GPR4 promotes ferroptosis in nucleus pulposus cells via MAPK-dependent mitophagy suppression

Inflammatory diseases are known to lower the pH value of the extracellular microenvironment. However, the role of proton-activated G protein-coupled receptors (GPCRs) in the progression of intervertebral disc degeneration (IVDD) remains largely unexplored. In this study, we identified GPR4 as a key regulator of IVDD progression. Our analysis revealed a significant upregulation of GPR4 in degenerative human intervertebral discs. The expression of GPR4 was assessed using immunohistochemistry (IHC). Functional assays, including flow cytometry, immunofluorescence and WB, were performed to evaluate the impact of GPR4 on ferroptosis. We observed that GPR4 expression was significantly elevated in tert-butyl hydroperoxide (TBHP)-treated degenerative nucleus pulposus cells. Proteomic sequencing further demonstrated that GPR4 overexpression altered gene expression related to the mitochondrial energy metabolism and ferritin homeostasis. Notably, knockdown of GPR4 via shRNA (shGPR4) enhanced the MAPK-mediated mitophagy while suppressing ferroptosis in TBHP-exposed NP cells. These findings suggested that GPR4 played a critical role in IVDD pathogenesis and may serve as a potential therapeutic target for mitigating disc degeneration.