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Activation of nuclear factor-kappa B by TNF promotes nucleus pulposus mineralization through inhibition of ANKH and ENPP1.

ABSTRACT: Spontaneous mineralization of the nucleus pulposus (NP) has been observed in cases of intervertebral disc degeneration (IDD). Inflammatory cytokines have been implicated in mineralization of multiple tissues through their modulation of expression of factors that enable or inhibit mineralization, including TNAP, ANKH or ENPP1. This study examines the underlying factors leading to NP mineralization, focusing on the contribution of the inflammatory cytokine, TNF, to this pathologic event. We show that human and bovine primary NP cells express high levels of ANKH and ENPP1, and low or undetectable levels of TNAP. Bovine NPs transduced to express TNAP were capable of matrix mineralization, which was further enhanced by ANKH knockdown. TNF treatment or overexpression promoted a greater increase in mineralization of TNAP-expressing cells by downregulating the expression of ANKH and ENPP1 via NF-κB activation. The increased mineralization was accompanied by phenotypic changes that resemble chondrocyte hypertrophy, including increased RUNX2 and COL10A1 mRNA; mirroring the cellular alterations typical of samples from IDD patients. Disc organ explants injected with TNAP/TNF- or TNAP/shANKH-overexpressing cells showed increased mineral content inside the NP. Together, our results confirm interactions between TNF and downstream regulators of matrix mineralization in NP cells, providing evidence to suggest their participation in NP calcification during IDD.

SUBMITTER: Krzyzanowska AK

PROVIDER: S-EPMC8050288 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Activation of nuclear factor-kappa B by TNF promotes nucleus pulposus mineralization through inhibition of ANKH and ENPP1.

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