Prolyl-4-hydroxylase Domain Protein 2 Controls NF-{kappa}B/p65 Transactivation and Enhances the Catabolic Effects of Inflammatory Cytokines on cells of the Nucleus Pulposus [Glycobiology and Extracellular Matrices]

January 29th, 2015 by Li, J., Yuan, W., Jiang, S., Ye, W., Yang, H., Shapiro, I. M., Risbud, M. V.

Prolyl-4-hydroxylase (PHD) proteins are key in sensing tissue hypoxia. In nucleus pulposus (NP) cells, our previous work has demonstrated that PHD isoforms have a differential contribution in controlling HIF-α degradation and activity. Recently we have shown that a regulatory relationship exists between PHD3 and inflammatory cytokines in NP cells. With respect to PHD2, the most abundant PHD isoform in NP cells, very little is known concerning its function and regulation under inflammatory conditions that characterize intervertebral disc degeneration. Here, we show that PHD2 is a potent regulator of the catabolic activities of TNF-α; silencing of PHD2 significantly decreased TNF-α-induced expression of catabolic markers including SDC4, MMP-3 and -13 and ADAMTS5 as well as several inflammatory cytokines and chemokines, while partially restoring aggrecan and collagen II expression. Use of NF-κB reporters with ShPHD2, SiHIF-1α, as well as p65-/-, PHD2-/- and PHD3-/- cells show that PHD2 serves as a co-activator of NF-κB/p65 signaling in HIF-1 independent fashion. Immunoprecipitation of endogenous and exogenously expressed tagged proteins as well as fluorescence microscopy indicates that following TNF-α treatment, PHD2 interacts with, and co-localizes with, p65. Conversely, loss-of-function experiments using lentivirally-delivered Sh-p65, Sh-IKKβ and NF-κB inhibitor confirmed that cytokine-dependent PHD2 expression in NP cells requires NF-κB signaling. These findings clearly demonstrate that PHD2 forms a regulatory circuit with TNF-α via NF-κB and thereby plays an important role in enhancing activity of this cytokine. We propose that during disc degeneration PHD2 may offer a therapeutic target to mitigate the deleterious actions of TNF-α, a key pro-inflammatory cytokine.
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