Involvement of protein kinase Czeta in interleukin-1beta induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-kappaB signaling in primary human osteoarthritic chondrocytes.
OBJECTIVE
Protein kinase Czeta (PKCzeta), 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 PKCzeta in interleukin-1beta (IL-1beta)-induced NF-kappaB 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 PKCzeta. Western blot analysis was used to evaluate phosphorylation of PKCzeta and NF-kappaB. 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 PKCzeta and NF-kappaB was induced by IL-1beta treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKCzeta suppressed IL-1beta-induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1beta-induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKCzeta and NO production. Furthermore, small interfering RNA- or short hairpin RNA-mediated knockdown of PKCzeta mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression.


CONCLUSIONS
Our results show that PKCzeta is involved in the regulation of IL-1beta-induced NF-kappaB signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKCzeta could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA.
Chockalingam
Priya S
PS
Department of Women's Health and Musculoskeletal Biology, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. pchockalinga@wyeth.com
Varadarajan
Usha
U
Sheldon
Richard
R
Fortier
Eric
E
LaVallie
Edward R
ER
Morris
Elisabeth A
EA
Yaworsky
Paul J
PJ
Majumdar
Manas K
MK
D051722:ADAM Proteins / Q000378:metabolism*; D000071121:ADAMTS4 Protein; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000473:pathology; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D011348:Procollagen N-Endopeptidase / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D000099221:Protein Kinase C zeta
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D010450:Endopeptidases; D053583:Interleukin-1beta; D016328:NF-kappa B; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D011348:Procollagen N-Endopeptidase; D011493:Protein Kinase C; D012333:RNA, Messenger; D000099221:Protein Kinase C zeta; C000603918:ADAMTS4 protein, human; C083641:aggrecanase