GPR4 aggravates ferroptosis in nucleus pulposus cells by inhibiting mitophagy via MAPK pathway activation

Abstract Inflammatory diseases are known to lower the pH of the extracellular microenvironment. However, the role of proton-activated G protein-coupled receptors (GPRs) 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 in 58 human disc samples (male: female = 31:27) and rat models (n = 3 per group) using western blotting (WB) and 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 (NPCs). Proteomic sequencing of primary NPCs further demonstrated that GPR4 overexpression altered gene expression related to mitochondrial energy metabolism and ferritin homeostasis. Notably, knockdown of GPR4 via shRNA (shGPR4) enhanced MAPK-mediated mitophagy while suppressing ferroptosis in TBHP-exposed NPCs. These findings suggest that GPR4 plays a critical role in IVDD pathogenesis and may serve as a potential therapeutic target for mitigating disc degeneration.