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

Abstract

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.