Nitric oxide enhances aggrecan degradation by aggrecanase in response to TNF-α but not IL-1β treatment at a post-transcriptional level in bovine cartilage explants

Abstract

The objective of this study was to determine the role of nitric oxide (NO) in tumor necrosis factor alpha (TNF-alpha)-induced matrix damage, compared to interleukin 1 beta (IL-1beta), in bovine cartilage explant cultures. Cartilage explants were subjected to treatment with TNF-alpha (100ng/ml), IL-1beta (10 ng/ml) and to the nitric oxide synthase inhibitor, N-methyl-arginine (L-NMA; 1.25 mM) for 26, 50 or 120 h (5 days). The collected medium was analyzed for sulfated glycosaminoglycan (sGAG), nitrate and nitrite, matrix metalloproteinase (MMP) activity by zymography, and aggrecan degradation by immunoblotting of aggrecan-G1 and aggrecan-G1-NITEGE fragments. RNA was extracted from the 26 and 50 h treated explants for real time quantitative PCR analyses. TNF-alpha and IL-1beta treatment caused a 3-5 fold increase in sGAG release with an increase in aggrecanase-specific aggrecan breakdown and an increase in nitrate and nitrite production. L-NMA treatment inhibited almost 50% of the sGAG release caused by TNF-alpha treatment, with concomitant decrease in the aggrecanase-specific-NITEGE neo-epitope of aggrecan released into the medium. No L-NMA effect was identified with IL-1beta. TNF-alpha and IL-1beta both increased a disintegrin and matrix metalloproteinase with thrombospondin motif (ADAMTS)4 and ADAMTS5 transcription with no effect by L-NMA, suggesting that NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha. TNF-alpha and IL-1beta both caused an increase in protease transcription (MMP-3, MMP-13, ADAMTS4 and ADAMTS5) and in pro-inflammatory enzymes, inducible nitric oxide synthase and cyclooxygenase (COX)-2, as well as a decrease in matrix protein transcription, including collagen II, aggrecan, fibromodulin and link protein (IL-1beta only), and an increase in MMP-3 and MMP-9 secretion. L-NMA had no effect on gene transcription or MMP secretion. NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-alpha treatment while having no effect on IL-1beta treated cartilage explants.