In this study, we focused on Wnt signaling to clarify the mechanism (upstream or downstream) underlying intervertebral disc degeneration. First, we examined the relation between Wnt signaling and the oxemic status of nucleus pulposus cells. Nucleus pulposus is an avascular tissue, and in some species, pO2
levels in large discs may be low. Oxygen tension is a powerful stimulus of nucleus pulposus cells, yet the net result of hypoxia--anabolic or catabolic--in nucleus pulposus cells is not known. In addition, the mechanism by which the nucleus pulposus cells survive in the hypoxic environment of the intervertebral disc remains largely unknown, and no evidence suggests how oxygen levels change with disc degeneration in vivo
. However, in vitro
studies demonstrate stimulatory effects of hypoxia on nucleus pulposus proliferation and matrix synthesis [35
]. It is very difficult to conclude that low oxygen would be bad for discs from all of these study data, because the existence of complex signaling networks is found in disc degeneration. However, in this study, we found a hypoxia-dependent increase in the expression of β-catenin and a concomitant increase in Wnt signaling activity. We demonstrated previously that activation of Wnt signaling elevates the gene expression and activity of aggrecan by the balance of catabolic and anabolic factors in nucleus pulposus cells. This is the key to understanding the biology of intervertebral disc maintenance and degeneration [26
]. Mwale et al.
] reported that low oxygen levels increase aggrecan mRNA expression in nucleus pulposus cells and that disc cell metabolism is not impaired at low oxygen concentrations. Our results indicate that effect of hypoxia on matrix synthesis of the nucleus pulposus cells might be directly mediated by Wnt signaling or indirectly by Wnt stimulation of other pathways, such as the hypoxia-inducible factor (HIF) pathway. However, this study was unable to distinguish whether this is a direct or an indirect effect. Kaidi et al.
] reported that Wnt signaling is mediated by the physical interaction between HIF-1α and β-catenin. Intriguingly, the β-catenin/HIF-1α interaction increases HIF transcriptional activity, which might help cells adapt to severe hypoxia. Risbud et al.
] reported that nucleus pulposus cells display robust and constitutive expression of both HIF-1α and HIF-2α, suggesting that these cells reside in a hypoxic environment. Future studies will address the role of the β-catenin/HIF pathways to determine whether regulation of Wnt signaling by hypoxic conditions is specific to nucleus pulposus cells.
Second, we investigated the expression of Klotho, a newly identified antiaging gene, and examined the relation in nucleus pulposus cells of Wnt signaling and Klotho.
Finally, we investigated the role of Klotho on aggrecan or collagen type-II expression and the proliferation of nucleus pulposus cells.
It is clear that the incidence of disc degeneration increases markedly with age. Several studies have focused on Klotho and its role in the aging phenotype. The Klotho protein is composed of a large extracellular domain (130 kDa), a transmembrane domain, and a very short intracellular domain (10 amino acids). The entire extracellular domain is released into the extracellular space and is detectable in blood, urine, and cerebrospinal fluid [40
]. Membrane Klotho functions as a receptor for a hormone that regulates excretion of phosphate and synthesis of active vitamin D in the kidney [41
]. Secreted Klotho regulates the activity of multiple growth factors, including insulin/insulin-like growth factor-1 (IGF-1) [11
], Wnt [28
], and transforming growth factor (TGF)-β1 [43
]. Liu et al.
] demonstrated that Klotho acts as a Wnt antagonist and suggested that prolonged Wnt stimulation may contribute to stem cell depletion and aging. They reported that secreted Klotho inhibits Wnt signaling activity by binding directly to Wnt ligands and preventing them from binding to their receptors. In this study, we performed molecular functional analysis to study the expression of Klotho in the intervertebral disc and to elucidate the signal crosstalk between Klotho and Wnt signaling; this crosstalk is attracting attention as a potential trigger for degeneration in the intervertebral disc. Interestingly, we demonstrated for the first time that the transcriptional activation of Klotho is associated with promoter demethylation by Wnt signaling. Accordingly, these experiments strongly suggested that in nucleus pulposus cells, activation of Wnt signaling induces an increase in Klotho promoter activity and gene and protein expression by demethylation. By contrast, activation of nucleus pulposus cells by secreted Klotho seems to suppress Wnt signaling. These results might support that Klotho is not simply an antagonist of Wnt signaling but that Wnt signaling and Klotho form a negative-feedback loop in nucleus pulposus cells.
We hypothesized that Wnt signaling might control nucleus pulposus homeostasis in normal intervertebral discs. However, although we know that, in general, Klotho is an antiaging gene, activation of Wnt signaling nevertheless resulted in induction of Klotho in nucleus pulposus cells. Consequently, Klotho inhibited aggrecan synthesis and proliferation of nucleus pulposus cells. This is unexpected, because suppression of aggrecan synthesis and proliferation of nucleus pulposus cells would appear to be potentially damaging to the intervertebral disc. The reason for these discrepant effects is not clear, but several possible explanations exist.
First, it is possible that Klotho interacts with signal pathways other than Wnt signaling. Klotho can affect the function of Wnt, TGF-β1, and IGF, which can affect the expression of aggrecan and collagen type II in different ways. Doi et al.
] reported that secreted Klotho suppresses TGF-β1 signaling by directly binding to type-II TGF-β receptor (TGFβR2) on the cell surface and preventing TGF-β1 binding to that receptor. TGF-β1 is the most potent inducer of matrix synthesis in nucleus pulposus cells. In addition, fibroblast growth factor (FGF) may act as a catabolic and antianabolic mediator in nucleus pulposus cells, stimulating matrix metalloproteinase (MMP)-13 expression and suppressing proteoglycan synthesis. Recently, Kuro-o [44
] reported that the most important function of the Klotho family is to regulate the function of the endocrine FGF family. Therefore, the final effect of Klotho on the aggrecan and collagen type II gene expression may be the result of the different effects.
Second, these mechanisms may be specific to the nucleus pulposus cells, because this cell is unique both embryologically and functionally. Moreover, although regulation of Wnt signaling may be tissue specific, the signaling pathway has been shown to modulate cellular proliferation or differentiation.
Third, these differences may relate to the age and species of the animal from which the cells are isolated and the environment in which the cell metabolism was studied. Other groups have shown that glycosaminoglycan production by nucleus pulposus and annulus fibrosus cells can vary, depending on the source of disc cells [45
Fourth, the Klotho gene encodes several isoforms in mammals, which include a membrane-bound and two secreted forms. Therefore, the results for this study might be from the different isoforms through different signaling pathways.
Future studies will address the role of Wnt signaling by using human samples to determine whether the regulation of cell growth and matrix synthesis by Klotho is specific to nucleus pulposus cells. Moreover, it is necessary to examine whether, in nucleus pulposus cells, Klotho affects the activity of several signaling pathways, including those of TGF, FGF, or the MMP family, which may participate in intervertebral disc degeneration. Wnt signals are regulated in various ways in response to various extracellular and intracellular signaling. However, DKK, which regulates extracellular Wnt signaling, did not inhibit the reporter activation of Klotho in this study. The DKK family comprises four subtypes, which affect Wnt signaling by combining with the collaboration receptor LRP5/6 of each Wnt-Fzd [46
]. The subtypes seem to influence the functional differences and types of cells that develop, although it is generally believed that only DKK1 and DKK4 inhibit Wnt signals. Our present results suggest that the action of the regulatory factor DKK outside of cells is not dependent on the expression of Klotho in nucleus pulposus cells.