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Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells
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Biochemical and Biophysical Research Communications 368 (2008) 852–857 www.elsevier.com/locate/ybbrc
Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells Tetsuya Moriue a, Junsuke Igarashi b,*, Kozo Yoneda a, Kozo Nakai a, Hiroaki Kosaka b, Yasuo Kubota a b
a Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan
Received 23 January 2008 Available online 11 February 2008
Abstract Reactive oxygen species including H2O2 lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H2O2. Treatment of cultured bovine aortic EC (BAEC) with H2O2 (750 lM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H2O2 were alike markedly attenuated by pretreatment with S1P (1 lM, 30 min). H2O2 induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H2O2-induced caspase-3 activation. Thus S1P attenuates H2O2-induced apoptosis of cultured BAEC, involving p38 MAP kinase. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Vascular endothelial cells; Apoptosis; Sphingolipids; Caspase-3; p38 MAP kinase
Sphingosine 1-phosphate (S1P) is a platelet-derived lipid mediator that elicits a wide array of physiological responses in various types of mammalian cells [1]. S1P is present in numerous biological fluids, including normal adult human serum, which contains several hundred nanoto micro-molar S1P concentrations [2]. Vascular endothelial cells (EC) represent a key target cell type of S1P, in which it exerts such diverse responses as proliferation, migration, vasorelaxation, and regulation of cell–cell junctions ultimately to promote angiogenesis [1]. Yet another key feature of S1P actions on EC is that this lipid is capable of protecting them from undergoing apoptosis induced by various deleterious stimuli [3–5]. Oxidative stress, defined as excess production of reactive oxygen species (ROS), often leads to EC damage. High concentrations of ROS, especially H2O2, are known to *
Corresponding author. Fax: +81 87 891 2101. E-mail address: [email protected] (J. Igarashi).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.155
occur in vasculature with activation of neutrophils or keratinocytes [6]. In turn, these concentrations of H2O2 lead EC to undergo apoptosis [7] and to lose their function and integrity. Such situations may have potential clinical relevance, for example because exposure to ultraviolet causes significant ROS production within in vivo skin [8]. Molecular mechanisms underlying H2O2-induced EC apoptosis have been characterized, which involve caspase3, a cysteine-dependent aspartate protease [9], as well as MAP kinases p38 and c-Jun N-terminal kinase (JNK) [7,10]. In the present study, we examined a hypothesis that S1P is capable of attenuating H2O2-induced DNA damage and activation of caspase-3, key features of apoptosis, in cultured EC. We also sought to explore mechanisms whereby S1P attenuates endothelial apoptosis elicited by H2O2. Our data implicate MAP kinase p38 as a point of control at which S1P counteracts H2O2-induced EC apoptotic responses.
T. Moriue et al. / Biochemical and Biophysical Research Communications 368 (2008) 852–857
Materials and methods Reagents. Antibodies specific to cleaved form of caspase-3, (phospho-) p38, and (phospho-) JNK were obtained from Cell Signaling Technologies (Beverly, MA). SB203580 and P600125 were from Calbiochem (Darmstadt, Germany). Cell Death Detection ELISAPlus kits were from Roche (Mannheim, Germany). ApopTag florescein in situ apoptosis detection kits were from Chemicon (Temecula, CA, USA). Other reagents derived from identical sources as we have previously described [11]. Cell culture and drug treatment. Cell culture and drug treatment were performed essentially as described [11]. Unless otherwise stated, bovine aortic EC (BAEC) between passages 5 and 7 were split at a ratio of 1:4 and used for experiments. At subconfluence, culture media were changed to Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS) and incubation proceeded overnight prior to all experiments to exclude the effects of residual S1P contained in FBS. SB203580 and P600125 were resolved in dimethyl sulfoxide (DMSO) and kept at 20 oC. Other drug treatments were performed exactly as described [11]. The final concentration of any solvent including DMSO did not exceed 0.1% (v/v) in any experiment. Enzyme-linked immunosorbent assay (ELISA). The degrees of intracellular DNA fragmentation were determined using cell death detection ELISAPlus kit essentially as described [12] following supplier’s protocol. All ELISA reactions were carried out with duplicate wells using a given cell lysate, and their averages were taken as a final result. A DNA–histone complex (supplied) and a lysis buffer blank were used as positive and negative controls in each assay, respectively. TUNEL staining. The degrees of DNA nick formation were determined in situ by means of TUNEL staining. BAEC were split at a ratio of 1:24 and were seeded onto gelatin-coated coverslips. At approximately 30% confluence 4 days after being split, cells were serum-starved overnight. They were washed with PBS on ice and were fixed with 1% paraformaldehyde in PBS (w/v) for 10 min at ambient temperature, followed by permeabilization with acetone/ethanol (1:2 in v/v) for 5 min at 20 oC. TUNEL staining was then performed with ApopTag florescein in situ apoptosis detection kit, according to the manufacturer’s instruction using a confocal laser microscope (Radiance 2100/Rainbow, BioRad, Hercules, CA) [13]. TUNEL-positive and negative cells were counted using five independent microscopic fields of 40 magnification in each coverslip. A total number of TUNEL-positive cells derived from five observed fields were summed up, in parallel with that of total cells; percentage of TUNEL positive cells over total was calculated in each coverslip by dividing the former number by the latter. Immunoblot analyses. Immunoblot analyses were performed exactly as described previously [11]. Statistics. All experiments were performed at least 3 times. Mean values for individual experiments are expressed as means ± SE. Statistical differences between two groups were analyzed by Student’s t test using Microsoft Excel. A p value less than 0.05 was considered statistically significant.
Results and discussion We first explored how H2O2 induces DNA fragmentation within BAEC, a key feature of apoptotic cell death, by means of ELISA. We treated BAEC with 1 mM of H2O2 up to 24 h. They were then subjected to ELISA in which the degrees of cytoplasmic DNA fragment formation were determined. As shown in Fig. 1A, this concentration of H2O2 started to increase DNA fragmentation of BAEC 2 h after the treatment with H2O2. The magnitude of DNA fragmentation peaked at 6 h after H2O2, reaching to 8.3-fold increase compared to t = 0, then returned to basal level within 24 h. Fig. 1B shows the results of dose– response study in which BAEC were treated for 6 h with
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increasing concentrations of H2O2. The degree of DNA fragmentation was maximum at 750 lM among the concentrations tested. These results demonstrate that H2O2 was capable of inducing DNA fragmentation of BAEC and that maximum response was obtained when they were treated with 750 lM of H2O2 for 6 h. We next examined whether or not S1P is able to attenuate apoptotic responses of BAEC to H2O2 treatment. Some BAEC had been pretreated with 1 lM of S1P for 30 min prior to H2O2 treatment (750 lM for 6 h), followed by ELISA. We note that similar concentrations of S1P can be found in adult human serum, especially in the presence of activated platelets [2], and also that in pathophysiological situations blood vessels can be exposed to hundred micromolar concentrations of H2O2 [6]. As indicated in Fig. 2A, pretreatment with S1P significantly attenuated H2O2-induced DNA fragmentation than that with vehicle. We utilized an independent approach to evaluate DNA damage of BAEC by subjecting them to TUNEL staining. As shown in left lower panel of Fig. 2B, significantly larger number of TUNEL-positive nuclei (green staining) were observed in H2O2-treated BAEC (arrowheads) compared with those without H2O2, showing that their DNA was nicked upon H2O2 treatment. Quantification of TUNELpositive nuclei revealed that the fraction of TUNEL-positive cells increased by 3.8-fold with H2O2 than basal. Significantly, when BAEC had been pretreated with 1 lM S1P, the fraction of TUNEL-positive cells decreased by 55.7% compared with those treated with H2O2 alone (Fig. 2B and C). Caspase-3 is a key ‘‘executer” protease of apoptotic processes of vascular EC [9]. We therefore examined the effects of H2O2/S1P on caspase-3 by immunoblot analyses of BAEC using an antibody specific to cleaved (activated) form of caspase-3. Fig. 2D demonstrates that H2O2 induces cleavage (activation) of caspase-3 protein, which was markedly attenuated when cells had been pre-incubated with S1P. A constitutive protein of BAEC, caveolin-1 [11], serves here as a loading control, which yields similar molecular mass as cleaved caspase-3, to ensure equal transfer and equivalent loading among protein samples. Together, these experiments indicated that S1P pretreatment reduced H2O2-induced apoptotic cell responses in BAEC, i.e., DNA fragmentation and cleavage/activation of caspase-3. A wide array of signaling machinery has been postulated as proximal molecules that connect cellular stimulation with execution of apoptosis. MAP kinases p38 and JNK have been previously implicated in H2O2-induced endothelial apoptosis [10,14]. We therefore sought to explore whether or not S1P attenuates H2O2-induced apoptosis by modulating activity of these molecules. We first evaluated the effects of H2O2/S1P on these MAP kinases by checking the degrees of phosphorylation. When BAEC were treated with H2O2, both MAP kinases underwent robust phosphorylation (activation) responses, as shown in the left half panel of Fig. 3A. Importantly, when they had been pretreated with S1P, the degrees of phosphoryla-
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T. Moriue et al. / Biochemical and Biophysical Research Communications 368 (2008) 852–857
Fig. 1. H2O2 induces DNA fragmentation in BAEC. Shown are the results of apoptotic cell death detection ELISA. (A) Results of time course study. BAEC were treated with 1 mM of H2O2 for the times indicated. Cells were then subjected to ELISA. The values determined at 405 nm in each time point were normalized to that obtained at t = 0. Each data point represents mean ± SE; N = 4. *p < 0.05 vs. t = 0. (B) BAEC were treated for 6 h using various concentrations of H2O2. The absorbance values obtained at each H2O2 concentration were normalized to that obtained with vehicle in each experiment; N = 4. *p < 0.05 vs. vehicle.
Fig. 2. S1P pretreatment reduces H2O2-induced apoptotic responses of BAEC. BAEC were treated with 750 lM of H2O2 or vehicle for 6 h. Some cells had been pretreated with 1 lM of S1P for 30 min. (A) Results of cell death detection ELISA. In each experiment, absorbance values obtained at 405 nm were normalized to that with H2O2 alone, which yielded maximum value of each experiment. Each data point represents mean ± SE; N = 4. (B) Representative images obtained in TUNEL assay, using confocal laser microscopy. Yellow arrowheads show TUNEL-positive signals. (C) The results of quantification of TUNEL assay. Fractions of TUNEL-positive cell numbers were determined in each preparation and expressed as percentage of the total. Each data point represents mean ± SE; N = 5. Upper half of (D) Representative images of immunoblots following H2O2/S1P treatment. Resulting membrane was probed for cleaved form of caspase-3 protein. It was re-probed for caveolin-1, which serves as a loading control. Lower half of (D) shows the results of densitometric analyses. Signals corresponding to cleaved form of caspase-3 were quantified and normalized against the value obtained with H2O2 alone (maximum) in each experiment. Each data point represents mean ± SE; N = 5.
T. Moriue et al. / Biochemical and Biophysical Research Communications 368 (2008) 852–857
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Fig. 3. S1P pretreatment attenuates H2O2-induced phosphorylation of p38 but not that of JNK in BAEC. (A) Representative images of immunoblots. BAEC were treated with 750 lM of H2O2 for the times indicated. Some cells had been treated with S1P (1 lM for 30 min). They were then probed for phosphorylated forms of p38 and JNK proteins. To confirm equal loading, they were re-probed for (total) p38 and JNK. (B and C) Results of densitometric analyses for phosphorylation responses of p38 and JNK, respectively. Signals corresponding to phosphorylated forms of p38 (or JNK) were quantified and normalized against the values obtained using antibodies specific to total p38 (or JNK). The resulting ratios of phospho/total p38 (or JNK) were then normalized to the basal value in each experiment. Each data point represents mean ± SE. Open and closed circles represent values obtained without and with pretreatment with S1P, respectively. *p < 0.05 versus H2O2 ( ). ; p < 0.05 versus S1P ( ). N = 5.
tion of only p38 MAP kinase, but not those of JNK phosphorylation, were attenuated (Fig. 3A–C). We then utilized a pharmacological inhibitor of p38 MAP kinase, SB203580. This agent markedly decreased H2O2-evoked cleavage of caspase-3 (Fig. 4A). In contrast, pretreatment with a JNK inhibitor P600125 (25 lM for 30 min) instead of SB203580 failed to attenuate H2O2-induced cleavage of caspase-3 (Fig. 4B). Note that SB203580 and P600125 under these conditions abrogated H2O2-induced phosphorylation/activation of p38 MAP kinase and JNK of BAEC, respectively (750 lM of H2O2 for 60 min, data not shown). When taken together, our results support a hypothesis that S1P attenuates H2O2-elicited caspase-3 activation at a level of, or proximal to, p38 MAP kinase, rather than JNK. Regulatory mechanisms of p38 MAP kinase by H2O2 appear to be cellular context-dependent and quite complex, involving various protein kinases like MAP kinase kinase MKK6, protein kinase D, and a protein kinase ASK-1, as well as protein phosphatases like MKP-1 [15]. Thus, precise molecular mechanisms whereby S1P interferes H2O2evoked activation of p38 MAP kinase in BAEC remain to be elucidated. Our findings that S1P is able to counteract H2O2-dependent apoptotic responses of BAEC are consistent with
earlier reports that imply S1P as a pro-survival sphingolipid metabolite. For example, S1P keeps various endothelial cell cultures from undergoing apoptosis, elicited by such diverse stimuli as serum starvation [3], tumor necrosis factor-a [4], and ceramide [5]. Interestingly, H2O2-induced apoptosis in EC can be recovered by cell stimulation other than S1P as well, for example by an angiogenic polypeptide growth factor angiopoietin-1 [16]. Thus, the present findings add another level of complexity to our understandings of vascular endothelial cell fate, modulated by oxidative stress and sphingolipids, as well as by other classes of molecules. We note that S1P also modulates apoptosis of skin cells, including fibroblasts and keratinocytes [17,18]. Because ROS are generated in skin at some dermatopathological situations, including UV irradiation [8] and wound formation [19], roles of S1P on cutaneous cell fate in the presence of excess ROS need to be determined. In this regard, a recent study by Kawanabe et al. provides an intriguing observation that addition of a high concentration of S1P has a therapeutic effect on wounded skin of diabetic, but not normal, mice [20]. While S1P has well-established pro-angiogenic properties [1] that would promote wound healing in these mice, our results raise a possibility that
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Fig. 4. SB203580, an inhibitor of p38 MAP kinase, attenuates H2O2-induced caspase-3 cleavage in BAEC. Upper part of (A) Representative images of immunoblots probed for cleaved form of caspase-3. BAEC were treated with H2O2 for 6 h, following pretreatment with SB203580 (0.4 lM for 30 min). Cells were subjected to immunoblot assays, followed by densitometry as above. In (B), cells had been pretreated with P600125 (25 lM for 30 min) prior to H2O2. Data are expressed as means ± SE, N = 4.
an anti-apoptotic potential of S1P could also contribute to promotion of wound healing processes. Additionally a novel immunosuppressant FTY-720, which, when phosphorylated, binds to specific S1P receptors and exerts beneficial effects on atopic dermatitis [21]. In conclusion, our results demonstrate that S1P, a platelet-derived sphingolipid mediator, attenuates H2O2-induced apoptosis of cultured vascular EC, involving caspase-3 and p38 MAP kinase. References [1] T. Hla, Physiological and pathological actions of sphingosine 1phosphate, Semin. Cell Dev. Biol. 15 (2004) 513–520. [2] Y. Yatomi, Y. Igarashi, L. Yang, N. Hisano, R. Qi, N. Asazuma, K. Satoh, Y. Ozaki, S. Kume, Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum, J. Biochem. (Tokyo) 121 (1997) 969–973. [3] Y.G. Kwon, J.K. Min, K.M. Kim, D.J. Lee, T.R. Billiar, Y.M. Kim, Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production, J. Biol. Chem. 276 (2001) 10627–10633. [4] P. Xia, L. Wang, J.R. Gamble, M.A. Vadas, Activation of sphingosine kinase by tumor necrosis factor-a inhibits apoptosis in human endothelial cells, J. Biol. Chem. 274 (1999) 34499–34505. [5] N. Hisano, Y. Yatomi, K. Satoh, S. Akimoto, M. Mitsumata, M.A. Fujino, Y. Ozaki, Induction and suppression of endothelial cell apoptosis by sphingolipids: a possible in vitro model for cell–cell interactions between platelets and endothelial cells, Blood 93 (1999) 4293–4299. [6] P.A. Ward, Mechanisms of endothelial cell killing by H2O2 or products of activated neutrophils, Am. J. Med. 91 (1991) 89S–94S. [7] H. Cai, Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences, Cardiovasc. Res. 68 (2005) 26–36. [8] T. Herrling, J. Fuchs, J. Rehberg, N. Groth, UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides, Free Radic. Biol. Med. 35 (2003) 59–67.
[9] K. Irani, Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling, Circ. Res. 87 (2000) 179–183. [10] N. Ali, M. Yoshizumi, K. Tsuchiya, M. Kyaw, Y. Fujita, Y. Izawa, S. Abe, Y. Kanematsu, S. Kagami, T. Tamaki, Ebselen inhibits p38 mitogen-activated protein kinase-mediated endothelial cell death by hydrogen peroxide, Eur. J. Pharmacol. 485 (2004) 127–135. [11] J. Igarashi, M. Miyoshi, T. Hashimoto, Y. Kubota, H. Kosaka, Hydrogen peroxide induces S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a platelet-derived lipid mediator, Am. J. Physiol. Cell Physiol. 292 (2007) C740– C748. [12] G.A. Walford, R.L. Moussignac, A.W. Scribner, J. Loscalzo, J.A. Leopold, Hypoxia potentiates nitric oxide-mediated apoptosis in endothelial cells via peroxynitrite-induced activation of mitochondriadependent and -independent pathways, J. Biol. Chem. 279 (2004) 4425–4432. [13] K. Yoneda, T. Furukawa, Y.J. Zheng, T. Momoi, I. Izawa, M. Inagaki, M. Manabe, N. Inagaki, An autocrine/paracrine loop linking keratin 14 aggregates to tumor necrosis factor alpha-mediated cytotoxicity in a keratinocyte model of epidermolysis bullosa simplex, J. Biol. Chem. 279 (2004) 7296–7303. [14] T. Machino, S. Hashimoto, S. Maruoka, Y. Gon, S. Hayashi, K. Mizumura, H. Nishitoh, H. Ichijo, T. Horie, Apoptosis signalregulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells, Crit. Care Med. 31 (2003) 2776–2781. [15] A.J. Whitmarsh, R.J. Davis, Role of mitogen-activated protein kinase kinase 4 in cancer, Oncogene 26 (2007) 3172–3184. [16] T. Murakami, H. Takagi, K. Suzuma, I. Suzuma, H. Ohashi, D. Watanabe, T. Ojima, E. Suganami, M. Kurimoto, H. Kaneto, Y. Honda, N. Yoshimura, Angiopoietin1 attenuates H2O2-induced SEK1/JNK phosphorylation through the PI3-kinase/Akt pathway in vascular endothelial cells, J. Biol. Chem. 280 (2005) 31841– 31849. [17] M. Manggau, D.S. Kim, L. Ruwisch, R. Vogler, H.C. Korting, M. Schafer-Korting, B. Kleuser, 1Alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate, J. Invest. Dermatol. 117 (2001) 1241–1249.
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[20] T. Kawanabe, T. Kawakami, Y. Yatomi, S. Shimada, Y. Soma, Sphingosine 1-phosphate accelerates wound healing in diabetic mice, J. Dermatol. Sci. 48 (2007) 53–60. [21] T. Kohno, T. Tsuji, K. Hirayama, K. Watabe, A. Matsumoto, T. Fujita, A novel immunomodulator, FTY720, prevents spontaneous dermatitis in NC/Nga mice, Biol. Pharm. Bull. 27 (2004) 1392– 1396.
Biochemical and Biophysical Research Communications 368 (2008) 852–857 www.elsevier.com/locate/ybbrc
Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells Tetsuya Moriue a, Junsuke Igarashi b,*, Kozo Yoneda a, Kozo Nakai a, Hiroaki Kosaka b, Yasuo Kubota a b
a Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-Cho, Kita-Gun, Kagawa 761-0793, Japan
Received 23 January 2008 Available online 11 February 2008
Abstract Reactive oxygen species including H2O2 lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H2O2. Treatment of cultured bovine aortic EC (BAEC) with H2O2 (750 lM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H2O2 were alike markedly attenuated by pretreatment with S1P (1 lM, 30 min). H2O2 induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H2O2-induced caspase-3 activation. Thus S1P attenuates H2O2-induced apoptosis of cultured BAEC, involving p38 MAP kinase. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Vascular endothelial cells; Apoptosis; Sphingolipids; Caspase-3; p38 MAP kinase
Sphingosine 1-phosphate (S1P) is a platelet-derived lipid mediator that elicits a wide array of physiological responses in various types of mammalian cells [1]. S1P is present in numerous biological fluids, including normal adult human serum, which contains several hundred nanoto micro-molar S1P concentrations [2]. Vascular endothelial cells (EC) represent a key target cell type of S1P, in which it exerts such diverse responses as proliferation, migration, vasorelaxation, and regulation of cell–cell junctions ultimately to promote angiogenesis [1]. Yet another key feature of S1P actions on EC is that this lipid is capable of protecting them from undergoing apoptosis induced by various deleterious stimuli [3–5]. Oxidative stress, defined as excess production of reactive oxygen species (ROS), often leads to EC damage. High concentrations of ROS, especially H2O2, are known to *
Corresponding author. Fax: +81 87 891 2101. E-mail address: [email protected] (J. Igarashi).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.155
occur in vasculature with activation of neutrophils or keratinocytes [6]. In turn, these concentrations of H2O2 lead EC to undergo apoptosis [7] and to lose their function and integrity. Such situations may have potential clinical relevance, for example because exposure to ultraviolet causes significant ROS production within in vivo skin [8]. Molecular mechanisms underlying H2O2-induced EC apoptosis have been characterized, which involve caspase3, a cysteine-dependent aspartate protease [9], as well as MAP kinases p38 and c-Jun N-terminal kinase (JNK) [7,10]. In the present study, we examined a hypothesis that S1P is capable of attenuating H2O2-induced DNA damage and activation of caspase-3, key features of apoptosis, in cultured EC. We also sought to explore mechanisms whereby S1P attenuates endothelial apoptosis elicited by H2O2. Our data implicate MAP kinase p38 as a point of control at which S1P counteracts H2O2-induced EC apoptotic responses.
T. Moriue et al. / Biochemical and Biophysical Research Communications 368 (2008) 852–857
Materials and methods Reagents. Antibodies specific to cleaved form of caspase-3, (phospho-) p38, and (phospho-) JNK were obtained from Cell Signaling Technologies (Beverly, MA). SB203580 and P600125 were from Calbiochem (Darmstadt, Germany). Cell Death Detection ELISAPlus kits were from Roche (Mannheim, Germany). ApopTag florescein in situ apoptosis detection kits were from Chemicon (Temecula, CA, USA). Other reagents derived from identical sources as we have previously described [11]. Cell culture and drug treatment. Cell culture and drug treatment were performed essentially as described [11]. Unless otherwise stated, bovine aortic EC (BAEC) between passages 5 and 7 were split at a ratio of 1:4 and used for experiments. At subconfluence, culture media were changed to Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS) and incubation proceeded overnight prior to all experiments to exclude the effects of residual S1P contained in FBS. SB203580 and P600125 were resolved in dimethyl sulfoxide (DMSO) and kept at 20 oC. Other drug treatments were performed exactly as described [11]. The final concentration of any solvent including DMSO did not exceed 0.1% (v/v) in any experiment. Enzyme-linked immunosorbent assay (ELISA). The degrees of intracellular DNA fragmentation were determined using cell death detection ELISAPlus kit essentially as described [12] following supplier’s protocol. All ELISA reactions were carried out with duplicate wells using a given cell lysate, and their averages were taken as a final result. A DNA–histone complex (supplied) and a lysis buffer blank were used as positive and negative controls in each assay, respectively. TUNEL staining. The degrees of DNA nick formation were determined in situ by means of TUNEL staining. BAEC were split at a ratio of 1:24 and were seeded onto gelatin-coated coverslips. At approximately 30% confluence 4 days after being split, cells were serum-starved overnight. They were washed with PBS on ice and were fixed with 1% paraformaldehyde in PBS (w/v) for 10 min at ambient temperature, followed by permeabilization with acetone/ethanol (1:2 in v/v) for 5 min at 20 oC. TUNEL staining was then performed with ApopTag florescein in situ apoptosis detection kit, according to the manufacturer’s instruction using a confocal laser microscope (Radiance 2100/Rainbow, BioRad, Hercules, CA) [13]. TUNEL-positive and negative cells were counted using five independent microscopic fields of 40 magnification in each coverslip. A total number of TUNEL-positive cells derived from five observed fields were summed up, in parallel with that of total cells; percentage of TUNEL positive cells over total was calculated in each coverslip by dividing the former number by the latter. Immunoblot analyses. Immunoblot analyses were performed exactly as described previously [11]. Statistics. All experiments were performed at least 3 times. Mean values for individual experiments are expressed as means ± SE. Statistical differences between two groups were analyzed by Student’s t test using Microsoft Excel. A p value less than 0.05 was considered statistically significant.
Results and discussion We first explored how H2O2 induces DNA fragmentation within BAEC, a key feature of apoptotic cell death, by means of ELISA. We treated BAEC with 1 mM of H2O2 up to 24 h. They were then subjected to ELISA in which the degrees of cytoplasmic DNA fragment formation were determined. As shown in Fig. 1A, this concentration of H2O2 started to increase DNA fragmentation of BAEC 2 h after the treatment with H2O2. The magnitude of DNA fragmentation peaked at 6 h after H2O2, reaching to 8.3-fold increase compared to t = 0, then returned to basal level within 24 h. Fig. 1B shows the results of dose– response study in which BAEC were treated for 6 h with
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increasing concentrations of H2O2. The degree of DNA fragmentation was maximum at 750 lM among the concentrations tested. These results demonstrate that H2O2 was capable of inducing DNA fragmentation of BAEC and that maximum response was obtained when they were treated with 750 lM of H2O2 for 6 h. We next examined whether or not S1P is able to attenuate apoptotic responses of BAEC to H2O2 treatment. Some BAEC had been pretreated with 1 lM of S1P for 30 min prior to H2O2 treatment (750 lM for 6 h), followed by ELISA. We note that similar concentrations of S1P can be found in adult human serum, especially in the presence of activated platelets [2], and also that in pathophysiological situations blood vessels can be exposed to hundred micromolar concentrations of H2O2 [6]. As indicated in Fig. 2A, pretreatment with S1P significantly attenuated H2O2-induced DNA fragmentation than that with vehicle. We utilized an independent approach to evaluate DNA damage of BAEC by subjecting them to TUNEL staining. As shown in left lower panel of Fig. 2B, significantly larger number of TUNEL-positive nuclei (green staining) were observed in H2O2-treated BAEC (arrowheads) compared with those without H2O2, showing that their DNA was nicked upon H2O2 treatment. Quantification of TUNELpositive nuclei revealed that the fraction of TUNEL-positive cells increased by 3.8-fold with H2O2 than basal. Significantly, when BAEC had been pretreated with 1 lM S1P, the fraction of TUNEL-positive cells decreased by 55.7% compared with those treated with H2O2 alone (Fig. 2B and C). Caspase-3 is a key ‘‘executer” protease of apoptotic processes of vascular EC [9]. We therefore examined the effects of H2O2/S1P on caspase-3 by immunoblot analyses of BAEC using an antibody specific to cleaved (activated) form of caspase-3. Fig. 2D demonstrates that H2O2 induces cleavage (activation) of caspase-3 protein, which was markedly attenuated when cells had been pre-incubated with S1P. A constitutive protein of BAEC, caveolin-1 [11], serves here as a loading control, which yields similar molecular mass as cleaved caspase-3, to ensure equal transfer and equivalent loading among protein samples. Together, these experiments indicated that S1P pretreatment reduced H2O2-induced apoptotic cell responses in BAEC, i.e., DNA fragmentation and cleavage/activation of caspase-3. A wide array of signaling machinery has been postulated as proximal molecules that connect cellular stimulation with execution of apoptosis. MAP kinases p38 and JNK have been previously implicated in H2O2-induced endothelial apoptosis [10,14]. We therefore sought to explore whether or not S1P attenuates H2O2-induced apoptosis by modulating activity of these molecules. We first evaluated the effects of H2O2/S1P on these MAP kinases by checking the degrees of phosphorylation. When BAEC were treated with H2O2, both MAP kinases underwent robust phosphorylation (activation) responses, as shown in the left half panel of Fig. 3A. Importantly, when they had been pretreated with S1P, the degrees of phosphoryla-
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Fig. 1. H2O2 induces DNA fragmentation in BAEC. Shown are the results of apoptotic cell death detection ELISA. (A) Results of time course study. BAEC were treated with 1 mM of H2O2 for the times indicated. Cells were then subjected to ELISA. The values determined at 405 nm in each time point were normalized to that obtained at t = 0. Each data point represents mean ± SE; N = 4. *p < 0.05 vs. t = 0. (B) BAEC were treated for 6 h using various concentrations of H2O2. The absorbance values obtained at each H2O2 concentration were normalized to that obtained with vehicle in each experiment; N = 4. *p < 0.05 vs. vehicle.
Fig. 2. S1P pretreatment reduces H2O2-induced apoptotic responses of BAEC. BAEC were treated with 750 lM of H2O2 or vehicle for 6 h. Some cells had been pretreated with 1 lM of S1P for 30 min. (A) Results of cell death detection ELISA. In each experiment, absorbance values obtained at 405 nm were normalized to that with H2O2 alone, which yielded maximum value of each experiment. Each data point represents mean ± SE; N = 4. (B) Representative images obtained in TUNEL assay, using confocal laser microscopy. Yellow arrowheads show TUNEL-positive signals. (C) The results of quantification of TUNEL assay. Fractions of TUNEL-positive cell numbers were determined in each preparation and expressed as percentage of the total. Each data point represents mean ± SE; N = 5. Upper half of (D) Representative images of immunoblots following H2O2/S1P treatment. Resulting membrane was probed for cleaved form of caspase-3 protein. It was re-probed for caveolin-1, which serves as a loading control. Lower half of (D) shows the results of densitometric analyses. Signals corresponding to cleaved form of caspase-3 were quantified and normalized against the value obtained with H2O2 alone (maximum) in each experiment. Each data point represents mean ± SE; N = 5.
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Fig. 3. S1P pretreatment attenuates H2O2-induced phosphorylation of p38 but not that of JNK in BAEC. (A) Representative images of immunoblots. BAEC were treated with 750 lM of H2O2 for the times indicated. Some cells had been treated with S1P (1 lM for 30 min). They were then probed for phosphorylated forms of p38 and JNK proteins. To confirm equal loading, they were re-probed for (total) p38 and JNK. (B and C) Results of densitometric analyses for phosphorylation responses of p38 and JNK, respectively. Signals corresponding to phosphorylated forms of p38 (or JNK) were quantified and normalized against the values obtained using antibodies specific to total p38 (or JNK). The resulting ratios of phospho/total p38 (or JNK) were then normalized to the basal value in each experiment. Each data point represents mean ± SE. Open and closed circles represent values obtained without and with pretreatment with S1P, respectively. *p < 0.05 versus H2O2 ( ). ; p < 0.05 versus S1P ( ). N = 5.
tion of only p38 MAP kinase, but not those of JNK phosphorylation, were attenuated (Fig. 3A–C). We then utilized a pharmacological inhibitor of p38 MAP kinase, SB203580. This agent markedly decreased H2O2-evoked cleavage of caspase-3 (Fig. 4A). In contrast, pretreatment with a JNK inhibitor P600125 (25 lM for 30 min) instead of SB203580 failed to attenuate H2O2-induced cleavage of caspase-3 (Fig. 4B). Note that SB203580 and P600125 under these conditions abrogated H2O2-induced phosphorylation/activation of p38 MAP kinase and JNK of BAEC, respectively (750 lM of H2O2 for 60 min, data not shown). When taken together, our results support a hypothesis that S1P attenuates H2O2-elicited caspase-3 activation at a level of, or proximal to, p38 MAP kinase, rather than JNK. Regulatory mechanisms of p38 MAP kinase by H2O2 appear to be cellular context-dependent and quite complex, involving various protein kinases like MAP kinase kinase MKK6, protein kinase D, and a protein kinase ASK-1, as well as protein phosphatases like MKP-1 [15]. Thus, precise molecular mechanisms whereby S1P interferes H2O2evoked activation of p38 MAP kinase in BAEC remain to be elucidated. Our findings that S1P is able to counteract H2O2-dependent apoptotic responses of BAEC are consistent with
earlier reports that imply S1P as a pro-survival sphingolipid metabolite. For example, S1P keeps various endothelial cell cultures from undergoing apoptosis, elicited by such diverse stimuli as serum starvation [3], tumor necrosis factor-a [4], and ceramide [5]. Interestingly, H2O2-induced apoptosis in EC can be recovered by cell stimulation other than S1P as well, for example by an angiogenic polypeptide growth factor angiopoietin-1 [16]. Thus, the present findings add another level of complexity to our understandings of vascular endothelial cell fate, modulated by oxidative stress and sphingolipids, as well as by other classes of molecules. We note that S1P also modulates apoptosis of skin cells, including fibroblasts and keratinocytes [17,18]. Because ROS are generated in skin at some dermatopathological situations, including UV irradiation [8] and wound formation [19], roles of S1P on cutaneous cell fate in the presence of excess ROS need to be determined. In this regard, a recent study by Kawanabe et al. provides an intriguing observation that addition of a high concentration of S1P has a therapeutic effect on wounded skin of diabetic, but not normal, mice [20]. While S1P has well-established pro-angiogenic properties [1] that would promote wound healing in these mice, our results raise a possibility that
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Fig. 4. SB203580, an inhibitor of p38 MAP kinase, attenuates H2O2-induced caspase-3 cleavage in BAEC. Upper part of (A) Representative images of immunoblots probed for cleaved form of caspase-3. BAEC were treated with H2O2 for 6 h, following pretreatment with SB203580 (0.4 lM for 30 min). Cells were subjected to immunoblot assays, followed by densitometry as above. In (B), cells had been pretreated with P600125 (25 lM for 30 min) prior to H2O2. Data are expressed as means ± SE, N = 4.
an anti-apoptotic potential of S1P could also contribute to promotion of wound healing processes. Additionally a novel immunosuppressant FTY-720, which, when phosphorylated, binds to specific S1P receptors and exerts beneficial effects on atopic dermatitis [21]. In conclusion, our results demonstrate that S1P, a platelet-derived sphingolipid mediator, attenuates H2O2-induced apoptosis of cultured vascular EC, involving caspase-3 and p38 MAP kinase. References [1] T. Hla, Physiological and pathological actions of sphingosine 1phosphate, Semin. Cell Dev. Biol. 15 (2004) 513–520. [2] Y. Yatomi, Y. Igarashi, L. Yang, N. Hisano, R. Qi, N. Asazuma, K. Satoh, Y. Ozaki, S. Kume, Sphingosine 1-phosphate, a bioactive sphingolipid abundantly stored in platelets, is a normal constituent of human plasma and serum, J. Biochem. (Tokyo) 121 (1997) 969–973. [3] Y.G. Kwon, J.K. Min, K.M. Kim, D.J. Lee, T.R. Billiar, Y.M. Kim, Sphingosine 1-phosphate protects human umbilical vein endothelial cells from serum-deprived apoptosis by nitric oxide production, J. Biol. Chem. 276 (2001) 10627–10633. [4] P. Xia, L. Wang, J.R. Gamble, M.A. Vadas, Activation of sphingosine kinase by tumor necrosis factor-a inhibits apoptosis in human endothelial cells, J. Biol. Chem. 274 (1999) 34499–34505. [5] N. Hisano, Y. Yatomi, K. Satoh, S. Akimoto, M. Mitsumata, M.A. Fujino, Y. Ozaki, Induction and suppression of endothelial cell apoptosis by sphingolipids: a possible in vitro model for cell–cell interactions between platelets and endothelial cells, Blood 93 (1999) 4293–4299. [6] P.A. Ward, Mechanisms of endothelial cell killing by H2O2 or products of activated neutrophils, Am. J. Med. 91 (1991) 89S–94S. [7] H. Cai, Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences, Cardiovasc. Res. 68 (2005) 26–36. [8] T. Herrling, J. Fuchs, J. Rehberg, N. Groth, UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides, Free Radic. Biol. Med. 35 (2003) 59–67.
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