Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration.
BACKGROUND
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.
University of Toronto and Toronto Western Hospital, Mc13-310, 399 Bathurst Street, Toronto M5T 2S8. mark.erwin@utoronto.ca
W Mark
WM
Erwin
Diana
D
Islam
Robert D
RD
Inman
Michael G
MG
Fehlings
Florence W L
FW
Tsui
D051722:ADAM Proteins / Q000235:genetics; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D015687:Cell Hypoxia; D002478:Cells, Cultured; D024043:Collagen Type II / Q000235:genetics; D017077:Culture Media, Conditioned / Q000378:metabolism / Q000494:pharmacology*; D004285:Dogs; D016326:Extracellular Matrix Proteins / Q000235:genetics; D053222:Fas Ligand Protein / Q000494:pharmacology; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D018960:Hyaluronan Receptors / Q000235:genetics; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics; D007403:Intervertebral Disc / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism / Q000517:prevention & control; D008297:Male; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D009672:Notochord / Q000166:cytology / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase / Q000235:genetics; D011509:Proteoglycans / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics; D000099249:Proteoglycan Link Protein
D051722:ADAM Proteins; D000071121:ADAMTS4 Protein; D053674:Aggrecans; D020169:Caspases; D024043:Collagen Type II; D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D053222:Fas Ligand Protein; D018960:Hyaluronan Receptors; D053583:Interleukin-1beta; D015850:Interleukin-6; D019278:Matrix Metalloproteinase 3; D011348:Procollagen N-Endopeptidase; D011509:Proteoglycans; D019715:Tissue Inhibitor of Metalloproteinase-1; D000099249:Proteoglycan Link Protein