The neural cell adhesion molecule NCAM plays an important role in axonal growth, learning, and memory. A signaling pathway has been elucidated in which clustering of the NCAM140 isoform in the neural plasma membrane stimulated the activating phosphorylation of mitogen‐activated protein kinases (MAPKs) and the transcription factor cyclic AMP response‐element binding protein (CREB). NCAM clustering transiently induced dual phosphorylation (activation) of the MAPKs ERK1 and ERK2 (extracellular signal‐regulated kinases) by a pathway regulated by the focal adhesion kinase p125fak, p59fyn, Ras, and MAPK kinase. CREB phosphorylation at serine 133 induced by NCAM was dependent in part on an intact MAPK pathway. c‐Jun N‐terminal kinase, which is associated with apoptosis and cellular stress, was not activated by NCAM. Inhibition of the MAPK pathway in rat cerebellar neuron cultures selectively reduced NCAM‐stimulated neurite outgrowth. These results define an NCAM signal transduction mechanism with the potential for modulating the expression of genes needed for axonal growth, survival, and synaptic plasticity. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 542–558, 1999

Objective. To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyro-phosphate in chondron pellet cultures. Methods. Chondron pellets were formed from 1.6 10 6 cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of 32 P-ATP to resting cultures. Results. Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophos-phate in resting cultures. Conclusion. The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydro-lase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chon-drocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophos-phate dihydrate crystal deposition diseases.

The role of the chondrocyte pericellular matrix (PCM) was examined in a three-dimensional chondrocyte culture system to determine whether retention of the native pericellular matrix could stimulate collagen and proteoglycan accumulation and also promote the formation of a mechanically functional hyaline-like neocartilage. Porcine chondrocytes and chondrons, consisting of the chondrocyte with its intact pericellular matrix, were maintained in pellet culture for up to 12 weeks. Sulfated glycosaminoclycans and type II collagen were measured biochemically. Immunocytochemistry was used to examine collagen localization as well as cell distribution within the pellets. In addition, the equilibrium compressive moduli of developing pellets were measured to determine whether matrix deposition contributed to the mechanical stiffness of the cartilage constructs. Pellets increased in size and weight over a 6-week period without apparent cell proliferation. Although chondrocytes quickly rebuilt a PCM rich in type VI collagen, chondron pellets accumulated significantly more proteoglycan and type II collagen than did chondrocyte pellets, indicating a greater positive effect of the native PCM. After 5 weeks in chondron pellets, matrix remodeling was evident by microscopy. Cells that had been uniformly distributed throughout the pellets began to cluster between large areas of interterritorial matrix rich in type II collagen. After 12 weeks, clusters were stacked in columns. A rapid increase in compressive strength was observed between 1 and 3 weeks in culture for both chondron and chondrocyte pellets and, by 6 weeks, both had achieved 25% of the equilibrium compressive stiffness of cartilage explants. Retention of the in vivo PCM during chondrocyte isolation promotes the formation of a mechanically functional neocartilage construct, suitable for modeling the responses of articular cartilage to chemical stimuli or mechanical compression.

The neural cell adhesion molecule NCAM plays an important role in axonal growth, learning, and memory. A signaling pathway has been elucidated in which clustering of the NCAM140 isoform in the neural plasma membrane stimulated the activating phosphorylation of mitogen-activated protein kinases (MAPKs) and the transcription factor cyclic AMP response-element binding protein (CREB). NCAM clustering transiently induced dual phosphorylation (activation) of the MAPKs ERK1 and ERK2 (extracellular signal-regulated kinases) by a pathway regulated by the focal adhesion kinase p125fak, p59fyn, Ras, and MAPK kinase. CREB phosphorylation at serine 133 induced by NCAM was dependent in part on an intact MAPK pathway. c-Jun N-terminal kinase, which is associated with apoptosis and cellular stress, was not activated by NCAM. Inhibition of the MAPK pathway in rat cerebellar neuron cultures selectively reduced NCAM-stimulated neurite outgrowth. These results define an NCAM signal transduction mechanism with the potential for modulating the expression of genes needed for axonal growth, survival, and synaptic plasticity.

We conclude that chondrons in 3-D culture respond to ATP by increasing [Ca(2+)]ic via P2Y2 receptor activation. Thus, ATP can pass through the agarose gel and the pericellular matrix, bind purinoceptors and increase intracellular Ca(2+) in a signaling response.

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Objective. To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures.Methods. Chondron pellets were formed from 1.6 ؋ 10 6 cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of ␥ 32 P-ATP to resting cultures. Results. Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures.Conclusion. The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.

Tissue transglutaminase (tTG) catalyzes a Ca' ' -dependent transglutaminase (TGase) activity which cross-links proteins and stabilizes many tissues [C.S. Greenberg et al. FASEB J. 5 (1991)