the effects of oxygen tension and antiaging factor klotho on wnt signaling in nucleus pulposus cells氧张力的影响和抗衰老的因素克罗索wnt信号在髓核细胞.pdf

Hiyama et al. Arthritis Research Therapy 2012, 14:R105 /content/14/3/R105 RESEARCH ARTICLE Open Access The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells Akihiko Hiyama1,2*, Fumiyuki Arai1,2, Daisuke Sakai1,2, Katsuya Yokoyama1,2 and Joji Mochida1,2 Abstract Introduction: The goals of this study were to examine the oxemic regulation of Wnt signaling to explore whether Wnt signaling accelerates the age-related degeneration of nucleus pulposus cells, and if so, to define the mechanism underlying this effect. We investigated the expression of Klotho, a newly identified antiaging gene, and whether its regulation is attributable to the suppression of Wnt signaling. Methods: Rat nucleus pulposus cells were cultured under normoxic (21% O ) or hypoxic (2% O ) conditions, and 2 2 the expression and promoter activity of Wnt signaling and Klotho were evaluated. The effect of Klotho protein was examined with transfection experiments, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, senescence-associated b-galactosidase staining, and cell-cycle analysis. To determine the methylation status of the Klotho promoter region, bisulfite genomic sequencing analysis was performed. Its relation with the activation of Wnt signaling was assessed. We also examined whether the expression of Klotho could block the effects of pathological Wnt expression in nucleus pulposus cells. Results: Nucleus pulposus cells exhibited increased b-catenin mRNA and protein under the hypoxic condition. Klotho protein was expressed in vivo, and protein and messenger RNA expression decreased under the hypoxic condition. Klotho treatment decreased cell proliferation and induced th