Regulation of chondrocyte phenotype by Bcl-2 and Bag-1

The anti-apoptosic protein Bcl-2 has been shown to function in roles unrelated to apoptosis in a variety of cell types. We have previously reported that loss of Bcl-2 expression alters chondrocyte morphology and modulates aggrecan expression via an apoptosis-independent pathway. Here we show that Bcl-2 is required for chondrocytes to maintain expression of a variety of cartilage-specific matrix proteins. Using quantitative, real-time PCR, we demonstrate that Bcl-2-deficient chondrocytes coordinately downregulate genes coding for hyaline cartilage matrix proteins including collagen II, collagen IX, aggrecan, and link protein. The decrease in steady-state level of these mRNA transcripts results, in part, from decreased RNA stability in Bcl-2-deficient chondrocytes. Transcriptional regulation is also likely involved because chondrocytes with decreased Bcl-2 levels show decreased expression of SOX9, a transcription factor necessary for expressing the major cartilage matrix proteins. In contrast, chondrocytes over-expressing Bcl-2 have a stable phenotype when subjected to loss of trophic factor signaling. These cells maintain high levels of SOX9, as well as the SOX9 targets collagen II and aggrecan. These results suggest that Bcl-2 is involved in a pathway important for maintaining a stable chondrocyte phenotype. It has been demonstrated that chondrocytes apoptosis increases in aged articular cartilage and we show here that expression of Bcl-2 decreases with age. The Bcl-2 binding protein Bag-1 is an important regulator of apoptosis for a variety of cell types, but little is known about its role in cartilage. Using immunostaining, we show that Bag-1 is expressed by growth plate chondrocytes and articular chondrocytes. Bag-1 expression in articular cartilage decreases with age whereas Bag-1 expression in the growth plate is generally maintained in aged animals. In the growth plate, Bag-1 is highest in pre-hypertrophic chondrocytes, just prior to terminal differentiation of the chondrocytes. The growth plate expression pattern of Bag-1 is similar to what has previously been reported for Bcl-2. In vitro studies show that Bag-1 protein expression is down-regulated by retinoic acid and by FGFb/TGFβ. These results suggest that Bag-1 is an important regulator of the chondrocyte phenotype and that both Bag-1 and Bcl-2 play critical roles in preventing chondrocyte apoptosis.