Biochem Biophys Res Commun. 2025 Apr 19;765:151857. doi: 10.1016/j.bbrc.2025.151857. Online ahead of print.
ABSTRACT
OBJECTIVE: To investigate the regulatory role of IP3R2 on mitochondrial function in retinal pigment epithelial cells during the early stage of hypoxic stress preceding apoptosis.
METHODS: ARPE-19 cell line was cultured in 1 % oxygen to establish an in vitro hypoxic model. The presence of hypoxia and absence of significant apoptosis in RPE cells were confirmed through hypoxia-inducible factor HIF-1α expression and apoptosis assays respectively. Mitochondrial function was evaluated using an ATP assay kit and flow cytometry. Immunoblotting was conducted to ascertain the expression levels of mitochondrial dynamics proteins (MFN2, DRP1, TOMM20) and mitochondrial calcium-related proteins (IP3R1, IP3R2, IP3R3, VDAC1). Mitochondrial morphology was observed using confocal microscopy. The impact of small interfering RNA (siRNA)-mediated IP3R2 knockdown on apoptosis and mitochondrial function was assessed in RPE cells.
RESULTS: Under hypoxic stress before the onset of apoptosis in RPE cells, mitochondrial dysfunction and significant increase in mitochondrial calcium flux were observed, accompanied by a notable upregulation of IP3R2 expression under hypoxia. Knockdown of IP3R2 during the pre-apoptotic stage further impaired RPE function under hypoxia.
CONCLUSION: IP3R2-mediated mitochondrial calcium overload is crucial for maintaining RPE function and mitochondrial homeostasis during the pre-apoptotic stage triggered by hypoxic stress.
PMID:40267840 | DOI:10.1016/j.bbrc.2025.151857