Maintenance of Differentiated Phenotype of Articular Chondrocytes by Protein Kinase C and Extracellular Signal-regulated Protein Kinase

Abstract

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.

αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

“…Our current finding is intriguing in that ␣v␤5 integrin plays a pivotal role in the dedifferentiation of chondrocytes through the activation of ERK signaling. Although the involvement of ERK signaling in dedifferentiation has been reported (47,48), our finding is novel in that we demonstrated that the ERK signaling is activated by ␣v␤5 integrin.…”

p38 kinase mediates nitric oxide-induced apoptosis of chondrocytes through the inhibition of protein kinase C ζ by blocking autophosphorylation

“…32 Briefly, cartilage slices were dissociated enzymatically for 6 h in 0.2% collagenase type II (381 U/mg solid, Sigma, St. Louis, MO, USA) in Dulbecco's modified Eagle's medium (DMEM) (Gibco-BRL, Rockville, MD, USA). The cells were plated on culture dishes at a density of 5 Â 10 4 cells/cm 2 in DMEM containing 10% bovine serum.…”

Identification and characterization of arginase II as a chondrocyte phenotype‐specific gene

“…Rabbit articular chondrocytes were isolated from knee joint cartilage slices of 2-week-old New Zealand white rabbits as described by Yoon et al (2002) (7). Briefly, cartilage slices were dissociated for 6 h in 0.2% collagenase type II in prolinefree Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL, Gaithersburg, MD).…”

“…For instance, both down-regulation of ERK1/2 and induction of p38 kinase activities are required for chondrogenic differentiation of mesenchymal cells (10,11), and activation of ERK1/2 in differentiated articular chondrocytes causes dedifferentiation (7). ERK1/2 and p38 kinase also oppositely regulate NOinduced dedifferentiation and apoptosis of chondrocytes.…”

“…Although differentiated chondrocytes show low activity of ERK, inhibition of basal activity of ERK by PD98059 treatment significantly increases type II collagen expression (7). In an attempt to understand the regulatory mechanisms of ERK signaling in type II collagen expression and thereby dedifferentiation of chondrocytes, we sought to identify and characterize genes up-regulated by the inhibition of ERK signaling in primary culture chondrocytes.…”