Expression of Proteinase-Activated Receptor-2 in the Intervertebral Disc

Study Design. Immunohistochemical and biochemical analyses of proteinase-activated receptor-2 (PAR-2) in rat and human intervertebral discs (IVDs). Objectives. To examine the expression and function of PAR-2 in rat IVD cells, and to determine if PAR-2 is expressed in human IVDs. Summary of Background Data. PAR-2 is a G protein-coupled receptor that contributes to the regulation of inflammatory reactions and the pathophysiology of inflammatory diseases, including arthritis. The expression of PAR-2 in the IVD has not been determined. Methods. PAR-2 expression by rat IVD cells and tissues was examined using immunohistochemistry and western blot. Rat anulus fibrosus cells in monolayer culture were used to examine the biologic role of PAR-2 in vitro. The effect of PAR-2-activating peptide (PAR-2(AP)) on the catabolic cascade was assessed by western blot and real-time PCR. The expression of PAR-2 by human IVD tissues at different stages of degeneration was determined by immunohistochemical analyses. Results. PAR-2 was expressed by rat IVD cells and in both anulus fibrosus and nucleus pulposus tissues, PAR-2 expression was up-regulated by interleukin-1 beta (IL-1 beta). PAR-2(AP) significantly increased the release of IL-1 beta into the medium. Although PAR-2(AP) had no direct effect on matrix metalloproteinase-3 (MMP-3) and MMP-13 mRNA levels, treatment with PAR-2(AP) significantly up-regulated the mRNA levels of a disintegrin and metalloproteinase with thrombospondin motif-4. The simultaneous administration of PAR-2(AP) and IL-1 beta synergistically up-regulated the mRNA levels of a disintegrin and metalloproteinase with thrombospondin motif-4, MMP-3, and MMP-13. The expression of PAR-2 was identified in human IVD tissues. The number of PAR-2-expressing cells was significantly elevated in advanced stages of IVD degeneration compared with those in early stages of degeneration. Conclusion. Our results demonstrate for the first time that IVD cells express PAR-2. The expression of PAR-2 is regulated by IL-1 beta stimulation. PAR-2 activation accelerates the expression of matrix-degrading enzymes. PAR-2 may play an important role in the cytokine-mediated catabolic cascade and consequently may be involved in IVD degeneration.