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Global Identification of Genes Related to Nutrient Deficiency in Intervertebral Disc Cells in an Experimental Nutrient Deprivation Model

PLoS ONE (2013) 8(3)
DOI: 10.1371/journal.pone.0058806
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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 p21Cip1 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 p27Kip1 and p53 protein expression was upregulated in serum-starved cells. p21Cip1 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. © 2013 Sudo et al.

Figures

  • Table 1. Highly significant (P,0.01) pathways based on KEGG database*.
  • Table 2. Genes with significant expression levels in serum-starved nucleus pulposus cells Only genes whose expression was significantly (P,0.05) up- or down-regulated at least 1.5 fold are shown.
  • Figure 1. Western blots of p15Ink4b, p16Ink4a, p21Cip1, p27Kip1, and p53 in rat nucleus pulposus cells. Cells were harvested after 6 or 48 h of serum starvation. Cells not subjected to serum starvation were used as untreated controls. b-actin was used as an internal control. (A) Representative western blot analysis. (B) Densitometry analyses were performed to quantify the levels of p21Cip1, p27Kip1, and p53 via normalization to beta-actin. Results are representative of three independent experiments. Values are expressed as the mean 6 SD (* = P,0.05). doi:10.1371/journal.pone.0058806.g001
  • Figure 2. Functional interaction between p21Cip1 and p53 in serum-deprived rat nucleus pulposus (NP) cells. Forty-eight hours after p53 short interfering RNA (siRNA) transfection, cells were serum-deprived and harvested after 48 h. (A) qRT-PCR analysis of p53 mRNA expression was performed using rat NP cells transfected with p53 siRNA and a scrambled negative control siRNA. Total RNA was extracted 48 h after transfection, and glyceraldehyde phosphate dehydrogenase (GAPDH) expression was used for normalization. The results are expressed as a percentage of the expression in control siRNA-transfected cells. (B) Representative western blot analysis of protein extracts from NP cells. (C) Densitometry analyses were performed to quantify the levels of p53 and p21Cip1 48 h after serum starvation via normalization to beta-actin. p21Cip1 expression was decreased in p53 siRNA-transfected cells before serum starvation (48 h after p53 siRNA transfection). However, there was no significant difference in the expression level of p21Cip1 among serum-starved only, control siRNA-transfected, and p53 siRNA-transfected cells at 48 h after serum starvation. Results are representative of three independent experiments. Values are expressed as the mean 6 SD (* = P,0.05 compared with control siRNA, reagent only, and untreated cells, ** = P,0.05 compared with untreated control and p53 siRNA, #= P,0.05 versus all other groups). doi:10.1371/journal.pone.0058806.g002
  • Figure 3. Functional interaction between p21Cip1 and caspase 3 in serum-deprived rat nucleus pulposus (NP) cells. Forty-eight hours after caspase 3 siRNA transfection, cells were serum-deprived (A) qRT-PCR analysis of caspase 3 mRNA expression was performed using rat NP cells transfected with caspase 3 siRNA and a scrambled negative control siRNA. Total RNA was extracted 48 h after transfection, and glyceraldehyde phosphate dehydrogenase (GAPDH) expression was used for normalization. The results are expressed as a percentage of the expression in control siRNA-transfected cells. (B) Caspase 3 expression was evaluated by immunofluorescence analysis. Caspase 3 expression decreased in cells transfected with caspase 3 siRNA. Bar = 200 mm (C) Representative western blot analysis of protein extracts from NP cells. (D) Densitometry analyses were performed to quantify the levels of caspase 3 and p21Cip1 48 h after serum starvation via normalization to beta-actin. p21Cip1 protein expression remained in caspase 3 siRNA-transfected cells, indicating that caspase 3 mediates p21Cip1 cleavage in serum-deprived NP cells. Results are representative of three independent experiments. Values are expressed as the mean 6 SD (* = P,0.05 compared with control siRNA, reagent only, and untreated cells, ** = P,0.05 compared with all other groups, #= P,0.05 versus untreated control and caspase 3 siRNA). doi:10.1371/journal.pone.0058806.g003
  • Table 3. Genes with significant expression levels in serum-starved nucleus pulposus cells transfected with caspase 3 siRNA.
  • Figure 4. Flow cytometric analysis of rat nucleus pulposus (NP) cells. Forty-eight hours after caspase 3 siRNA transfection, cells were serum-deprived and harvested after 48 h. (A) Representative graphs showing the cell cycle. (B) Comparison of the cell cycle in the G1 and S phase. (C) The dual parametric dot plots combining annexin V-FITC and PI fluorescence show the early apoptotic cells (FITC+/PI -), and the late apoptotic cells (FITC+/PI +). (D) Percentage of (early+late) apoptotic cells. Results are representative of three independent experiments. Values are expressed as the mean 6 SD (* = P,0.05 versus all other groups, ** = P,0.05 versus serum-starved only and control siRNA). doi:10.1371/journal.pone.0058806.g004

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Sudo, H., Yamada, K., Iwasaki, K., Higashi, H., Ito, M., Minami, A., & Iwasaki, N. (2013). Global Identification of Genes Related to Nutrient Deficiency in Intervertebral Disc Cells in an Experimental Nutrient Deprivation Model. PLoS ONE, 8(3). https://doi.org/10.1371/journal.pone.0058806

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