Arthritis Res Ther. 2011;13(6):R215. Epub 2011 Dec 29.

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration.

Erwin WM, Islam D, Inman RD, Fehlings MG, Tsui FW.

Source

University of Toronto and Toronto Western Hospital, Mc13-310, 399 Bathurst Street, Toronto M5T 2S8. mark.erwin@utoronto.ca

Abstract

INTRODUCTION:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

PMID:: 22206702; [PubMed - in process]
PMCID:: PMC3334668

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Figure 1

Flow Cytometry Analysis of NCCM-Induced Protection from Apoptosis in NP Cells. (A) Representative sample of flow cytometry analysis of bovine NP cell death (Propridium Iodide (PI) (Y axis) and Annexin V (X axis) staining. All experiments were performed in triplicate. NCCM was generated from notochordal cells from six canine specimens. (a) 0% (basal medium) control, (b) IL-1β + FasL, (c) IL-1β + FasL + NCCM (b) The use of IL-1β + FasL causes an increase in the PI+ and Annexin V+ cells (upper right quadrant = dead cells) as compared to either (a) or (c). PI+ and Annexin V- = necrosis; PI + and Annexin V+ = apoptotic cell death. Treatment with NCCM (c) results in far fewer cells that are PI+ and Annexin V+ with an appearance that is almost equivalent to baseline (a). The black oval in (b) represents the cell population within the IL-1β + FasL treated cells that are 'rescued' by NCCM since these increasingly Annexin V+ and progressively PI+ cells are not apparent in the NCCM-treated groups. Triplicate experiments are presented in graphically in Figure 1 (b). (B) Summary of flow cytometry NP cell death assays (triplicate experiments as depicted in Figure 1 (a) above. The dark bars depict the percentage of all dead cells (Annexin V+ plus PI+) and the white bars depict the percentage of the total dead cells for each condition that died via apoptosis (Annexin V+ but PI-). There is little death in the untreated (0% (basal) media) cells but there is a marked increase in cell death when treated with IL-1β + FasL. This increase in both total cell death (from 2.5% to 8.8%) and apoptotic cell death (from 31.69% to 46.2%) is brought almost to baseline by the use of NCCM (apoptotic cell death is brought back to a level that is identical to the untreated cells). The differences between IL-1β + FasL and IL-1β + FasL + NCCM in terms of both total and apoptotic cell death are statistically significant at P = .016 and P = .0002 respectively (see asterisk indicating significance). Treatment with IL-1β + FasL + NCCM results in the most cell death as a function of apoptotic, not necrotic cell death. Abbreviations: IL-1β (Interleukin-1beta) FasL (Fas Ligand), NCCM (notochordal cell conditioned medium),

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration

Arthritis Res Ther. Arthritis Res Ther;13(6):R215-R215.

Figure 2

Activated caspase assays for NP cell apoptosis. (a) Activated caspase-8 activity for all experimental conditions; there is marginally increased activity in NCCM + IF and further elevated activity for BCCM but no suppression of activated caspase-8 activity for any of the treatment groups. (b) Activated caspase-9 activity is upregulated in the 2% +IF group and markedly down-regulated by NCCM (P < .01) and also (although to a lesser extent) by BCCM (P < .01). (c) Levels of activated caspase-3 for 2%, 2% + IL-1β + FasL, NCCM + IL-1β + FasL and BCCM + IL-1β + FasL treated cells. Here the use of NCCM in the presence of IL-1β + FasL reduces activated caspase-3 activity to almost baseline - precisely as demonstrated using flow cytometry and AnnexinV and PI labeling. Interestingly, there is no protection from apoptosis using conditioned medium obtained from bovine NP cells (BCCM) indicating that anti-apoptotic signaling is uniquely conferred by NCCM (P < .01). The suppression of both activated caspase-3 and -9 by treatment with NCCM indicates an anti-apoptotic effect of NCCM upon NP cells in the presence of IL-1β + FasL that is not accounted for by the other treatment groups. RLU (depicted on the 'Y' axis) is the fluorometric activity that occurs in the presence of caspase activation that releases a substrate for luciferase that in turn is determined using fluorometric methods. In each case 2% NCCM was generated from pooled notochordal cells obtained from the IVDs of five non-chondrodystrophic canines (eight to nine discs/animal). Bovine NP cells were obtained from the caudal discs of five to six three-year old steers. The 2% BCCM was generated from a separate pool of bovine caudal discs similarly obtained, seeded within alginate beads and conditioned medium developed over three days. Abbreviations: IL-1β (Interleukin-1beta) FasL (Fas ligand), NCCM (notochordal cell conditioned medium), BCCM (Bovine Cell Conditioned Medium), NP cells (nucleus pulposus cells), RLU (Relative Luminescence Units).

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration

Arthritis Res Ther. Arthritis Res Ther;13(6):R215-R215.

PubMed

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Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration.

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