Involvement of protein kinase Cζ in interleukin-1β induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-κB signaling in primary human osteoarthritic chondrocytes

Objective. Protein kinase C zeta (PKC zeta), an atypical PKC, has been found to be transcriptionally upregulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC zeta in interleukin-1 beta (IL-1 beta)-induced NF-kappa B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods. Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC zeta. Western blot analysis was used to evaluate phosphorylation of PKC zeta and NF-kappa B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results. Phosphorylation of PKC zeta and NF-kappa B was induced by IL-1 beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCC suppressed IL-1 beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1 beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC zeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKC zeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion. Our results show that PKC zeta is involved in the regulation of IL-1 beta-induced NF-kappa B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKC zeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.