Hydrogen sulfide reduces RAGE toxicity through inhibition of its dimer formation

Abstract RAGE is important in the development of neurodegenerative diseases. The present study was designed to investigate the effect of hydrogen sulfide (H 2 S, 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 H 2 S 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 H 2 S in brain cells. Both Western blots and split GFP complementation analysis demonstrate that NaHS reduced H 2 O 2 -enhanced RAGE dimerization. Immunofluorescence analysis shows that H 2 O 2 up-regulated the membrane expression of wild-type RAGE. However, H 2 O 2 –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 H 2 O 2 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 H 2 S may directly S-sulfhydrate the C259/C301 residues. Our data suggest that H 2 S 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.