Chondrocyte cell death mediated by reactive oxygen species-dependent activation of PKC-betaI.

Signals generated by the extracellular matrix (ECM) promote cell survival. We have shown that chondrocytes detached from their native ECM and plated without serum at low density on poly-l-lysine undergo significant cell death that is associated with the production of reactive oxygen species (ROS). No cell death or ROS production was observed when cells were plated on fibronectin under the same conditions. Cell death on poly-l-lysine could be completely inhibited with the addition of either antioxidants or inhibitors of specific protein kinase C (PKC) isoforms including PKC-betaI. PKC-betaI was noted to translocate from the cytosol to the particulate membrane after plating on poly-l-lysine, and this translocation was inhibited by the addition of an antioxidant. Time-course analyses implicated endogenous ROS production as a secondary messenger leading to PKC-betaI activation and subsequent chondrocyte cell death. Cell survival on poly-l-lysine was significantly improved in the presence of oligomycin or DIDS, suggesting that ROS production occurred via complex V of the electron transport chain of the mitochondria and that ROS were released to the cytosol via voltage-dependent anion channels. Together, these results represent a novel mechanism by which ROS can initiate cell death through the activation of PKC-betaI.

IGF-I stimulation of proteoglycan synthesis by chondrocytes requires activation of the PI 3-kinase pathway but not ERK MAPK.

The IGF-I (insulin-like growth factor-I) signalling pathway responsible for regulation of proteoglycan synthesis in chondrocytes has not been defined and is the focus of the present study. Chondrocytes isolated from normal human articular cartilage were stimulated with IGF-I in monolayer culture or in suspension in alginate. IGF-I activated members of both the PI3K (phosphoinositide 3-kinase) pathway and the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway. The PI3K inhibitors LY294002 and wortmannin blocked IGF-I-stimulated Akt phosphorylation without blocking ERK phosphorylation and this was associated with complete inhibition of proteoglycan synthesis. A decrease in IGF-I-stimulated proteoglycan synthesis was also observed upon inhibition of mTOR (mammalian target of rapamycin) and p70S6 kinase, both of which are downstream of Akt. The MEK (MAPK/ERK kinase) inhibitors PD98059 and U0126 blocked IGF-I-stimulated ERK phosphorylation but did not block the phosphorylation of Akt and did not decrease proteoglycan synthesis. Instead, in alginate- cultured chondrocytes, the MEK inhibitors increased IGF-I-stimulated proteoglycan synthesis when compared with cells treated with IGF-I alone. This is the first study to demonstrate that IGF-I stimulation of the PI3K signalling pathway is responsible for the ability of IGF-I to increase proteoglycan synthesis. Although IGF-I also activates the ERK/MAPK pathway, ERK activity is not required for proteoglycan synthesis and may serve as a negative regulator.