The effects of oxygen tension and antiaging factor Klotho on the Wnt singaling in nucleus pulposus cells

Akihiko Hiyama,Fumiyuki Arai,Katsuya Yokoyama,Joji Mochida,Daisuke Sakai

doi:10.1186/ar3830

Abstract

IntroductionThe 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.MethodsRat nucleus pulposus cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and 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 β-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.ResultsNucleus pulposus cells exhibited increased β-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 the quiescence of nucleus pulposus cells. In addition, Klotho treatment inhibited expression of β-catenin gene and protein compared with untreated control cells.ConclusionsThese data indicate that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells. These results suggest that the expression of Klotho is regulated by the balance between upregulation and downregulation of Wnt signaling.

Highlights

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
The b-catenin mRNA level was threefold higher in nucleus pulposus cells cultured under the hypoxic (2% O2) compared with the normoxic condition (21% O2) (P < 0.05) (Figure 1C)
To confirm the dependence of b-catenin expression on oxygen tension, as shown in Figure 1D, we showed that total b-catenin level and the nuclear translocation of b-catenin increased to a greater extent in nucleus pulposus cells cultured under the hypoxic condition than in cells cultured under the normoxic condition

Summary

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. To clarify the mechanism underlying low-back pain, the molecular mechanisms involved in intervertebral disc degeneration must be identified. The O2 levels in the nucleus pulposus may be 1% to 5%, and disc-cell metabolism can vary with O2 concentration. The activity of disc cells is very sensitive to changes in extracellular oxygen and pH. A little information is known about the effect of oxygen tension on nucleus pulposus cells [13]. More data are needed to determine whether a low oxygen tension is beneficial or detrimental in the culture of nucleus pulposus cells

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