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

Abstract

Objective Protein kinase Cζ (PKCζ), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKCζ in interleukin-1β (IL-1β)–induced NF-κB 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 PKCζ. Western blot analysis was used to evaluate phosphorylation of PKCζ and NF-κB. 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 PKCζ and NF-κB was induced by IL-1β treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCζ suppressed IL-1β–induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1β–induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCζ and NO production. Furthermore, small interfering RNA– or short hairpin RNA–mediated knockdown of PKCζ mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKCζ is involved in the regulation of IL-1β–induced NF-κB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCζ could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.