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

• Published in: Osteoarthritis and cartilage 2015-06, Vol.23 (6), p.985-995
• Main Authors: van Caam, A, Blaney Davidson, E, Garcia de Vinuesa, A, van Geffen, E, van den Berg, W, Goumans, M.-J, ten Dijke, P, van der Kraan, P
• Format: Article
• Language: English
• Subjects: Animals; BMP-9; Cartilage, Articular - cytology; Cartilage, Articular - metabolism; Cattle; Cells, Cultured; Chondrocyte; Chondrocytes - drug effects; Chondrocytes - metabolism; Chondrocytes - pathology; Extracellular Matrix Proteins - pharmacology; Gene Expression Regulation - drug effects; Growth Differentiation Factor 2 - antagonists & inhibitors; Growth Differentiation Factor 2 - pharmacology; Hypertrophy; Ligands; Phosphorylation - drug effects; Rheumatology; Signal Transduction - drug effects; Smad signaling; Smad1 Protein - metabolism; Smad2 Protein - metabolism; Smad5 Protein - metabolism; TGF-beta1; Transforming Growth Factor beta - pharmacology
• ISSN: 1063-4584; 1522-9653
• DOI: 10.1016/j.joca.2015.02.007

Summary Objective 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. Design 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. 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 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. 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 TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype.