title: Prolyl-4-hydroxylase domain protein 2 controls NF-κB/p65 transactivation and enhances the catabolic effects of inflammatory cytokines on cells of the nucleus pulposus.
abstract: Prolyl-4-hydroxylase (PHD) proteins are key in sensing tissue hypoxia. In nucleus pulposus (NP) cells, our previous work demonstrated that PHD isoforms have a differential contribution in controlling hypoxia-inducible factor (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, MMP-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, shows 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 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 proinflammatory cytokine.
authors: From the Department of Orthopaedic Surgery and the Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors: Jun
authors: J
authors: Li
authors: the Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors: Wen
authors: W
authors: Yuan
authors: the Department of Orthopaedic Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors: Shuai
authors: S
authors: Jiang
authors: From the Department of Orthopaedic Surgery and the Department of Orthopaedic Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-Sen University, Guangzhou 510120, China, and.
authors: Wei
authors: W
authors: Ye
authors: From the Department of Orthopaedic Surgery and the Department of Orthopaedics, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, China.
authors: Hao
authors: H
authors: Yang
authors: From the Department of Orthopaedic Surgery and the Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107.
authors: Irving M
authors: IM
authors: Shapiro
authors: From the Department of Orthopaedic Surgery and the Graduate Program in Cell and Developmental Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107, makarand.risbud@jefferson.edu.
authors: Makarand V
authors: MV
authors: Risbud
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D053583:Interleukin-1beta / Q000276:immunology*; D007403:Intervertebral Disc / Q000166:cytology*; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000276:immunology; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology*; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000276:immunology*; D017208:Rats, Wistar; D015398:Signal Transduction; D051996:Transcription Factor RelA / Q000235:genetics* / Q000276:immunology; D015533:Transcriptional Activation; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology*
chemical_list: D053583:Interleukin-1beta; D016328:NF-kappa B; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D011393:Procollagen-Proline Dioxygenase; C580731:Egln1 protein, rat; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases
keywords: Cytokine Action; Hypoxia-inducible Factor (HIF); Intervertebral Disc; NF-kappa B (NF-KB); Nucleus Pulposus; Prolyl Hydroxylase; Tumor Necrosis Factor (TNF)
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