Cadherin 2 is required for intervertebral disc homeostasis and maintenance of vacuolated phenotype in nucleus pulposus cells

Abstract

Introduction Integrity of the nucleus pulposus (NP) has been implicated in the function and homeostasis of intervertebral disc (IVD). Understanding the regulation of NP cells would contribute to their engineering and development of therapeutics for treating IVD degeneration. Studies in mouse models indicate that early events of IVD degeneration involve segregation of the notochordal NP cell clusters (1), suggesting that disc degeneration may be associated with cell adhesion molecule activities. Cadherins are transmembrane glycoproteins that mediate calcium dependent cell adhesion (2). Based on microarray analysis, we have revealed specific expression of Cdh2 gene, encoding cadherin 2/N-cadherin, in rodent NP cells, suggesting a potential regulatory role of cadherins in IVD homeostasis. To date, the function of cadherin 2 in IVD and its relationship to IVD degeneration remain elusive. We hypothesize that cadherin 2 has a regulatory role in NP cells and that a deregulation of its activities has adverse effects on IVD homeostasis. We aimed to study the expression pattern of cadherin 2 in rodent discs during development, aging, and degeneration, and to investigate its function in the NP via a gene and protein ablation strategies. Results By immunofluorescence, cadherin 2 was weakly detected in murine embryonic notochord and newborn NP. At 3- and 6-month old, strong cadherin 2 signals were specifically detected as foci along the cell-cell junctions of the vacuolated NP cells (notochordal cells). Annulus fibrosus showed no signals. In aged IVD, the notochordal cells were replaced by small chondrocyte-like cells with lower expression of cadherin 2. In puncture-induced degenerative rodent discs, the notochordal NP cells were replaced by rounded chondrocyte-like cells, showing reduced level of cadherin 2 expression. Human NP showed heterogeneous cadherin 2 expression. By injecting cadherin 2 antibody into rat NP to perturb its function in vivo, cadherin 2 expression was reduced along with reduction of disc height. Compared with the IgG injection control, cadherin 2 antibody ablation group showed a transformation of the notochordal NP cells into less vacuolated chondrocyte-like phenotype with upregulation of collagen II. Cdh2 conditional knockout mice (CKO) showed absence of cadherin 2 and a loss of vacuolated phenotype in the NP cells (Fig. 1A), displaying significantly smaller body size by 1-month old (Fig. 1B). Moreover, the mutant exhibited irregular annulus organization and reduced disc height in IVD (Fig. 1C). Conclusion Our study suggests cadherin 2 as a marker of notochordal NP cells. The lower expression of cadherin 2 in the aged and puncture-injured rodent IVD substantiates its association with IVD degeneration. The reduction of cadherin 2 positive NP cells in mature human IVD is consistent with gradual loss of notochordal cells after the first decade of life. Our parallel in vivo studies of gene/protein ablation support that cadherin 2 is essential to the maintenance of a vacuolated phenotype of the notochordal NP cells, and that a loss of cadherin 2 function may initiate degenerative changes in the IVD. In summary, our study implicates an important role of cadherin 2 in notochordal cell function and regulating IVD homeostasis.