Nitric oxide regulates cartilage destruction by causing dedifferentiation and apoptosis of chondrocytes. We investigated the role of the mitogen-activated protein kinase subtypes, extracellular signal-regulated protein kinase (ERK)-1/2, and p38 kinase in NO-induced apoptosis of rabbit articular chondrocytes and their involvement in dedifferentiation. Generation of NO with sodium nitroprusside (SNP) caused dedifferentiation, as indicated by the inhibition of type II collagen expression and proteoglycan synthesis. NO additionally caused apoptosis, accompanied by p53 accumulation and caspase-3 activation. SNP treatment stimulated activation of ERK-1/2 and p38 kinase. Inhibition of ERK-1/2 with PD98059 rescued SNP-induced dedifferentiation but enhanced apoptosis up to 2-fold, whereas inhibition of p38 kinase with SB203580 enhanced dedifferentiation, with significant blockage of apoptosis. The stimulation of apoptosis by ERK inhibition was accompanied by increased p53 accumulation and caspase-3 activity, whereas the inhibitory effect of p38 kinase blockade was associated with reduced p53 accumulation and caspase-3 activity. Our results indicate that NO-induced p38 kinase functions as an induction signal for apoptosis and in the maintenance of chondrocyte phenotype, whereas ERK activity causes dedifferentiation and operates as an anti-apoptotic signal. NO generation is less proapoptotic in chondrocytes that are dedifferentiated by serial monolayer culture or phorbol ester treatment. NO-induced p38 kinase activity is low in dedifferentiated cells compared with that in differentiated chondrocytes, with lower levels of p53 accumulation and caspase-3 activity. Our findings collectively suggest that ERK-1/2 and p38 kinase oppositely regulate NO-induced apoptosis of chondrocytes, in association with p53 accumulation, caspase-3 activation, and differentiation status.

The differentiated phenotype of chondrocyte is rapidly lost during in vitro culture by a process designated "dedifferentiation." In this study, we investigate the roles of protein kinase C (PKC) and extracellular signalregulated protein kinase (ERK) in the maintenance of the differentiated chondrocyte phenotype. Chondrocytes isolated from rabbit articular cartilage underwent dedifferentiation upon serial monolayer culture with cessation of type II collagen expression and proteoglycan synthesis, which was reversed by culturing dedifferentiated cells in alginate gel. The expression pattern of PKC␣ was essentially the same as that of type II collagen during de-and redifferentiation, in that expression was decreased during dedifferentiation and increased during redifferentiation. In contrast to PKC␣, ERK activity increased 15-fold during dedifferentiation. This enhanced activity was terminated during redifferentiation. Down-regulation of PKC␣ in passage 0 chondrocytes resulted in dedifferentiation. However, overexpression of PKC␣ did not affect type II collagen levels, suggesting that PKC␣ expression is not sufficient to maintain the differentiated phenotype. However, inhibition of ERK by PD98059 enhanced type II collagen expression and proteoglycan synthesis in passage 0 cells, retarded dedifferentiation during monolayer cultures, and reversed dedifferentiation caused by downregulation of PKC. Unlike PKC-dependent ERK regulation of chondrogenesis, PKC and ERK independently modulated chondrocyte dedifferentiation, as confirmed by observations that PKC down-regulation and ERK inhibition did not alter ERK phosphorylation and PKC expression, respectively. In addition, expression of Ncadherin, ␣-catenin, and ␤-catenin, which are oppositely regulated to type II collagen during phenotype alterations, were modulated by PKC and ERK during chondrogenesis but not dedifferentiation, supporting distinct mechanisms for the regulation of chondrocyte differentiation and maintenance of differentiated phenotype by these two protein kinases.

During limb development, epithelial cells in the apical ectodermal ridge keep the underlying mesenchymal cells in a proliferative state preventing differentiation by secreting signaling molecules such as epidermal growth factor (EGF). We investigated the molecular mechanism of the EGF effect on the regulation of micromass culture-induced chondrogenesis of chick limb bud mesenchymal cells as a model system. We found that expression and tyrosine phosphorylation of the EGF receptor was increased transiently during chondrogenesis. Exogenous EGF inhibited chondrogenic differentiation of mesenchymal cells, and this effect was reversed by the EGF receptor inhibitor AG1478. EGF treatment also inhibited the expression and activation of protein kinase C-␣, whereas it activated Erk-1 and inhibited p38 mitogen-activated protein kinase, all of which appeared to be involved in the EGF-induced inhibition of chondrogenesis. Stimulation of the EGF receptor blocked precartilage condensation and altered the expression of cell adhesion molecules such as N-cadherin and integrins ␣ 5 and ␤ 1 . All these EGF effects were reversible by AG1478. The data indicate that EGF negatively regulate chondrogenesis of chick limb bud mesenchymal cells by inhibiting precartilage condensation and by modulating signaling pathways including those of protein kinase C-␣, Erk-1, and p38 mitogen-activated protein kinase.

Chondrogenesis of mesenchymal cells during in vitro micromass

WD repeats are implicated in protein-protein interactions and regulate a wide variety of cellular functions, including chromatin remodeling and transcription. The WD repeats of the Bub3 and Cdc20 kinetochore proteins are important for the physical interactions of these proteins with Mad2 and BubR1 to yield a kinetochore protein complex capable of delaying anaphase by inhibiting ubiquitin ligation via the anaphase-promoting complex/cyclosome. Here, we show that Bub3 and Cdc20 form a complex with histone deacetylases; this interaction appears to confer transcriptional repressor activity in a heterologous DNA-binding context. In addition, inhibition of Bub3 and Cdc20 expression significantly impairs interphase cell cycle. These results indicate that Bub3 and Cdc20 play additional roles in the integration of cell cycle arrest as transcriptional repressors.