4‐Hydroxynonenal‐induced GPR109A (HCA₂ receptor) activation elicits bipolar responses, Gαi‐mediated anti‐inflammatory effects and Gβγ‐mediated cell death

Background and Purpose In this study, we examined the possibility that 4‐hydroxynonenal (4‐HNE) acting as a ligand for the HCA 2 receptor (GPR109A) elicits both anti‐inflammatory and cell death responses. Experimental Approach Agonistic activity of 4‐HNE was determined by observing the inhibition of cAMP generation in CHO‐K1‐GPR109A‐G i cell line, using surface plasmon resonance (SPR) binding and competition binding assays with [ 3 H]‐niacin. 4‐HNE‐mediated signalling pathways and cellular responses were investigated in cells expressing GPR109A and those not expressing these receptors. Key Results Agonistic activity of 4‐HNE was stronger than that of niacin or 3‐OHBA at inhibiting forskolin‐induced cAMP production and SPR binding affinity. In ARPE‐19 and CCD‐841 cells, activation of GPR109A by high concentrations of the agonists 4‐HNE (≥10 μM), niacin (≥1000 μM) and 3‐OHBA (≥1000 μM) induced apoptosis accompanied by elevated Ca 2+ and superoxide levels. This 4‐HNE‐induced cell death was blocked by knockdown of GPR109A or NOX4 genes, or treatment with chemical inhibitors of G βγ (gallein), intracellular Ca 2+ (BAPTA‐AM), NOX4 (VAS2870) and JNK (SP600125), but not by the cAMP analogue 8‐CPT‐cAMP. By contrast, low concentrations of 4‐HNE, niacin and 3‐OHBA down‐regulated the expression of pro‐inflammatory cytokines IL‐6 and IL‐8. These 4‐HNE‐induced inhibitory effects were blocked by a cAMP analogue but not by inhibitors of G βγ ‐downstream signalling molecules. Conclusions and Implications These results revealed that 4‐HNE is a strong agonist for GPR109A that induces G αi ‐dependent anti‐inflammatory and G βγ ‐dependent cell death responses. Moreover, the findings indicate that specific intracellular signalling molecules, but not GPR109A, can serve as therapeutic targets to block 4‐HNE‐induced cell death.