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Hydrogen sulfide-induced apoptosis of human aorta smooth muscle cells via the activation of mitogen-activated protein kinases and caspase-3

Department of Physiology, College of Medicine, Cardiovascular Research Group, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

¹Correspondence: Department of Physiology, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, SK S7N 5E5, Canada. E-mail: wangrui{at}duke.usask.ca

SPECIFIC AIMS

H₂S is endogenously generated in mammalian cells. Its role in regulating physiological functions of neuronal and cardiovascular systems has been shown, but little is known about the actions of H₂S on cell proliferation and growth in general and on apoptosis of vascular smooth muscle cells (SMC) in particular. The specific aims of this study were to examine the physiological role of H₂S in regulation of cellular apoptosis and underlying cellular signaling transduction pathways.

PRINCIPAL FINDINGS

1. Both exogenous and endogenous H₂S affect apoptosis of human aortic SMCs
Using Trypan blue exclusion test and LDH release assay, we found that the application of H₂S to human aortic smooth muscle cells (HASMC) did not induce significant cell necrotic death. Whether H₂S induced HASMC apoptosis was subsequently examined. Nuclei of untreated control cells were uniformly stained by Hoechst 33258 and the cells exhibited normal morphology. When incubating cells with 500 µM H₂S, the number of condensed apoptotic nuclei increased dramatically, and cells showed morphological changes typical of apoptosis. After incubation with various concentrations of H₂S, cells were processed for TUNEL assay. The number of TUNEL-positive cells increased after exposure to H₂S. H₂S-induced apoptosis in HASMCs was concentration dependent with significant apoptosis detected at concentrations of 200 µM and higher. Induction of HASMC apoptosis by H₂S was further confirmed by internucleosomal DNA fragmentation. In the presence of H₂S at different concentrations for 12 h, oligonucleosomal DNA fragments formed ladders, which became visible with 20 and 50 µM H₂S and very evident with 200 and 500 µM H₂S.

To determine the role of endogenous H₂S in the apoptotic process of HASMCs, HASMCs were first treated with DL-propargylglycine (PPG) to inhibit activity of cystathionine -lyase (CSE), which is the H₂S-generating enzyme in vascular smooth muscle cells. PPG treatment alone (10–20 mM) did not induce apoptotic changes. One hour pretreatment with PPG significantly enhanced the apoptotic effect of exogenously applied H₂S. H₂S at 50 µM started to induce apoptosis of HASMCs in the presence of PPG and this effect of H₂S became significant at 100 µM (Fig. 1 A). Exogenous H₂S at 100 µM induced 5.2 ± 0.7% apoptosis of HASMCs in the absence of PPG treatment (P>0.05), whereas after PPG treatment exogenous H₂S at 100 µM induced a significant 11.7 ± 0.7% apoptosis (P<0.05). H₂S-induced apoptosis of HASMCs was not altered by either albumin or glutathoine (GSH) at millimolar concentrations (Fig. 1B, C ).

View larger version (14K): [in this window] [in a new window]   Figure 1. The role of endogenous H2S in the apoptotic process of HASMCs. HASMCs were pretreated with the indicated concentrations of PPG for 1 h before addition of H2S. The extent of apoptosis was evaluated 12 h later by Hoeschst 33258 and fluorescence microscopy, expressed as the percentage of apoptotic nuclei of total number of cell nuclei. Interaction of PPG and exogenous H2S on cellular apoptosis was studied in the absence (A) or presence (B) of 1 mg/mL albumin or 5 mM glutathione (C). *P< 0.05, n = 3.

2. H₂S sequentially activated ERK and caspase-3 signaling pathways
To determine whether mitogen-activated protein kinases (MAPK) were phosphorylated and activated by H₂S in HASMCs, lysates obtained from HASMCs with different H₂S treatment regimes were subjected to Western blot analysis using anti-phospho-MAPK antibodies. Treatment of HASMCs with H₂S resulted in strong activation of ERK and p38 MAPK. ERK activation appeared during the first 15 min of H₂S treatment, and peaked at 2 h followed by a slow decline (Fig. 2 A). A similar but delayed stimulatory effect of H₂S was observed on p38 MAPK. H₂S maximally activated p38 MAPK within 2 h of application. JNK activity was constant during the 12 h period of culture with H₂S. The total amount of MAPK protein remained unchanged with H₂S stimulation. Activation of ERK and p38 MAPK by H₂S in HASMC was also concentration-dependent. At concentration as low as 20 µM, H₂S activated ERK and p38 MAPK (Fig. 2B ).

View larger version (52K): [in this window] [in a new window]   Figure 2. Western blot determination of the H2S-induced phosphorylation of different MAPK. A) Time course of the activation of different MAPK induced by H2S (500 µM) in HASMCs. B) Concentration-dependent activation of ERK and p38 MAPK by 2 h treatment with H2S in HASMCs. Results are representative of 3 independent experiments.

Caspase-3 is an effector caspase and one of the main enzymes involved in the apoptotic process. H₂S induced a dramatic increase in caspase-3 activity in a concentration-dependent manner. Addition of AC-DEVD-CHO (10 µM), a specific caspase-3 inhibitor, completely inhibited activation of caspase-3 by H₂S in HASMCs. H₂S-induced caspase-3 activation was partly and significantly decreased by U0126 (20 µM), a selective inhibitor of the MEK/ERK signaling pathway, but not by SB203580 (a specific p38 MAPK inhibitor). Pretreatment of HASMCs with AC-DEVD-CHO (10 µM) for 1 h did not alter the effects of H₂S on the activation of ERK and p38 MAPK. These results demonstrate that H₂S first activated ERK and the latter stimulated caspase-3 in HASMCs.

Treatment of HASMCs with different concentrations of H₂S for 12 h did not significantly alter expression of Bax protein. We observed no difference in the expression of Bcl-2 protein between H₂S-treated and nontreated HASMCs.

3. Blockade of H₂S-induced apoptosis by inhibiting ERK and caspase-3 activation
Inhibition of caspase-3 with AC-DEVD-CHO (10 µM) alone had no effect on cell apoptosis. This treatment significantly reduced H₂S-induced apoptosis of HASMCs by 57.0 ± 0.3% (P<0.05). Application of U0126 (20 µM) also inhibited H₂S-induced apoptosis of HASMCs by 52.1 ± 0.3% (P<0.05). Inhibiting p38 MAPK activation with SB203580 (40 µM) did not alter the apoptotic effect of H₂S.

CONCLUSIONS AND SIGNIFICANCE

H₂S can be generated in blood vessels in a reaction catalyzed mainly by CSE. Abnormal metabolism and functions of endogenous H₂S may impact on vascular contractile status and structural remodeling of blood vessel walls under different pathophysiological conditions. Proliferation and apoptosis of vascular SMCs are important cellular events of vascular remodeling. In this study, for the first time, we described the novel physiological effect of H₂S on apoptosis of human vascular smooth muscle cells. The following summarizes the most significant and novel findings. 1) We demonstrated that H₂S induced apoptosis of human aortic smooth muscle cells at physiologically relevant concentrations. This finding enlarges the family of gasotransmitters by adding H₂S to join two established members (i.e., nitric oxide and carbon monoxide). 2) After inhibiting endogenous H₂S production, exogenous H₂S induced even more significant apoptosis, which was not altered by the presence of albumin or glutathione. Therefore, the endogenous H₂S level is critical for the apoptotic process of smooth muscle cells. A desensitization mechanism of the apoptotic signaling system 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. 3) We presented evidence that the proapoptotic effect of H₂S was mediated by the activation of ERK, but not that of p38 MAPK or JNK. This study reveals the novel mechanism underlying the vascular effects of a novel physiological endogenous gas. 4) We showed that activation of ERK by H₂S was accompanied by increased caspase-3 activity. Inhibition of caspase-3 attenuated the H₂S-induced cell apoptosis. This discovery further elucidates the downstream targets of H₂S-ERK interaction.

The MAPK family represents important signal transduction machinery and occupies a central position in cell growth, differentiation, and programmed cell death. Different MAPK are activated by different stimuli, target different downstream molecules, and therefore perform different functions. In the present study, we provided evidence that activation of ERK is important for H₂S-induced apoptosis of HASMCs. Both ERK and p38 MAPK were activated at an early stage of H₂S incubation. Down-regulation of ERK activity inhibited H₂S-induced apoptosis. However, inhibition of p38 MAPK did not alter the apoptotic sensitivity of HASMCs to H₂S. Thus, only ERK is involved in apoptosis of H₂S-treated HASMCs. The protective effect of ERK inhibitor U0126 against H₂S suggests that ERK plays a major role in the induction of apoptosis evoked by H₂S. Involvement of ERK in the process of cell apoptosis has been shown in many cell types. Given that U0126 also inhibits MEK, a tyrosine/threonine kinase that phosphorylates and activates ERK, it is possible that MEK and further upstream molecules such as Raf and Ras may be subjected to H₂S modulation.

Some apoptosis inducers have been shown to activate caspase-3, which is an important effector caspase. Caspase-3 can mediate MAPK activation in TNF- or Fas-induced apoptosis, and caspase inhibitors attenuate MAPK activation, indicating a close causative relationship between MAPK and caspase activation. Here, we report that activation of ERK in H₂S-treated HASMCs was accompanied by caspase-3 activation. Our observations that the caspase-3 inhibitor AC-DEVD-CHO partly blocked H₂S-induced programmed cell death, but not ERK activation, suggest that ERK activity may precede activation of caspase-3 in H₂S-induced cell death. This hypothesis is rationalized since inhibition of ERK leads to decreased caspase-3 activity. Further experiments are needed to investigate the mechanisms by which ERK activates caspase-3 in the process of apoptosis of HASMCs.

Our study demonstrates for the first time that endogenous H₂S is not only a vasorelaxant or a messenger in neuronal system, but also an important endogenous modulator of cellular apoptosis via activation of the MAPK pathway. H₂S may first activate ERK through various mechanisms. Phosphorylated and activated ERK then transduces the apoptotic signal to its downstream enzyme cascades, eventually activating caspase-3 (Fig. 3 ). Identification of the missing links in this cascade will lead to a refined understanding of the cellular and molecular mechanisms underlying H₂S-induced cellular apoptosis. These findings will help to significantly advance our understanding of H₂S biology and physiology. Novel mechanisms for many diseases linked to H₂S-related abnormal cellular proliferation and apoptosis may be revealed.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic signal transduction pathways underlying the H2S-induced apoptosis. "p-" prefix indicates a phosphorylated form.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2279fje;

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