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

Physiological and excessive mechanical compression of articular cartilage activates Smad2/3P signaling

Item does not contain fulltextOBJECTIVE: Transforming growth factor beta (TGF-beta) in articular cartilage can signal via two routes, the ALK5/Smad2/3P and the ALK1/Smad1/5/8P route, the first being protective and the latter favoring chondrocyte terminal differentiation. Since biomechanical factors are known to play an essential role in osteoarthritis (OA) initiation and progression, we investigated if excessive mechanical compression can alter TGF-beta signaling in cartilage shifting it from ALK5/Smad2/3P to ALK1/Smad1/5/8P pathway, favoring terminal differentiation of chondrocytes. DESIGN: Articular cartilage explants were harvested from bovine metacarpophalangeal joints. After equilibration, explants were subjected to unconfined dynamic mechanical compression (1 Hz) with 3 MPa (physiological) or 12 MPa (excessive) stress. After different time intervals samples were frozen and mRNA levels of selected genes were examined using real-time polymerase chain reaction. RESULTS: In articular cartilage compressed with 3 MPa and also 12 MPa stress the expression of Smad2/3P responsive genes bSerpine1, bSmad7 and bAlk5 was up-regulated, whereas the expression of Smad1/5/8P responsive gene bId1 was down-regulated. Furthermore, the expression of bTgfb1 was significantly up-regulated in both compression groups. When ALK5/Smad2/3P pathway was blocked with a selective ALK4/5/7 inhibitor, the effect of excessive mechanical compression on bSmad7 and bAlk5 expression was prevented. CONCLUSIONS: Here we show that excessive mechanical compression alone is not able to shift TGF-beta signaling toward the ALK1/Smad1/5/8P pathway. In contrast, we show that mechanical compression not only with physiological but also with excessive stress can activate Smad2/3P signaling, which is known to be protective for articular cartilage and to block chondrocyte terminal differentiation

Expression of TGF-beta Signaling Regulator RBPMS (RNA-Binding Protein With Multiple Splicing) Is Regulated by IL-1beta and TGF-beta Superfamily Members, and Decreased in Aged and Osteoarthritic Cartilage

Item does not contain fulltextOBJECTIVE: RNA-binding protein with multiple splicing (RBPMS) has been shown to physically interact with Smads and enhance transforming growth factor-beta (TGF-beta)-mediated Smad2/3 transcriptional activity in mammalian cells. Objective of this study was to examine whether expression of RBPMS is regulated by interleukin-1beta (IL)-1beta and TGF-beta superfamily growth factors and whether expression of RBPMS is altered during aging and experimental osteoarthritis. METHODS: Expression of RBPMS protein was investigated in chondrocyte cell lines of murine (H4) and human (G6) origin using Western blot analysis. Regulation of RBPMS expression in H4 chondrocytes at mRNA level was done by reverse transcriptase-quantitative polymerase chain reaction. Furthermore, characterization of Smad signaling pathways regulating RBPMS expression was performed by blocking studies using small molecule inhibitors or by transfection studies with adenoviral vector constructs (constitutive-active ALK1 and constitutive-active ALK5). Expression of RBPMS in cartilage of different age groups of C57BL/6N mice (6 months and 20 months) and in a surgically induced osteoarthritis (OA) mouse model was analyzed using immunohistochemistry. RESULTS: RBPMS was shown to be expressed in chondrocytes and cartilage of murine, human, and bovine origin. TGF-beta inhibited RBPMS expression while BMP2 and IL-1beta increased its expression. TGF-beta-induced inhibition was blocked by ALK5 inhibitor. Overexpression of ca-ALK1 stimulated RBPMS expression. Moreover, RBPMS expression was found to be reduced with ageing and in OA pathogenesis. CONCLUSIONS: Expression of RBPMS in chondrocytes is regulated by TGF-beta superfamily members and IL-1beta, indicating a counter-regulatory mechanism. Expression of RBPMS, in cartilage and its reduction during ageing and OA might suggest its potential role in the maintenance of normal articular cartilage

The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFbeta1

Item does not contain fulltextOBJECTIVE: In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorss1 (TGFbeta1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFbeta1 signaling was also studied. DESIGN: Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFbeta1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFbeta/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. RESULTS: In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFbeta1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFbeta/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFbeta1. CONCLUSIONS: This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFbeta1. This observation underlines the importance of TGFbeta1 in maintenance of chondrocyte phenotype