDC5 cells with mechanotransductive perturbations

Chondrocyte phenotype is preserved when cells are round and the actin cytoskeleton is cortical. Conversely, these cells rapidly dedifferentiate in vitro with increased mechanoactive Rho signaling, which increases cell size and causes large actin stress fiber to form. While the effects of Rho on chondrocyte phenotype are well established, the molecular mechanism is not yet fully elucidated. Yap, a transcriptional co-regulator, is regulated by Rho in a mechanotransductive manner and can suppress chondrogenesis in vivo. Here, we sought to elucidate the relationship between mechanoactive Rho and Yap on chondrogenic gene expression. We first show that decreasing mechanoactive state through Rho inhibition results in a broad increase in chondrogenic gene expression. Next, we show that Yap and its co-regulator Taz, are negative regulators of chondrogenic gene expression, and removal of these factors promotes chondrogenesis even in environments that promote cell spreading. Finally, we establish that Yap/Taz is essential for translating Rho-mediated signals to negatively regulate chondrogenic gene expression, and that its removal negates the effects of increased Rho signaling. Together, these data indicate that Rho is a mechanoregulator of chondrogenic differentiation, and that its impact on chondrogenic expression is exerted principally through mechanically-induced translocation and activity of Yap and Taz. Overall design: ATDC5 cells cultured in chemically defined chondrogenic media under the following conditions: Rho inhibition for 3 days with C3 transferase, Rho promotion using LPA for 3 days, siRNA to Yap1/Wwtr1 five days post-transfections, and cells seeded for two days at low density on fibronectin coated glass, 55 kPa polyacrylamide gels and 5 kPa polyacrylamide gels.