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• W2001860318 abstract "Cystathionine γ-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 μm) 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. Cystathionine γ-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 μm) 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. Cystathionine γ-lyase (CSE, 5The abbreviations used are: CSE, cystathionine γ-lyase; Ad-CSE, adenovirus containing CSE gene; Ad-lacZ, adenovirus containing β-galactosidase gene; CBS, cystathionine β-synthase; DTT, dithiothreitol; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HASMC, human aorta smooth muscle cell; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; m.o.i., multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PPG, dl-propargylglycine; siRNA, short-interfering RNA; SREBP-1c, sterol regulatory element-binding protein-1c; TUNEL, terminal deoxyribonucleotidyltransferase-mediated dUTP nick-end labeling.5The abbreviations used are: CSE, cystathionine γ-lyase; Ad-CSE, adenovirus containing CSE gene; Ad-lacZ, adenovirus containing β-galactosidase gene; CBS, cystathionine β-synthase; DTT, dithiothreitol; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HASMC, human aorta smooth muscle cell; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; m.o.i., multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); PPG, dl-propargylglycine; siRNA, short-interfering RNA; SREBP-1c, sterol regulatory element-binding protein-1c; TUNEL, terminal deoxyribonucleotidyltransferase-mediated dUTP nick-end labeling. EC 4.4.1.1) is a key pyridoxal 5′-phosphate-dependent enzyme in the trans-sulfuration pathway, which uses l-cysteine to produce hydrogen sulfide (H2S), a novel and important gasotransmitter (1.Erickson P.F. Maxwell I.H. Su L.J. Baumann M. Glode L.M. Biochem. J. 1990; 269: 335-340Crossref PubMed Scopus (111) Google Scholar, 2.Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1516) Google Scholar, 3.Wang R. Antioxid. Redox. Signal. 2003; 5: 493-501Crossref PubMed Scopus (416) Google Scholar). Endogenous productions of H2S in different organs and tissues as well as the circulatory concentration of H2S have been elucidated, and the physiological importance of H2S has gained increasing recognition (2.Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1516) Google Scholar, 3.Wang R. Antioxid. Redox. Signal. 2003; 5: 493-501Crossref PubMed Scopus (416) Google Scholar, 4.Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1614) Google Scholar, 5.Zhao W. Ndisang J.F. Wang R. Can. J. Physiol. Pharmacol. 2003; 81: 848-853Crossref PubMed Scopus (202) Google Scholar).Diabetes is a spectrum of clinical conditions arising from relative or absolute insulin deficiency with decreased functional beta cell mass (6.Mathis D. Vence L. Benoist C. Nature. 2001; 414: 792-798Crossref PubMed Scopus (755) Google Scholar). Any change in beta cell mass must reflect an imbalance between proliferation (neogenesis or replication) and cell death (necrosis or apoptosis) (7.Scaglia L. Cahill C.J. Finegood D.T. Bonner-Weir S. Endocrinology. 1997; 138: 1736-1741Crossref PubMed Scopus (318) Google Scholar). Excessive loss of beta cells constitutes one of the causes of diabetes, and apoptosis is considered to be the main mode of beta cell death in type I and type II diabetes (8.Lipson K.L. Fonseca S.G. Urano F. Curr. Mol. Med. 2006; 6: 71-77Crossref PubMed Scopus (56) Google Scholar). In recent years, pathophysiological implications of the CSE/H2S system in diabetes have been reported (9.Jia X. Yang W. Jakic Z. Wang R. Wu L. Can. J. Cardiol. 2004; 20: 56Google Scholar, 10.Yusuf M. Kwong-Huat B.T. Hsu A. Whiteman M. Bhatia M. Moore P.K. Biochem. Biophys. Res. Commun. 2005; 333: 1146-1152Crossref PubMed Scopus (154) Google Scholar). Endogenous production of H2S together with the expression of CSE and cystathionine β-synthase (CBS), another H2S-producing enzyme, was identified in rat pancreatic tissues (9.Jia X. Yang W. Jakic Z. Wang R. Wu L. Can. J. Cardiol. 2004; 20: 56Google Scholar, 10.Yusuf M. Kwong-Huat B.T. Hsu A. Whiteman M. Bhatia M. Moore P.K. Biochem. Biophys. Res. Commun. 2005; 333: 1146-1152Crossref PubMed Scopus (154) Google Scholar). CSE mRNA expression and H2S formation in the rat pancreas were significantly increased after diabetes induction by streptozotocin injection (10.Yusuf M. Kwong-Huat B.T. Hsu A. Whiteman M. Bhatia M. Moore P.K. Biochem. Biophys. Res. Commun. 2005; 333: 1146-1152Crossref PubMed Scopus (154) Google Scholar). Pancreatic H2S level in Zuker diabetic fatty rats was significantly higher than that in nondiabetic Zuker fatty rats (9.Jia X. Yang W. Jakic Z. Wang R. Wu L. Can. J. Cardiol. 2004; 20: 56Google Scholar). Inhibition of CSE activity by dl-propargylglycine (PPG) significantly decreased production of H2S and increased plasma insulin levels in Zuker diabetic fatty rats (9.Jia X. Yang W. Jakic Z. Wang R. Wu L. Can. J. Cardiol. 2004; 20: 56Google Scholar). Increased CSE and CBS activity or decreased homocysteine levels in diabetic animals and human have also been observed in different laboratories (10.Yusuf M. Kwong-Huat B.T. Hsu A. Whiteman M. Bhatia M. Moore P.K. Biochem. Biophys. Res. Commun. 2005; 333: 1146-1152Crossref PubMed Scopus (154) Google Scholar, 11.Veldman B.A. Vervoort G. Blom H. Smits P. Diabete Med. 2005; 22: 301-305Crossref PubMed Scopus (30) Google Scholar, 12.Wijekoon E.P. Hall B. Ratnam S. Brosnan M.E. Zeisel S.H. Brosnan J.T. Diabetes. 2005; 54: 3245-3251Crossref PubMed Scopus (80) Google Scholar, 13.Ratnam S. Maclean K.N. Jacobs R.L. Brosnan M.E. Kraus J.P. Brosnan J.T. J. Biol. Chem. 2002; 277: 42912-42918Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In vitro studies showed that incubation of INS-1E cells (an insulin-secreting beta cell line) with H2S drastically decreased insulin secretion because of the activation of plasma ATP-sensitive potassium (KATP) channels (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). Given the importance of pancreatic β cell mass for the pathogenesis of diabetes and altered endogenous pancreatic production of H2S in diabetes, it becomes imperative to understand whether the apoptotic status of pancreatic β cells is regulated by H2S and the underlying signaling cascade.In this study, we overexpressed the CSE gene in INS-1E cells using a highly effective replication-deficient adenovirus expression system. The successful adenovirus-mediated overexpression of CSE was confirmed by measuring CSE protein levels and endogenous H2S production. Cell survival was examined and compared after the increased CSE expression. Whether CSE overexpression-induced changes in cell survival were because of overproduced H2S was determined and compared with the effect of exogenously applied H2S. Finally, the involvement of the mitogen-activated protein kinase (MAPK) pathway and endoplasmic reticulum (ER) stress on the effect of the CSE/H2S system on INS-1E cell apoptosis was examined. Our findings support the hypothesis that H2S plays a fundamental role in regulating pancreatic β cell apoptosis.EXPERIMENTAL PROCEDURESCell Culture−INS1-E cells derived from a rat insulinoma (kindly provided by Dr. C. B. Wollheim, Geneva, Switzerland) were cultured with RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum, 1 mm sodium pyruvate, 50 μm 2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin, as described previously (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). The experiments were performed when the cells reached 70-80% confluence between passages 56 and 70. In all studies, cells were first incubated in the serum-free medium for 12 h and then 10% serum added together with different treatments.Construction of a Recombinant CSE Adenovirus and Gene Transfer−Recombinant adenovirus containing the CSE gene (Ad-CSE) was prepared as described previously (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). The recombinant adenovirus encoding bacterial β-galactosidase (Ad-lacZ) derived from the same vector was used as control. For adenoviral transfection, subconfluent INS-1E cells were incubated with Ad-CSE or Ad-lacZ at 50 multiplicities of infection (m.o.i.) in serum-free media (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). After 4 h of incubation, media were removed, and cells were incubated in appropriate media for another 44 h.Cellular Viability Assays−Cell viabilities were assessed based on conversion of trazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, according to manufacturer’s instruction (CellTiter96®, Promega, Madison, WI). Briefly, cells at equal number were plated onto each well of 96-well plates for 24 h. At different time points after treatment with different concentrations of H2S or transfection with adenovirus, 0.5 mg/ml MTT was added to each well. The cells were then cultured at 37 °C for 4 h, and absorbance of formazan products at 570 nm was measured in a Multiskan spectrum microplate spectrophotometer (Thermo Labsystems, Frankin, MA). The cells incubated with control medium were considered 100% viable.Apoptosis Assays−Apoptosis was monitored by measuring nuclear condensation by Hoechst 33258 staining and DNA fragmentation by the terminal deoxyribonucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), and DNA electrophoresis was performed on 1.8% agarose gel containing 0.5 μg/ml ethidium bromide as described previously (15.Yang G. Sun X. Wang R. FASEB J. 2004; 18: 1782-1784Crossref PubMed Scopus (262) Google Scholar).GSH Level Measurement−GSH levels were determined by using the high pressure liquid chromatography method described as before (16.Wu L. Noyan Ashraf M.H. Facci M. Wang R. Paterson P.G. Ferrie A. Juurlink B.H. Proc. Natl. Acad. Sci. U. S. A. 2004; 18: 7094-7099Crossref Scopus (239) Google Scholar). Protein content was determined by using the bicinchoninic acid procedure with bovine serum albumin as reference.Measurement of H2S Production and Concentration−H2S production rate was measured as described previously (4.Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1614) Google Scholar, 17.Siegel M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (368) Google Scholar). Briefly, after different treatments, the cells were collected and homogenized in 50 mm ice-cold potassium phosphate buffer (pH 6.8). The flasks containing reaction mixture (100 mm potassium phosphate buffer, 10 mml-cysteine, 2 mm pyridoxal 5′-phosphate, and 10% (w/v) cell homogenates) and center wells containing 0.5 ml 1% zinc acetate and a piece of filter paper (2 × 2 cm) were flushed with N2 and incubated at 37 °C for 90 min. The reaction was stopped by adding 0.5 ml of 50% trichloroacetic acid, and the flasks were incubated at 37 °C for another 60 min. The contents of the center wells were transferred to test tubes each containing 3.5 ml of water. Then 0.5 ml of 20 mm N,N-dimethyl-p-phenylenediamine sulfate in 7.2 m HCl and 0.5 ml 30 mm FeCl3 in 1.2 m HCl were added. The absorbance of the resulting solution at 670 nm was measured 20 min later with a Multiskan spectrum microplate spectrophotometer.To measure H2S concentration, 200 μl of culture media from each treatment were collected and added to microcentrifuge tubes containing zinc acetate (1% w/v, 600 μl) to trap H2S. After 5 min, the reaction was terminated by adding 400 μl of N,N-dimethyl-p-phenylenediamine sulfate (20 μm in 7.2 m HCl) and 400 μl of FeCl3 (30 mm in 1.2 m HCl). After the mixture was kept in the dark for 20 min, 300 μl of trichloroacetic acid (10% w/v) was added to precipitate any protein that might be present in the culture media. Subsequently, the mixture was centrifuged at 10,000 × g for 10 min. H2S in the sampled culture media interacts with N,N-dimethyl-p-phenylenediamine sulfate to form methylene blue, and the absorbance of the resulting solution was determined at 670 nm (17.Siegel M. Anal. Biochem. 1965; 11: 126-132Crossref PubMed Scopus (368) Google Scholar). H2S concentration in the culture media was calculated against the calibration curve of standard H2S solutions.CSE Activity Measurement−CSE activity was determined by a sensitive method using a colorimetric assay for the determination of pyruvate formation (18.Yang G. Wu L. Wang R. FASEB J. 2006; 20: 553-555Crossref PubMed Scopus (270) Google Scholar). Briefly, after different treatments the cells were collected and homogenized in 20 mm potassium phosphate buffer (pH 7.8), and the homogenate was centrifuged at 12,000 × g for 20 min at 4 °C. Twenty microliters of supernatants were added into 170 μl of 100 mm Tris phosphate buffer (pH 8.0) containing 2.35 mm EDTA, 35 μm pyridoxal 5′-phosphate, and 13.7 mm β-chloro-l-alanine, and the mixture was incubated at 37 °C for 15 min. The reaction was terminated by adding 200 μl of 6.5 mm PPG. After 5 min, the resulting solution was well mixed with 1.4 ml of color-producing reagent containing 0.1 m PIPES (pH 6.4), 0.82 mm thiamine pyrophosphate, 6.86 mm MgCl2, 0.27 units/ml peroxidase, 0.27 mm N-(carboxymethylamino)-4,4′-bis(dimethylamino)-diphenylamine, and 10.97 units/ml pyruvate oxidase, and incubated at 37 °C for 10 min. The mixture containing no cell sample was used as a blank control. The absorbance of green dye at 727 nm was measured in a Multiskan spectrum microplate spectrophotometer. The CSE-specific activity was expressed as the optical density unit of absorbance at 727 nm per mg of protein.Western Blot Analysis−Cultured cells were harvested and lysed. Equal amounts of proteins were boiled and separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane as described previously (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). The primary antibody dilutions were 1:500 for anti-CSE antibody or BiP, 1:1000 for phosphorylated or total extracellular signal-regulated kinase (ERK), p38 MAPK, or c-Jun N-terminal kinase (JNK), and 1:5000 for β-actin. Horseradish peroxidase-conjugated secondary antibody was used at 1:5000. The immunoreactions were visualized by ECL and exposed to x-ray film (Kodak Scientific Imaging film).Short Interfering RNA (siRNA) Transfection−Pre-designed CHOP-targeted siRNA (CHOP-siRNA) was purchased from Ambion (Austin, TX). Negative siRNA (Neg-siRNA), a 21-nucleotide RNA duplex with no known sequence homology with all the genes, was also from Ambion. Transfection of siRNA into INS-1E cells was achieved using the siPORT™ lipid transfection agent from Ambion (14.Yang W. Yang G. Jia X. Wu L. Wang R. J. Physiol. (Lond.). 2005; 569: 519-531Crossref Scopus (413) Google Scholar). Briefly, the cells were plated overnight to form 60-70% confluent monolayers. CHOP-siRNA or Neg-siRNA at different concentrations and transfection reagent complex were added to the cells in serum-free medium for 4 h. Fresh normal growth medium was then added, and the cells were incubated for another 20 h. As control, Neg-siRNA was used to transfect INS-1E cells.Determination of mRNA Level by Real-time PCR−After treatment with H2S or transfection with adenovirus or siRNA, the cells were harvested from 100-mm culture dishes. Total RNA was prepared using TriReagent (Molecular Research Center, Cincinnati) and DNA-free kit (Ambion), and then 2 μg of total RNA was reversetranscribed into cDNA with avian myeloblastosis virus reverse transcriptase using random hexamer primers according to manufacturer’s protocol (Roche Applied Science). Controls containing no reverse transcriptase were used to safeguard for genomic DNA contamination in each sample. Real-time PCR was performed in an iCycler iQ apparatus (Bio-Rad) associated with the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix, as described previously (19.Yang G. Cao K. Wu L. Wang R. J. Biol. Chem. 2004; 279: 49199-49205Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Briefly, all PCRs were performed in a volume of 20 μl, using 96-well optical-grade PCR plates and an optical sealing tape. The cycling was conducted at 95 °C for 90 s followed by 38 cycles of 95 °C for 10 s and at 60 °C for 20 s. The primers of CSE (GenBank™ accession number AY032875) were 5′-AGCGATCACACCACA-GACCAAG-3′ (sense, position 432-453) and 5′-ATCAGCACCCAGAGCCAAAGG-3′ (antisense, position 589-609). These primers produced a product of 178 bp. The primers of BiP (GenBank™ accession number M14050) were 5′-TCCGGCGTGAGGTAGAAAAG-3′ (sense, position 422-441) and 5′-GAGTAGATCCGCCAACCAGAACAA-3′ (antisense, position 635-658) with a product of 236 bp. The primers of CHOP (GenBank™ accession number BC100664) were 5′-CAGAGGTCACAAGCACCT-3′ (sense, position 338-355) and 5′-TCCCTGGTCAGGCGCTC-3′ (antisense, position 562-578) with a product of 240 bp. The primers of SREBP-1c (Gen-Bank™ accession number XM213329) were 5′-GGAGCCATGGATTGCACATT-3′ (sense, position 95-115) and 5′-AGGAAGGCTTCCAGAGAGGA-3′ (antisense, position 267-286) with a product of 191 bp. The primers of β-actin were purchased from Ambion, which produce a product of 295 bp. A standard curve was constructed using a series of dilution of total RNA (Ambion) transcribed to cDNA using the same protocol outlined above to confirm the same amplifying efficiency in the PCR. A standard melting curve analysis was performed using the following thermal cycling profile: 95 °C for 10 s, 55 °C for 15 s, and ramping to 95 °C at 1° increments to confirm the absence of primer dimers. Relative mRNA quantification was calculated by using the arithmetic formula “2-ΔΔCT” (18.Yang G. Wu L. Wang R. FASEB J. 2006; 20: 553-555Crossref PubMed Scopus (270) Google Scholar), where ΔCT is the difference between the threshold cycle of a given target cDNA and an endogenous reference β-actin cDNA. Based on the calculated ΔCT value, the target mRNA level in the treated carotid arteries was subsequently expressed as the percentage of that in the untreated controls.Reagents and Chemicals−H2S stock solution was freshly prepared by directly bubbling distilled water with pure H2S gas (Praxair) to make the saturated H2S solution (0.09 m at 30 °C) (4.Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1614) Google Scholar). H2S stock solution was diluted to different concentrations into cell culture medium, and the pH of medium was adjusted to 7.4. The anti-CSE antibody was homemade. Briefly, synthesized and conjugated targeted peptide VGLEDEQDLLEDLD (GenBank™ accession number AAL99218.1, position 377-390) was used to immunize male New Zealand rabbits. After the titer of anti-CSE antisera reached 1:4,000, the antisera were purified by protein G affinity. Purified antibody was eluted with 0.1 m glycine buffer (pH 2.5) and stored at -80°C for later use. The anti-MAPK antibodies and U0126 were obtained from New England Biolabs (Camarillo, CA). Anti-BiP antibody was from StressGen (San Diego, CA). Horseradish peroxidase-conjugated goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody were from Bio-Rad. All other chemicals were from Sigma or New England Biolabs.Statistical Analysis−All data are expressed as means ± S.E. and represent at least three independent experiments. Statistical comparisons were made using Student’s t test or one-way analysis of variance followed by a post hoc analysis (Tukey test) where applicable. Significance level was set at p < 0.05.RESULTSOverexpression of CSE in INS-1E Cells−Transfection efficiency was first determined using the control adenovirus, Ad-lacZ. INS-1E cells were transfected with Ad-lacZ at an m.o.i. ranging from 10 to 200 for 48 h. At an m.o.i. of 50, >90% cells showed nuclear staining for β-galactosidase (data not shown). All subsequent experiments were performed at an m.o.i. of 50. The Ad-CSE-transfected cells or nontransfected control cells did not exhibit any intrinsic β-galactosidase activity or falsepositive staining.After 48 h of transfection of INS-1E cells with Ad-CSE, the incubation medium was removed, and cell lysates were prepared. As shown in Fig. 1A, CSE mRNA expression level in Ad-CSE-transfected cells was 1.87 ± 0.21 times higher than that in Ad-lacZ-transfected cells or control cells (p < 0.05). Significant increase in CSE proteins was also detected after transfection with Ad-CSE (Fig. 1B). As expected, the cells transfected with Ad-CSE exhibited marked increases in CSE activity and H2S production rate, 4.2 ± 0.4 and 2.4 ± 0.5 times that in Ad-lacZ-transfected cell (p < 0.05) (Fig. 1, C and D). The concentrations of H2S of culture media, in which different types of INS-1E cells were incubated, were further tested. It was found Ad-CSE-transfected cells produced and released much higher concentration of H2S in their culture media than that of control cells or Ad-LacZ-transfected cells (p < 0.05). H2S concentration of the culture medium of control cells is 17.4 ± 1.6 μm, although it is 31.0 ± 1.3 μm for CSE overexpressed cells (Fig. 1E).Effects of Exogenous Applied H2S and CSE Overexpression on Cell Viability and Apoptosis−As shown in Fig. 2A, exogenously applied H2S at 50-200 μm reduced cell viability in a dose-dependent manner, and the cell viability with 100 μm H2S treatment was only 78.9 ± 1.9% that in the absence of H2S (p < 0.05). The cells transfected with Ad-CSE also had reduced cell viability in comparison with that of Ad-lacZ-transfected or control cells. At 48 h after Ad-CSE transfection, cell viability was 80.9 ± 2.6% that observed in Ad-lacZ-transfected cells (p < 0.05) (Fig. 2B).FIGURE 2Exogenously applied H2S or CSE overexpression decreased viability of INS-1E cells. A, changes in cell viability induced by H2S. After the cells were incubated with different concentrations of H2S for 12 h, cell viability was analyzed by MTT assay. *, p < 0.05 versus that in the absence of H2S. n = 4 for each experiment. B, changes in cell viability induced by Ad-CSE transfection. After the cells were transfected at 50 m.o.i. with Ad-lacZ or Ad-CSE for 48 h, cell viability was analyzed by MTT assay. *, p < 0.05 versus Ad-lacZ-transfected cells or the control (virus-free). n = 3 for each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The nuclei of untreated control cells with normal morphology (Fig. 3A, panel a) were uniformly stained by Hoechst 33258 (Fig. 3A, panel d). When incubated with 100 μm H2S, the cells exhibited increased condensed apoptotic nuclei and the morphological changes typical of apoptosis (Fig. 3A, panels b and e). Similar apoptotic changes occurred in Ad-CSE-transfected cells (Fig. 3A, panels c and f). Pro-apoptotic effect of H2S was further evidenced by TUNEL assay. TUNEL-positive staining increased in H2S-treated cells (Fig. 3A, panel h) and Ad-CSE-transfected cells (Fig. 3A, panel i) compared with the control cells (Fig. 3A, panel g). INS-1E cell apoptosis induced by H2S and Ad-CSE was also confirmed by internucleosomal DNA fragmentation. Oligonucleosomal DNA fragmentation was observed in the presence of 100 μm H2S or transfection of Ad-CSE for 48 h but not in control cells or Ad-lacZ-transfected cells (Fig. 3B). Ammonium and pyruvate, another two products of CSE-catalyzed cysteine degradation, had no effect on cell viability and apoptosis at 100 μm (data not shown).FIGURE 3Exogenously applied H2S or CSE overexpression induced apoptosis of INS-1E cells. A, after incubation with 100 μm H2S for 12 h or transfection at 50 m.o.i. with Ad-CSE for 48 h, the cells were fixed and processed for Hoechst 33258 staining or TUNEL assay. Cell morphologies are shown in panels a-c observed under light microscope. Stained nuclear chromatin with Hoechst 33258 are shown in panels d-f. TUNEL staining results are presented in panels g-i. Arrows indicate representative apoptotic cells. Scale bar represents 20 μm. B, oligonucleosomal DNA fragmentation induced by H2S (100 μm) treatment for 12 h or Ad-CSE transfection for 48 h. MW, molecular weight. The results are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effects of Exogenously Applied H2S or CSE Overexpression on the MAPK Pathway−The MAPK pathway is an important signal mechanism for cell growth regulation. Whether MAPKs were phosphorylated and activated by exogenously applied H2S or CSE, overexpression in INS-1E cells was examined using anti-phospho-MAPK antibodies. The same blots were later stripped and re-probed with an antibody that recognized both phosphorylated and unphosphorylated forms of MAPK. As shown in Fig. 4A, ERK1/2 activation was decreased during the first 30 min of H2S treatment and continued to decline for 4 h. Conversely, p38 MAPK activation was induced by H2S during the first 30 min, peaked at 2 h, and later subsided (Fig. 4A). Reduced ERK1/2 activation and increased p38 MAPK activation were also observed in Ad-CSE-transfected cells compared with Ad-lacZ-transfected cells and the control cells (Fig. 4B). JNK activity, however, was quiescent during the period of H2S treatment or Ad-CSE transfection (Fig. 4, A and B). The total amounts of ERK1/2, p38 MAPK, and JNK proteins remained unchanged with H2S stimulation or Ad-CSE transfection. U0126 (an inhibitor of MEK/ERK) and SB203580 (a p38 MAPK inhibitor) were used in the following experiments. As shown in Fig. 4C, 10 μm U0126 augmented the inhibitory effect of H2S on ERK1/2 activity but had no effect on H2S-induced phosphorylation of p38 MAPK. SB203580 at 20 μm completely inhibited H2S-induced phosphorylation of p38 MAPK but had no effect on the phosphorylation of ERK1/2 (Fig. 4C).FIGURE 4Activation of MAPK pathway by H2S in INS-1E cells. A, exogenously applied H2S (100 μm) decreased ERK but increased p38 MAPK phosphorylation, detected with Western blot" @default.
• W2001860318 created "2016-06-24" @default.
• W2001860318 creator A5007660396 @default.
• W2001860318 creator A5026617963 @default.
• W2001860318 creator A5042263308 @default.
• W2001860318 creator A5043861758 @default.
• W2001860318 date "2007-06-01" @default.
• W2001860318 modified "2023-10-17" @default.
• W2001860318 title "H2S, Endoplasmic Reticulum Stress, and Apoptosis of Insulin-secreting Beta Cells" @default.
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