Dataset Information

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells.

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% 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 ?-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 ?-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 ?-catenin gene and protein compared with untreated control cells.

Conclusions

These 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.

SUBMITTER: Hiyama A

PROVIDER: S-EPMC3446482 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells.

Hiyama Akihiko A Arai Fumiyuki F Sakai Daisuke D Yokoyama Katsuya K Mochida Joji J

Arthritis research & therapy 20120502 3

<h4>Introduction</h4>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.<h4>Methods</h4>Rat nucleus pulposus cells were cultured under normoxic (21% O2) or ...[more]

PMID: 22551380

Similar Datasets

Project description:INTRODUCTION: Increased expression of the proinflammatory cytokine TNF-? in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-?-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-? and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-? in nucleus pulposus cells. METHODS: Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-? were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-? expression in nucleus pulposus cells. RESULTS: TNF-? stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3'-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-? expression and promoter activity. Conversely, the suppression of TNF-? promoter activity using a ?-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-? activation; these effects were not observed for DKK-1 or DKK-2. CONCLUSIONS: Here, we have demonstrated that Wnt signaling regulates TNF-? and that Wnt signaling and TNF-? form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-? expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.

Project description:BackgroundThe aims of this study were to investigate the gene expression of CCN family members in rat intervertebral disc (IVD) cells and to examine whether Wnt-β-catenin signaling regulates the expression of CCN family 2 (CCN2)/connective tissue growth factor (CTGF) in rat nucleus pulposus (NP) cells.MethodsThe gene expression of CCN family members were assessed in rat IVD cells using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression pattern of CCN2 was also assessed in rat IVD cells using western blot and immunohistochemical analyses. Gain-of-function and loss-of-function experiments were performed to identify the mechanisms by which Wnt-β-catenin signaling influences the activity of the CCN2 promoter. To further determine if the mitogen-activated protein kinase (MAPK) pathway is required for the Wnt-β-catenin signaling-induced regulation of CCN2 expression in the NP cells, CCN2 expression was analyzed by reporter assay, RT-PCR and western blot analysis.ResultsCCN2 messenger RNA (mRNA) and protein were expressed in rat IVDs. Expression of CCN2 was significantly higher than for mRNA of other CCN family members in both rat NP and annulus fibrosus (AF) cells. The relative activity of the CCN2 promoter decreased 24 h after treatment with 6-bromoindirubin-3'-oxime (1.0 μM) (0.773 (95% 0.735, 0.812) P = 0.0077) in NP cells. In addition, treatment with the WT-β-catenin vector (500 ng) significantly decreased CCN2 promoter activity (0.688 (95% 0.535, 0.842) P = 0.0063), whereas β-catenin small interfering RNA (500 ng) significantly increased CCN2 promoter activity (1.775 (95% 1.435, 2.115) P < 0.001). Activation of Wnt-β-catenin signaling decreased the expression of CCN2 mRNA and protein by NP cells. Regulation of CCN2 by Wnt-β-catenin signaling involved the MAPK pathway in rat NP cells.ConclusionsThis study shows that Wnt-β-catenin signaling regulates the expression of CCN2 through the MAPK pathway in NP cells. Understanding the balance between Wnt-β-catenin signaling and CCN2 is necessary for developing therapeutic alternatives for the treatment of IVD degeneration.

Project description:Low back pain is a common ailment that affects millions of individuals each year and is linked to degeneration of the intervertebral discs in the spine. Intervertebral disc degeneration is known to result from an imbalance in anabolic and catabolic activity by disc cells. Due to the avascular nature of the intervertebral disc, oxygen deficiency may occur in the central nucleus pulposus (NP). The resulting hypoxia affects matrix regulation and energy metabolism of disc cells, although the mechanisms are not fully understood. This study investigates in vitro glucose consumption and gene expression by NP cells over time under varying oxygen tensions. Notochordal porcine NP cells were cultured in agarose discs at 21, 5, or 1% oxygen tension for 1, 5, or 10 days. The expression of 10 key matrix genes, as well as Brachyury (T), by NP cells was analyzed using RT-PCR. Glucose consumption was measured using a two-point method. Results show that culture time and oxygen tension significantly affect glucose consumption rates by porcine NP cells. There were also significant changes in T expression based on oxygen level and culture time. The 1% oxygen tension had a significantly higher T expression on day 10 than the other two groups, which may indicate a better maintenance of the notochordal phenotype. MMP 1 and 13 expression increased over time for all groups, while only the 5% group showed an increase over time for MMP 3. TIMP expression followed the direction of MMPs but to a lesser magnitude. Five percent and twenty-one percent oxygen tensions led to decreases in anabolic gene expression while 1% led to increases. Oxygen concentration and culture time significantly impacted glucose consumption rate and the gene expression of matrix regulatory genes with hypoxic conditions most accurately maintaining the proper NP phenotype. This information is valuable not only for understanding disc pathophysiology, but also for harnessing the potential of notochordal NP cells in therapeutic applications.

Project description:Background: Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro, human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD.

Dataset Information

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells.

Introduction

Methods

Results

Conclusions

Publications

The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets