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The klotho gene was identified as an "aging-suppressor" gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in klotho mutant mice.Four-week-old klotho mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, klotho and WT: n = 12, each) or not containing EPA (control food, klotho and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in klotho mice after 4 weeks of control food, but they were not elevated in klotho mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (NOX4), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of klotho mice. Activity of NOX was also significantly higher in SMCs of klotho mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of klotho mice.EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in klotho mutant mice.

To compare induction of the aggrecanases (ADAMTS‐1, ADAMTS‐4, ADAMTS‐5, ADAMTS‐8, ADAMTS‐9, and ADAMTS‐15) by interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) in chondrocyte‐like OUMS‐27 cells and human chondrocytes, and to determine the mechanism of induction of the most responsive aggrecanase gene.OUMS‐27 cells were stimulated for different periods of time and with various concentrations of IL‐1β and/or TNFα. Human chondrocytes obtained from osteoarthritic joints and human skin fibroblasts were also stimulated with IL‐1β and/or TNFα. Total RNA was extracted, reverse transcribed, and analyzed by quantitative real‐time polymerase chain reaction and Northern blotting. ADAMTS‐9 protein was examined by Western blotting, and the role of the MAPK signaling pathway for ADAMTS9 induction in IL‐1β–stimulated OUMS‐27 cells was investigated.IL‐1β increased messenger RNA (mRNA) levels of ADAMTS4, ADAMTS5, and ADAMTS9 but not ADAMTS1 and ADAMTS8. The fold increase for ADAMTS9 mRNA was greater than that for mRNA of the other aggrecanase genes. The increase of ADAMTS9 mRNA by IL‐1β stimulation was greater in chondrocytes than in fibroblasts. The combination of IL‐1β and TNFα had a synergistic effect, resulting in a considerable elevation in the level of ADAMTS9 mRNA. ADAMTS‐9 protein was also induced in IL‐1β–stimulated OUMS‐27 cells. The MAPK inhibitors SB203580 and PD98059 decreased ADAMTS9 up‐regulation in OUMS‐27 cells.ADAMTS9 is an IL‐1β– and TNFα‐inducible gene that appears to be more responsive to these proinflammatory cytokines than are other aggrecanase genes. Furthermore, these cytokines had a synergistic effect on ADAMTS9. Together with the known ability of ADAMTS‐9 to proteolytically degrade aggrecan and its potential to cleave other cartilage molecules, the data suggest that ADAMTS‐9 may have a pathologic role in arthritis. [ABSTRACT FROM AUTHOR]