Notochordal Cells Protect Nucleus Pulposus Cells from Degradation and Apoptosis: Implications for the Mechanisms of Intervertebral Disk Degeneration

Introduction Degenerative disk disease (DDD) is an extremely common and costly health care condition which to date has no curative strategy which would be greatly aided by an ability to re-establish equilibrium between catabolic and anabolic tissue remodeling. 1 The nonchondrodystrophic (NCD) canine is unique amongst the canine subspecies in which this animal retains a high notochordal cell population throughout life, and is relatively resistant to the development of DDD and an emerging body of evidence indicates that notochordal cells confer anabolic capacity upon nucleus pulposus (NP) cells and that their absence is associated with susceptibility to degenerative changes. 2–5 Regulation of cellular turnover is vital to tissue homeostasis and is dependent upon a highly regulated form of programmed cell death (apoptosis). Some cells, classified as type I cells, function independently of the mitochondria and signal via Fas-induced apoptotic cell death involving the Caspase-8 pathway. Type II cells (such as NP cells) have a critical reliance upon the mitochondria whereby apoptosis is mediated via Caspase-9. 8,9 Here, we demonstrate that notochordal cell-conditioned medium or “NCCM” is capable of protecting NP cells from matrix protein degradation and proinflammatory cytokine secretion induced by IL-1β and FasL. Furthermore, IL-1β and FasL-mediated cell death is inhibited by NCCM via the inhibition of activated caspases -9 and -3. Materials and Methods We obtained NP cells from bovine caudal disks as per our established methods to be used as the “target” cells in our experiments. We also developed bovine NP cell conditioned medium (BCCM) in exactly the same fashion as we obtained bovine NP “target” cells but in the case of BCCM cultured the bovine NP cells within alginate beads. We developed conditioned medium from the NPs of lower thoracic and entire lumbar IVDs from 11 nonchondrodystrophic canines and after overnight digestion seeded the cells within alginate beads for subsequent culture. Conditioned medium was generated from both nonchondrodystrophic canine notochordal cells (NCCM) and bovine NP cells (BCCM). We then determined the ability of NCCM or BCCM to protect bovine NP cells from cell death in the presence of IL-1β + Fas ligand (FasL) as well as the ability of NCCM to protect the expression of salient extracellular matrix genes and other molecules using qRT-PCR methods. We used flow cytometry and activated caspase assays to determine protection from cell death and to discern the apoptotic pathways involved. Results NCCM inhibits bovine NP cell apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1β and IL-1β + Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen typeII, CD44, link protein and TIMP-1) and downregulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4 is increased. NCCM also protects against IL-1 + FasL-mediated downregulation of Ankexpression. Furthermore, NCCM downregulates the expression of IL-6by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells. Conclusion We have demonstrated in this study that NCCM protects against NP apoptosis via suppression of activated caspase-9 and -3/7. Possible mechanisms include stabilization of the mitochondrial membrane via inhibition of Bcl-2 activity, BID activation, or through the P53 growth factor-related pathway. It may be that components of NCCM suppress the formation of the apoptoses and in doing so they suppress the formation of activated caspase-3 thereby preventing apoptotic cell death. The novel finding of the activation of the Ankgene by NCCM under degenerative/death-inducing conditions suggests a possible role for this gene in the maintenance of NP cell phenotype. These areas are under examination by our group. The results of the present study provide evidence in vitro that notochordal cell-secreted soluble factors provide essential molecular signals that mediate apoptosis and degradation of NP cells induced by IL-1β + Fas-L. Harnessing the regenerative capacity of these cells and the important factors they secrete may lay the cornerstone of biological therapy for the treatment of degenerative disk disease. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Goetzel RZ, Hawkins K, Ozminkowski RJ, Wang S. The health and productivity cost burden of the “top 10” physical and mental conditions affecting six large U.S. employers in 1999. Journal of Occupational and Environmental Medicine 2003;45(1):5–14 Aguiar DJ, Johnson SL, Oegema TR. Notochordal cells interact with nucleus pulposus cells: regulation of proteoglycan synthesis. Experimental Cell Research 1999;,246:129–137 Oegema Jr. TR. The role of disk cell heterogeneity in determining disk biochemistry: a speculation. Biochemical Society Transactions 2002; 30(6):839–844 Erwin WM, Inman RD: Notochord cells regulate intervertebral disk chondrocyte proteoglycan production and cell proliferation. Spine 2006; 31(10):1094–1099 Erwin WM, Ashman K, O'Donnell P, Inman RD. Nucleus pulposus notochord cells secrete connective tissue growth factor and upregulate proteoglycan expression by intervertebral disk chondrocytes. Arthritis and Rheumatism 2006;54(12):3859–3867 Ozoren Nesrin, El-Deiry Wafik S. Defining Characteristics of Types I and II Apoptotic Cells in Response to TRAIL. Neoplasia 2002;4(6):551–557 Krammer Peter H: CD95's deadly mission in the immune system. Nature 2000;407:789–795

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