Protein kinase C zeta as a mediator of NF-kappa B signal transduction in articular chondrocytes and its potential relationship to osteoarthritis

Protein kinase C zeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in a subset of cell types. A study of gene expression in articular chondrocytes from human osteoarthritis (OA) patients revealed that PKCzeta· is transcriptionally upregulated in human OA articular cartilage samples compared to normal cartilage. IL-1 treatment of normal chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. To evaluate whether these degradative signaling pathways in chondrocytes could be modulated by manipulation of PKCzeta activity, chondrocyte cell-based in vitro assays were developed and refined to allow testing of these catabolic pathways with pharmacological agents and with viral vectors. One such assay was a novel pellet culture assay in which primary chondrocytes were cultured under conditions that were optimized to retain the cell's chondrocytic phenotype for several weeks, allowing the synthesis and accumulation of associated cartilage extracellular matrix proteins that could serve as both an indicator of the chondrocyte anabolic state and also as a substrate for matrix degradative factors following treatment of the cells to various anabolic and catabolic agents. Chondrocyte pellet cultures treated with TNF or IL-1 showed significantly increased NF-kappaB transcriptional activity resulting in induction of ADAMTS-4 (Aggrecanase-1) and ADAMTS-5 (Aggrecanase-2) expression. Coincident with this increased aggrecanase expression was a considerable increase in proteoglycan degradation from the extracellular matrix. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Go 6976, and was unaffected by the PKCzeta-sparing inhibitors calphostin C and chelerythrine chloride. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNF- and IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures resulting in preservation of the extracellular matrix. In addition, adenovirus-mediated overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that can cause catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.