Trophoblast H2S Maintains Early Pregnancy via Regulating Maternal-Fetal Interface Immune Hemostasis.

CONTEXT: Dysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive. OBJECTIVE: To determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion. DESIGN: First trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo. RESULTS: CBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors. CONCLUSION: CBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

Inhibition of hydrogen sulfide production by gene silencing attenuates inflammatory activity of LPS-activated RAW264.7 cells.

Hydrogen sulfide is an inflammatory mediator and is produced by the activity of the enzyme cystathionine γ-lyase (CSE) in macrophages. Previously, pharmacological inhibition of CSE has been reported to have conflicting results, and this may be due to the lack of specificity of the pharmacological agents. Therefore, this study used a very specific approach of small interfering RNA (siRNA) to inhibit the production of the CSE in an in vitro setting. We found that the activation of macrophages by lipopolysaccharide (LPS) resulted in higher levels of CSE mRNA and protein as well as the increased production of proinflammatory cytokines and nitric oxide (NO). We successfully used siRNA to specifically reduce the levels of CSE mRNA and protein in activated macrophages. Furthermore, the levels of proinflammatory cytokines in LPS-activated macrophages were significantly lower in siRNA-transfected cells compared to those in untransfected controls. However, the production levels of NO by the transfected cells were higher, suggesting that CSE activity has an inhibitory effect on NO production. These findings suggest that the CSE enzyme has a crucial role in the activation of macrophages, and its activity has an inhibitory effect on NO production by these cells.

Hydrogen sulfide is an endogenous potentiator of T cell activation.

H(2)S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H(2)S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H(2)S production. Consistent with its increased production, H(2)S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H(2)S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H(2)S potentiate TCR-induced activation. Nanomolar levels of H(2)S (50-500 nM) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H(2)S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nM (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H(2)S via increased expression of cystathionine γ-lyase and cystathionine ß-synthase. Disrupting this response by silencing these H(2)S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nM H(2)S. Thus, H(2)S represents a novel autocrine immunomodulatory molecule in T cells.

Down-regulation of endogenous hydrogen sulphide pathway in nasal mucosa of allergic rhinitis in guinea pigs.

BACKGROUND: The present study was designed to explore the possible changes in endogenous hydrogen sulphide (H(2)S), a novel gasotransmitter, on the pathogenesis of allergic rhinitis (AR). METHODS: AR guinea pig model was established by nasal ovalbumin sensitisation. Guinea pigs were divided into four groups: Saline control, AR sensitised, sodium hydrosulphide (NaHS) treated, and propargylglycine (PPG) treated group. The frequency of sneezing and nose rubbing was recorded. Leukocyte infiltration in nasal lavage fluid (NLF) and plasma H(2)S level were measured. Expression of Cystathionine-beta-synthase (CBS) and Cystathionine-gamma-lyase (CSE) mRNA as H(2)S-producing enzymes in nasal mucosa was determined by real time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). RESULTS: The frequency of sneezing and nose rubbing, and levels of leukocyte infiltration in NLF were higher than those of control (P<0.01), but plasma H(2)S in sensitised guinea pigs was lower than those of control (P<0.05). From the results of RT-PCR, it was found that the expression of CSE was higher than CBS in nasal mucosa, and in sensitised guinea pigs it was lower than that of control (P<0.05). NaHS successfully increased the level of H(2)S and alleviated the symptoms of AR accompanied by up-regulation of CSE as compared with AR group (P<0.05). PPG significantly suppressed the expression of CSE and decreased the H(2)S level, yet also aggravated the symptoms of AR. CONCLUSION: H(2)S level may be negatively correlated with the process of inflammation and positively correlated with expression of CSE in nasal mucosa. The endogenous H(2)S pathway is down-regulated in AR.

Age-associated oxidative damage leads to absence of gamma-cystathionase in over 50% of rat lenses: relevance in cataractogenesis.

Oxidative damage to lens proteins and glutathione depletion play a major role in the development of senile cataract. We previously found that a deficiency in gamma-cystathionase activity may be responsible for glutathione depletion in old lenses. The aims of this study were: (1) to investigate the mechanism that causes the age-related deficiency in gamma-cystathionase activity in the eye lens, and (2) to determine the role of gamma-cystathionase deficiency in cataractogenesis. Two populations of old rats were found, one (56%) whose lenses lacked gamma-cystathionase activity and the rest that exhibited detectable enzyme activity. gamma-Cystathionase protein was absent in lenses from old rats without gamma-cystathionase activity. Oxidative stress targeted gamma-cystathionase in the eye lens upon aging, since the enzyme contained more carbonyl groups in old lenses than in young ones. gamma-Cystathionase mRNA was also markedly reduced in old lenses, thus contributing to the age-associated deficiency in gamma-cystathionase. Inhibition of gamma-cystathionase activity caused glutathione depletion in lenses and led to cataractogenesis in vitro. In conclusion, the lack of gamma-cystathionase activity in over 50% of old lenses is due to decreased gene expression and proteolytic degradation of the oxidized enzyme. This results in a high risk for the development of senile cataract.

Murine cystathionine gamma-lyase: complete cDNA and genomic sequences, promoter activity, tissue distribution and developmental expression.

Cystathionine gamma-lyase (CSE) is the last key enzyme in the trans-sulphuration pathway for biosynthesis of cysteine from methionine. Cysteine could be provided through diet; however, CSE has been shown to be important for the adequate supply of cysteine to synthesize glutathione, a major intracellular antioxidant. With a view to determining physiological roles of CSE in mice, we report the sequence of a complete mouse CSE cDNA along with its associated genomic structure, generation of specific polyclonal antibodies, and the tissue distribution and developmental expression patterns of CSE in mice. A 1.8 kb full-length cDNA containing an open reading frame of 1197 bp, which encodes a 43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb mouse genomic fragment was obtained by lambda genomic library screening. It contained promoter regions, 12 exons, ranging in size from 53 to 579 bp, spanning over 30 kb, and exon/intron boundaries that were conserved with rat and human CSE. The GC-rich core promoter contained canonical TATA and CAAT motifs, and several transcription factor-binding consensus sequences. The CSE transcript, protein and enzymic activity were detected in liver, kidney, and, at much lower levels, in small intestine and stomach of both rats and mice. In developing mouse liver and kidney, the expression levels of CSE protein and activity gradually increased with age until reaching their peak value at 3 weeks of age, following which the expression levels in liver remained constant, whereas those in kidney decreased significantly. Immunohistochemical analyses revealed predominant CSE expression in hepatocytes and kidney cortical tubuli. These results suggest important physiological roles for CSE in mice.

Identification of probasin-related antigen as cystathionine gamma-lyase by molecular cloning.

We reported previously that a monoclonal antibody against probasin (rat prostatic secretory protein) recognizes a 40-kDa protein localized in rat liver and kidney. The protein (probasin-related antigen, PRB-RA) may participate in a specific differentiated function of these tissues. To clarify the molecular nature of PRB-RA, a series of cDNA clones coding for the protein were isolated from a rat liver expression library using an affinity-purified polyclonal antibody. The amino acid sequence deduced from the determined cDNA sequence included sequences identical with those of proteolytic fragments of PRB-RA, which covered about 70% of the deduced sequence. Northern blot hybridization of poly(A)+ RNA isolated from rat tissues showed the presence of predominant and minor mRNA species of about 2.0 and 4.3 kilobases, respectively, in the liver and kidney. A sequence homology search revealed that PRB-RA is almost completely identical to rat cystathionine gamma-lyase (cystathionase) and that it does not show overall homology with probasin. Three candidates for an epitope common to probasin and PRB-RA were found on close examination of the amino acid sequences of the two proteins. A synthetic peptide, TYFRRI, corresponding to one of the candidates, neutralized the reactivity of the anti-probasin monoclonal antibody to both probasin and PRB-RA on Western blot analysis. These results show that PRB-RA/cystathionase is neither structurally nor functionally related to probasin except for a common epitope and that cystathionase, a cystein-producing enzyme, is localized in urinary tubular epithelial cells in a highly restricted region of the kidney in addition to in liver parenchymal cells.

Cystathionine gamma-lyase of Saccharomyces cerevisiae: structural gene and cystathionine gamma-synthase activity.

Purification of Saccharomyces cerevisiae cystathionine gamma-lyase (gamma-CTLase) was hampered by the presence of a protein migrating very close to it in various types of column chromatography. The enzyme and the contaminant were nevertheless separated by polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that they are coded for by CYS3 (CYI1) and MET17 (MET25), respectively, leading to the conclusion that CYS3 is the structural gene for gamma-CTLase and that the contaminant is O-acetylserine/O-acetylhomoserine sulfhydrylase (OAS/OAH SHLase). Based on these findings, we purified gamma-CTLase by the following strategy: (1) extraction of OAS/OAH SHLase from a CYS3-disrupted strain; (2) preparation of antiserum against it; (3) identification of a strain devoid of the OAS/OAH SHLase protein using this antiserum; and (4) extraction of gamma-CTLase from this strain. Purified gamma-CTLase had cystathionine gamma-synthase (gamma-CTSase) activity if O-succinylhomoserine, but not O-acetylhomoserine, was used as substrate. From this notion we discuss the evolutional relationship between S. cerevisiae gamma-CTLase and Escherichia coli gamma-CTSase.

[Gamma-cystathionase: the non-ideal gel filtration high performance liquid chromatography. Physico-chemical and immunochemical characteristics of the enzyme]. / Gamma-tsistationaza: vysokoéffektivnaia zhidkostnaia khromatografiia v rezhime neideal'noi gel'-fil'tratsii. Fiziko-khimicheskaia i immunokhimicheskaia kharakteristika fermenta.

The rapid method for gamma-cystathionase purification was developed. It is based on the non-ideal gel filtration HPLC. The isolated homogeneous enzyme was used for immunization and immunosorbent preparation. A monospecific polyclonal antibody was prepared. The substrate specificity of the isolated enzyme was studied.

Use of monoclonal antibody to increase sensitivity and specificity in quantitative immunodiffusion assays.

A mouse monoclonal antibody has been produced which recognizes human liver gamma-cystathionase, Radioiodination of the monoclonal antibody facilitated its use in combination with non-specific precipitating rabbit antisera in classical immunodiffusion assays. The technique may have broad applicability in the detection and quantitation of rare antigens which are difficult to purify but easily recognizable in immunodiffusion assays. It may also be used for the initial detection of monoclonal antibodies to unique antigens of interest.

Reduced gamma-cystathionase protein content in human malignant leukemia cell lines as measured by immunoassay with monoclonal antibody.

A murine hybrid cell line has been produced which secretes immunoglobulin G2b with specificity for human gamma-cystathionase (EC 4.2.1.15). The antibody has been iodinated and used in combination with quantitative immunoelectrophoresis in an assay which is capable of detecting as little as 1.5 ng enzyme protein. Human lymphoblastic leukemia cell lines CEM and Laz-221 contain undetectable enzyme protein, corresponding to their behavior as cysteine auxotrophs. In contrast, nonmalignant lymphoblastoid lines contain easily detectable enzyme protein which correlates with their behavior as cysteine prototrophs. Other malignant leukocyte cell lines contained detectable but variable amounts of enzyme protein, suggesting that the enzyme may be a useful marker of cellular differentiation.

L-cyst(e)ine requirements of malignant cells and progress toward depletion therapy.

The L-cyst(e)ine requirements of normal and malignant cells are reviewed and expanded within the context of establishing whether the measurement of gamma-cystathionase levels constitutes a predictive test for tumor sensitivity to L-cyst(e)ine depletion. The ability of both purified L-cysteine desulfhydrase and gamma-cystathionase to inhibit the growth of the L-cystine-dependent L1210 leukemia in culture is presented, as well as approaches to circumvent the limitations of these enzymes for in vivo therapy. The ability of proparagylglycine to inhibit L-cysteine biosynthesis in vivo is reviewed for its possible use in combination therapy. In addition, the ability of poly D,L-alanine modification of Escherichia coli L-asparaginase to increase the plasma half-life in mice tenfold as well as to decrease the immunogenicity of the enzyme is presented.

Vitamin B6-responsive and -unresponsive cystathioninuria: two variant molecular forms.

Cystathionase activity in a lymphoid cell line extracts from a vitamin B6-responsive patient with cystathioninuria was increased strikingly by pyridoxal phosphate. Immunodiffusion with antiserum to human hepatic cystathionase showed identity between this cystathionase protein and cystathionase from an extract of normal lymphoid cells. Neither an increase in cystathionase activity nor immunochemical identity was found using extract of cells from a B6-unresponsive patient.