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 ␣5␤1 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.Formation of cartilage is initiated by differentiation of mesenchymes into chondrocytes. The differentiation process requires proliferation of chondrogenic competent cells that subsequently undergo precartilage condensation (1-3). Precartilage condensation is characterized by cells that are more densely packed in specific regions. This precartilage condensation is an important prerequisite for the initiation of chondrogenesis in mesenchymes in vivo. It becomes evident that several adhesion molecules including cadherins (4-7), integrins (8, 9), and extracellular matrix (ECM) 1 components (9-12) regulate precartilage condensation. Limb mesenchymes undergo spontaneous differentiation to chondrocytes in vitro when cultured at a high seeding density such as micromass culture in the presence of serum. Precartilage condensation similar to that formed in vivo during chondrogenesis occurs in micromass cultures of limb mesenchymes (13). The cellular aggregates that form in early micromass cultures subsequently differentiate into cartilage nodules, which express cartilage-specific molecules such as type II collagen (6, 14-16). However, the signal transduction pathways involved in precartilage condensation and initiated by condensation are not well characterized.We have previously shown that protein kinase C (PKC) positively regulates chondrogenesis of mesenchymes (17,18). PKC is a multigene family composed of 11 known isoforms (19,20). Multiple PKC isoforms su...

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Although regulation of chondrogenesis and cartilage development by Wnt signaling is well established, the function of Wnt in the maintenance and destruction of cartilage remains largely unknown. Here we investigated the involvement and regulatory mechanisms of Wnt signaling in cartilage destruction. We found that interleukin-1␤, the primary pro-inflammatory cytokine involved in cartilage destruction, induces expression of Wnt-5a and -7a in primary culture articular chondrocytes. The level of ␤-catenin was also increased in chondrocytes of arthritic cartilage, suggesting the association of Wnt/␤-catenin signaling with arthritic cartilage destruction. In addition, our results show that Wnt-7a induces dedifferentiation and inhibits NO-induced apoptosis of primary culture articular chondrocytes. Wnt-7a induces dedifferentiation of articular chondrocytes by stimulating transcriptional activity of ␤-catenin, whereas NO-induced apoptosis is inhibited via the activation of cell survival signaling, such as phosphatidylinositol 3-kinase and Akt, which block apoptotic signaling cascade. Our results collectively suggest that Wnt-7a is associated with cartilage destruction by regulating the maintenance of differentiation status and the apoptosis of articular chondrocytes via different mechanisms.

Leukotriene B 4 is a potent chemoattractant known to be involved mainly in inflammation, immune responses, and host defense against infection, although the exact signaling mechanisms by which it exerts its effects are not well understood. Here we show that exogenous leukotriene B 4 induces reactive oxygen species (ROS) generation via a Rac-dependent pathway, and that stable expression of Rac N17 , a dominant negative Rac1 mutant, completely blocks leukotriene B 4 -induced ROS generation. In addition, leukotriene B 4 -induced ROS generation is selectively blocked by inhibition of ERK or cytosolic phospholipase A 2 , but not p38 kinase, which is indicative of its dependence on ERK activation and synthesis of arachidonic acid. Consistent with those findings, leukotriene B 4 Rac-dependently stimulates ERK and cytosolic phospholipase A 2 activity, and transient transfection with plasmid expressing Rac V12 , a constitutively activated Rac1 mutant, also dose-dependently stimulates ERK activity. Our findings suggest that ERK and cytosolic phospholipase A 2 are situated downstream of Rac, and we conclude that Rac, ERK, and cytosolic phospholipase A 2 all play pivotal roles in mediating the ROS generation that appears to be a prerequisite for leukotriene B 4 -induced chemotaxis and cell proliferation. LTs1 are potent biological mediators of inflammation generated from arachidonic acid via the 5-LO pathway (1, 2). Among them, LTB 4 is one of the most potent chemoattractants known, acting mainly on neutrophils and eosinophils, but also on mast cells and endothelial cells (3)(4)(5). LTB 4 stimulates a number of cellular functions in addition to chemotaxis, including release of lysosomal enzymes and production of ROS (6 -8); it also promotes cell adhesion to vascular endothelial cells and transmigration, which amplifies inflammatory responses. Although LTB 4 -induced leukocyte recruitment is thought to play a protective role in the host defense against various pathogens, it is also involved in the pathogenesis of such inflammatory diseases as bronchial asthma (9, 10), inflammatory bowel diseases (11, 12), and psoriasis (13,14).Despite many reports on the cellular functions of LTB 4 , the exact signaling pathway along which its biological activities are transduced remains largely unknown. It is known, however, that LTB 4 acts via two G protein-coupled receptors, . The former is a high affinity LTB 4 receptor expressed mainly in polymorphonuclear leukocytes, whereas the latter is a ubiquitous, low affinity receptor whose expression is highest in spleen (17, 18). The details of the cellular functions of BLT1 and BLT2 are still largely unknown. Recently, however, LTB 4 -induced chemotaxis was shown to be completely inhibited in cells pretreated with PTX (100 ng/ml), indicating the participation of a PTX-sensitive G protein in LTB 4 signaling to chemotaxis (16). LTB 4 also elicits increases in intracellular free Ca 2ϩ and inositol 1,4,5-triphosphate, but these are apparently not involved in the chemotactic response by LTB 4 (15). ...