Young-Mee Yoon

The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1. 7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin alpha5beta1 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.

Chondrogenesis of mesenchymal cells during in vitro micromass culture requires the generation of cyclic adenosine monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase A (PKA). In this study, we investigated the regulatory activity of PKA during chondrogenesis of chick limb bud mesenchymal cells. PKA activity was high in 1-day and 2-day cultures, which was followed by a slight decrease in 4-day and 5-day old cultures. Inhibition of PKA blocked chondrogenesis. It did not affect precartilage condensation, but it blocked the progression from the precartilage condensation stage to cartilage nodule formation. The PKA inhibition-induced blockage of chondrogenesis was accompanied by an altered expression of N-cadherin. Although expression of N-cadherin was detected during the early period of chondrogenesis, it became reduced as chondrogenesis proceeded. Still, inhibition of PKA maintained expression of N-cadherin throughout the micromass culture period. The inhibition of PKA did not affect expression of protein kinase C-alpha (PKCalpha), PKCepsilon, PKCdelta, and PKClambda/iota, which are the isoforms expressed in differentiating mesenchymal cells. However, PKA inhibition completely blocked activation of PKCalpha. Because PKC activity regulates N-cadherin expression and chondrogenesis, the PKA-mediated regulation of PKCalpha appears to be responsible for the PKA regulation of N-cadherin expression and chondrogenesis. Taken together, our results suggest that PKA regulates chondrogenesis by activating PKCalpha at the stage of post-precartilage condensation.