Figure 2.

Cartilage injury leads to activation of transforming growth factor β–activated kinase 1 (TAK‐1), and inhibition of TAK‐1 abolishes injury‐dependent inflammatory gene expression. A, Porcine metacarpophalangeal (MCP) joints were equilibrated at 37°C for 1 hour. Articular cartilage was dissected and either snap‐frozen or kept in serum‐free medium for the indicated times. Cartilage was then washed with 1× phosphate buffered saline and lysed in radioimmunoprecipitation assay buffer. Lysates were analyzed by Western blotting (WB) for phospho–TAK‐1 (ph‐T187), phospho‐JNK, and ERK. B, A TAK‐1 activity assay was carried out as described in Materials and Methods to confirm TAK‐1 activation. IP = immunoprecipitation; ph‐MBP = phospho–myelin basic protein. C and D, Porcine MCP joints were injected with 5 μM TAK‐1 inhibitor 5z‐7‐oxozeanol (OXO) or DMSO (vehicle [V]), as described in Materials and Methods, and kept at 37°C for 1 hour. Joints were then opened and cartilage was dissected and snap‐frozen (time point 0 minutes) or cultured for the indicated times with or without TAK‐1 inhibitor. Cartilage lysates were analyzed for phospho‐JNK and total ERK (T‐ERK) (C) and for IκB and total ERK (D). E, Cartilage was snap‐frozen (time point 0 hours) or kept at 37°C for 4 hours with or without 5 μM TAK‐1 inhibitor (OXO). RNA was extracted from the tissue and used in quantitative reverse transcriptase–polymerase chain reaction studies of a panel of typical inflammatory response genes. Bars show the mean ± SEM of 3 independent experiments. ∗ = P < 0.05; ∗∗ = P < 0.01, by unpaired t‐test. NS = not significant; COX‐2 = cyclooxygenase 2; IL‐6 = interleukin‐6; MMP‐3 = matrix metalloproteinase 3; iNOS = inducible nitric oxide synthase; TNF = tumor necrosis factor. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39965/abstract.