The Expression of Chordin in the Intervertebral Disc

Introduction: Bone morphogenetic proteins (BMPs) are signalling molecules that belong to the transforming growth factor-β (TGF-β) superfamily of proteins that were initially identified by their capacity to induce endochondral bone formation. During early embryogenesis of both vertebrates and invertebrates, antagonism between BMPs and several unrelated secreted factors including chordin is a general mechanism by which the dorso-ventral axis is established [1]. High affinity binding of chordin sequesters the BMP ligands in the extracellular space, preventing interactions with their membrane receptors, resulting in the patterning of the mesoderm and induction of neural tissue in the adjacent ectoderm. Chordin contains four von Willebrandt factor type C (vWC) repeats or cysteine-rich (CR) domains, which are the sites for the specific interaction with BMP-2,-4 and -7. Chordin is also inactivated in vivo by the matrix metalloprotease BMP1/tolloid which cleaves chordin from the chordin-BMP complex and releases free BMP to the extracellular space [2]. The chordin gene (CHRD) has also been shown to be expressed in fetal as well as in adult tissues, suggesting functions in late embryogenesis and adult physiology [3]. Chordin is highly expressed in undifferentiated chondrocytes where it acts as an inhibitor of cell differentiation. It’s levels decline in mature cartilage tissue and is upregulated in the osteoarthritic joint cartilage [4]. However, chordin expression has not been described fully in the intervertebral disc. Here we report for the first time on the expression of chordin in the intervertebral disc (IVD).Materials and Methods:Bovine and Human specimens were processed for histology and immunohistochemistry (IHC), using formalin fixation and paraffin embedding. Samples were checked for orientation and inclusion of anulus fibrosus (AF) and nucleus pulposus (NP), and scored for degree of degeneration using a published histological 12 point scale [5]. IVD scoring 1-3 were graded “normal” 4-6 were graded “mildly degenerate” 7-9 were graded “moderately degenerate” and 9-12 “severely degenerate”. 5μm paraffin wax sections were dewaxed in xylene, rehydrated in IMS and washed in dH2O. Antigen retrieval was carried out using 0.01% (w/v) chymotrypsin (Sigma) for 20 min at 370C. Following washing in 1xTBS, endogenous peroxidase was blocked using 3% (v/v) H202 for 15 mins and washed in 1xTBS. Non-specific binding sites were blocked at room temperature for 30 min with 10% (w/v) donkey serum/2% (w/v) BSA (Sigma) and sections incubated overnight at 40C with goat polyclonal primary antibodies against chordin (Santacruz, Cat No SC-18265 ) at a 1:100 dilution in 10% (w/v) donkey serum/2% (w/v) BSA. Negative controls involved replacement of the antibody with goat IgG (Dako) at equal protein concentrations. After washing in 1xTBS, sections were incubated in a 1:400 dilution of biotinylated donkey anti-goat antiserum (Santa Cruz Biotechnology), for 1 hour at room temperature. Disclosure of secondary antibody binding was by the ABC-HRP complex (Vector) technique with 3,3'-Diaminobenzidine tetrahydrochloride (DAB) solution (Sigma). Sections were counterstained with Mayer’s haematoxylin (Raymond A Lamb), washed in water, dehydrated in IMS, taken through xylene and mounted in XAM (BDH) ready for viewing by microscopy.Results: The localisation of chordin in bovine tissue by IHC (Fig.1 A-D) revealed that both notochordal cells and the majority of NP cells present in the NP, (including NP with no notochordal cells present) strongly expressed this protein. In addition, bovine AF and articular cartilage (AC) cells did not demonstrate any immunoreactivity for the chordin antibody. Examination of chordin expression in normal and degenerate human disc samples confirmed the localised expression of chordin to the NP cells of the IVD. However, unlike bovine AC, chordin was also immunoreactive for human AC cells in normal and osteoarthritis (OA) tissues. Interestingly though this study identified a trend in chordin expression with stage of degeneration, which was found to significantly decrease as degeneration progresses (Fig.1 E). Discussion: This study has shown for the first time that chordin is expressed by the native chondrocyte-like cells of the IVD. Interestingly these results demonstrate the loss of chordin expression in human degenerate cells suggesting the potential use of chordin as a marker for healthy NP cells. Furthermore this raises questions about the role of chordin in the establishment and maintenance of a healthy NP cell. We know that chordin is an antagonist of BMP signaling and prevents BMP interaction with its receptors thereby inhibiting cell growth and differentiation. Therefore the loss of chordin in these degenerate tissues may allow BMP to act as a growth factor on degenerate cells in an attempt to repair the degenerate disc. In addition, bovine samples show that chordin appears to be specifically expressed in both notochordal cells and NP cells which also raises interesting questions about the origin of NP cells, and lends weight to the argument that NP cells are derived from the notochord.