4-Hydroxynonenal-induced GPR109A (HCA ₂ receptor) activation elicits bipolar responses, G _αi -mediated anti-inflammatory effects and G _βγ -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.