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 C (PKC ), 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 in interleukin-1␤ (IL-1␤)-induced NF-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 . Western blot analysis was used to evaluate phosphorylation of PKC and NF-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 and NF-B was induced by IL-1␤ treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC suppressed IL-1␤-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1␤-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC and NO production. Furthermore, small interfering RNA-or short hairpin RNA-mediated knockdown of PKC mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion. Our results show that PKC is involved in the regulation of IL-1␤-induced NF-B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2.Therefore, inhibition of PKC could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.Osteoarthritis (OA) is a degenerative joint disorder characterized by destruction and loss of articular cartilage as a result of an imbalance between catabolic and anabolic cartilage matrix metabolism. It is the most common form of arthritis. Chondrocytes are the single cell type present in cartilage, and they play an important role in the pathogenesis of OA. They are the primary source of type II collagen and aggrecan, the most important components of the cartilage matrix. Cartilage loss in OA is characterized by matrix degradation and chondrocyte death.In OA, catabolic factors, including interleukin-1 (IL-1) and tumor necrosis factor (TNF), have been implicated in stimulating chondrocytes to produce matrix-degrading enzymes including aggrecanases (ADAMTS-4 and ADAMTS-5) and other metalloproteinases, resulting in degradation of aggrecan and collagen, respectively (1,2). Catabolic cytokines have also been demonstrated to induce synthesis of type 2 nitric oxide synthase (NOS2), resulting in the formation of nitric oxide (NO), an inducer of chondrocyte apoptosis (3). Studies of inducible NOS-null mice and investiga-

An Atypical Protein Kinase C (PKCζ) Plays a Critical Role in Lipopolysaccharide-Activated NF-κB in Human Peripheral Blood Monocytes and Macrophages

“…Recently, several studies have indicated the role of PKC in NF-B activation (32,33). Our data suggest that in human peripheral blood monocyte LPS activates PKC, which provides signal acting downstream of GTPase RhoA and which mediates LPS-induced NF-B activation.…”

Protein Kinase C Isoforms ζ and ι Mediate Collagenase Expression and Cartilage Destruction via STAT3- and ERK-dependent c-fos Induction

“…nPKC␦ and aPKC isoforms were analyzed because these have previously been investigated in relation to MMP expression (35)(36)(37). Fig.…”

“…aPKC appears to regulate both NFB and Janus kinase (JAK)/STAT pathways in inflammation (34). aPKC is reported to be required for NFB activation in chondrocytes, is up-regulated in OA (35), and its inhibition reduces aggrecanase-1 expression (36). nPKC␦ has been implicated in cross-talk with mitogen-activated protein kinases (MAPKs) for MMP13 expression (37).…”

Adaptor Proteins and Ras Synergistically Regulate IL-1-Induced ADAMTS-4 Expression in Human Chondrocytes

“…The potent inhibition of ADAMTS-4 mRNA expression and activity by the antioxidants Trolox and NDGA supports the involvement of ROS production by 5-lipoxygenase or NADPH oxidase during IL-1 induction of ADAMTS-4 (31,58). ADAMTS-4 is partly regulated by NF-B transcription factor as its induction in OA synovial fibroblasts is inhibited by the overexpression of the endogenous NF-B inhibitor, IB␣ (18,19,60). IL-1-stimulated NF-B is known to be more active in the superficial layer of OA cartilage (9).…”

“…Blocking aggrecanolysis in the interglobular domain by genetic means resulted in abrogation of cartilage erosion (17). Although implication of protein kinase C in aggrecanase has been demonstrated (18,19), the detailed mechanism of IL-1 signal transduction leading to ADAMTS-4 gene induction and the role of adaptor proteins in particular is not known. By RNA interference-mediated genetic knockdown, we demonstrate that MyD88, IRAK-1, and TRAF6, as well as Ras-driven reactive oxygen species (ROS) culminating on NF-B transcription factor synergistically regulate IL-1-induced ADAMTS-4 gene expression.…”