Hydrogen Sulfide Suppresses Keratinocyte Migration in Scratch Assay

Cell migration is crucial for maintaining physiological functions in the body and is driven by mechanical, electrical, or chemical stimuli. Hydrogen sulfide (H2S), a gaseous signaling molecule, has been shown to regulate cellular migration in a cell type and context-dependent manner. Keratinocytes are the predominant cell-type in the epidermis and drive re-epithelialization during wound healing. However, the effect of H2S on keratinocyte migration remains incompletely defined. Here, we demonstrate that H2S suppresses keratinocyte migration in a serum-restricted in vitro scratch assay. Intracellular H2S levels were modulated using fast-releasing donors H2S donors (NaHS or Na2S) to achieve supraphysiological levels or by siRNA knockdown of endogenous H2S-producing enzymes cystathionine-gamma-lyase (CGL) and cystathionine-beta-synthase (CBS) to obtain infraphysiological levels. Treatment with NaHS or Na2S (10 µM - 5 mM, 24 h) reduced gap closure in a dose-dependent fashion, with negligible effects at <100 µM and progressive suppression at higher concentrations. Conversely, siRNA knockdown of CGL or CBS boosted keratinocyte migration by 30% versus scrambled siRNA control. Supplementation with a slow-release H2S donor rescued the siCGL phenotype, restoring migration to control levels. Across experiments, intracellular H2S level is negatively associated with keratinocyte migration. These findings suggest that H2S can restrain keratinocyte motility under serum-restricted conditions, and that reports of accelerated wound closure with H2S donors in vivo may reflect effects on other cell types and/or inflammatory pathways rather than direct enhancement of keratinocyte migration.