Canonical Wnt signaling skews TGF-β signaling in chondrocytes towards signaling via ALK1 and Smad 1/5/8.

Both Wnt signaling and TGF-β signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-β signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-β signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-β signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway. Stimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-β-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-β signaling via Smad 2/3, measured by the functional CAGA12-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation. Our data show that canonical Wnt signaling is able to skew TGF-β signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.

van den Bosch MH ，Blom AB ，van Lent PL ，van Beuningen HM ，Blaney Davidson EN ，van der Kraan PM ，van den Berg WB ... -  《-》

ALK1 opposes ALK5/Smad3 signaling and expression of extracellular matrix components in human chondrocytes.

Finnson KW ，Parker WL ，ten Dijke P ，Thorikay M ，Philip A ... -  《-》

Increase in ALK1/ALK5 ratio as a cause for elevated MMP-13 expression in osteoarthritis in humans and mice.

Blaney Davidson EN ，Remst DF ，Vitters EL ，van Beuningen HM ，Blom AB ，Goumans MJ ，van den Berg WB ，van der Kraan PM ... -  《-》

The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFβ1.

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 (TGFβ1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFβ1 signaling was also studied. Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFβ1 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 TGFβ/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. 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 TGFβ1 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 TGFβ/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 TGFβ1. This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype.

van Caam A ，Blaney Davidson E ，Garcia de Vinuesa A ，van Geffen E ，van den Berg W ，Goumans MJ ，ten Dijke P ，van der Kraan P ... -  《-》

Transforming growth factor-β stimulates Smad1/5 signaling in pulmonary artery smooth muscle cells and fibroblasts of the newborn mouse through ALK1.

The intracellular signaling mechanisms through which TGF-β regulates pulmonary development are incompletely understood. Canonical TGF-β signaling involves Smad2/3 phosphorylation, Smad2/3·Smad4 complex formation and nuclear localization, and gene regulation. Here, we show that physiologically relevant TGF-β1 levels also stimulate Smad1/5 phosphorylation, which is typically a mediator of bone morphogenetic protein (BMP) signaling, in mouse pup pulmonary artery smooth muscle cells (mPASMC) and lung fibroblasts and other interstitial lung cell lines. This cross-talk mechanism likely has in vivo relevance because mixed Smad1/5/8·Smad2/3 complexes, which are indicative of TGF-β-stimulated Smad1/5 activation, were detected in the developing mouse lung using a proximity ligation assay. Although mixed Smad complexes have been shown not to transduce nuclear signaling, we determined that TGF-β stimulates nuclear localization of phosphorylated Smad1/5 and induces the expression of prototypical BMP-regulated genes in the mPASMC. Small-molecule kinase inhibitor studies suggested that TGF-β-regulated Smad1/5 phosphorylation in these cells is mediated by TGF-β-type I receptors, not BMP-type I receptors, but possibly the accessory activin-like kinase (ALK1) receptor. Although work by others suggested that ALK1 is expressed exclusively in endothelial cells in the vasculature, we detected ALK1 mRNA and protein expression in mPASMC in vitro and in mouse pup lungs. Moreover, using an antimurine ALK1 antibody and mPASMC, we determined that ALK1 regulates Smad1/5 phosphorylation by TGF-β. Together, these studies characterize an accessory TGF-β-stimulated BMP R-Smad signaling mechanism in interstitial cells of the developing lung. They also indicate the importance of considering alternate Smad pathways in studies directed at determining how TGF-β regulates newborn lung development.

Zhang H ，Du L ，Zhong Y ，Flanders KC ，Roberts JD Jr ... -  《-》