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Hydrogen sulphide (H2S), a newly identified gasotransmitter, can be endogenously produced by cystathionine gamma-lyase (CSE) in the cardiovascular system. This study investigated the role of the CSE/H2S system on lipid overload-induced lipotoxicity and cardiac senescence. Lipid overload in rat cardiomyocyte cells (H9C2) promoted intracellular accumulation of lipid, oxidative stress, mitochondrial dysfunctions, lipid peroxidation and inhibited cell viability, all of which could be reversed by exogenously applied H2S. Further data revealed that H2S protected H9C2 cells from lipid overload-induced senescence by altering the expressions of lipid metabolism-related genes and inhibiting cellular acetyl-CoA and global protein acetylation. Enhancement of protein acetylation abolished the protective role of H2S on cardiac senescence. In vivo, knockout of the CSE gene strengthened cardiac lipid accumulation, protein acetylation, and cellular ageing in high fat diet-fed mice. Taken together, the CSE/H2S system is capable of maintaining lipid homeostasis and cellular senescence in heart cells under lipid overload.

Cystathionine gamma-lyase (CSE)-derived hydrogen sulfide (H

Hydrogen sulfide (H2 S) can be endogenously produced and belongs to the class of signaling molecules known as gasotransmitters. Cystathionine gamma-lyase (CSE)-derived H2 S is implicated in the regulation of cell differentiation and the aging process, but the involvements of the CSE/H2 S system in myogenesis upon aging and injury have not been explored. In this study, we demonstrated that CSE acts as a major H2 S-generating enzyme in skeletal muscles and is significantly down-regulated in aged skeletal muscles in mice. CSE deficiency exacerbated the age-dependent sarcopenia and cardiotoxin-induced injury/regeneration in mouse skeletal muscle, possibly attributed to inefficient myogenesis. In contrast, supplement of NaHS (an H2 S donor) induced the expressions of myogenic genes and promoted muscle regeneration in mice. In vitro, incubation of myoblast cells (C2C12) with H2 S promoted myogenesis, as evidenced by the inhibition of cell cycle progression and migration, altered expressions of myogenic markers, elongation of myoblasts, and formation of multinucleated myotubes. Myogenesis was also found to upregulate CSE expression, while blockage of CSE/H2 S signaling resulted in a suppression of myogenesis. Mechanically, H2 S significantly induced the heterodimer formation between MEF2c and MRF4 and promoted the binding of MEF2c/MRF4 to myogenin promoter. MEF2c was S-sulfhydrated at both cysteine 361 and 420 in the C-terminal transactivation domain, and blockage of MEF2c S-sulfhydration abolished the stimulatory role of H2 S on MEF2c/MRF4 heterodimer formation. These findings support an essential role for H2 S in maintaining myogenesis, presenting it as a potential candidate for the prevention of age-related sarcopenia and treatment of muscle injury.

Hydrogen sulfide (H2S), a multifunctional gasotransmitter, participates in a wide range of cellular signal transduction and pathophysiological processes. Cystathionine gamma-lyase (CSE) acts as a major H2S-generating enzyme in peripheral organs and tissues. As a cysteine-rich and heavy metal-binding protein, metallothionein-1 (MT-1) is known to protect cells from various environmental stresses. Here we demonstrated that exposure of cadmium (Cd) induced oxidative stress, depleted intracellular thiols, and stimulated apoptotic cell death in mouse myoblast cells. CSE expression and H2S production were significantly enhanced by Cd treatment. NaHS, a well-known H2S donor, at physiologically relevant concentration significantly alleviated Cd-induced damage in both myoblasts and mouse skeletal muscles. In contrast, down-regulation of CSE/H2S system deteriorated Cd-stimulated oxidative stress and cell death. Exposure of the cells to Cd lead to increased expressions of metal regulatory transcription factor 1 and MT-1, while siRNA-mediated MT-1 knockdown alleviated Cd-induced CSE expression and caused more oxidative stress and cell death. In addition, H2S post-translationally modified MT-1 by S-sulfhydration and stabilized zinc-protein complex. Taken together, these data suggest that CSE/H2S system would protect myoblasts and skeletal muscles from Cd-induced damage by S-sufhydrating MT-1.

Hydrogen sulfide (H

Hydrogen sulfide (HBoth in vitro cell model and in vivo animal model of lipid accumulation were used in this study. Western blotting and real-time PCR were used for analysis of protein and mRNA expression. Acetyl-CoA was analyzed by a coupled enzyme assay, and lipid accumulation was observed with Oil Red O staining.Incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and HCSE/H

Hydrogen sulfide (H2S) is mostly produced by cystathionine-gamma-lyase (CSE) in vascular system and it inhibits the proliferation of vascular smooth muscle cells (SMCs). Insulin-like growth factor-1 (IGF-1), via its receptor (IGF-1R), exerts multiple physiological and pathophysiological effects on the vasculature, including stimulating SMC proliferation and migration, and inhibiting SMC apoptosis. Since H2S and IGF-1/IGF-1R have opposite effects on SMC proliferation, it becomes imperative to better understand the interaction of these two signaling mechanisms on SMC proliferation. SMCs isolated from small mesenteric arteries of CSE knockout (KO) and wild-type (WT) mice were used in the present study. The effects of IGF-1 and H2S on SMC proliferation were evaluated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assays. Protein expression was determined by western blot, and H2S-induced protein S-sulfhydration was assessed with a modified biotin switch assay. We found that IGF-1 dose-dependently increased the proliferation of both WT-SMCs and KO-SMCs, and this effect was more significant in KO-SMCs. Supplement of sodium hydrosulfide (NaHS) inhibited IGF-1-induced cell proliferation, while this effect was abolished by blocking IGF-1/IGF-1R signaling with picropodophyllin (PPP) or knocking out of the expression of IGF-1R. H2S significantly down-regulates the expression of IGF-1R, stimulates IGF-1R S-sulfhydration, and attenuates the binding of IGF-1 with IGF-1R. This study provides novel insight on the involvement of IGF-1/IGF-1R in H2S-inhibited SMC proliferation and suggests H2S-based innovative treatment strategies for proliferative cardiovascular diseases such as atherosclerosis.

Aims Hydrogen sulfide (H2S) as a novel gasotransmitter can be endogenously produced in liver by cystathionine gamma-lyase (CSE). The dysfunctions of CSE/H2S system have been linked to various liver diseases. Acetyl-CoA is the key intermediate from the metabolism of lipid. This study examined the roles of H2S in hepatic acetyl-CoA and lipid metabolism. Materials and methods Both in vitro cell model and in vivo animal model of lipid accumulation were used in this study. Western blotting and real-time PCR were used for analysis of protein and mRNA expression. Acetyl-CoA was analyzed by a coupled enzyme assay, and lipid accumulation was observed with Oil Red O staining. Key findings Incubation of human liver carcinoma (HepG2) cells with a mixture of free fatty acids (FFAs) or high glucose reduced CSE expression and H2S production, promoted intracellular accumulation of acetyl-CoA and lipid. Supply of exogenous NaHS or cysteine reduced acetyl-CoA contents and lipid accumulation, while blockage of CSE activity promoted intracellular lipid accumulation. Furthermore, H2S blocked FFAs-induced transcriptions of de novo lipogenesis, inflammation, and fibrosis-related genes. In vivo, knockout of CSE gene stimulated more hepatic acetyl-CoA and lipid accumulation in mice induced by high-fat choline-deficient diet. The expressions of lipogenesis, inflammation, and fibrosis-related genes were significantly higher in liver tissues from CSE knockout mice when compared with wild-type mice. Significance CSE/H2S system is indispensable for maintaining the homeostasis of acetyl-CoA and lipid accumulation and protecting from the development of inflammation and fibrosis in liver under excessive caloric ingestion.

Hydrogen sulfide (HHuman primary hepatocellular carcinoma cell line (HepG2) and doxorubicin (Dox)-resistant cells were used in this study. Cell survival was analyzed by MTT, Annexin V-FITC/propidium iodide staining and clonogenic assay. Western blotting was used for analysis of protein expression, and immunoprecipitation was used to determine interactions of LXR/RXR.The expression of HOur study provides a novel solution for reversing drug resistance in cancer cells by targeting H

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H

Aims: Hydrogen sulfide (H2S) inhibits the proliferation of vascular smooth muscle cells (VSMCs). However, how cystathionine-gamma-lyase (CSE), a major enzyme that produces H2S, is regulated remains unknown. Whether calcium-sensing receptor (CaSR) inhibits the proliferation of VSMCs by regulating the endogenous CSE/H2S pathway in diabetic rat has not been previously investigated. Methods and Results: The morphological and ultrastructure alterations were tested by transmission electron microscopy, changes in the H2S concentration and the relaxation of the mesenteric secondary artery loop of diabetic rats were determined by Multiskan spectrum microplate spectrophotometer and isometric force transducer. Additionally, the expression levels of CaSR, CSE and Cyclin D1 in the mesenteric arteries of rats were examined by western blotting. The intracellular calcium concentration, the expression of p-CaMK II (phospho-calmodulin kinases II), CSE activity, the concentration of endogenous H2S and the proliferation of cultured VSMCs from rat thoracic aortas were measured by using confocal microscope, western blotting, microplate spectrophotometer, MTT and BrdU, respectively. The VSMC layer thickened, the H2S concentration dropped, the relaxation of the mesenteric secondary artery rings weakened, and the expression of CaSR and CSE decreased whereas the expression of Cyclin D1 increased in diabetic rats compared with the control group. The [Ca2+]i of VSMCs increased upon treatment with CaSR agonists (10 µM Calindol and 2.5 mM CaCl2), while it decreased upon administration of calhex231, U73122 and 2-APB. The expression of p-CaMK II and CSE increased upon treatment with CaSR agonists in VSMCs. CSE activity and the endogenous H2S concentration decreased in response to high glucose, while it increased with treatment of CaSR agonists. The proliferation rate increased in response to high glucose, and CaSR agonists or NaHS significantly reversed the proliferation of VSMCs caused by high glucose. Conclusions: Our results demonstrated that CaSR regulated the endogenous CSE/H2S pathway to inhibit the proliferation of VSMCs in both diabetic and high glucose models.

The gasotransmitter role of H2S in mammalian has been extensively studied, and cystathionine gamma-lyase (CSE) is the major H2S-producing enzyme in vascular system. Dysregulation of CSE/H2S system was found in various pathophysiological conditions. MicroRNA (miRNA) and short interfering RNA (siRNA) are known to inhibit gene expression by mRNA degradation and/or translation repression. The regulation of CSE expression by miRNA and siRNA has been reported recently, but the off-target effect of miRNA and the lower knockdown efficiency of siRNA have shadowed the application of these approaches. In the present study, we designed CSE-specific miRNAs based on the rules of miRNA–mRNA complementary and human CSE cDNA sequence. The CSE-specific miRNAs significantly inhibited CSE expression and H2S production, increased reactive oxygen species generation, and induced proliferation of human aorta smooth muscle cells (HASMCs). The designed CSE-specific miRNAs specifically targeted on CSE gene as evidenced by the direct inhibition of luciferase activity from reporter gene containing human CSE 3′-UTR sequence. The expression of other genes, such as estrogen receptor α, heme oxygenase 1, specificity protein 1, and 3-mercaptopyruvate sulfurtransferase, was not affected by the CSE-specific miRNAs. Compared with CSE-siRNAs, CSE-specific miRNAs displayed significantly higher efficacy in suppressing CSE expression and H2S production. miR-143, a highly expressed miRNA in vascular system, down-regulated the expressions of CSE as well as other genes, such as insulin-like growth factor binding protein 5 and kruppel-like factor 4. miR-143 suppressed H2S production but had no effect on HASMC proliferation. In conclusion, CSE-specific miRNAs designed in our study provide a highly effective research tool for regulating CSE expression and H2S production. These CSE-specific miRNAs have potential as being novel therapeutic agents for treating vascular disorders related to abnormal oxidative stress and SMC growth.

Abnormal hepatic lipid metabolism is a key component of fatty liver development with excess fat deposition in the liver through steatosis. Cystathionine gamma-lyase (CSE) is one of the enzymes that catalyze hydrogen sulfide (H2S) production in the liver. The aim of the present study was to investigate the role of CSE/H2S in hepatic regulation of cholesterol and fatty acid metabolism. Wild-type (WT) and CSE knockout (CSE-KO) mice fed with high-fat diet (HFD) were analyzed for liver morphological and biochemical changes. HFD feeding of CSE-KO mice, not WT mice, markedly increased cholesterol levels in plasma and livers, and the sizes of the liver and gall bladder. Typical histological and biochemical changes of fatty liver disease were found in CSE-KO mice with damaged liver functions. The levels of plasma and liver triglyceride were significantly lower in HFD-fed CSE-KO mice than in HFD-fed WT mice. Moreover, the expression of nuclear receptors transcriptional factors, especially LXRα, in the liver was decreased in both control diet- or HFD-fed CSE-KO mice. Decreased expression of CYP7A1, an LXRα targeted gene, halted catabolism of cholesterol into bile and subsequently led to cholesterol accumulation in the liver and gall bladder. Since deficiency in CSE/H2S pathway results in high susceptibility to HFD-induced fatty liver, targeting at CSE/H2S pathway in the liver may represent a novel strategy against the development of fatty liver damage.

Hydrogen sulfide (H2S) has been recognized as an important gasotransmitter analogous to nitric oxide and carbon monoxide. Cystathionine gamma-lyase (CSE)-derived H2S is implicated in the regulation of insulin resistance and glucose metabolism, but the involvement of CSE/H2S system in energy homeostasis and fat mass has not been extensively explored. In this study, a potential functional role of the CSE/H2S system in in vitro adipocyte differentiation and in vivo adipogenesis and the underlying mechanism was investigated. CSE expression and H2S production were increased during adipocyte differentiation, and that the pattern of CSE mRNA expression was similar to that of CCAAT/enhancer-binding protein (C/EBP) β and δ, two key regulators for adipogenesis. C/EBPβ and γ bind to the CCAAT box in CSE promoter and stimulate CSE gene transcription. H2S induced PPARγ transactivation activity by S-sulfhydrating all the cysteine residues in the DNA binding domain and stimulated adipogenesis. High fat diet-induced fat mass was lost in CSE deficient mice, and exogenously applied H2S promoted fat mass accumulation in fruit flies. In conclusion, CSE/H2S system is essential for adipogenesis and fat mass accumulation through enhancement of PPARγ function in adipocytes. This study suggests that the CSE/H2S system is involved in the pathogenesis of obesity in mice.

Hydrogen sulfide (H

Background— Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H 2 S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H 2 S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H 2 S in atherosclerosis development. Methods and Results— Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N -acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. Conclusions— Endogenously synthesized H 2 S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H 2 S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H 2 S pathway is an important therapeutic target for protection against atherosclerosis.

Hydrogen sulfide (H2 S) and nitric oxide (NO) are major gasotransmitters produced in endothelial cells (ECs), contributing to the regulation of vascular contractility and structural integrity. Their interaction at different levels would have a profound impact on angiogenesis. Here, we showed that H2 S and NO stimulated the formation of new microvessels. Incubation of human umbilical vein endothelial cells (HUVECs-926) with NaHS (a H2 S donor) stimulated the phosphorylation of endothelial NO synthase (eNOS) and enhanced NO production. H2 S had little effect on eNOS protein expression in ECs. L-cysteine, a precursor of H2 S, stimulated NO production whereas blockage of the activity of H2 S-generating enzyme, cystathionine gamma-lyase (CSE), inhibited this action. CSE knockdown inhibited, but CSE overexpression increased, NO production as well as EC proliferation. LY294002 (Akt/PI3-K inhibitor) or SB203580 (p38 MAPK inhibitor) abolished the effects of H2 S on eNOS phosphorylation, NO production, cell proliferation and tube formation. Blockade of NO production by eNOS-specific siRNA or nitro-L-arginine methyl ester (L-NAME) reversed, but eNOS overexpression potentiated, the proliferative effect of H2 S on ECs. Our results suggest that H2 S stimulates the phosphorylation of eNOS through a p38 MAPK and Akt-dependent pathway, thus increasing NO production in ECs and vascular tissues and contributing to H2 S-induced angiogenesis.

H2S, a third member of gasotransmitter family along with nitric oxide and carbon monoxide, exerts a wide range of cellular and molecular actions in our body. Cystathionine gamma-lyase (CSE) is a major H2S-generating enzyme in our body. Aging at the cellular level, known as cellular senescence, can result from increases in oxidative stress. The aim of this study was to investigate how H2S attenuates oxidative stress and delays cellular senescence.Here we showed that mouse embryonic fibroblasts isolated from CSE knockout mice (CSE KO-MEFs) display increased oxidative stress and accelerated cellular senescence in comparison with MEFs from wild-type mice (WT-MEFs). The protein expression of p53 and p21 was significantly increased in KO-MEFs, and knockdown of p53 or p21 reversed CSE deficiency-induced senescence. Incubation of the cells with NaHS (a H2S donor) significantly increased the glutathione (GSH) level and rescued KO-MEFs from senescence. Nrf2 is a master regulator of the antioxidant response, and Keap1 acts as a negative regulator of Nrf2. NaHS S-sulfhydrated Keap1 at cysteine-151, induced Nrf2 dissociation from Keap1, enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream genes, such as glutamate-cysteine ligase and GSH reductase.These results provide a mechanistic insight into how H2S signaling mediates cellular senescence induced by oxidative stress.H2S protects against cellular aging via S-sulfhydration of Keap1 and Nrf2 activation in association with oxidative stress.

L-type voltage-dependent Ca(2+) channels (VDCCs) in pancreatic beta cells play a critical role in regulating insulin secretion. The gasotransmitter H(2)S is mostly generated from L-cysteine in pancreatic beta cells by cystathionine γ-lyase (CSE) and has been reported to inhibit insulin release by opening ATP-sensitive K(+) channels. However, whether and how H(2)S affects VDCCs in beta cells is unknown.The whole-cell patch-clamp technique was used to record VDCCs in beta cells from Cse (also known as Cth)-knockout (KO) and wild-type (WT) mice. Insulin secretion from pancreatic islets and endogenous H(2)S production in pancreas were measured.The H(2)S donor NaHS reversibly decreased L-type VDCC current density in a concentration-dependent fashion in WT pancreatic beta cells, and the current density was further inhibited by nifedipine. Furthermore, NaHS inhibited the channel recovery from depolarisation-induced inactivation, but did not shift the current-voltage (I-V) relationship. ACS67, another H(2)S donor, also inhibited L-type VDCCs in beta cells. Inhibiting CSE activity with DL-propargylglycine increased the basal L-channel activity of beta cells from WT mice, but not that of beta cells from Cse-KO mice. Beta cells from Cse-KO mice displayed higher L-type VDCC density than those from WT mice. Insulin secretion from pancreatic islets was elevated in Cse-KO mice compared with WT mice. NaHS dose-dependently inhibited glucose-stimulated insulin secretion, which was further inhibited by nifedipine. Bay K-8644 increased glucose-stimulated insulin secretion, but this was counteracted by NaHS and nifedipine.Exogenous and endogenous H(2)S inhibit L-type VDCC activity and pancreatic insulin secretion, constituting a novel mechanism for the regulation of insulin secretion by the CSE/H(2)S system.

Hydrogen sulfide (H(2)S) has emerged as an important gasotransmitter, offering protection against ischemia-reperfusion damage to the heart. Cystathionine gamma-lyase (CSE) is believed to be the major H(2)S-generating enzyme in the heart. Quite contrary to the general contemplation, CSE protein in cardiac tissues has not been convincingly detected and it has become an issue of controversy. In the present study, we isolated cardiac tissues from wild type (WT) and CSE knockout mice or the rat. CSE expression at transcriptional and translational levels were assayed by RT-PCR and Western Blotting with five different antibodies (four commercial products and one homemade), respectively. Cardiac H(2)S production rate was also examined. Our data validated the expression of CSE mRNA in the heart of WT mice or rats, not in CSE KO mice. Using all 5 different anti-CSE antibodies, we could not detect CSE proteins in mouse or rat cardiac tissues or in cultured rat cardiomyocytes. On the other hand, CSE protein was detectable in liver tissues from WT mice with the expected molecular mass of 43.6 kDa. H(2)S production rate of heart tissues in CSE KO mice was significantly decreased compared with that in WT mice. In the presence of an CSE inhibitor, D,L-propargylglycine, H(2)S production rate of heart tissues from WT mice was inhibited by approximately 80%. It appears that CSE mediates mostly endogenous H(2)S production in heart tissues. However, the available anti-CSE antibodies could not detect CSE proteins in rat and mouse heart tissues or rat cardiomyocytes.

Cystathionine gamma-lyase (CSE) is the major H(2)S-generating enzyme in vascular smooth muscle cells (SMCs). CSE/H(2)S system contributes to the maintenance of SMC phenotype, and transcript factor specificity protein-1 (SP1) is a critical regulator of CSE expression during SMC differentiation. The involvements of microRNA-21 (miR-21) in cardiovascular pathophysiology have been known, however miR-21 regulation of CSE and SP1 as well as SMC phenotype are uncertain. Using quantitative real-time PCR, we demonstrated that the expression of miR-21 was upregulated in dedifferentiated human aorta SMCs (HASMCs) and injured mouse carotid arteries. To determine the potential roles of miR-21 in SP1-mediated CSE gene expression and SMC phenotypic change, we showed that miR-21 expression was upregulated by miR-21 precursor. Interestingly, miR-21 overexpression significantly repressed the protein expressions of both CSE and SP1, inhibited H(2)S production, stimulated SMC proliferation, and reduced SMC differentiation marker gene expression, respectively. The mRNA expression of CSE but not SP1 was inhibited by miR-21 precursor. Blockage of SP1 binding by mithramycin or inhibition of CSE activity by DL-propargylglycine did not change miR-21 expression. We further demonstrated that miR-21 repressed SP1 protein expression by directly targeting at SP1 3' untranslational regions, which in turn downregulated CSE mRNA expression and stimulated SMC proliferation. Take together, these results suggest that miR-21 participates in CSE/H(2)S-mediated-SMC differentiation by targeting SP1.

The physiological and pathological roles of hydrogen sulfide (H(2)S) in the regulation of cardiovacular functions have been recognized. Vascular smooth muscle cells (SMCs) express cystathionine gamma-lyase (CSE) and produce significant amount of H(2)S. Although growing evidence demonstated the anti-atherosclerotic effect of H(2)S, less is known about the contribution of the endogenous CSE/H(2)S pathway to the development of vascular remodeling. This study investigated the roles of the CSE/H(2)S pathway on SMC migration and neoimtimal formation by using CSE knockout (KO) mice. SMCs and aortic explants isolated from CSE KO mice exhibited more migration and outgrowth compared with that from wild-type (WT) mice, and exogenously applied NaHS (a H(2)S donor) at 100 μM significantly inhibited SMC migration and outgrowth. SMCs became more elongated and spread in the absence of CSE, and fibronectin significantly stimulated adhesion and migration of SMCs from CSE KO mice (KO-SMCs) in comparison with SMCs from WT mice (WT-SMCs). The expressions of α5- and β1-integrins were significantly higher in KO-SMCs, and functional blocking of α5β1-integrin effectively abrogated KO-SMC migration. CSE deficiency also enhanced matrix metalloproteinase-2 (MMP-2) expression, and the selective blocking of MMP-2 decreased KO-SMC migration. NaHS treatment decreased both the expressions of α5- and β1-integrins and MMP-2. We further found that the expressions of α5- and β1-integrins as well as MMP-2, were stimulated by fibronectin, and that the blockage of α5β1-integrin reduced but overexpression of α5β1-integrin induced MMP-2 expression in both WT-SMCs and KO-SMCs. We also noticed that CSE deficiency in mice led to increased neointima formation in carotid arteries 4 weeks after ligation, which were attenuated by NaHS administration. In conclusion, inhibition of SMC migration by H(2)S may be a novel target for the treatment of vascular occlusive disorder.

Hydrogen sulfide (H(2)S) is a novel gasotransmitter that regulates cell proliferation and other cellular functions. Sulforaphane (SFN) is a sulfur-containing compound that exhibits anticancer properties, and young sprouts of broccoli are particularly rich in SFN. There is consistent epidemiological evidence that the consumption of sulfur-containing vegetables, such as garlic and cruciferous vegetables, may help reduce the occurrence of prostate cancer. Here we found that a large amount of H(2)S is released when SFN is added into cell culture medium or mixed with mouse liver homogenates, respectively. Both SFN and NaHS (a H(2)S donor) decreased the viability of PC-3 cells (a human prostate cancer cell line) in a dose-dependent manner, and supplement of methemoglobin or oxidized glutathione (two H(2)S scavengers) reversed SFN-reduced cell viability. We further found both cystathionine gamma-lyase (CSE) and cystathionine beta-synthase are expressed in PC-3 cells and mouse prostate tissues. H(2)S production in prostate tissues from CSE knockout mice was only 20% of that from wild-type mice, suggesting CSE is a major H(2)S-producing enzyme in prostate. CSE overexpression enhanced H(2)S production and inhibited cell viability in PC-3 cells. In addition, both SFN and NaHS activated p38 mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase (JNK). Pre-treatment of PC-3 cells with methemoglobin decreased SFN-stimulated MAPK activities. Suppression of both p38 MAPK and JNK reversed H(2)S- or SFN-reduced viability of PC-3 cells. Our results demonstrated that H(2)S mediates the inhibitory effect of SFN on the proliferation of PC-3 cells, which suggests that H(2)S-releasing diet or drug might be beneficial in the treatment of prostate cancer.

Reduced β-cell mass and increased activities of ATP-sensitive K(+) channels in pancreatic β cells are associated with the pathogenesis of diabetes. Cystathionine γ-lyase (CSE) is a major hydrogen sulfide (H(2)S)-producing enzyme in pancreatic β cells. Herein, we examine the effects of genetic and pharmacologic ablation of CSE on β-cell functions and their correlation with streptozotocin (STZ)-induced diabetes. Compared with wild-type mice, CSE knockout (CSE KO) mice that received STZ injections exhibited a delayed onset of diabetic status. The application of dl-propargylglycine (PPG) to inhibit CSE activity protected wild-type mice from STZ-induced hyperglycemia and hypoinsulinemia. STZ significantly increased pancreatic H(2)S production in wild-type mice but not in CSE KO mice. STZ induced more apoptotic β-cell death in wild-type mice than in CSE KO mice. STZ exposure decreased the viability of cultured INS-1E cells, which was partly reversed by PPG co-treatment. STZ also significantly stimulated H(2)S production in cultured INS-1E cells. In addition, STZ stimulated ATP-sensitive K(+) currents in pancreatic β cells from wild-type mice but not in the presence of PPG or in β cells from CSE KO mice. Sodium hydrosulfide injection instantly increased blood glucose, decreased plasma insulin, and deteriorated glucose tolerance in mice. Take together, these results provide evidence that the CSE/H(2)S system plays a critical role in regulating β-cell functions.

Cystathionine γ-lyase (CSE) catalyses the endogenous production of hydrogen sulphide (H(2)S) in pancreatic beta cells, and H(2)S has been shown to inhibit insulin release from these cells. As altered pancreatic H(2)S production modulated by glucose has been previously shown, we hypothesised that the Cse gene could be regulated by glucose level in insulin-secreting cells.The effects of glucose on CSE protein level and mRNA level were analysed in INS-1E cells. Glucose effect on Cse promoter activity was tested by constructing a proximal Cse promoter vector including specificity protein 1 (Sp1) consensus sequence.High glucose (20 mmol/l) inhibited H(2)S production in INS-1E cells and freshly isolated rat pancreatic islets. Cse mRNA expression, CSE activity and protein abundance were also profoundly reduced by high glucose. The involvement of SP1 in basal and high-glucose-regulated CSE production was demonstrated. Sp1-knockdown abolished a large portion of CSE production at basal glucose. Phosphorylation of SP1 stimulated by high glucose was inhibited by p38 mitogen-activated protein kinase (MAPK) inhibitors SB203580 and SB202190. After blocking p38 MAPK phosphorylation, the inhibitive effects of high glucose on CSE protein production and promoter activity in INS-1E cells were also virtually abolished.Glucose stimulates the phosphorylation of SP1 via p38 MAPK activation, which leads to decreased Cse promoter activity and subsequent downregulation of Cse gene expression. Inhibited H(2)S production through glucose-mediated CSE activity and production alterations may be involved in the fine control of glucose-induced insulin secretion.

Butyrate is a short-chain fatty acid that arrests growth of various types of cells. H(2)S can be endogenously produced by cystathionine gamma-lyase (CSE) or cystathionine beta-synthase (CBS) or both in colonic tissues. In this study, we observed endogenous H(2)S production in a colon cancer cell line (WiDr) and colonic tissues through the activity of both CSE and CBS. After 24 h of incubation of WiDr cells, butyrate increased cell production of H(2)S and upregulated CBS and CSE expressions. Both butyrate and NaHS (a H(2)S donor) decreased cell viability in a dose-dependent manner. Blockade of CBS, but not CSE, decreased butyrate-stimulated H(2)S production and reversed butyrate-inhibited cell viability. In addition, NaHS treatment stimulated the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), but not c-Jun N-terminal kinase (JNK). Inhibition of the phosphorylation of either p38 MAPK or ERK did not abolish NaHS-induced cell death. Butyrate treatment increased the phosphorylation of ERK, not p38 MAPK and JNK, but inhibition of ERK and p38 MAPK phosphorylation did not inhibit butyrate-reduced cell viability. In conclusion, butyrate regulates endogenous H(2)S production by stimulating CBS expression in colon cancer cells, but butyrate and H(2)S inhibit cancer cell growth through different mechanisms.

Hydrogen sulfide (H(2)S) has been traditionally known for its toxic effects on living organisms. The role of H(2)S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H(2)S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H(2)S production and insulin release were also assayed. It was found that H(2)S was generated in rat pancreas islets, catalyzed predominantly by cystathionine gamma-lyase (CSE). Pancreatic CSE expression and H(2)S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H(2)S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.

Aims: Hydrogen sulfide (H2S) exerts a wide range of actions in the body, especially in the modulation of mitochondrial functions. The normal replication of mitochondrial DNA (mtDNA) is critical for cellular energy metabolism and mitochondrial biogenesis. The aim of this study was to investigate whether H2S affects mtDNA replication and the underlying mechanisms. We hypothesize that H2S maintains mtDNA copy number via inhibition of Dnmt3a transcription and TFAM promoter methylation. Results: Here, we demonstrated that deficiency of cystathionine gamma-lyase (CSE), a major H2S-producing enzyme, reduces mtDNA copy number and mitochondrial contents, and it inhibits the expressions of mitochondrial transcription factor A (TFAM) and mitochondrial marker genes in both smooth muscle cells and aorta tissues from mice. Supply of exogenous H2S stimulated mtDNA copy number and strengthened the expressions of TFAM and mitochondrial marker genes. TFAM knockdown diminished H2S-enhanced mtDNA copy number. In addition, CSE deficiency induced the expression of DNA methyltransferase 3a (Dnmt3a) and TFAM promoter DNA methylation, and H2S repressed Dnmt3a expression, resulting in TFAM promoter demethylation. We further found that H2S S-sulfhydrates transcription repressor interferon regulatory factor 1 (IRF-1) and enhances the binding of IRF-1 with Dnmt3a promoter after reduced Dnmt3a transcription. H2S had little effects on the expression of Dnmt1 and Dnmt3b as well as on ten-eleven translocation methylcytosine dioxygenase 1, 2, and 3. Innovation: A sufficient level of H2S is able to inhibit TFAM promoter methylation and maintain mtDNA copy number. Conclusion: CSE/H2S system contributes to mtDNA replication and cellular bioenergetics and provides a novel therapeutic avenue for cardiovascular diseases. Antioxid. Redox Signal. 23, 630–642.

The physiological and biomedical importance of hydrogen sulfide (H2S) has been fully recognized in the cardiovascular system as well as in the rest of the body. In blood vessels, cystathionine γ-lyase (CSE) is a major H2S-producing enzyme expressed in both smooth muscle and endothelium as well as periadventitial adipose tissues. Regulation of H2S production from CSE is controlled by a complex integration of transcriptional, posttranscriptional, and posttranslational mechanisms in blood vessels. In smooth muscle cells, H2S regulates cell apoptosis, phenotypic switch, relaxation and contraction, and calcification. In endothelial cells, H2S controls cell proliferation, cellular senescence, oxidative stress, inflammation, etc. H2S interacts with nitric oxide and acts as an endothelium-derived relaxing factor and an endothelium-derived hyperpolarizing factor. H2S generated from periadventitial adipose tissues acts as an adipocyte-derived relaxing factor and modulates the vascular tone. Extensive evidence has demonstrated the beneficial roles of the CSE/H2S system in various blood vessel diseases, such as hypertension, atherosclerosis, and aortic aneurysm. The important roles signaling in the cardiovascular system merit further intensive and extensive investigation. H2S-releasing agents and CSE activators will find their great applications in the prevention and treatment of blood vessel-related disorders.

Hydrogen sulfide (H2S) is a highly diffusible gasotransmitter, that influence cellular and organ functions by a number of different mechanisms. Cystathionine gamma-lyase (CSE) is one major H2S-producing enzyme with L-cysteine as the main substrate in mammalian cells. Since the discovery of endogenously-produced H2S as a biological mediator, there has been an explosion of interest in CSE expression and regulation. CSE expression and activity and ultimately the amount of H2S synthesis is controlled by a complex integration of transcriptional, post-transcriptional and post-translational mechanisms. Considering the key role that CSE/H 2 S system plays in both health and diseases, a better understanding of the regulation of CSE/H 2 S system will help us to develop novel and more effective strategies to target CSE and alter H2S production inside cells. In this review, we summarize the altered expression and activity of CSE and abnormal H2S production in various pathophysiological conditions. The current knowledge on the signaling and regulatory pathways for CSE expression and H2S production are also elucidated. As such, our understanding of the pathogenesis of human diseases will be better achieved and the corresponding new therapy can be devised.

Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using Magnetic Twisting Cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H2S in the range of 10–275 µM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.

Hydrogen sulfide (H2S) is an endogenous gasotransmitter with physiologic functions similar to nitric oxide and carbon monoxide. Exogenous treatment with H2S can induce a reversible hypometabolic state, which can protect organs from ischemia/reperfusion injury, but whether cystathionine γ-lyase (CSE), which produces endogenous H2S, has similar protective effects is unknown. Here, human renal tissue revealed abundant expression of CSE, localized to glomeruli and the tubulointerstitium. Compared with wild-type mice, CSE knockout mice had markedly reduced renal production of H2S, and CSE deficiency associated with increased damage and mortality after renal ischemia/reperfusion injury. Treatment with exogenous H2S rescued CSE knockout mice from the injury and mortality associated with renal ischemia. In addition, overexpression of CSE in vitro reduced the amount of reactive oxygen species produced during stress. Last, the level of renal CSE mRNA at the time of organ procurement positively associated with GFR 14 days after transplantation. In summary, these results suggest that CSE protects against renal ischemia/reperfusion injury, likely by modulating oxidative stress through the production of H2S.

Mounting evidence has established hydrogen sulfide (H(2)S) as an important gasotransmitter with multifaceted physiological functions. The aim of the present study was to investigate the role of H(2)S on glucose utilization, glycogen synthesis, as well as gluconeogenesis in both HepG(2) cells and primary mouse hepatocytes. Incubation with NaHS (a H(2)S donor) impaired glucose uptake and glycogen storage in HepG(2) cells via decreasing glucokinase activity. Adenovirus-mediated cystathionine γ-lyase (CSE) overexpression increased endogenous H(2)S production and lowered glycogen content in HepG(2) cells. Glycogen content was significantly higher in liver tissues from CSE knockout (KO) mice compared to that from wild type (WT) mice in fed condition. Glucose consumption was less in primarily cultured hepatocytes isolated from WT mice than those from CSE KO mice, but more glucose was produced by hepatocytes via gluconeogenesis and glycogenolysis pathways in WT mice than in CSE KO mice. NaHS treatment reduced the phosphorylation of AMP-activated protein kinase, whereas stimulation of AMP-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside reversed H(2)S-impaired glucose uptake. H(2)S-increased glucose production was likely through increased phosphoenolpyruvate carboxykinase activity. In addition, insulin at the physiological range inhibited CSE expression, and H(2)S decreased insulin-stimulated phosphorylation of Akt in HepG(2) cells. CSE expression was increased, however, in insulin-resistant state induced by exposing cells to high levels of insulin (500 nm) and glucose (33 mm) for 24 h. Taken together, these data suggest that the interaction of H(2)S and insulin in liver plays a pivotal role in regulating insulin sensitivity and glucose metabolism.

Cystathionine γ-lyase (CSE) is one of the major enzymes producing hydrogen sulfide (H 2 S) in lungs, participating in the regulation of respiratory functions. The role of CSE-derived H 2 S in eosinophil-dominant inflammation in allergic diseases has been unclear. The objective of this study was to explore the protective role of H 2 S against allergen-induced airway hyperresponsiveness (AHR) and inflammation. CSE expression and H 2 S production rate were assessed in mouse lung tissues with ovalbumin (OVA)-induced acute asthma. AHR, airway inflammation, and Th2 response in wild-type (WT) mice were compared with those in CSE gene knockout (KO) mice. The effect of NaHS, an exogenous H 2 S donor, was also evaluated on these parameters. CSE expression was absent and H 2 S production rate was significantly lower in the lungs of CSE KO mice when compared with WT littermates. OVA challenge decreased lung CSE expression and H 2 S production in WT mice. CSE deficiency resulted in aggravated AHR, increased airway inflammation, and elevated levels of Th2 cytokines such as IL-5, IL-13, and eotaxin-1 in bronchoalveolar lavage fluid after OVA challenge. The aforementioned alterations were reversed by exogenous H 2 S treatment. More importantly, NaHS supplement rescued CSE KO mice from the aggravated pathological process of asthma. The CSE/H 2 S system plays a critical protective role in the development of asthma. A new therapeutic potential for asthma via targeting CSE/H 2 S metabolism is indicated.

In the study, we investigated how exogenous H(2)S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50 mg/kg) and the daily administration of 100 μM NaHS (sodium hydrosulfide, an H(2)S donor) in the diabetes + NaHS treatment group. At the end of 4, 8, and 12 weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H(2)S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.

Although many types of ancient bacteria and archea rely on hydrogen sulfide (H 2 S) for their energy production, eukaryotes generate ATP in an oxygen-dependent fashion. We hypothesize that endogenous H 2 S remains a regulator of energy production in mammalian cells under stress conditions, which enables the body to cope with energy demand when oxygen supply is insufficient. Cystathionine γ-lyase (CSE) is a major H 2 S-producing enzyme in the cardiovascular system that uses cysteine as the main substrate. Here we show that CSE is localized only in the cytosol, not in mitochondria, of vascular smooth-muscle cells (SMCs) under resting conditions, revealed by Western blot analysis and confocal microscopy of SMCs transfected with GFP-tagged CSE plasmid. After SMCs were exposed to A23187, thapsigargin, or tunicamycin, intracellular calcium level was increased, and CSE translocated from the cytosol to mitochondria. CSE was coimmunoprecipitated with translocase of the outer membrane 20 (Tom20) in mitochondrial membrane. Tom20 siRNA significantly inhibited mitochondrial translocation of CSE and mitochondrial H 2 S production. The cysteine level inside mitochondria is approximately three times that in the cytosol. Translocation of CSE to mitochondria metabolized cysteine, produced H 2 S inside mitochondria, and increased ATP production. Inhibition of CSE activity reversed A23187-stimulated mitochondrial ATP production. H 2 S improved mitochondrial ATP production in SMCs with hypoxia, which alone decreased ATP production. These results suggest that translocation of CSE to mitochondria on specific stress stimulations is a unique mechanism to promote H 2 S production inside mitochondria, which subsequently sustains mitochondrial ATP production under hypoxic conditions.

The integrated stress response mediated by eukaryotic translation initiation factor 2α (eIF2α) phosphorylation maintains cellular homeostasis under endoplasmic reticulum (ER) stress. eIF2α phosphorylation induces activating transcription factor 4 (ATF4), a basic leucine zipper transcription factor that regulates the expression of genes responsible for amino acid metabolism, cellular redox state, and anti-stress responses. Cystathionine γ-lyase (CSE) and cystathionine β-synthase are critical enzymes in the transsulfuration pathway, which also regulate cellular redox status by modulating glutathione (GSH) levels. To determine the link between the integrated stress response and the transsulfuration pathway, we used homocysteine (Hcy) as an inducer of eIF2α phosphorylation and ATF4 gene induction. Mouse embryonic fibroblasts (MEFs) lacking ATF4 (ATF4(-/-)) had reduced GSH levels and increased reactive oxygen species and were susceptible to apoptotic cell death under normal culture conditions. Further, ATF4(-/-) MEFs were more sensitive to Hcy-induced cytotoxicity and showed significantly reduced intracellular GSH levels associated with apoptosis. ATF4(-/-) MEFs could be rescued from l-Hcy-induced apoptosis by β-mercaptoethanol medium supplementation that increases cysteine levels and restores GSH synthesis. ATF4(-/-) MEFs showed little or no CSE protein but did express cystathionine β-synthase. Further, ER stress-inducing agents, including tunicamycin and thapsigargin, induced the expression of CSE in ATF4(+/+) MEFs. Consistent with ATF4(-/-) MEFs, CSE(-/-) MEFs showed significantly greater apoptosis when treated with tunicamycin, thapsigargin, and l-Hcy, compared with CSE(+/+) MEFs. Liver and kidney GSH levels were also reduced in CSE(-/-) mice, suggesting that CSE is a critical factor in GSH synthesis and may act to protect the liver and kidney from a variety of conditions that cause ER stress.

Hydrogen sulfide (H(2)S) can be endogenously generated from cystathionine gamma-lyase (CSE) in cardiovascular system, offering a cardiovascular protection. It is also known that the lower risk of cardiovascular diseases in female is partially attributed to the protective effect of estrogen. The current study explores the interaction of H(2)S and estrogen on smooth muscle cell (SMC) growth. In the present study, we found that the proliferation of cultured vascular SMCs isolated from wild-type mice (WT-SMCs) was inhibited, but that from CSE gene knockout mice (CSE-KO-SMCs) increased, by estrogen treatments. The expression of estrogen receptor α (ERα), but not ERβ, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H(2)S markedly increased ERα but not ERβ expression. In addition, the inhibition of ER activation and knockdown of ERα expression in WT-SMCs or the overexpression of ERα in CSE-KO-SMCs reversed the respective effects of estrogen on cell proliferation. The expression of cyclin D1 was reduced in WT-SMCs but increased in CSE-KO-SMCs after estrogen treatments, which was reversed by knockdown of ERα in WT-SMCs or overexpression of ERα in CSE-KO-SMCs, respectively. The overexpression of cyclin D1 in WT-SMCs or knockdown of cyclin D1 expression in CSE-KO-SMCs reversed the effects of estrogen on cell proliferation. These results suggest that H(2)S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERα/cyclin D1 pathways.

Hydrogen sulfide (H(2)S) plays a crucial role in human and animal physiology. Its ubiquity and versatile properties have recently caught the attention of plant physiologists and biochemists. Two cysteine desulfhydrases (CDes), L-cysteine desulfhydrase and D-cysteine desulfhydrase, were identified as being mainly responsible for the degradation of cysteine in order to generate H(2)S. This study investigated the expression regulation of these genes and their relationship to drought tolerance in Arabidopsis. First, the expression pattern of CDes in Arabidopsis was investigated. The expression levels of CDes gradually increased in an age-dependent manner. The expression of CDes was significantly higher in stems and cauline leaves than in roots, rosette leaves and flowers. Second, the protective effect of H(2)S against drought was evaluated. The expression pattern of CDes was similar to the drought associated genes induced by dehydration, and H(2)S fumigation was found to stimulate further the expression of drought associated genes. Drought also significantly induced increased H(2)S production, a process that was reversed by re-watering. In addition, seedlings after treatment with NaHS (a H(2)S donor) showed a higher survival rate and displayed a significant reduction in the size of the stomatal aperture compared to the control. These findings provide evidence that H(2)S, as a gasotransmitter, improves drought resistance in Arabidopsis.

Cystathionine γ-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H(2)S. Phenotypic switching of SMCs is affected by endogenous H(2)S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H(2)S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H(2)S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to +140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H(2)S at 100 μM stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H(2)S system is essential for maintenance of SMC phenotype.

Cystathionine gamma-lyase (CSE)-derived H2S plays an important role in regulating cell growth. Lack of CSE expression results in development of hypertension. The current study compared proliferation of smooth muscle cells derived from CSE gene knockout mice (SMCs-KO) with that of wild-type mice (SMCs-WT). Methods and results Cell proliferation was assessed by bromodeoxyuridine incorporation. Gene expression was analysed by western blot- ting, real-time PCR, and microarray analysis. Enhanced cell proliferation was detected in SMCs-KO and in the media of the aorta from CSE KO mice. SMCs-KO underwent significantly more apoptosis than SMCs-WT when treated with exogenous H2S (100 mM). CSE KO mice showed much lower level of phosphorylated extracellular signal- regulated kinase (ERK1/2) in mesentery arteries compared with those of WT mice, and exogenous H2S induced more phosphorylation of ERK1/2 in SMCs-KO compared with that in SMCs-WT. Decreased p21 Cip/WAF-1 but increased cyclin D1 expression was observed in isolated SMCs and vascular tissues from CSE KO mice, and exogen- ous H2S caused more increase in p21 Cip/WAF-1 expression and more decrease in cyclin D1 expression in SMCs-KO than in SMCs-WT. The transcriptional expression of calcitonin receptor-like, intergrin beta 1, and heparin-binding epidermal growth factor-like growth factor was also significantly increased in the aorta of CSE KO mice. Conclusion SMCs-KO display an increased proliferation rate in vitro and in vivo, and these cells are more susceptible to apoptosis induced by exogenous H2S at physiologically relevant concentrations. These cellular effects of H2S are mediated by phosphorylation of ERK1/2 and altered expression of cyclin D1 and p21 Cip/WAF-1 .

Studies of nitric oxide over the past two decades have highlighted the fundamental importance of gaseous signaling molecules in biology and medicine. The physiological role of other gases such as carbon monoxide and hydrogen sulfide (H 2 S) is now receiving increasing attention. Here we show that H 2 S is physiologically generated by cystathionine γ-lyase (CSE) and that genetic deletion of this enzyme in mice markedly reduces H 2 S levels in the serum, heart, aorta, and other tissues. Mutant mice lacking CSE display pronounced hypertension and diminished endothelium-dependent vasorelaxation. CSE is physiologically activated by calcium-calmodulin, which is a mechanism for H 2 S formation in response to vascular activation. These findings provide direct evidence that H 2 S is a physiologic vasodilator and regulator of blood pressure.

O2chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine β-synthase and cystathionine γ-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a Po2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar ∼10-mV depolarization. These studies are consistent with H2S involvement in O2sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to Po2and may provide an exquisitely sensitive, yet simple, O2sensor in a variety of tissues.

Endogenous hydrogen sulfide (H(2)S), generated from homocysteine metabolism mainly catalyzed by cystathionine gamma-lyase (CSE), possesses important functions in the cardiovascular system. In this study, we investigated the role of H(2)S during the pathogenesis of neointimal formation induced by balloon injury in rats. CSE mRNA levels were reduced by 86.5% at 1 week and 64.0% at 4 weeks after balloon injury compared with the uninjured controls. CSE activity was also correspondingly reduced. Endogenous production of H(2)S in the injured carotid artery was significantly inhibited at 1 week and 4 weeks after balloon injury. Treatment with NaHS (a donor of H(2)S) enhanced methacholine-induced vasorelaxation of balloon-injured artery. More importantly, treatment with NaHS significantly inhibited neointima formation (0.15 +/- 0.01 mm(2) versus 0.21 +/- 0.01 mm(2), P0.001) of the balloon-injured carotid arteries and reduced the intima/media ratio (1.05 +/- 0.07 versus 1.43 +/- 0.06, P0.001). A significant decrease in vascular smooth muscle cell proliferation was demonstrated by bromodeoxyuridine incorporation at day 7 after injury. In conclusion, CSE expression and H(2)S production are reduced during the development of balloon injury-induced neointimal hyperplasia, and treatment with NaHS significantly reduces neointimal lesion formation.

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 microm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 microm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival.

Hydrogen sulfide (H2S) is a highly diffusible gasotransmitter, that influence cellular and organ functions by a number of different mechanisms. Cystathionine gamma-lyase (CSE) is one major H2S-producing enzyme with L-cysteine as the main substrate in mammalian cells. Since the discovery of endogenously-produced H2S as a biological mediator, there has been an explosion of interest in CSE expression and regulation. CSE expression and activity and ultimately the amount of H2S synthesis is controlled by a complex integration of transcriptional, post-transcriptional and post-translational mechanisms. Considering the key role that CSE/H 2 S system plays in both health and diseases, a better understanding of the regulation of CSE/H 2 S system will help us to develop novel and more effective strategies to target CSE and alter H2S production inside cells. In this review, we summarize the altered expression and activity of CSE and abnormal H2S production in various pathophysiological conditions. The current knowledge on the signaling and regulatory pathways for CSE expression and H2S production are also elucidated. As such, our understanding of the pathogenesis of human diseases will be better achieved and the corresponding new therapy can be devised.