p53-Pathway activity and apoptosis in hydrogen sulfide-exposed stem cells separated from human gingival epithelium

Calenic B, Yaegaki K, Ishkitiev N, Kumazawa Y, Imai T, Tanaka T. p53-Pathway activity and apoptosis in hydrogen sulfide-exposed stem cells separated from human gingival epithelium. J Periodont Res 2013; 48: 322-330. (C) 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective Hydrogen sulfide (H2S) is a volatile sulfur compound responsible for physiological halitosis. H2S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H2S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H2S . Material and Methods OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H2S for 24 and 48h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR. Results The levels of apoptosis were significantly increased following incubation in the presence of H2S, especially after 48h (36.95 +/- 1.91% vs. 4.77 +/- 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 +/- 9.84units/mL vs. 1.5 +/- 0units/mL and 32.22 +/- 10.23units/mL vs. 0.15 +/- 0units/mL, respectively, at 48h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased. Conclusion Following incubation with H2S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.