p38 Kinase-dependent and -independent Inhibition of Protein Kinase C ζ and -α Regulates Nitric Oxide-induced Apoptosis and Dedifferentiation of Articular Chondrocytes

Abstract

In articular chondrocytes, nitric oxide (NO) production triggers dedifferentiation and apoptotic cell death that is regulated by the converse functions of two mitogen-activated protein kinase subtypes, extracellular signal-regulated kinase (ERK) and p38 kinase. Since protein kinase C (PKC) transduces signals that influence differentiation, survival, and apoptosis of various cell types, we investigated the roles and underlying molecular mechanisms of action of PKC isoforms in NO-induced dedifferentiation and apoptosis of articular chondrocytes. We report here that among the expressed isoforms, activities of PKC␣ and -were reduced during NO-induced dedifferentiation and apoptosis. Inhibition of PKC␣ activity was independent of NO-induced activation of ERK or p38 kinase and occurred due to blockage of expression. On the other hand, PKC activity was inhibited as a result of NO-induced p38 kinase activation and was observed prior to proteolytic cleavage by a caspase-mediated process to generate enzymatically inactive fragments. Inhibition of PKC␣ or -activities potentiated NO-induced apoptosis, whereas ectopic expression of these isoforms significantly reduced the number of apoptotic cells and blocked dedifferentiation. Ectopic expression of PKC␣ or -did not affect p38 kinase or ERK but inhibited the p53 accumulation and caspase-3 activation that are required for NO-induced apoptosis of chondrocytes. Therefore, our results collectively indicate that p38 kinase-independent and -dependent inhibition of PKC␣ and -, respectively, regulates NO-induced apoptosis and dedifferentiation of articular chondrocytes.

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

“…Consistent with previous observations, 15 we found addition of the NO donor, SNP, to primary monolayer cultures of rabbit articular chondrocytes transiently activated p38 kinase (Figure 1a,upper panel). NO production also caused a decrease in protein level and activity of PKCz.…”

“…This is consistent with our previous demonstration that NO inhibits PKCz activity and stimulates subsequent proteolysis by caspase-3. 15 Inhibition of NO-induced p38 kinase activation by the specific inhibitor SB203580 20 . Levels of p38 kinase and PKCz proteins were determined by Western blot analysis, and the activities were determined by immune complex kinase assays using ATF-2 and MBP as p38 kinase and PKCz substrates, respectively.…”

“…The data in (a-c) represent mean and standard deviation, and in (d) and (e) represent a typical result from at least four independent experiments p38 kinase regulation of PKCf during apoptosis J-S Kim et al with NO-induced apoptosis requiring downregulation of PKCz. [15][16][17][18][19] The chondrocyte pro-and antiapoptotic functions of p38 kinase and PKCz, respectively, are also observed in other cell types. [21][22][23][24] Evidence that PKCz provides survival signals in chondrocytes is supported by the observations that ectopic expression of wild-type or constitutively active PKCz inhibited NO-induced apoptosis of articular chondrocytes, whereas inhibition of PKCz activity by PS-PKCz potentiated NO-induced apoptosis.…”

“…[21][22][23][24] Evidence that PKCz provides survival signals in chondrocytes is supported by the observations that ectopic expression of wild-type or constitutively active PKCz inhibited NO-induced apoptosis of articular chondrocytes, whereas inhibition of PKCz activity by PS-PKCz potentiated NO-induced apoptosis. 15 Currently, underlying molecular mechanism leading to the inhibition of p38 kinase-induced apoptosis of chondrocyte by PKCz is not known. Our previous study indicated that p38 kinase causes chondrocyte apoptosis by accumulating p53 protein in two different ways, transcriptional activation by NF-kB and post-translational stabilization.…”

“…13,14 PKCz functions as a survival signal during NO-induced apoptosis of articular chondrocytes since NO-induced chondrocyte apoptosis requires inhibition of PKCz. [15][16][17][18][19] The PKCz inhibition occurs as a result of NOinduced p38 kinase activation and the inhibition of PKCz activity is followed by proteolytic cleavage via the caspase-3-dependent pathway. 15 Our previous observation 15 that inhibition of caspase-3 did not rescue the inhibition of PKCz activity although cleavage of PKCz was significantly abrogated clearly indicated that inhibition of PKCz activity precedes its cleavage by caspase-3.…”