Gene Expression Profiling of Early Intervertebral Disk Degeneration

Abstract

Introduction Early degeneration of the IVD involves a change in the nucleus pulposus (NP) cell population, characterized by the replacement/differentiation of the native notochordal cells (NCs) by/into chondrocyte-like cells (CLCs). In this respect, the NC has received considerable attention as a potential NP progenitor cell and as a target to realize disk regeneration.1,2 The canine species is affected by naturally occurring IVD degeneration, like humans.3 Moreover, with regard to the maintenance of NCs and the occurrence of IVD degeneration, the canine species reveals a unique phenomenon. In chondrodystrophic dogs, NCs are replaced by CLCs before the age of 1 year, with a concurrent onset of IVD degeneration at all spinal levels. By contrast, in nonchondrodystrophic dogs the NC remains the predominant cell type of the NP during the majority of life; replacement of NCs by CLCs and concurrent IVD degeneration only occur at old age. The aim of this study is to investigate gene expression profiles in the process of early NP degeneration, thereby revealing potential biomolecular signaling pathways for IVD regeneration. Materials and Methods Canine IVD samples from chondrodystrophic and nonchondrodystrophic dogs were classified into (1) NP rich in NCs, (2) NP containing both NCs and CLC-cells (Mixed), and (3) early degenerated NP rich in CLCs (Fig. 1). Gene expression profiling of the NP of these three groups was performed using a two-color cDNA-microarray, with subsequent validation (qPCR and immunohistochemistry) in IVD samples and cultures of primary NCs. Results Early IVD degeneration involved significant regulations of various pathways, including extracellular matrix (ECM) remodeling, plasmin, and plasminogen activator urokinase. Bone Morphogenetic Protein (BMP)-, and Wnt/β-catenin-signaling and cytoskeleton remodeling. Wnt/β-catenin signaling plays a crucial role in maintaining the notochordal fate during embryogenesis and stem-cell fate determination, but is also involved in IVD degeneration and regeneration. Whether the differential expression of Wnt/β-catenin-signaling is associated with degeneration or is an attempt to repair the damaged IVD remains uncertain. Both NCs and CLCs showed nuclear and cytoplasmic expression of the Wnt effector protein β-catenin4; axin2 gene expression, a specific read-out for Wnt signaling, revealed a dual response: in the initial stages of NP degeneration tended to increase (Mixed group), but diminished with ongoing degeneration (CLC-rich group). A concurrent increase in Wnt inhibitory factor 1 gene expression was observed, which may be a significant factor involved in the downregulation of canonical Wnt signaling in the chondrodystrophic dogs.5 Caveolin-1, which is known to regulate canonical Wnt signaling,6 emerged as a significantly regulated gene. NCs in culture showed robust caveolin-1 gene and protein expression and IVD degeneration involved significant decrease in Caveolin-1 gene and protein expression. Conclusion Early IVD degeneration involves significant alterations in biomolecular pathways involved in ECM synthesis, cell-matrix interactions, and cytoskeletal remodeling. Subclinical, early IVD degeneration is associated with diminished Wnt/β-catenin signaling activity. In this process, caveolin-1 expression appears to be a relevant factor in NC physiology and thus maintenance of the healthy NP. I confirm having declared any potential conflict of interest for all authors listed on this abstract No Disclosure of Interest None declared Risbud MV, Shapiro IM. Notochordal cells in the adult intervertebral disc: new perspective on an old question. Critical Reviews in Eukaryotic Gene Expression 2011;21(1):29–41 Erwin WM. The enigma that is the nucleus pulposus cell: the search goes on. Arthritis Research and Therapy 2010;12(3):118 Bergknut N, et al. The dog as an animal model for intervertebral disc degeneration? Spine (Phila Pa 1976) 2011; Epub ahead of print Smolders LA, et al. Basal canonical Wnt signaling of the notochordal cells is upregulated during the process of intervertebral disc degeneration. Journal of orthopaedic Research 2011; Epub ahead of print Surmann-Schmitt C, et al. Wif-1 is expressed at cartilage-mesenchyme interfaces and impedes Wnt3a-mediated inhibition of chondrogenesis. Journal of cell Science 2009;122(Pt 20):3627–3637 Yamamoto H, et al. Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin. Developmental Cell 2006;11(2):213–223