The Extracellular Calcium-Sensing Receptor as a Contributor in Human Cartilaginous Endplate Degeneration

Introduction Intervertebral discs (IVDs) are virtually avascular tissue relying on the diffusion of nutrients and metabolites from blood vessels in the outer annulus fibrosus, and in the vertebral bodies that partially penetrate the subchondral plate, and terminate at the cartilaginous endplate (CEP). Degeneration of CEP is viewed as a catalyst to IVD degeneration, as it impedes nutrient diffusion to the disc. Similar to articular cartilage, degeneration of CEP involves decreased proteoglycan and type II collagen (Col II) content, thinning, increased type X collagen (Col X), and advanced calcification. Although biological repair strategies of the degenerative disc involving supplementation of growth factors are being pursued, disc nutrition is an important factor in considering their success in disc repair. The extracellular calcium-sensing receptor (CaSR) is a G protein-coupled receptor, and currently, is the only known mechanism in sensing both local and systemic calcium fluctuations. CaSR is the principle regulator of parathyroid hormone synthesis and secretion from the parathyroid gland, and in addition to its expression in other calcium-handling tissues (intestine, kidney), evidence suggests a direct involvement in bone and cartilage development. Recently, CaSR has been shown to modulate vascular calcification; however, its function in the calcification of other tissues remains unknown. Objectives Calcification of CEP is suggested to be a catalyst in the degeneration of IVDs. However, the mechanism(s) of CEP degeneration are currently unknown. We propose that CaSR activation modulates the degeneration of CEP, by increasing the expression of catabolic factors in CEP chondrocytes, resulting from the accumulation of ionic calcium in the milieu. Materials and Methods Human CEPs were obtained from donor IVDs of Thompson grades 2, 3, and 4 through organ donations within 24 hours of death. CEP chondrocytes were isolated from tissue by sequential digestion with pronase followed by collagenase. Cells were expanded for 7 days under standard cell culture conditions. Immunohistochemistry was performed on CEP tissue to validate the grade and expression of CaSR. Free calcium levels were also measured and compared between grades. Immunocytochemistry, Western blotting, and MAPK signaling were performed on cultured CEP chondrocytes of various grades to demonstrate expression of CaSR and degenerative markers, including the function of the receptor. CEP cells of various grades were cultured in medium containing either high calcium (5 mM Ca 2+ ), CaSR allosteric agonist (cinacalcet, 1 µM), or regular growth medium (control, 1 mM Ca 2+ ) for 7 days, and monitored for changes in markers of degeneration and CaSR expression. Results Ionic calcium content in CEP tissue increased significantly with IVD degeneration (Fig. A). Interestingly, immunohistochemistry on CEP tissue also revealed an increase in the expression of CaSR that paralleled the degree of IVD degeneration. Immunocytochemistry and Western blotting on isolated CEP cells demonstrated an upregulation of CaSR (Fig. B) and type X collagen that varied with Thompson grades. The expression of matrix metalloproteinase (MMP)-13 was also increased with degeneration; however, the expression of Col II and the proteoglycan aggrecan (Agg) were inversely related to Thompson grade. The magnitude of CaSR signaling in CEP cells when stimulated with 5 mM Ca 2+ , as determined by ERK phosphorylation, was significantly enhanced in grade 4 versus grade 2 CEP chondrocytes. To determine a role of CaSR in the degeneration of CEP, we cultured Thompson grade 2 CEP chondrocytes in 1 mM Ca 2+ (control medium), 5 mM Ca 2+ or cinacalcet (allosteric CaSR agonist, 1 µM) for 7 days. Western blot analysis on lysate from G2 CEP cells treated with 5 mM Ca 2+ or cinacalcet demonstrated significant increases in the expression of Col X and MMP-13 (Figs. D and E), however, decreased expression of Col II and Agg was observed (Fig. C). Conclusion CaSR has been described as a mediator in ectopic calcification, and its expression is upregulated in CEP of degenerative discs. We provide evidence that CaSR upregulation may be a hallmark of CEP calcification. Activation of CaSR either by its agonist, calcium or cinacalcet, lead to increases in the synthesis of degenerative factors (Col X and MMP-13), and decreases in the production of matrix proteins (Col II and Agg). An understanding on the mechanism(s) of CEP degeneration is key when considering growth factor substitution in IVD repair, as disc nutrition may be an important factor in determining their success. Disclosure of Interest None declared References Lotz JC, Fields AJ, Liebenberg EC. The role of the vertebral end plate in low back pain. Global Spine J 2013;3(3):153–164 Brown EM. Role of the calcium-sensing receptor in extracellular calcium homeostasis. Best Pract Res Clin Endocrinol Metab 2013;27(3):333–343

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