Dataset Information

Global identification of genes related to nutrient deficiency in intervertebral disc cells in an experimental nutrient deprivation model.

ABSTRACT:

Background

Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an experimental nutrient deprivation model.

Methodology/principal findings

Rat NP cells were subjected to serum starvation. Global gene expression was profiled by microarray analysis. Confirmation of the selected genes was obtained by real-time polymerase chain reaction array analysis. Western blotting was used to confirm the expression of selected genes. Functional interactions between p21(Cip1) and caspase 3 were examined. Finally, flow cytometric analyses of NP cells were performed. Microarray analysis revealed 2922 differentially expressed probe sets with ?1.5-fold changes in expression. Serum starvation of NP cells significantly affected the expression of several genes involved in DNA damage checkpoints of the cell cycle, including Atm, Brca1, Cdc25, Gadd45, Hus1, Ppm1D, Rad 9, Tp53, and Cyclin D1. Both p27(Kip1) and p53 protein expression was upregulated in serum-starved cells. p21(Cip1) expression remained in NP cells transfected with short interfering RNA targeting caspase 3 (caspase 3 siRNA). Both G1 arrest and apoptosis induced by serum starvation were inhibited in cells transfected with caspase 3 siRNA.

Conclusions/significance

Nutrient deprivation in NP cells results in the activation of a signaling response including DNA damage checkpoint genes regulating the cell cycle. These results provide novel possibilities to improve the success of intervertebral disc regenerative techniques.

SUBMITTER: Sudo H

PROVIDER: S-EPMC3592817 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Global identification of genes related to nutrient deficiency in intervertebral disc cells in an experimental nutrient deprivation model.

Sudo Hideki H Yamada Katsuhisa K Iwasaki Koji K Higashi Hideaki H Ito Manabu M Minami Akio A Iwasaki Norimasa N

PloS one 20130308 3

<h4>Background</h4>Intervertebral disc degeneration is a significant cause of degenerative spinal diseases. Nucleus pulposus (NP) cells reportedly fail to survive in large degenerated discs with limited nutrient availability. Therefore, understanding the regulatory mechanism of the molecular response of NP cells to nutrient deprivation may reveal a new strategy to treat disc degeneration. This study aimed to identify genes related to nutrient deprivation in NP cells on a global scale in an exper ...[more]

PMID: 23520533

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Project description:The expression of Interleukin-1ß (IL-1ß) and its antagonist and Interleukin-1 receptor antagonist (IL-1Ra) are correlated with greater human intervertebral disc (IVD) degeneration, suggesting that elevated IL-1β activity promotes disc degeneration. Many in vitro studies support such a mechanistic relationship, whereas few in vivo investigations have been reported. The present study tests the effect of increased IL-1β activity on intervertebral disc in mice with an IL-1Ra gene deletion. IL-1Ra-/- mice and wild-type (WT) C57Bl6J mice were examined at 3 and 12 months of age. Caudal IVD segments were evaluated for disc degeneration by histopathology, functional testing, and inflammatory gene expression relevant to IL-1β pathways. To test differences in injury response, pinprick annular puncture was performed on IL-1Ra-/- and WT mice and evaluated similarly. IL-1Ra-/- IVDs had significantly worse histopathology at 3 months compared to WT controls, but not at 12 months. IL-1Ra-/- IVDs exhibited significantly more viscous mechanical properties than WT IVDs. qPCR revealed downregulation of inflammatory genes at 3 and 12 months in IL-1Ra-/- IVDs, with concomitant downregulation of anabolic and catabolic genes. Annular puncture yielded no appreciable differences between 2-week and 6-week post-injured WT and IL1-Ra-/- IVDs in histopathology or biomechanics, but inflammatory gene expression was sharply downregulated in IL-1Ra-/- mice at 2 weeks, returning by 6 weeks post injury. In the present study, IL-1Ra deletion resulted in increased IVD histopathology, inferior biomechanics, and transiently decreased pro-inflammatory cytokine gene expression. The histopathology of IL-1Ra-/- IVDs on a C57BL/6J background is less severe than a previous report of IL1Ra-/- on a BALB/c background, yet both strains exhibit IVD degeneration, reinforcing a mechanistic role of IL-1β signaling in IVD pathobiology. Despite a pro-inflammatory environment, the annular puncture was no worse in IL-1Ra-/- mice, suggesting that response to injury involves pathways other than inflammation. Overall, this study supports the hypothesis that IL-1β-driven inflammation is important in IVD degeneration.

Project description:Background: Intervertebral disc degeneration impairs the quality of patients lives. Even though there has been development of many therapeutic strategies, most of them remain unsatisfactory due to the limited understanding of the mechanisms that underlie the intervertebral disc degeneration. Questions/purposes: This study is meant to identify the key modules and hub genes related to the annulus fibrosus in intervertebral disc degeneration (IDD) through: (1) constructing a weighted gene co-expression network; (2) identifying key modules and hub genes; (3) verifying the relationships of key modules and hub genes with IDD; and (4) confirming the expression pattern of hub genes in clinical samples. Methods: The Gene Expression Omnibus provided 24 sets of annulus fibrosus microarray data. Differentially expressed genes between the annulus fibrosus of degenerative and non-degenerative intervertebral disc samples have gone through the Gene Ontology (GO) and pathway analysis. The construction of a gene network and classification of genes into different modules were conducted through performing Weighted Gene Co-expression Network Analysis. The identification of modules and hub genes that were most related to intervertebral disc degeneration was proceeded. In order to verify the relationships of the module and hub genes with intervertebral disc degeneration, Ingenuity Pathway Analysis was operated. Clinical samples were adopted to help verify the hub gene expression profile. Results: One thousand one hundred ninety differentially expressed genes were identified. Terms and pathways associated with intervertebral disc degeneration were presented by GO and pathway analysis. The construction of a Weighted Gene Coexpression Network was completed and clustering differentially expressed genes into four modules was also achieved. The module with the lowest P-value and the highest absolute correlation coefficient was selected and its relationship with intervertebral disc degeneration was confirmed by Ingenuity Pathway Analysis. The identification of hub genes and the confirmation of their expression profile were also realized. Conclusions: This study generated a comprehensive overview of the gene networks underlying annulus fibrosus in intervertebral disc degeneration. Clinical Relevance: Modules and hub genes identified in this study are highly associated with intervertebral disc degeneration, and may serve as potential therapeutic targets for intervertebral disc degeneration.

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Project description:Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.Microarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip(R) Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.Microarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed > or =100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.This study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.

Dataset Information

Global identification of genes related to nutrient deficiency in intervertebral disc cells in an experimental nutrient deprivation model.

Background

Methodology/principal findings

Conclusions/significance

Publications

Global identification of genes related to nutrient deficiency in intervertebral disc cells in an experimental nutrient deprivation model.

Similar Datasets

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