Fig. 3. Transcriptomic profile induced by TRPV4 activation.

(A) Engineered cartilage tissue constructs were made from isolated primary porcine chondrocytes cast into agarose hydrogels. Tissue constructs were cultured in nutrient-rich medium before deformational mechanical loading or GSK101 pharmacologic stimulation (red, 3 hours per round) following the indicated time course and cartilage construct harvest (arrows). (B) Forty-one genes were differentially up-regulated in response to TRPV4 activation, and levels returned back to baseline after 12 to 20 hours after loading (n = 3 per treatment/time point). (C) cAMP- and calcium-responsive transcription factors were immediately and highly regulated by both mechanical loading and GSK101 stimulation (red arrows indicate removal from loading). (D) Pathway analysis based on transcription activity suggests that both inflammatory and anabolic pathways are strongly regulated by TRPV4 activation. (E) Analysis of gene target response after all bouts of mechanical loading and all bouts of GSK101 stimulation produces a list of distinctly TRPV4-sensitive genes. (F) TRPV4-responsive targets from the microarray analysis were confirmed by qPCR (n = 2 to 3), and a one-tailed t test was used to test whether loaded or GSK101 groups were significantly up-regulated with treatment. *P < 0.05.