A role for transcription factors CREB and ATF-2 in modulation of chondrocyte differentiation

The long bones of the vertebrate skeleton are formed through generation of a cartilage anlage from mesenchymal cells, through intensive proliferation, condensation and differentiation of these precursor cells. This cartilage model is replaced by bone, gradually, in a multi-step process called endochondral ossification. The cells comprising the cartilage tissue (chondrocytes) are progressing through different stages of proliferation, hypertrophy and terminal differentiation followed by apoptosis and matrix calcification. Their fate is controlled by a large number of local and systemic molecules, some driving the cells towards maturation, others preventing them from differentiation. Previous studies have characterized retinoic acid, thyroid hormone and bone morphogenetic proteins (BMPs) as stimulators of terminal differentiation while transforming growth factor-beta (TGF-β) and parathyroid hormone related peptide (PTHrP) are inhibitors of maturation. While TGF-β and PTHrP are known to be critical regulators of chondrocyte proliferation and differentiation, the signaling pathways through which these factors act remain to be elucidated. This study investigates the transcription factors that can be activated in TGF-β and PTHrP signaling, as well as their interaction with the BMP signaling cascade. This study demonstrates that both transcription factors CREB and AP-1 are critical to PTHrP signaling in chondrocytes while transcription factors ATF-2 and Smad3 are essential components of TGF-β signaling. In addition, we show that signals transduced by CREB, but not ATF-2, are capable of antagonizing BMP-signaling in chondrocytes through activation of a negative feedback loop involving BMP-inhibitory Smad6. PTHrP treatment leads to rapid CREB and AP-1 activation, with important consequences on chondrocyte phenotype. In the presence of dominant negative inhibitors of CREB and c-Fos, PTHrP effects on chondrocyte maturation and proliferation are suppressed, and chondrocyte maturation is accelerated. TGF-β treatment leads to ATF-2, but not CREB activation, and subsequently to PTHrP induction and inhibition of chondrocyte maturation. The BMPs signaling pathway consists in activation of receptor regulated Smads 1 and 5 and repression from the inhibitory Smad6. Discovery of a CREB, but not ATF-2, binding site in the Smad6 promoter confers PTHrP control on BMP-driven chondrocyte maturation. Altogether, these results evidentiate an important role for transcription factors CREB and ATF-2 in chondrocyte development.