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Pro-apoptotic effect of endogenous H₂S on human aorta smooth muscle cells

Guangdong Yang^*, Lingyun Wu and Rui Wang^*,1

^* Department of Physiology,
Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Canada

¹Correspondence: Office of Vice President (Research), Lakehead University, 955 Oliver Rd., Thunder Bay, Ontario, Canada P7B 5E1. E-mail: rwang{at}lakeheadu.ca

SPECIFIC AIMS

Cystathionine gamma-lyase (CSE, EC4.4.1.1) is a key enzyme in the trans-sulfuration pathway, which mainly contributes to the endogenous production of H₂S in vasculature and heart, and the CSE/H₂S system has been shown to play an important role in regulating cellular functions in different systems. Previous study from our laboratory has shown that exogenous H₂S at physiologically relevant concentration induces human aorta smooth muscle cell (HASMC) apoptosis via the activation of mitogen-activated protein kinases (MAPKs) and caspase-3 (G. Yang et al., FASEB J. 18, 1782–1784, 2004), but little is known about cellular consequences of an elevated CSE expression as well as the associated increases in endogenously produced H₂S in cardiovascular system. The specific aim of this study was to test the hypothesis that endogenously produced H₂S plays a fundamental role in cell proliferation and survival.

PRINCIPAL FINDINGS

1. CSE overexpression increases H₂S production and induces apoptosis of HASMCs
Using a highly effective replication-deficient adenovirus expression system, we overexpressed CSE gene in HASMCs. The expression of CSE in recombinant CSE adenovirus (Ad-CSE) transfected cells was considerably higher than that observed in recombinant adenovirus encoding bacterial ß-galactosidase (Ad-lacZ) transfected cells or control cells. At MOI = 100, expression of CSE in Ad-CSE transfected cells was 30.2 ± 2.6-fold higher than that observed in Ad-lacZ transfected cells or control cells. Dose-dependent increase in the amounts of CSE proteins was detected after infection with increasing multiplicity of infection (MOI) by Ad-CSE. Marked overexpression was observed in cells 24 h after infection with Ad-CSE at 100 MOI, and this increase remained stable for at least 72 h. Cells infected with Ad-CSE at 100 MOI exhibited a marked increase in CSE activity as reflected by cellular levels of pyruvate (31.9±1.2 OD₇₂₇/mg protein) and H₂S production rate (3.8±0.8 nmol/g/min) compared with Ad-lacZ infected cell (2.5±0.1 OD₇₂₇/mg protein and 0.5±0.1 nmol/g/min, P <0.05, respectively) or control cells (2.6±0.1 OD₇₂₇/mg protein and 0.6±0.2 nmol/g/min, P<0.05, respectively). These results suggest that infection with Ad-CSE greatly increased the amount of functional CSE proteins in the transfected HASMCs.

To determine whether infection with Ad-CSE inhibited proliferation and induced apoptosis of HASMCs, cells were infected with Ad-lacZ or Ad-CSE at 100 MOI for different time. One day after gene transfer, cells infected with Ad-CSE exhibited significant reduction in cell growth rate compared with Ad-lacZ infected cells or the control. At the third day, the number of Ad-CSE infected cells was only 81.0% of the control cells. Anti-proliferative effect of CSE overexpression was also evidenced by cell viability assay. At 72 h, cell viability of Ad-CSE transfected cells was 81.7% of that observed in Ad-lacZ transfected cells. We also observed that overexpression of CSE increased the apoptotic cell death of HASMCs. After 72 h of transfection, the nuclei of Ad-lacZ infected cells exhibited normal morphology; however, the number of apoptotic nuclei in Ad-CSE infected cells increased drastically, and cells showed the morphological changes typical of apoptosis. The number of TUNEL positive nuclei in Ad-CSE infected cells was 7.4-fold greater than that in Ad-lacZ infected cells (Fig. 1 A).

View larger version (13K): [in this window] [in a new window]   Figure 1. Effect of CSE overexpression on apoptosis of HASMCs. A) Quantitative analysis of apoptosis induced by CSE overexpression. After infection at 100 MOI with Ad-lacZ or Ad-CSE for 72 h, cells were fixed and processed by TUNEL assay. TUENL-positive cells were quantified and expressed as % of total cells in each group. P < 0.05 vs. Ad-lacZ infected cells or the control. B) Activation of caspase-3 by CSE overexpression. Cells were infected at the indicated MOI with Ad-lacZ or Ad-CSE for 72 h, then cells were collected and processed for caspase-3 activation analysis. *P< 0.05 vs. Ad-lacZ infected cells or the control. A, B) Data represent 3 independent experiments.

Caspase-3 is an effector caspase and one of the main enzymes involved in the apoptotic process. Infection of HASMCs with Ad-CSE resulted in a significant increase in caspase-3 activities in a dose-dependent manner (Fig. 1B ). In contrast, infection of HASMCs with Ad-lacZ at 50–100 MOI failed to stimulate caspase-3 activity. Only at 200 MOI did Ad-lacZ lead to an increase in caspase-3 activity, but this increase was insignificant compared with that seen at 200 MOI with Ad-CSE.

2. CSE overexpression affects MAPKs and cell cycle proteins
Infection of HASMCs with Ad-CSE, but not of Ad-lacZ, resulted in a marked accumulation of phosphorylated ERK and p38 MAPK. Strong ERK activation appeared at 24 h, persisted up to 72 h; strong p38 MAPK activation was observed at 48 h followed by a decline. Neither phosphorylated JNK nor total amount of MAPK was affected. The expression of cyclin D1 was decreased and that of p21^Cip/WAK-1 significantly increased in Ad-CSE infected cells compared with cells infected with Ad-lacZ or control cells, and at 72 h, expression level of cyclin D1 and p21^Cip/WAK-1 was respectively 0.55- and 1.70-fold that in Ad-lacZ infected cells. All these results indicate that MAPK, cyclin D1, and p21^Cip/WAK-1 were involved in CSE overexpression-mediated cell growth change.

3. Effect of CSE inactivation on HASMC apoptosis
To more rigorously explore the effect of H₂S on HASMC apoptosis, we used RNA interference (RNAi) approach to knock down endogenous CSE gene in HASMCs. After inhibition of CSE mRNA expression by 40.6 ± 4.8% and CSE activity by 70.5 ± 3.7% (Fig. 2 A, B), the application of exogenous H₂S (100 µM) significantly increased the apoptosis of HASMCs (Fig. 2C ). The other two end products of CSE-catalyzed reaction, ammonium and pyruvate, at 100 µM each failed to stimulate HASMC apoptosis.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effect of CSE inactivation on HASMC apoptosis. A) CSE mRNA level was decreased in CSE-siRNA infected cells. After infection with 30 nM CSE specific siRNA (CSE-siRNA) or negative control siRNA (Neg-siRNA) for 24 h, cells were collected and processed for real-time PCR analysis. The CSE mRNA level was normalized to that of an internal standard ß-actin. The CSE mRNA level in control cells were considered as 100%. *P < 0.05 vs. Neg-siRNA infected cells or the control. B) CSE activity was significantly decreased in CSE-siRNA infected cells. After infection with 30 nM CSE-siRNA or Neg-siRNA for 24 h, cells were collected for measuring CSE activity. *P < 0.05 vs. Neg-siRNA infected cells or the control. C) Exogenous H2S induced apoptosis of HASMCs. Cells were first infected with 30 nM CSE-siRNA or Neg-siRNA for 24 h, exogenous H2S at 100 µM was added for another 12 h. *P < 0.05. A–C) Data from 3 independent experiments. C) Exogenous H2S treatment down-regulated cyclin D1 and up-regulated p21Cip/WAK-1. After incubated with 100 µM H2S, cells were collected and subjected to Western blot using specific antibodies. The data represent one typical experiment from 3 independent experiments.

To investigate whether exogenous H₂S at physiological relevant concentrations affects the expression of cyclin D1 and p21^Cip/WAK-1, HASMCs were incubated with 100 µM H₂S for 2 h. Our results showed that exogenous H₂S also up-regulated p21^Cip/WAK-1 and down-regulated cyclin D1 (Fig. 2D ). All these results indicated that CSE overexpression-induced cell apoptosis might be mainly due to increased production of H₂S. Moreover, incubating HASMCs with exogenously applied H₂S at 100 µM for 12 h had no significant effect on expression of CSE or endogenous H₂S production rate.

CONCLUSION AND SIGNIFICANCE

There has been recent increased interest in the physiological and pathophysiological roles of the CSE/H₂S pathway in cardiovascular systems. Abnormal metabolism and functions of endogenous H₂S may affect vascular contractile status and structural remodeling of blood vessel walls under different pathophysiological conditions. Vascular smooth muscle cell proliferation and apoptosis induced by various growth factors or stress can lead to a variety of pathological processes including atherosclerosis, hypertension, and restenosis after balloon angioplasty. Using adenovirus-mediated gene transfer, CSE expression was up-regulated in HASMCs. The expressed CSE proteins were functional, as demonstrated by a 12.3-fold increase in CSE activity and 6.2-fold increase in endogenous H₂S production rate after infection with Ad-CSE. The overexpression of CSE in HASMCs significantly attenuates the rate of cell proliferation. This effect is accompanied by a significant increase in the rate of SMC apoptosis, as demonstrated by condensed nuclei, positive TUNEL staining, and increased caspase-3 activation. Although infection of vascular SMCs with higher titer of the control adenovirus (Ad-lacZ) also induced caspase-3 activation, the increase in caspase-3 activity in CSE overexpressed cells is specific at lower titer of the adenovirus transfection.

The mechanisms by which CSE stimulates apoptosis of HASMCs are not entirely clear. Ammonium and pyruvate do not induce apoptosis in HASMCs. H₂S, a main product of the CSE-catalyzed reaction, was recently reported to induce apoptosis at physiologically relevant concentration in HASMCs. In the present study, it was found that the induction of apoptosis by CSE overexpression is associated with a significant increase in H₂S production rate, suggesting that CSE-catalyzed formation of H₂S may mediate the apoptotic response. After inhibiting endogenous CSE gene expression by _DL-propargylglycine (PPG) or with CSE specific siRNA, exogenous administration of H₂S at 100 µM significantly stimulates cell apoptosis. Endogenous H₂S production and CSE expression were not changed by exogenously applied H₂S. Therefore, the endogenous H₂S level is critical for the apoptotic process of smooth muscle cells. A desensitization mechanism of apoptotic signaling pathways resulting from the endogenous basal level of H₂S may exist. By minimizing the endogenous level of H₂S, the real sensitivity of HASMCs to H₂S stimulation may be unmasked.

The MAPK superfamily is involved in cellular survival and apoptotic responses, including ERK, JNK, and p38 MAPK. Here, we provide evidence that a significant increase in ERK and p38 MAPK activities was induced in Ad-CSE infected cells, but not in Ad-lacZ infected cells or control cells. It is reasoned that these two MAPK may be involved in CSE overexpression-mediated cell growth changes. These findings are in line with some previous reports that ERK and p38 MAPK were also involved in CSE-overexpressed HEK-293 cells, H₂S-treated HASMCs or intestinal epithelial cells (IEC-18). Cell cycle transitions are controlled by the action of cyclin-dependent kinases (cdk) and their activating subunits, the cyclins. The kinase activity of this cyclin/cdk complex can be negatively regulated by cdk inhibitors, such as p21^Cip/WAK-1, which inhibit cyclin D1 activation and thereby prevent cyclin D1/CRM1 association. Both cyclin D1 and p21^Cip/WAK-1 play important roles in regulating cellular differentiation, DNA repair, cell senescence and apoptosis. Our data provide evidence that inhibition of growth and induction of apoptosis of HASMCs by CSE overexpression was accompanied by marked down-regulation of cyclin D1 and up-regulation of p21^Cip/WAK-1. These results suggest that CSE overexpression-mediated growth inhibition and induction of apoptosis of HASMCs were due to activation of MAPK and selective cell cycle proteins. To our knowledge, our study for the first time demonstrates the involvement of the MAPK-cyclin D1-p21^Cip/WAK-1 machinery during cell growth inhibition and apoptosis of HASMCs induced by CSE overexpression.

In conclusion, we have demonstrated that adenovirus transduction of HASMCs with the CSE gene leads to growth inhibition and apoptosis, which appears to be mediated by the overproduced H₂S. Increased expression of ERK, p38 MAPK and p21^Cip/WAK-1, and down-regulation of cyclin D1 were all observed in CSE-overexpressed cell and exogenous H₂S-treated cells (Fig. 3 ). Collectively, the ability of the CSE/H₂S pathway to alter cell growth suggests that genetic approaches to manipulate CSE expression and H₂S production may provide a novel therapeutic avenue in the treatment of the CSE/H₂S disorder-related diseases.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic signal transduction pathways underlying the H2S-induced cell growth changes. The prefix "p-" indicates a phosphorylated form.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4712fje;

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