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Objective We previously identified PAPSS2 as a transcriptional target of TGF-β in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-β responsive gene, PRG4. In this study, TGF-β-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. Design Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3. Western blot and real-time quantitative RT-PCR were used to measure changes in protein and mRNA levels in response to treatment. Results Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-β treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-β signaling. Furthermore, Sox9 was required for full TGF-β-mediated induction of Papss2. In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-β although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-β. Conclusions TGF-β stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-β-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-β-mediated gene regulation in chondrocytes.

ObjectivePrg4, also known as Lubricin, acts as a joint/boundary lubricant. Prg4 has been used to prevent surgically induced osteoarthritis (OA) in mice. Surgically induced OA serves as a good model for post-traumatic OA but is not ideal for recapitulating age-related OA. Reduced expression of the TGF-β type II receptor (TGFβR2) is associated with age-related OA in clinical samples, so we previously characterized a mouse model that exhibits OA due to expression of a mutated dominant-negative form of TGFβR2 (DNIIR). Prg4 expression was significantly reduced in DNIIR mice. Furthermore, we showed that Prg4 was a transcriptional target of TGF-ß via activation of Smad3, the main signal transducing protein for TGF-ß. The objective of the present study was to determine whether maintenance of Prg4, a down-stream transcriptional target of TGF-ß, prevents OA associated with attenuated TGF-ß signaling in mice.DesignWild-type, DNIIR, and bitransgenic mice that express both DNIIR and Prg4, were compared. Mice were assessed with a foot misplacement behavioral test, μCT, histology, and Western blot.ResultsCompared to DNIIR mice, bitransgenic DNIIR+Prg4 mice missed 1.3 (0.4, 2.1) fewer steps while walking (mean difference (95% confidence interval)), exhibited a cartilage fibrillation score that was 1.8 (0.4, 3.1) points lower, exhibited cartilage that was 28.2 (0.5, 55.9) μm thicker, and exhibited an OARSI score that was 6.8 (-0.9, 14.5) points lower. However, maintenance of Prg4 expression did not restore levels of phosphorylated Smad3 in DNIIR mice, indicating Prg4 does not simply stimulate TGF-ß signaling.ConclusionsOur results indicate that maintenance of Prg4 expression prevents OA progression associated with reduced TGF-β signaling in mice. Since there was no evidence that Prg4 acts by stimulating the TGF-ß signaling cascade, we propose that Prg4, a transcriptional target of TGF-ß, attenuates OA progression through its joint lubrication function.

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Reduced transforming growth factor beta (TGF-β) signaling is associated with osteoarthritis (OA). TGF-β is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage.A potential approach for treating OA is to identify the factors that act downstream of TGF-β's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissueengineered cartilage model with reduced TGF-β signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-β that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-β regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-β type I receptor inhibitor, SB431542, was used to attenuate TGF-β signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-β receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-β signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-β signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage. K E Y W O R D Sanabolic, cartilage, osteoarthritis, scaffoldless tissue engineering, Sox9, TGF-β