Cystathionine γ-Lyase–Hydrogen Sulfide Induces Runt-Related Transcription Factor 2 Sulfhydration, Thereby Increasing Osteoblast Activity to Promote Bone Fracture Healing

Abstract

Aims: Hydrogen sulfide (H2S) plays an essential role in bone formation, in part, by inhibiting osteoclast differentiation, maintaining mesenchymal stem cell osteogenesis ability, or reducing osteoblast injury. We aimed to investigate the role of H2S in osteoblast function and its possible molecular target. Results: In this study, we found that cystathionine c-lyase (CSE) majorly contributed to endogenous H2S production in the primary osteoblast. Overexpressed CSE increased osteoblast differentiation and maturation with higher bone morphogenetic protein 2 and osteopontin expression, alkaline phosphatase activity, and calcium nodule formation; in contrast, knockdown of CSE had opposite effects. Runt-related transcript factor 2 (RUNX2) is required for osteoblast biologic function. CSE-H2S increased nuclear RUNX2 accumulation, DNA binding activity, and target gene transcription. Protein sulfhydration is a common signal by H2S. We confirmed that RUNX2 was also sulfhydrated by H2S. This chemical modification enhanced RUNX2 transactivation, which was blocked by dithiothreitol (DTT, sulfhydration remover). Mutation of two cysteine sites in the runt domain of RUNX2 abolished H2S-induced RUNX2 sulfhydration and transactivation. In a bone -fracture rat model, overexpressed CSE promoted bone healing, which confirmed the effect of CSE-H2S on osteoblasts. Innovation: CSE-H2S is a dominant H2S generation system in osteoblasts and promotes osteoblast activity by the RUNX2 pathway, with RUNX2 sulfhydration as a novel transactivation regulation. Conclusion: CSE-H2S sulfhydrated RUNX2 enhanced its transactivation and increased osteoblast differentiation and maturation, thereby promoting bone healing. Antioxid. Redox Signal. 27, 742-753.

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…The H 2 S donor GYY4137 induced the same sulfhydrationdependent induction, whereas mutation of these Cys residues on RUNX2 or treatment with the CSE inhibitor dlpropargylglycine prevented RUNX2 sulfhydration, nuclear localization, and transactivation. Delivery of CSE using an adenoviral vector facilitated healing of fractured femurs in treated rats (315). The contrast between this study and those already mentioned, suggesting inhibition of osteopontin expression by H 2 S, may reflect a biphasic dose response, wherein physiological H 2 S induces osteopontin but supraphysiological concentrations are inhibitory.…”

“…Treatment with 100 lM NaHS significantly decreased proliferation of VSMCs via an ERK1/ERK2-dependent pathway and partially inhibited the induction of osteopontin by homocysteine. In contrast, endogenous H 2 S produced by CSE induces differentiation and maturation of primary osteoblasts, resulting in induction of osteopontin and BMP2 expression (315). This is mediated by sulfhydration of the transcription factor RUNX2 at Cys 123 and Cys 132 and results in elevated BMP2, osteopontin, and osteocalcin expression (Fig.…”

Hydrogen sulfide inhibits calcification of heart valves; implications for calcific aortic valve disease

“…Haematoxylin and eosin (upper panels), von Kossa (middle panels), CSE (second middle panels), and α smooth muscle actin (α-SMA; lower panels) staining was performed on aortic valves of mice fed a normal diet (first column; N = 5), on a high-fat diet (second column, N = 9); and on a high-fat diet treated with AP72 (third column; N = 5). Comparison (>) designates the calcified regions, and arrow indicates CSE positive cells CSE-produced endogenous sulfide enhanced its transactivation (Zheng et al, 2017). This effect is in contrast to our observations that endogenously produced or exogenously administered sulfide inhibited nuclear translocation of RUNX2, suggesting that the anti-calcification effects of H 2 S are more likely due to the inhibition of phosphate uptake, and the inhibition of RUNX2 translocation in our model is likely to be a secondary effect of this, as we proposed above.…”

Hydrogen sulfide lowers hyperhomocysteinemia dependent on cystathionine γ lyase S‐sulfhydration in ApoE‐knockout atherosclerotic mice

“…Nitrosylation of GAPDH inhibits, but sulfhydration enhances, its activity (Mustafa et al, ). Knockout of CSE (Mustafa et al, ), or inhibition of CSE activity (Zheng et al, ) lowered protein sulfhydration, which suggests that CSE might be a key enzyme for sulfhydration. In our experimental conditions, H 2 S donor treatment increased CSE protein expression and its sulfhydration but decreased its nitrosylation.…”