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
| Autor: | Anna L. Stevens, Cameron A. Wheeler, Steven R. Tannenbaum, Alan J. Grodzinsky |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
Cartilage
Articular Electrophoresis Time Factors Transcription Genetic Blotting Western Interleukin-1beta Biomedical Engineering In Vitro Techniques Interleukin 1 Nitric Oxide Article Nitric oxide Glycosaminoglycan 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Rheumatology Endopeptidases Animals Orthopedics and Sports Medicine Aggrecans Collagenases Enzyme Inhibitors Aggrecan Nitrites 030304 developmental biology Aggrecanase Glycosaminoglycans 030203 arthritis & rheumatology 0303 health sciences Nitrates biology Tumor necrosis factor alpha Reverse Transcriptase Polymerase Chain Reaction Tumor Necrosis Factor-alpha ADAMTS Molecular biology Bovine Cartilage Extracellular Matrix Nitric oxide synthase ADAMTS4 chemistry biology.protein Cattle Matrix degradation Nitric Oxide Synthase |
| Popis: | Summary Objective The objective of this study was to determine the role of nitric oxide (NO) in tumor necrosis factor alpha (TNF-α)-induced matrix damage, compared to interleukin 1 beta (IL-1β), in bovine cartilage explant cultures. Methods Cartilage explants were subjected to treatment with TNF-α (100ng/ml), IL-1β (10ng/ml) and to the nitric oxide synthase inhibitor, N -methyl-arginine (l-NMA; 1.25mM) for 26, 50 or 120h (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 50h treated explants for real time quantitative PCR analyses. Results TNF-α and IL-1β 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-α 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-1β. TNF-α and IL-1β 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-α. TNF-α and IL-1β 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-1β only), and an increase in MMP-3 and MMP-9 secretion. l-NMA had no effect on gene transcription or MMP secretion. Conclusion NO regulates aggrecanase activity at a post-transcriptional level in response to TNF-α treatment while having no effect on IL-1β treated cartilage explants. |
| Databáze: | OpenAIRE |
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