Involvement of H-Ras and reactive oxygen species in proinflammatory cytokine-induced matrix metalloproteinase-13 expression in human articular chondrocytes

Proinflammatory cytokines such as interleukin-1 beta (11,10) and tumor necrosis factor alpha (TNF-alpha) enhance degradation of cartilage-specific, type II collagen by matrix metalloproteinase-13 (MMP-13). We investigated the previously unknown role of H-Ras and reactive oxygen species (ROS) in the cytokine induction of MMP-13 gene expression in human articular chondrocytes by using pharmacological inhibitors, RNA interference (RNAi) and antioxidants. Manumycin A, an inhibitor of H-Ras farnesylation by famesyltransferase, suppressed IL-1 beta- and TNF-alpha-induced MMP-13 mRNA and protein expression. Small interfering RNA (siRNA)-mediated H-Ras silencing down-regulated MMP-13 mRNA and protein induction by IL-beta and TNF-alpha. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase/NOX) inhibitor, diphenyleneiodonium (DPI) suppressed cytokine-induced MMP-13 expression and superoxide production. Apocynin, another NOX inhibitor, also diminished MMP-13 induction. Deoxyglucose an antimetabolite of glucose metabolism reduced MMP-13 increase. Role of NOX-mediated ROS production was reaffirmed by the observation that the antioxidants, trolox, nordihydroguaiaretic acid (NDGA), quercetin and resveratrol downregulated cytokine-induced MMP-13 mRNA and protein expression. These results provide strong pharmacological and genetic evidence for the implication of H-Ras and NADPH oxidase-generated superoxide production in MMP-beta gene regulation by IL-beta and TNF-alpha. These proteins could be potentially targeted for therapeutic inhibition of MMP-13-driven cartilage erosion by using H-Ras and NOX inhibitors and antioxidants. (C) 2011 Elsevier Inc. All rights reserved.