TGFbeta1-induced SMAD2/3 and SMAD1/5 phosphorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity.

Contains fulltext : 173935.pdf (publisher's version ) (Open Access)BACKGROUND: Dysregulated transforming growth factor beta (TGFbeta) signaling is implicated in osteoarthritis development, making normalizing TGFbeta signaling a possible therapy. Theoretically, this can be achieved with small molecule inhibitors specifically targeting the various TGFbeta receptors and downstream mediators. In this study we explore in primary chondrocytes the use of small molecule inhibitors to target TGFbeta-induced pSmad1/5/9-, pSmad2/3- and TGFbeta-activated kinase 1 (TAK1)-dependent signaling. METHOD: Primary bovine chondrocytes and explants were isolated from metacarpophalangeal joints. To modulate TGFbeta signaling the activin receptor-like kinase (ALK)1/2/3/6 inhibitor LDN-193189, the ALK4/5/7 inhibitor SB-505124 and the TAK1 inhibitor (5Z)-7-Oxozeaenol were used. pSmad1/5 and pSmad2 were measured using western blot analysis and TGFbeta1-induced gene expression was measured using quantitative real time PCR (qPCR). RESULTS: In chondrocytes, TGFbeta1 strongly induced both pSmad1/5 and pSmad2. Remarkably, LDN-193189 did not inhibit TGFbeta-induced pSmad1/5. In contrast, SB-505124 did inhibit both TGFbeta-induced Smad2 and Smad1/5 phosphorylation. Furthermore, (5Z)-7-Oxozeaenol also profoundly inhibited TGFbeta-induced pSmad2 and pSmad1/5. Importantly, both SB-505124 and (5Z)-7-Oxozeaenol did not significantly inhibit constitutively active ALK1, making an off-target effect unlikely. Additionally, LDN-193189 was able to potently inhibit BMP2/7/9-induced pSmad1/5, showing its functionality. On gene expression, LDN-193189 did not affect TGFbeta1-induced regulation, whereas both SB-505124 and (5Z)-7-Oxozeaenol did. Similar results were obtained in cartilage explants, although pSmad1/5 was not strongly induced by addition of TGFbeta1. CONCLUSION: Our data suggest that ALK5 kinase activity plays a central role in both TGFbeta-induced Smad1/5 and Smad2/3 phosphorylation, making it difficult to separate both pathways with the use of currently available small molecule inhibitors. Furthermore, our data regarding (5Z)-7-Oxozeaenol suggest that TAK1 facilitates Smad-dependent signaling