Attenuation of the degenerative effects of endothelin-1 on cartilaginous endplate cells by the endothelin receptor antagonist BQ-123 via the wnt/β-catenin signaling pathway.

BACKGROUND: Context: Endothelin-1 (ET-1) is an inflammatory mediator associated with cartilage endplate (CEP) degeneration in the intervertebral disc. SOX9 is down-regulated during CEP degeneration, along with its targets, collagen II and aggrecan. Wnt/β-catenin signaling is associated with CEP degeneration and a downstream target of SOX9; however, the precise mechanism of CEP degeneration and the role of ET-1 are largely unknown.

PURPOSE: The purpose of the study was to evaluate the influence of the endothelin A receptor antagonist, BQ-123, on ET-1-induced effects on cartilaginous endplate cells (CECs) associated with CEP degeneration via the Wnt/β-catenin signaling pathway.

STUDY DESIGN/SETTING: The influence of ET-1 on the expression levels of collagen II, aggrecan, and SOX9 in CECs, and the effect of BQ-123 in this context, were investigated.

METHODS: To establish a model for CEP degeneration, three lumbar discs (L3/4, L4/5, and L5/6 levels) in New Zealand white rabbits were punctured close to the vertebral endplate using a 14G needle. Intervertebral disc degeneration was evaluated by MRI four weeks after vertebral endplate injury. CECs were then isolated from the degenerated CEPs, to allow evaluation of the role of ET-1 and BQ-123, and investigate their effects on the Wnt/β-catenin signaling pathway. The expression of ET-1 in CECs from degenerated CEPs was analyzed by immunofluorescent staining. Changes in the levels of collagen II, aggrecan, and SOX9 were evaluated in CECs by real-time polymerase chain reaction (PCR) and western blotting. The Wnt/β-catenin signaling pathway was also investigated by western blotting.

RESULTS: After four weeks, intervertebral discs with vertebral endplate injury exhibited clear signs of disc degeneration. Immunofluorescent staining showed that ET-1 was expressed in the cytoplasm of CECs. ET-1 stimulation significantly inhibited the expression of collagen II, aggrecan, and SOX9 in CECs, while BQ-123 increased the levels of these three molecules. In addition, ET-1 stimulation increased the expression of β-catenin, Cyclin D1, and Dvl1 in the Wnt/β-catenin signaling pathway of CECs from degenerated discs, and reduced the expression of GSK-3β, whereas BQ-123 had the opposite effect.

CONCLUSIONS: ET-1 can reduce levels of collagen II, aggrecan, and SOX9 in CECs through activation of the Wnt/β-catenin signaling pathway, whereas BQ-123 attenuates these negative effects, highlighting a new molecular mechanism with potential for exploitation for treatment of CEP degeneration.