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Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells
Biochemical and Biophysical Research Communications xxx (2015) 1e6
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Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells Bin Huang a, b, c, d, Chang-Ting Chen e, Chi-Shia Chen a, Yun-Ming Wang f, Hsyue-Jen Hsieh e, **, Danny Ling Wang g, * a
Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan d Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan e Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan f Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan g Institute of Medical Science, College of Medicine, Tzu Chi University, Hualien County 97004, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2015 Accepted 22 July 2015 Available online xxx
Laminar shear flow triggers a signaling cascade that maintains the integrity of endothelial cells (ECs). Hydrogen sulfide (H2S), a new gasotransmitter is regarded as an upstream regulator of nitric oxide (NO). Whether the H2S-generating enzymes are correlated to the enzymes involved in NO production under shear flow conditions remains unclear as yet. In the present study, the cultured ECs were subjected to a constant shear flow (12 dyn/cm2) in a parallel flow chamber system. We investigated the expression of three key enzymes for H2S biosynthesis, cystathionine-g-lyase (CSE), cystathionine-b-synthase (CBS), and 3-mercapto-sulfurtransferase (3-MST). Shear flow markedly increased the level of 3-MST. Shear flow enhanced the production of H2S was determined by NBD-SCN reagent that can bind to cysteine/ homocystein. Exogenous treatment of NaHS that can release gaseous H2S, ECs showed an increase of phosphorylation in AktS473, ERKT202/Y204 and eNOSS1177. This indicated that H2S can trigger the NOproduction signaling cascade. Silencing of CSE, CBS and 3-MST genes by siRNA separately attenuated the phosphorylation levels of AktS473 and eNOSS1177 under shear flow conditions. The particular mode of shear flow increased H2S production. The interplay between H2S and NO-generating enzymes were discussed in the present study. © 2015 Elsevier Inc. All rights reserved.
Keywords: Shear flow Hydrogen sulfide Nitric oxide 3-Mercapto-sulfurtransferase NBD-SCN Endothelial cell
1. Introduction Shear flow is a mechanical stressor that can be sensed by mechano-sensors located on membranes of endothelial cells (ECs). It triggers a network of signaling pathways in the effects of anti-
Abbreviations: eNOS, endothelial nitric oxide synthase; CSE, cystathionine-glyase; CBS, cystathionine-b-synthase; 3-MST, 3-mercapto-sulfurtransferase; NBDSCN, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole; Akt, protein kinase B; ERK, extracellular signal-regulated kinase. * Corresponding author. Institute of Medical Science, College of Medicine, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan. ** Corresponding author. Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan. E-mail addresses: [email protected] (H.-J. Hsieh), [email protected]. tw (D.L. Wang).
atherosclerogenesis and vascular homeostasis [1]. Among the various shear-flow induced signaling molecules, reactive oxygen species (ROS)/reactive nitrogen species (RNS) are most discussed [2]. Under irregular shear flow, an elevated level of ROS increases the risk of atherogenesis [3]. However, during laminar shear flow, reversible ROS such as nitric oxide (NO) is abundantly generated through protein kinase B (Akt)-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) ser-1177 residues [4,5]. NO can temporarily bind to the catalytic sites of enzymes, also known as S-nitrosylation, and then protects enzymes from irreversible inactivation caused by strong oxidative modifications [6]. Through the suppression of NADPH oxidase (Nox), an enzyme that promotes ROS synthesis, laminar shear flow can reduce the formation of superoxide and oxidative stress in arteries [7]. Under disturbed flow, the level of thioredoxin-1 is increased and leads to the
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Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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B. Huang et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e6
formation of inflammatory signaling in ECs [8]. The mechanism of how NO/ROS modifies proteins and thus modulates the function of these proteins and the physiological responses of ECs, is still an important issue of research [9e11]. In addition to NO, the toxic gas hydrogen sulfide (H2S) has been recognized as a novel vasodilator for several years [12]. Cellular H2S can be produced by the conversion of cysteine with three enzymes: cystathionine-g-lyase (CSE), cystathionine-b-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) [13]. Mutation of H2S-producing enzymes result in some disorders such as neural degeneration, skeletal abnormalities, increased risks of thromboembolism, and early onset of atherosclerosis [14]. With H2S treatment, the production of adhesion molecules and the proliferation of vascular smooth muscle cells (VSMCs) can be suppressed and then prevents the formation of atherosclerotic lesions [15]. Increasing evidence demonstrates that hydrogen sulfide (H2S) decreases the generation of ROS/RNS and leads to the proliferation and migration of ECs, indicating that H2S is a ROS scavenger that protects ECs from hydrogen peroxide-induced injury [16,17]. H2S would increase the expression of microRNA-21 (miR-21) and then attenuates myocardial injury through the suppression of the inflammasome, a macromolecular assembly that is implicated in many pathogenic processes [18]. As a result, H2S has been a potent target in preventing heart failure, atherosclerosis, neuron degeneration, and cell aging [19,20]. A crosstalk between NO and H2S in the cardiovascular system has been proposed [21,22]. The signaling responses after H2S treatment shares a high similarity with laminar shear flow in cases of anti-oxidative stress, anti-inflammation and anti-atherosclerosis [12]. H2S might, therefore, be implicated as a shear flow-triggered signaling molecule. To elucidate this issue, the measurement of endogenous levels of H2S is important. Even though several approaches were designed to measure H2S, the accuracy and a long operation time were unsatisfying [23,24]. Hence, a fluorescence probe was applied, using the reaction of 4-nitro-7thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with cysteine and homocysteine [22,25]. The cysteine/homocysteine then can be converted to H2S via CSE, CBS and 3-MST [14]. Protein kinase B (Akt), an upstream enzyme activating eNOS can prevent cell apoptosis by inhibiting the action of caspase-9 and forkhead box O3 (FOXO3) transcription factor [26]. Interestingly, the Akt/FOXO3 pathway is also implicated in a H2S-mediated vascular homeostasis [27]. Extracellular signal-regulated kinase (ERK) is a member of mitogen-activated kinase (MAPK) that functions in the p38 MAPK/ERK pathway and its regulation either by mechanical shear flow or hydrogen sulfide have been broadly discussed [28,29]. Our particular objectives were to study shear flow-induced H2S by using fluorescent probes that can bind to cysteine/homocysteine and the possible implications of Akt, ERK and eNOS in the downstream signaling by siRNA silencing of CBS, CSE and 3-MST enzymes. 2. Materials and methods 2.1. Cell culture and shear flow treatment Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications, Inc. (San Diego, CA, USA). HUVECs were cultured in M199 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 100 U/ml penicillin/streptomycin, 2.5 mg/ml amphotericin B, 20% fetal bovine serum (FBS), and 20% endothelial cell growth medium (Lonza, MD, USA). HUVECs from passages 3e4 were cultured on glass slides and exposed to shear flow (12 dyn/cm2) for 30 min in a well-defined
parallel plate flow chamber system [10]. 2.2. Applications of fluorescent probe and flow cytometry Under shear flow, the ECs were coincubated with 5 mM of 4nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) for 30 min. Cysteine/homocysteine, that represents the level of H2S were observed by fluorescence microscopy (lex 460 nm, lem 550 nm; € ttingen, Germany) [25]. After fluorescence Axiovert 40 CFL, Zeiss, Go microscopic observations, the ECs were washed twice with PBS buffer and detached by tryptic reaction. ECs were collected by centrifugation and then resuspended in PBS buffer. The fluorescence was immediately measured by the Accuri C6 flow cytometer (BD Bioscience, San Diego, CA, USA) with excitation and emission settings of 488 and 530 nm, respectively. The fluorescence strength was obtained from 1 104 cells and statistically calculated from three repeats. 2.3. Cell lysis and protein extraction After treatment, ECs were washed with cord buffer: [NaCl (0.14 M), KCl (4 mM), glucose (11 mM), HEPES (10 mM, pH 7.4)] and then lysed with 100 ml of lysis buffer: [Hepes (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM) and CHAPS (0.4%, w/v)]. After centrifugation, proteinaceous supernatant was collected and protein concentrations were determined with BCA assay reagents (Thermo Fisher Scientific, Waltham, MA, USA). 2.4. Western blot analysis Forty micrograms of cell lysate with various treatments were mixed with equal volumes of sample buffer [TriseHCl (62.5 mM, pH 6.8), SDS (3%, w/v), 2-mercaptoethanol (5%, v/v), glycerol (10%, v/ v)], and then separated by SDS-PAGE. The gel was transferred to PVDF membranes (Millipore, Billerica, MA, USA) and immunoblotted with antibodies: CBS (1:1000, Abnova, Taipei, Taiwan), CSE (1:1000, Abnova), 3-MST (1:1000, Abcam, Cambridge, UK), Akt (1:2000, BD Biosciences), pAktS473 (1:3000, Cell Signaling Tech., Lane Danvers, MA, USA), ERK (1:2000, BD Biosciences), pERKT202/ Y204 (1:1500, Millipore), eNOS (1:3000, Cell Signaling Tech.), peNOSS1177 (1:2000, Cell Signaling Tech.). The membranes were visualized with the SuperSignal West Femto reagent (Thermo Fisher Scientific) on X-ray films. The images on X-ray films were scanned using a digital scanner (Microtek International Inc., Hsinchu, Taiwan) and the density was calculated by the Progenesis Samespots v2.0 software (NonLinear Dynamics, Newcastle, UK). 2.5. siRNA transfection The CBS and 3-MST siRNA were generated by Dharmacon ONTARGET plus SMART pool human CBS #L-008617-00 and #L010119-00 (Thermo Fisher Scientific). The CSE siRNA sequence was designed as sense strand 50 -GGUUAUUUAUCCUGGGCUGdTdT-30 and anti-sense strand 50 -CAGCCCAGGAUAAAUAACCdTdT-30 (MDBio Inc., Taipei, Taiwan). ECs were co-transfected with siRNA using TurboFect™ (Thermo Fisher Scientific) and then cultured on glass slides for subsequent shear flow treatment. 2.6. Data analysis All the data were collected and statistically calculated from three repeats. Statistical significance with increased level (shown by *p < 0.05, **p < 0.01) or decreased level (#p < 0.05, ##p < 0.01) was evaluated by using ANOVA with post-hoc Tukey HSD test. Since the exposure time would affect the visibility of resulting images on
Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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Fig. 1. Shear flow increases the expression of 3-MST enzyme. (A) After shear flow of 12 dyn/cm2 for 30 min and 60 min, the expressions of three enzymes, CBS, CSE and 3-MST were investigated by western blot. (B) The relative folds of three protein levels that were compared to the control treatment were shown as mean ± S.E. from three repeats. Statistical significance (**p < 0.01, ns ¼ no significance) was evaluated by ANOVA with post-hoc Tukey HSD test.
X-ray films in Figs. 3 and 4, the phosphorylation level were shown as the phosphorylated form/total protein and then normalized by the fold change of treatments compared to the control.
expression of cysteine/homocysteine (Fig. 2A). Cysteine/homocysteine was markedly produced from 54.7 % to 86.4 % within 30 min under the stimulation of shear flow (12 dyn/cm2). However, the production of cysteine/homocysteine was decreased at 60 min (Fig. 2BeD).
3. Results 3.1. Shear flow increases the expression of 3-MST and the endogenous level of cysteine/homocysteine Under shear flow conditions, 3-MST exhibited a higher expression level from 30 to 60 min individually as compared to CBS and CSE (Fig. 1). An NBD-SCN fluorescent probe was injected into a defined parallel plate flow chamber system to monitor the
3.2. H2S enhances the phosphorylation levels of Akt, ERK and eNOS After exogenous treatment of various concentrations of NaHS (20, 60 and 100 mM) for 30 and 60 min, the relative folds of the phosphorylated AktS473, ERKT202/Y204 and eNOSS1177 were analyzed by western blot and were normalized to the control treatment. The phosphorylation of AktS473 was dominantly enhanced by 60 mM of
Fig. 2. Shear flow promotes the production of cysteine/homocysteine in endothelial cells. (A) Application of fluorescent NBD-SCN probe in a shear flow device. (B) The production of cysteine/homocysteine in ECs treated with shear flow (12 dyn/cm2, 30 min and 60 min) was observed by fluorescent microscopy (lex 460 nm, lem 550 nm). (C, D) The fluorescent strength was obtained from 1 104 cells by flow cytometry and then statistically calculated. The level of cysteine/homocysteine was shown as means ± S.E. from three repeats. Statistical significance (**p < 0.01, #p < 0.05) was analyzed by using ANOVA with post-hoc Tukey HSD test. Bar ¼ 30 mm.
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Fig. 3. NaHS increases the phosphorylation of Akt, ERK and eNOS. (A) ECs treated with different concentrations of NaHS (20, 60 and 100 mM) for 30 and 60 min separately were subjected to the western blot analysis for detecting the phosphorylation levels of Akt, ERK and eNOS. The phosphorylation levels were shown as pAktS473/Akt, pERKT202/Y204/ERK and peNOSS1177/eNOS and were indicated under the figure panels. (B) The phosphorylation fold of each protein under different NaHS concentrations was normalized statistically by NaHS/control. Data were shown as means ± S.E. from three repeats. Statistical significance (*p < 0.05, **p < 0.01, #p < 0.05) was analyzed by using ANOVA with post-hoc Tukey HSD test.
NaHS at 30e60 min. As for ERKT202/Y204, the phosphorylation level was substantially activated by 60 and 100 mM of NaHS at 30e60 min. With a similar expression profile to ERKT202/Y204, the phosphorylation level of eNOSS1177 also showed a significant increase (Fig. 3). 3.3. Deficiency in H2S-producing enzymes reduces shear flowinduced Akt and eNOS phosphorylation The ECs separately defected in the expressions of CBS, CSE and 3-MST by siRNA were applied to monitor the phosphorylation levels of AktS473, ERKT202/Y204, and eNOSS1177 (Fig. 4A). Shear flowincreased phosphorylation of AktS473, ERKT202/Y204 and eNOSS1177 was dramatically inhibited once the H2S biosynthetic proteins CBS and 3-MST were silenced by siRNA. However, under the silencing of CSE, only AktS473 and eNOSS1177 were suppressed in their phosphorylation levels (Fig. 4B). 4. Discussion H2S is a gaseous molecule that has been characterized for its importance in vascular homeostasis recently. To investigate the production of H2S under laminar shear flow, a western blot analysis
of the H2S-generating enzymes was performed. We found that only 3-MST showed a significant increase from 30 to 60 min, while the levels of CBS and CSE were not changed (Fig. 1). This is coinciding with the finding that 3-MST is the major H2S-producing enzyme in endothelial cells [30]. Coincident to the knowledge that gaseous molecules bear a rapid chemical reaction character in their cellular responses, we found that H2S precursor, cysteine/homocysteine can be produced within 30 min merely under a moderate laminar shear flow of 12 dyn/cm2, using an NBD-SCN fluorescent probe (Fig. 2). However, with an extended period of shear flow, the cysteine/homocysteine level was decreased. The reason could be the frequent exposure to relatively high concentrations of H2S leading to a desensitization of the response to H2S stimulation [13]. The majority of endogenous H2S is likely to be released or stored according to the pH value of the cells [31]. Consequently, whether shear flow mediates Ca2þ distribution and affects the stored/free form of H2S are worthy of further study. In the present study, 60 mM of NaHS were used to investigate variable expressions of Akt and ERK (Fig. 3). This concentration was similar to the annotated cellular concentrations of H2S and also similar to reports about vascular experiments [22,32]. In the study of angiogenesis, H2S can stimulate the phosphorylation of eNOS through p38 MAPK and Akt-dependent pathways
Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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Fig. 4. Shear flow-induced H2S generation increases NO production through the Akt-eNOS pathway. (A) The shear flow-enhanced phosphorylations of AktS473, ERKT202/Y204 and eNOSS1177 were evaluated as H2S-producing genes and silenced by siRNA. (B) The relative folds of protein phosphorylation from shear and static conditions were normalized statistically by siRNA/Scramble. Data were shown as means ± S.E. from three repeats. Statistical significances (**p < 0.01, ##p < 0.01) were analyzed by using ANOVA with post-hoc Tukey HSD test.
[33]. This indicates that Akt and ERK are simultaneously implicated in the H2S response. In the present study, the gene silencing of the H2S-producing genes, CBS, CSE and 3-MST by siRNA were applied to determine the relative expressions of Akt, ERK and eNOS under shear flow. Following the concern that the resulting images on the X-ray film are affected by the variation of exposure time, the phosphorylation level of each treatment was normalized to the control treatment. The phosphorylation of AktS473 and eNOSS1177 was seriously decreased when all the three H2S-producing enzymes were silenced. The knockdown of CBS and 3-MST decreased the phosphorylation of ERKT202/Y204. However, the knockdown of CSE was unexpectedly enhancing the phosphorylation of ERKT202/ Y204 as compared to a scramble treatment (Fig. 4). Thus, the hypothesis that Akt, compared to ERK, is more likely a downstream signal transmitter for shear flow-generated H2S, we proposed this issue for further study. A similar result was also suggested in the study of the PI3K inhibitor which was blocking phosphorylation of eNOS and Akt, but not ERK in response to shear stress [34]. By using a fluorescent probe and gene silencing, we here firstly demonstrate that shear flow can upregulate 3-MST/cysteine/homocysteine bioactivity and then triggers the protein signaling cascade in the Akt-NO pathway. Acknowledgments This study was supported by grants (NSC102-2320-B-320-010) from the Ministry of Science and Technology of Taiwan. The study was partially supported by the Kaohsiung Medical University “Aim for the Top Universities Grant, grant No. KMU-TP103C00, KMUTP103E00 and KMU-O104003”, and Ten Chan General Hospital, Chung-Li and KMU Joint Research Project (ST102004). We thank Dr. Hans-Uwe Dahms for critically reading an earlier version of the manuscript.
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Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells Bin Huang a, b, c, d, Chang-Ting Chen e, Chi-Shia Chen a, Yun-Ming Wang f, Hsyue-Jen Hsieh e, **, Danny Ling Wang g, * a
Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Center for Biomarkers and Biotech Drugs, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Center for Infectious Disease and Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan d Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan e Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan f Department of Biological Science and Technology, Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan g Institute of Medical Science, College of Medicine, Tzu Chi University, Hualien County 97004, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 July 2015 Accepted 22 July 2015 Available online xxx
Laminar shear flow triggers a signaling cascade that maintains the integrity of endothelial cells (ECs). Hydrogen sulfide (H2S), a new gasotransmitter is regarded as an upstream regulator of nitric oxide (NO). Whether the H2S-generating enzymes are correlated to the enzymes involved in NO production under shear flow conditions remains unclear as yet. In the present study, the cultured ECs were subjected to a constant shear flow (12 dyn/cm2) in a parallel flow chamber system. We investigated the expression of three key enzymes for H2S biosynthesis, cystathionine-g-lyase (CSE), cystathionine-b-synthase (CBS), and 3-mercapto-sulfurtransferase (3-MST). Shear flow markedly increased the level of 3-MST. Shear flow enhanced the production of H2S was determined by NBD-SCN reagent that can bind to cysteine/ homocystein. Exogenous treatment of NaHS that can release gaseous H2S, ECs showed an increase of phosphorylation in AktS473, ERKT202/Y204 and eNOSS1177. This indicated that H2S can trigger the NOproduction signaling cascade. Silencing of CSE, CBS and 3-MST genes by siRNA separately attenuated the phosphorylation levels of AktS473 and eNOSS1177 under shear flow conditions. The particular mode of shear flow increased H2S production. The interplay between H2S and NO-generating enzymes were discussed in the present study. © 2015 Elsevier Inc. All rights reserved.
Keywords: Shear flow Hydrogen sulfide Nitric oxide 3-Mercapto-sulfurtransferase NBD-SCN Endothelial cell
1. Introduction Shear flow is a mechanical stressor that can be sensed by mechano-sensors located on membranes of endothelial cells (ECs). It triggers a network of signaling pathways in the effects of anti-
Abbreviations: eNOS, endothelial nitric oxide synthase; CSE, cystathionine-glyase; CBS, cystathionine-b-synthase; 3-MST, 3-mercapto-sulfurtransferase; NBDSCN, 4-nitro-7-thiocyanatobenz-2-oxa-1,3-diazole; Akt, protein kinase B; ERK, extracellular signal-regulated kinase. * Corresponding author. Institute of Medical Science, College of Medicine, Tzu Chi University, No. 701, Sec. 3, Zhongyang Rd., Hualien 97004, Taiwan. ** Corresponding author. Department of Chemical Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan. E-mail addresses: [email protected] (H.-J. Hsieh), [email protected]. tw (D.L. Wang).
atherosclerogenesis and vascular homeostasis [1]. Among the various shear-flow induced signaling molecules, reactive oxygen species (ROS)/reactive nitrogen species (RNS) are most discussed [2]. Under irregular shear flow, an elevated level of ROS increases the risk of atherogenesis [3]. However, during laminar shear flow, reversible ROS such as nitric oxide (NO) is abundantly generated through protein kinase B (Akt)-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) ser-1177 residues [4,5]. NO can temporarily bind to the catalytic sites of enzymes, also known as S-nitrosylation, and then protects enzymes from irreversible inactivation caused by strong oxidative modifications [6]. Through the suppression of NADPH oxidase (Nox), an enzyme that promotes ROS synthesis, laminar shear flow can reduce the formation of superoxide and oxidative stress in arteries [7]. Under disturbed flow, the level of thioredoxin-1 is increased and leads to the
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Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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formation of inflammatory signaling in ECs [8]. The mechanism of how NO/ROS modifies proteins and thus modulates the function of these proteins and the physiological responses of ECs, is still an important issue of research [9e11]. In addition to NO, the toxic gas hydrogen sulfide (H2S) has been recognized as a novel vasodilator for several years [12]. Cellular H2S can be produced by the conversion of cysteine with three enzymes: cystathionine-g-lyase (CSE), cystathionine-b-synthetase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) [13]. Mutation of H2S-producing enzymes result in some disorders such as neural degeneration, skeletal abnormalities, increased risks of thromboembolism, and early onset of atherosclerosis [14]. With H2S treatment, the production of adhesion molecules and the proliferation of vascular smooth muscle cells (VSMCs) can be suppressed and then prevents the formation of atherosclerotic lesions [15]. Increasing evidence demonstrates that hydrogen sulfide (H2S) decreases the generation of ROS/RNS and leads to the proliferation and migration of ECs, indicating that H2S is a ROS scavenger that protects ECs from hydrogen peroxide-induced injury [16,17]. H2S would increase the expression of microRNA-21 (miR-21) and then attenuates myocardial injury through the suppression of the inflammasome, a macromolecular assembly that is implicated in many pathogenic processes [18]. As a result, H2S has been a potent target in preventing heart failure, atherosclerosis, neuron degeneration, and cell aging [19,20]. A crosstalk between NO and H2S in the cardiovascular system has been proposed [21,22]. The signaling responses after H2S treatment shares a high similarity with laminar shear flow in cases of anti-oxidative stress, anti-inflammation and anti-atherosclerosis [12]. H2S might, therefore, be implicated as a shear flow-triggered signaling molecule. To elucidate this issue, the measurement of endogenous levels of H2S is important. Even though several approaches were designed to measure H2S, the accuracy and a long operation time were unsatisfying [23,24]. Hence, a fluorescence probe was applied, using the reaction of 4-nitro-7thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) with cysteine and homocysteine [22,25]. The cysteine/homocysteine then can be converted to H2S via CSE, CBS and 3-MST [14]. Protein kinase B (Akt), an upstream enzyme activating eNOS can prevent cell apoptosis by inhibiting the action of caspase-9 and forkhead box O3 (FOXO3) transcription factor [26]. Interestingly, the Akt/FOXO3 pathway is also implicated in a H2S-mediated vascular homeostasis [27]. Extracellular signal-regulated kinase (ERK) is a member of mitogen-activated kinase (MAPK) that functions in the p38 MAPK/ERK pathway and its regulation either by mechanical shear flow or hydrogen sulfide have been broadly discussed [28,29]. Our particular objectives were to study shear flow-induced H2S by using fluorescent probes that can bind to cysteine/homocysteine and the possible implications of Akt, ERK and eNOS in the downstream signaling by siRNA silencing of CBS, CSE and 3-MST enzymes. 2. Materials and methods 2.1. Cell culture and shear flow treatment Human umbilical vein endothelial cells (HUVECs) were obtained from Cell Applications, Inc. (San Diego, CA, USA). HUVECs were cultured in M199 medium (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA) supplemented with 100 U/ml penicillin/streptomycin, 2.5 mg/ml amphotericin B, 20% fetal bovine serum (FBS), and 20% endothelial cell growth medium (Lonza, MD, USA). HUVECs from passages 3e4 were cultured on glass slides and exposed to shear flow (12 dyn/cm2) for 30 min in a well-defined
parallel plate flow chamber system [10]. 2.2. Applications of fluorescent probe and flow cytometry Under shear flow, the ECs were coincubated with 5 mM of 4nitro-7-thiocyanatobenz-2-oxa-1,3-diazole (NBD-SCN) for 30 min. Cysteine/homocysteine, that represents the level of H2S were observed by fluorescence microscopy (lex 460 nm, lem 550 nm; € ttingen, Germany) [25]. After fluorescence Axiovert 40 CFL, Zeiss, Go microscopic observations, the ECs were washed twice with PBS buffer and detached by tryptic reaction. ECs were collected by centrifugation and then resuspended in PBS buffer. The fluorescence was immediately measured by the Accuri C6 flow cytometer (BD Bioscience, San Diego, CA, USA) with excitation and emission settings of 488 and 530 nm, respectively. The fluorescence strength was obtained from 1 104 cells and statistically calculated from three repeats. 2.3. Cell lysis and protein extraction After treatment, ECs were washed with cord buffer: [NaCl (0.14 M), KCl (4 mM), glucose (11 mM), HEPES (10 mM, pH 7.4)] and then lysed with 100 ml of lysis buffer: [Hepes (250 mM, pH 7.7), EDTA (1 mM), neocuproine (0.1 mM) and CHAPS (0.4%, w/v)]. After centrifugation, proteinaceous supernatant was collected and protein concentrations were determined with BCA assay reagents (Thermo Fisher Scientific, Waltham, MA, USA). 2.4. Western blot analysis Forty micrograms of cell lysate with various treatments were mixed with equal volumes of sample buffer [TriseHCl (62.5 mM, pH 6.8), SDS (3%, w/v), 2-mercaptoethanol (5%, v/v), glycerol (10%, v/ v)], and then separated by SDS-PAGE. The gel was transferred to PVDF membranes (Millipore, Billerica, MA, USA) and immunoblotted with antibodies: CBS (1:1000, Abnova, Taipei, Taiwan), CSE (1:1000, Abnova), 3-MST (1:1000, Abcam, Cambridge, UK), Akt (1:2000, BD Biosciences), pAktS473 (1:3000, Cell Signaling Tech., Lane Danvers, MA, USA), ERK (1:2000, BD Biosciences), pERKT202/ Y204 (1:1500, Millipore), eNOS (1:3000, Cell Signaling Tech.), peNOSS1177 (1:2000, Cell Signaling Tech.). The membranes were visualized with the SuperSignal West Femto reagent (Thermo Fisher Scientific) on X-ray films. The images on X-ray films were scanned using a digital scanner (Microtek International Inc., Hsinchu, Taiwan) and the density was calculated by the Progenesis Samespots v2.0 software (NonLinear Dynamics, Newcastle, UK). 2.5. siRNA transfection The CBS and 3-MST siRNA were generated by Dharmacon ONTARGET plus SMART pool human CBS #L-008617-00 and #L010119-00 (Thermo Fisher Scientific). The CSE siRNA sequence was designed as sense strand 50 -GGUUAUUUAUCCUGGGCUGdTdT-30 and anti-sense strand 50 -CAGCCCAGGAUAAAUAACCdTdT-30 (MDBio Inc., Taipei, Taiwan). ECs were co-transfected with siRNA using TurboFect™ (Thermo Fisher Scientific) and then cultured on glass slides for subsequent shear flow treatment. 2.6. Data analysis All the data were collected and statistically calculated from three repeats. Statistical significance with increased level (shown by *p < 0.05, **p < 0.01) or decreased level (#p < 0.05, ##p < 0.01) was evaluated by using ANOVA with post-hoc Tukey HSD test. Since the exposure time would affect the visibility of resulting images on
Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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Fig. 1. Shear flow increases the expression of 3-MST enzyme. (A) After shear flow of 12 dyn/cm2 for 30 min and 60 min, the expressions of three enzymes, CBS, CSE and 3-MST were investigated by western blot. (B) The relative folds of three protein levels that were compared to the control treatment were shown as mean ± S.E. from three repeats. Statistical significance (**p < 0.01, ns ¼ no significance) was evaluated by ANOVA with post-hoc Tukey HSD test.
X-ray films in Figs. 3 and 4, the phosphorylation level were shown as the phosphorylated form/total protein and then normalized by the fold change of treatments compared to the control.
expression of cysteine/homocysteine (Fig. 2A). Cysteine/homocysteine was markedly produced from 54.7 % to 86.4 % within 30 min under the stimulation of shear flow (12 dyn/cm2). However, the production of cysteine/homocysteine was decreased at 60 min (Fig. 2BeD).
3. Results 3.1. Shear flow increases the expression of 3-MST and the endogenous level of cysteine/homocysteine Under shear flow conditions, 3-MST exhibited a higher expression level from 30 to 60 min individually as compared to CBS and CSE (Fig. 1). An NBD-SCN fluorescent probe was injected into a defined parallel plate flow chamber system to monitor the
3.2. H2S enhances the phosphorylation levels of Akt, ERK and eNOS After exogenous treatment of various concentrations of NaHS (20, 60 and 100 mM) for 30 and 60 min, the relative folds of the phosphorylated AktS473, ERKT202/Y204 and eNOSS1177 were analyzed by western blot and were normalized to the control treatment. The phosphorylation of AktS473 was dominantly enhanced by 60 mM of
Fig. 2. Shear flow promotes the production of cysteine/homocysteine in endothelial cells. (A) Application of fluorescent NBD-SCN probe in a shear flow device. (B) The production of cysteine/homocysteine in ECs treated with shear flow (12 dyn/cm2, 30 min and 60 min) was observed by fluorescent microscopy (lex 460 nm, lem 550 nm). (C, D) The fluorescent strength was obtained from 1 104 cells by flow cytometry and then statistically calculated. The level of cysteine/homocysteine was shown as means ± S.E. from three repeats. Statistical significance (**p < 0.01, #p < 0.05) was analyzed by using ANOVA with post-hoc Tukey HSD test. Bar ¼ 30 mm.
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Fig. 3. NaHS increases the phosphorylation of Akt, ERK and eNOS. (A) ECs treated with different concentrations of NaHS (20, 60 and 100 mM) for 30 and 60 min separately were subjected to the western blot analysis for detecting the phosphorylation levels of Akt, ERK and eNOS. The phosphorylation levels were shown as pAktS473/Akt, pERKT202/Y204/ERK and peNOSS1177/eNOS and were indicated under the figure panels. (B) The phosphorylation fold of each protein under different NaHS concentrations was normalized statistically by NaHS/control. Data were shown as means ± S.E. from three repeats. Statistical significance (*p < 0.05, **p < 0.01, #p < 0.05) was analyzed by using ANOVA with post-hoc Tukey HSD test.
NaHS at 30e60 min. As for ERKT202/Y204, the phosphorylation level was substantially activated by 60 and 100 mM of NaHS at 30e60 min. With a similar expression profile to ERKT202/Y204, the phosphorylation level of eNOSS1177 also showed a significant increase (Fig. 3). 3.3. Deficiency in H2S-producing enzymes reduces shear flowinduced Akt and eNOS phosphorylation The ECs separately defected in the expressions of CBS, CSE and 3-MST by siRNA were applied to monitor the phosphorylation levels of AktS473, ERKT202/Y204, and eNOSS1177 (Fig. 4A). Shear flowincreased phosphorylation of AktS473, ERKT202/Y204 and eNOSS1177 was dramatically inhibited once the H2S biosynthetic proteins CBS and 3-MST were silenced by siRNA. However, under the silencing of CSE, only AktS473 and eNOSS1177 were suppressed in their phosphorylation levels (Fig. 4B). 4. Discussion H2S is a gaseous molecule that has been characterized for its importance in vascular homeostasis recently. To investigate the production of H2S under laminar shear flow, a western blot analysis
of the H2S-generating enzymes was performed. We found that only 3-MST showed a significant increase from 30 to 60 min, while the levels of CBS and CSE were not changed (Fig. 1). This is coinciding with the finding that 3-MST is the major H2S-producing enzyme in endothelial cells [30]. Coincident to the knowledge that gaseous molecules bear a rapid chemical reaction character in their cellular responses, we found that H2S precursor, cysteine/homocysteine can be produced within 30 min merely under a moderate laminar shear flow of 12 dyn/cm2, using an NBD-SCN fluorescent probe (Fig. 2). However, with an extended period of shear flow, the cysteine/homocysteine level was decreased. The reason could be the frequent exposure to relatively high concentrations of H2S leading to a desensitization of the response to H2S stimulation [13]. The majority of endogenous H2S is likely to be released or stored according to the pH value of the cells [31]. Consequently, whether shear flow mediates Ca2þ distribution and affects the stored/free form of H2S are worthy of further study. In the present study, 60 mM of NaHS were used to investigate variable expressions of Akt and ERK (Fig. 3). This concentration was similar to the annotated cellular concentrations of H2S and also similar to reports about vascular experiments [22,32]. In the study of angiogenesis, H2S can stimulate the phosphorylation of eNOS through p38 MAPK and Akt-dependent pathways
Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115
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Fig. 4. Shear flow-induced H2S generation increases NO production through the Akt-eNOS pathway. (A) The shear flow-enhanced phosphorylations of AktS473, ERKT202/Y204 and eNOSS1177 were evaluated as H2S-producing genes and silenced by siRNA. (B) The relative folds of protein phosphorylation from shear and static conditions were normalized statistically by siRNA/Scramble. Data were shown as means ± S.E. from three repeats. Statistical significances (**p < 0.01, ##p < 0.01) were analyzed by using ANOVA with post-hoc Tukey HSD test.
[33]. This indicates that Akt and ERK are simultaneously implicated in the H2S response. In the present study, the gene silencing of the H2S-producing genes, CBS, CSE and 3-MST by siRNA were applied to determine the relative expressions of Akt, ERK and eNOS under shear flow. Following the concern that the resulting images on the X-ray film are affected by the variation of exposure time, the phosphorylation level of each treatment was normalized to the control treatment. The phosphorylation of AktS473 and eNOSS1177 was seriously decreased when all the three H2S-producing enzymes were silenced. The knockdown of CBS and 3-MST decreased the phosphorylation of ERKT202/Y204. However, the knockdown of CSE was unexpectedly enhancing the phosphorylation of ERKT202/ Y204 as compared to a scramble treatment (Fig. 4). Thus, the hypothesis that Akt, compared to ERK, is more likely a downstream signal transmitter for shear flow-generated H2S, we proposed this issue for further study. A similar result was also suggested in the study of the PI3K inhibitor which was blocking phosphorylation of eNOS and Akt, but not ERK in response to shear stress [34]. By using a fluorescent probe and gene silencing, we here firstly demonstrate that shear flow can upregulate 3-MST/cysteine/homocysteine bioactivity and then triggers the protein signaling cascade in the Akt-NO pathway. Acknowledgments This study was supported by grants (NSC102-2320-B-320-010) from the Ministry of Science and Technology of Taiwan. The study was partially supported by the Kaohsiung Medical University “Aim for the Top Universities Grant, grant No. KMU-TP103C00, KMUTP103E00 and KMU-O104003”, and Ten Chan General Hospital, Chung-Li and KMU Joint Research Project (ST102004). We thank Dr. Hans-Uwe Dahms for critically reading an earlier version of the manuscript.
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Please cite this article in press as: B. Huang, et al., Laminar shear flow increases hydrogen sulfide and activates a nitric oxide producing signaling cascade in endothelial cells, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.07.115