Transforming growth factor beta induced protein (TGFBI), a novel player in osteoarthritis

Purpose: As the most common form of chronic joint diseases, osteoarthritis (OA) represents a significant public health issue associated with a high economic burden. Transforming Growth Factor β (TGFβ) has been identified as a key regulator pathway in cartilage homeostasis. Its dysregulation, notably in bone marrow mesenchymal stem cells (MSCs), has been associated with the onset of OA. We therefore aimed at identifying members of the TGFβ pathway that are secreted by MSCs and could be dysregulated in the disease. Based on a mass spectrometry analysis of MSCs secretome, we selected Transforming Growth Factor β-Induced (TGFβI), a poorly studied extracellular matrix protein. Methods: Human cartilage, bone, infrapatellar fat pad and bone marrow-derived MSCs were obtained from OA patients and healthy donors after informed consent. Cartilage and bone tissues were incubated for 24h and TGFβI quantified in tissues and supernatants. Chondrogenic differentiation of MSCs was induced by culture in micropellet for 21 days. Transfection of siRNA directed against TGFβI was performed at day 3 and day 1 before induction of MSC differentiation. TGFβI expression was analyzed by RT-qPCR, IHC and ELISA. Primary human articular chondrocytes were isolated from OA patients and cultured till passage 3 for the dedifferentiation protocol or passage 1 for IL1β -induced OA-like chondrocytes. IL1β treatment was done for 24h and medium was replaced for another 24h before cell recovery. For siRNA experiment, chondrocyte markers were quantified at day 7. For recombinant protein approaches, different doses of TGFβI were added for 72h. We used the in vivo model of Collagenase-Induced Osteoarthritis (CIOA) to recover tibial epiphyses for RNA extraction or limbs for IHC staining. Results: TGFβI mRNA and protein were both highly expressed in human MSCs as compared to joint tissues. Interestingly, TGFβI was downregulated in OA MSCs as compared to MSCs from healthy subjects. We therefore analyzed TGFβI during chondrogenesis of MSCs and found a 10 fold increase of mRNA transcripts at days 1 and 2 after differentiation induction. TGFβI protein was detected in supernatants by ELISA and on histological sections of micropellets. Using a loss-of-function approach, we showed that down-regulation of TGFβI in MSCs induced to differentiate resulted in reduced expression of all chondrocyte markers, type IIB collagen, aggrecan, MMP13 and type X collagen by day 21. In human chondrocytes induced to dedifferentiate through passages, decreased expression of collagen type IIB and aggrecan was associated with decrease of TGFβI. TGFβI silencing in chondrocytes resulted in decreased levels of type II collagen while addition of recombinant TGFβI led to up-regulation of the hypertrophic markers Runx2, MMP13 and type X collagen. Addition of IL1β on chondrocytes to upregulate OA markers further increase the expression of TGFβI at mRNA and protein levels. Importantly, we found that TGFβI mRNA and protein were also significantly increased in cartilage and bone tissues recovered from OA donors as compared to healthy subjects while it was decreased in infrapatellar fat pad. In the murine models, histological analysis revealed that TGFβI was predominantly found in chondro/osteophytes. Conclusions: Our results indicate that up-regulation of TGFβI is required during the early stages of chondrogenesis and its expression helps maintaining cartilage homeostasis. However, its upregulation in OA chondrocytes is associated with hypertrophy and mineralization markers. Its tight regulation seems therefore needed to sustain cartilage ECM integrity.