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Type II TGFβ receptor modulates chondrocyte phenotype.

ABSTRACT: Aging is one of the major risk factors of osteoarthritis. This pathology during which chondrocytes undergo modifications of their phenotype may result from alteration of transforming growth factor β (TGFβ) signaling. This study investigates the role of TGFβ response in the process of chondrocyte dedifferentiation/redifferentiation. Dedifferentiation was induced by successive passages of human articular chondrocytes, whereas their redifferentiation was performed by three-dimensional culture in alginate. Human mesenchymal stem cells were obtained from bone marrow and differentiated into chondrocyte-like phenotype by three-dimensional culture, embedded in the same scaffold. Protein and mRNA levels were analyzed by Western blot and real-time reverse transcription PCR. Regulatory mechanism was investigated using specific inhibitors (mithramycin), mRNA silencing or decoy oligonucleotides, and expression vectors. Chondrocyte dedifferentiation interfered with TGFβ signaling by decreasing TβRII mRNA and protein levels and subsequent TGFβ response. TβRII ectopic expression in passaged chondrocytes permitted to increase the expression of several matrix genes, such as aggrecan or type II collagen. Redifferentiation of passaged chondrocytes permitted to restore, at least in part, TβRII expression and was related to differentiation of human bone marrow mesenchymal stem cells toward chondrocytes, where both specific protein 1 (Sp1) and TβRII mRNA levels were increased. Moreover, Sp1 manipulation by silencing or ectopic expression and pharmacologic inhibition revealed a link between expression levels of this transcriptional factor, which is crucial for constitutive expression of TβRII in cartilage, and TGFβ response. Therefore, these data permit us to suggest an important role of TβRII expression in the maintenance of chondrocyte phenotype, which is altered with age, and bring new insights in our understanding of chondrogenesis process.

SUBMITTER: Bauge C

PROVIDER: S-EPMC3705098 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The fibrocartilage chondrocyte phenotype has been recognized to attribute to osteoarthritis (OA) development. These chondrocytes express genes related to unfavorable OA outcomes, emphasizing its importance in OA pathology. BMP7 is being explored as a potential disease-modifying molecule and attenuates the chondrocyte hypertrophic phenotype. On the other hand, BMP7 has been demonstrated to relieve organ fibrosis by counteracting the pro-fibrotic TGFβ-Smad3-PAI1 axis and increasing MMP2-mediated Collagen type I turnover. Whether BMP7 has anti-fibrotic properties in chondrocytes is unknown. Human OA articular chondrocytes (HACs) were isolated from end-stage OA femoral cartilage (total knee arthroplasty; n = 18 individual donors). SW1353 cells and OA HACs were exposed to 1 nM BMP7 for 24 h, after which gene expression of fibrosis-related genes and fibrosis-mediating factors was determined by RT-qPCR. In SW1353, Collagen type I protein levels were determined by immunocytochemistry and western blotting. PAI1 and MMP2 protein levels and activity were measured with an ELISA and activity assays, respectively. MMP2 activity was inhibited with the selective MMP-2 inhibitor OA-Hy. SMAD3 activity was determined by a (CAGA)12-reporter assay, and pSMAD2 levels by western blotting. Following BMP7 exposure, the expression of fibrosis-related genes was reduced in SW1353 cells and OA HACs. BMP7 reduced Collagen type I protein levels in SW1353 cells. Gene expression of MMP2 was increased in SW1353 cells following BMP7 treatment. BMP7 reduced PAI1 protein levels and -activity, while MMP2 protein levels and -activity were increased by BMP7. BMP7-dependent inhibition of Collagen type I protein levels in SW1353 cells was abrogated when MMP2 activity was inhibited. Finally, BMP7 reduced pSMAD2 levels determined by western blotting and reduced SMAD3 transcriptional activity as demonstrated by decreased (CAGA)12 luciferase reporter activity. Our data demonstrate that short-term exposure to BMP7 decreases the fibrocartilage chondrocyte phenotype. The BMP7-dependent reduction of Collagen type I protein expression seems MMP2-dependent and inhibition of Smad2/3-PAI1 activity was identified as a potential pathway via which BMP7 exerts its anti-fibrotic action. This indicates that in chondrocytes BMP7 may have a double mode-of-action by targeting both the hypertrophic as well as the fibrotic chondrocyte phenotype, potentially adding to the clinical relevance of using BMP7 as an OA disease-modifying molecule.

Project description:The transforming growth factor-β (TGFβ) family plays an important role in many developmental processes and when mutated often contributes to various diseases. Marfan syndrome is a genetic disease with an occurrence of approximately 1 in 5,000. The disease is caused by mutations in fibrillin, which lead to an increase in TGFβ ligand activity, resulting in abnormalities of connective tissues which can be life-threatening. Mutations in other components of TGFβ signaling (receptors, Smads, Schnurri) lead to similar diseases with attenuated phenotypes relative to Marfan syndrome. In particular, mutations in TGFβ receptors, most of which are clustered at the C-terminal end, result in Marfan-like (MFS-like) syndromes. Even though it was assumed that many of these receptor mutations would reduce or eliminate signaling, in many cases signaling is active. From our previous studies on receptor trafficking in C. elegans, we noticed that many of these receptor mutations that lead to Marfan-like syndromes overlap with mutations that cause mis-trafficking of the receptor, suggesting a link between Marfan-like syndromes and TGFβ receptor trafficking. To test this hypothesis, we introduced three of these key MFS and MFS-like mutations into the C. elegans TGFβ receptor and asked if receptor trafficking is altered. We find that in every case studied, mutated receptors mislocalize to the apical surface rather than basolateral surface of the polarized intestinal cells. Further, we find that these mutations result in longer animals, a phenotype due to over-stimulation of the nematode TGFβ pathway and, importantly, indicating that function of the receptor is not abrogated in these mutants. Our nematode models of Marfan syndrome suggest that MFS and MFS-like mutations in the type II receptor lead to mis-trafficking of the receptor and possibly provides an explanation for the disease, a phenomenon which might also occur in some cancers that possess the same mutations within the type II receptor (e.g. colon cancer).

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Type II TGFβ receptor modulates chondrocyte phenotype.

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