Endogenous hydrogen sulfide is involved in osteogenic differentiation in human periodontal ligament cells

Objective Endogenous hydrogen sulfide (H2S) has recently emerged as an important intracellular gaseous signaling molecule within cellular systems. Endogenous H2S is synthesized from l -cysteine via cystathionine β-synthase and cystathionine γ-lyase and it regulates multiple signaling pathways in mammalian cells. Indeed, aberrant H2S levels have been linked to defects in bone formation in experimental mice. The aim of this study was to examine the potential production mechanism and function of endogenous H2S within primary human periodontal ligament cells (PDLCs). Design Primary human PDLCs were obtained from donor molars with volunteer permission. Immunofluorescent labeling determined expression of the H2S synthetase enzymes. These enzymes were inhibited with D,L-propargylglycine or hydroxylamine to examine the effects of H2S signaling upon the osteogenic differentiation of PDLCs. Gene and protein expression levels of osteogenic markers in conjunction with ALP staining and activity and alizarin red S staining of calcium deposition were used to assay the progression of osteogenesis under different treatment conditions. Cultures were exposed to Wnt3a treatment to assess downstream signaling mechanisms. Results In this study, we show that H2S is produced by human PDLCs via the cystathionine β-synthase/cystathionine γ-lyase pathway to promote their osteogenic differentiation. These levels must be carefully maintained as excessive or deficient H2S levels temper the observed osteogenic effect by inhibiting Wnt/β-catenin signaling. Conclusions These results demonstrate that optimal concentrations of endogenous H2S must be maintained within PDLCs to promote osteogenic differentiation by activating the Wnt/β-catenin signaling cascade.