Dataset Information

HIF-1? and HIF-2? degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc.

ABSTRACT: Studies of many cell types show that levels of hypoxia inducible factor (HIF)-1? and HIF-2? are primarily controlled by oxygen-dependent proteasomal degradation, catalyzed by HIF prolyl-hydroxylases (PHDs). However, in the hypoxic niche of the intervertebral disc, the mechanism of HIF-? turnover in nucleus pulposus cells is not yet known. We show that in nucleus pulposus cells HIF-1? and HIF-2?, degradation was mediated through 26S proteasome irrespective of oxygen tension. It is noteworthy that HIF-2? degradation through 26S proteasome was more pronounced in hypoxia. Surprisingly, treatment with DMOG, a PHD inhibitor, shows the accumulation of only HIF-1? and induction in activity of its target genes, but not of HIF-2?. Loss and gain of function analyses using lentiviral knockdown of PHDs and overexpression of individual PHDs show that in nucleus pulposus cells only PHD2 played a limited role in HIF-1? degradation; again HIF-2? degradation was unaffected. We also show that the treatment with inhibitors of lysosomal proteolysis results in a strong accumulation of HIF-1? and to a much smaller extent of HIF-2? levels. It is thus evident that in addition to PHD2 catalyzed degradation, the HIF-1? turnover in nucleus pulposus cells is primarily regulated by oxygen-independent pathways. Importantly, our data clearly suggests that proteasomal degradation of HIF-2? is not mediated by a classical oxygen-dependent PHD pathway. These results for the first time provide a rationale for the normoxic stabilization as well as the maintenance of steady-state levels of HIF-1? and HIF-2? in nucleus pulposus cells.

SUBMITTER: Fujita N

PROVIDER: S-EPMC3260409 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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HIF-1α and HIF-2α degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc.

Fujita Nobuyuki N Chiba Kazuhiro K Shapiro Irving M IM Risbud Makarand V MV

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20120201 2

Studies of many cell types show that levels of hypoxia inducible factor (HIF)-1α and HIF-2α are primarily controlled by oxygen-dependent proteasomal degradation, catalyzed by HIF prolyl-hydroxylases (PHDs). However, in the hypoxic niche of the intervertebral disc, the mechanism of HIF-α turnover in nucleus pulposus cells is not yet known. We show that in nucleus pulposus cells HIF-1α and HIF-2α, degradation was mediated through 26S proteasome irrespective of oxygen tension. It is noteworthy that ...[more]

PMID: 21987385

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Project description:The relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration.We cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O2) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS.NCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells.Notochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.

Project description:STUDY DESIGN:Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE:To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE:Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS:IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS:A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS:This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

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Dataset Information

HIF-1? and HIF-2? degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc.

Publications

HIF-1α and HIF-2α degradation is differentially regulated in nucleus pulposus cells of the intervertebral disc.

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