Hydrogen sulfide reduces RAGE toxicity through inhibition of its dimer formation

RAGE is important in the development of neurodegenerative diseases. The present study was designed to investigate the effect of hydrogen sulfide (H₂S, an endogenous gaseous mediator) on the cytotoxicity caused by RAGE activation during the chronic oxidative stress. Aβ_1-42 decreased cell viability and induced cell senescence in SH-SY5Y cells. Treatment with advanced glycation end products (AGEs) induced cell injury in HEK293 cells stably expressing RAGE (HEK293-RAGE) and stimulated inflammatory responses in SH-SY5Y cells. Pretreatment of SH-SY5Y cells with an H₂S donor, NaHS, significantly attenuated the above harmful effects caused by Aβ_1-42 or AGEs. Western blotting analysis shows that oxidative stress enhanced RAGE protein expression which was attenuated by either NaHS or over-expression of cystathionine β-synthase (CBS), a critical enzyme for producing H₂S in brain cells. Both Western blots and split GFP complementation analysis demonstrate that NaHS reduced H₂O₂-enhanced RAGE dimerization. Immunofluorescence analysis shows that H₂O₂ up-regulated the membrane expression of wild-type RAGE. However, H₂O₂-enhanced expression of the RAGE harboring C259S/C310S double mutation (DM-RAGE) was observed in the endoplasmic reticulum. Treatment with NaHS attenuated the effects of H₂O₂ on the protein expression of WT-RAGE, but not that of DM-RAGE. Cycloheximide chase and ubiquitination assays show that NaHS reduced the half-life of WT-RAGE to a similar level of DM-RAGE. S-sulfhydration assay with the tag-switch technique demonstrate that H₂S may directly S-sulfhydrate the C259/C301 residues. Our data suggest that H₂S reduces RAGE dimer formation and impairs its membrane stability. The lowered plasma membrane abundance of RAGE therefore helps to protect cells against various RAGE mediated pathological effects.