Articular Chondrocyte Maturation Is Induced by Shifting Signaling Dominance from Thetgf-beta to the Bmp Pathway

Introduction. Maintenance of the articular surface depends on the function of articular chondrocytes (ACs) which produce matrix and are constrained from undergoing terminal maturation. Under some circumstances, such as in osteoarthritis, these constraints are lost and ACs enter the maturational program that occurs during endochondral ossification. The aim of this study was to identify these unknown suppressive constraints, with the goal of understanding the molecular processes at work during inappropriate stimulation of AC hypertrophy. To address this aim, a novel cell model has been developed which utilizes 5-azacytidine (Aza) to induce maturation in cultured ACs. Aza is known to un-mask expression of silenced genes in a number of cell models by replacing cytidine in the DNA, thus blocking the suppressive effect of cytidine methylation on gene transcription. ACs, which do not spontaneously express markers of maturation such as colX, Ihh and alkaline phosphatase, have been induced to express this marker by treatment with Aza. Since it is well established that TGF-beta and BMP-2 are primary regulators of the respective suppression or induction of chondrocyte maturation, we hypothesize that Aza-induction of maturation is facilitated via a shift in signaling dominance from TGF-beta to BMP. Using Aza as a tool, we have attempted to identify molecular mechanisms that constrain AC maturation. Specifically, we have i) compared Aza effects with those induced by over-expressing the BMP-2 signaling molecule Smad1 or by blocking signaling through the TGFbeta Smads (2 and 3) using a dominant negative (A) approach, ii) examined the impact of Aza on the expression of the TGF-beta Smads (2 and 3), the BMP-2 Smads (1 and 5), and the ubiquitin E3 ligase Smurf2, a protein that initiates the degradation of Smad2 and 3, and iii) used a pulse/chase method to measure the rate of Smad2 and 3 degradation following Aza treatment. Our findings suggest that blockade of TGFbeta signaling and enhancement of BMP signaling is sufficient to induce maturation of ACs and may represent an underlying mechanism that facilitates aberrant maturation of these cells during disease.