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Cardiovascular Research 2006 69(4):908-915; doi:10.1016/j.cardiores.2005.11.023
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Copyright © 2005, European Society of Cardiology

Endoplasmic reticulum Ca2+ depletion induces endothelial cell apoptosis independently of caspase-12

Tomoyasu Nakanoa, Hiroshi Watanabeb,*, Mariko Ozekia, Masayoshi Asaia, Hideki Katoha, Hiroshi Satoha and Hideharu Hayashia

aDepartment of Internal Medicine III, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
bDepartment of Clinical Pharmacology and Therapeutics, Hamamatsu University School of Medicine. 1-20-1 Handayama, Hamamatsu, 431-3192, Japan

* Corresponding author. Tel.: +81 53 435 2267; fax: +81 53 434 2910. Email address: hwat{at}hama-med.ac.jp

Received 2 September 2005; revised 7 November 2005; accepted 17 November 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: Apoptosis of endothelial cells is considered an initial step in the development of atherosclerosis. Recent studies have indicated that depletion of the endoplasmic reticulum (ER) Ca2+ content plays an important role in apoptosis. Caspase-12 is a key signal in ER stress-induced apoptosis. However, it is not known whether the depletion of ER Ca2+ is linked to caspase-12 signalling in endothelial cells. Here we have investigated the interaction of Ca2+ signalling and caspase-12 cleavage in apoptosis of endothelial cells.

Methods: Cytosolic Ca2+ concentration ([Ca2+]i) of primary porcine aortic endothelial cells was measured using fura-2/AM. Apoptosis was assessed by DNA fragmentation, and cleavage of caspase-12 using Western blotting techniques.

Results: Thapsigargin (5 µM), an inhibitor of the ER Ca2+-ATPase, depleted ER Ca 2+ content, increased [Ca2+]i, cleaved caspase-12, and induced apoptosis. Bradykinin (10 nM) also increased [Ca2+]i but did not cleave caspase-12 or induce apoptosis. However, when intracellular Ca2+ was chelated with BAPTA/AM (100 µM), bradykinin caused ER Ca2+ depletion and apoptosis without accompanying caspase-12 cleavage. A non-selective caspase inhibitor, z-VAD.fmk (100 µM), inhibited apoptosis and cleavage of caspase-12 stimulated by thapsigargin, while a calpain inhibitor, MDL 28170 (120 µM), inhibited caspase-12 cleavage but not apoptosis.

Conclusions: Thus, increases in intracellular Ca2+ concentration are not sufficient for the induction of apoptosis in endothelial cells, and ER Ca2+ depletion appears to induce apoptosis independently of caspase-12.

KEYWORDS Apoptosis; Calcium; Caspase-12; Endothelial cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Endothelial cell apoptosis is now considered an initial step in the development of atherosclerosis; it is associated with decreased NO production and increased superficial endothelial denudation leading to endothelial intercellular gap formation, which facilitates adhesion of monocytes and platelets [1]. In many cell types endoplasmic reticulum (ER) stress has been shown to cause apoptosis [2–4]. The ER is the site of folding and assembly of membrane proteins and plays a very important role in the regulation of intracellular Ca2+ homeostasis [5,6]. Conditions that alter protein folding or calcium homeostasis in the ER trigger a signalling cascade known as the unfolded protein response (UPR) [7]. If the stress on ER is excessive or prolonged, UPR initiates the apoptotic cell-death cascade [2,8]. However, the cellular factors responsible for ER-stress-induced apoptosis in endothelial cells have not been completely elucidated.

Caspases, a family of cysteine-dependent aspartate-specific proteases, are critical mediators of apoptosis. Fourteen members of caspase family have been identified that are widely expressed in a variety of tissues and cell types. Caspases normally exist in cells as proenzymes, which may be activated through recruitment into activating complexes or direct proteolytic cleavage by another caspase [9]. Among the caspase family members, caspase-12 is a key signal involved in ER stress-induced apoptosis [2]. Caspase-12 is localized on the cytoplasmic side of the ER and is specifically activated in cells treated with ER stress agents, but not in cells treated with death cytokines or intrinsic apoptotic stimuli. It has been shown that cells from caspase-12-deficient mice are resistant to apoptosis triggered by the known ER stress agents [2].

Thapsigargin (TG), a sesquiterpene lactone tumor promoter derived from the plant Thapsia garganica, is known to be a strong inducer of ER stress that causes cleavage of caspase-12. TG is a selective inhibitor of the ER Ca2+-ATPase [10]. TG inhibits the uptake of Ca2+ into the ER, leading to depletion of ER Ca2+ content, which stimulates the entry of extracellular Ca2+ [10–12].

Previous studies demonstrated that elevation of cytosolic Ca2+ concentration ([Ca2+]i) is an important component of the signalling pathways leading to apoptosis [13–15]. However, in some cell types, such as lymphoma cells, it has been reported that the depletion of ER Ca2+ stores plays an important role in apoptosis rather than the increase in [Ca2+]i [3,4]. Thus, it is not entirely clear which components of the Ca2+ signalling cascade are important to trigger apoptosis in endothelial cells.

In this study, we investigated the effect of ER Ca2+ depletion on apoptosis and the relationship between caspase-12 cleavage and ER Ca2+ depletion in primary cultured porcine aortic endothelial cells. Our data suggest that increased [Ca2+]i is not sufficient for the induction of apoptosis in endothelial cells. In addition, the ER Ca2+ depletion appears to cause apoptosis independently of the cleavage of caspase-12.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1 Cell culture
Porcine aortic endothelial cells (ECs) were isolated and cultured, as previously described [16], by gently scraping the intima of the descending part of porcine aortas. After centrifugation at 250 x g for 10 min in Medium 199 (M-199), the pellet of ECs was purified from this suspension, resuspended in M-199 medium with Earle's salts, and supplemented with 100 IU/ml penicillin G, 100 µM streptomycin, and 20% newborn calf serum (NCS). Cells were then seeded onto culture dishes or polybiphenyl dishes fixed on glass coverslips, and incubated at 37 °C in 5% CO2. Due to known loss of signaling components in cells of late passages, only cells from the first to second passages were used. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

2.2 Measurement of cytosolic Ca2+ concentration ([Ca2+]i)
[Ca2+]i was measured in individual ECs as described previously [17,18]. Cells were incubated for 45 min in a modified Tyrode's solution (pH 7.4 at 25 °C) containing NaCl (150 mM), KCl (2.7 mM), KH2PO4 (1.2 mM), MgSO4 (1.2 mM), CaCl2 (1.0 mM), and HEPES (10 mM) with 10% NCS and 2 µM fura-2/AM, a fluorescence Ca2+ indicator. The cells were subsequently washed 3 times with the modified Tyrode's solution to remove the dye. Experiments were performed at 25 °C. The fura-2 absorption shift that occurs upon binding was determined by scanning the excitation spectra between 340 and 380 nm while monitoring emission at 510 nm. The resultant fluorescence images were analyzed every 30 s from the individual cells with a fluorescence analyzer (Aqua-cosmos) using an ultra high-sensitivity television camera (CCD). The fluorescence ratio (F340/F380) was obtained by dividing, after background subtraction, the 340 nm by the 380 nm images on a pixel-by-pixel basis.

2.3 Western blotting
After addition of ER stress stimuli, including TG, or BK and BAPTA/AM, cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated for 30 min on ice in PBS containing 1% Triton X-100. Cells were harvested by scraping and centrifuged 10,000 x g at 4 °C for 10 min. Supernatants were collected and heated with SDS sample buffer, separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. After blocking in Tris-buffered saline (10 mM Tris, pH 7.5, 100 mM, NaCl) containing Tween 20 (0.1% vol/vol), and non-fat dry milk (1% vol/vol) for 1 h, the membrane was probed with antibody against caspase-12 (1:333) for 8 h at 4 °C. Proteins bound to primary antibody were probed with secondary antibody (1:5000 for 1 h at room temperature), conjugated with horseradish peroxidase and visualized by enhanced chemiluminescence using a commercially available kit (Amersham Biosciences, Buckinghamshire, UK). Densitometric analysis was performed using the public domain NIH image 1.56 program.

2.4 DNA fragmentation
Approximately 2 x 106 cells were grown in 100 mm Petri dishes. After treatment with agents, the cells were scraped off using a rubber policeman, centrifuged at 2500 x g for 5 min, and resuspended in 100 µl of lysis buffer: containing 10 mM Tris–HCl, 10 mM EDTA and 0.5% Triton X-100, pH 7.4. After incubation on ice for 10 min, lysates were centrifuged at 15,000 x g for 20 min at 4 °C. The supernatants were supplemented with 4 µl of RNase A (10 mg/ml) and then incubated at 37 °C for 1 h. Next, 4 µl of proteinase K (10 mg/ml) was added and incubation was continued at 37 °C for 1 h. Twenty microliters of 5 M NaCl and 120 µl of isopropanol were added and the mixture was kept overnight at –20 °C. After centrifugation at 15,000 x g for 15 min, DNA pellets were resuspended in 10 mM Tris–HCl (pH 7.4) containing 1 mM EDTA. The DNA samples were exposed to electrophoresis on 2% agarose gel for 30 min at 100 V. The gel was stained with ethidium bromide and photographed under UV transillumination.

2.5 Materials
M-199, NCS, trypsin, penicillin, and streptomycin were purchased from Invitrogen Corp. (Carlsbad, CA, USA). Fura-2/AM was obtained from Dojindo (Kumamoto, Japan). BK, TG, benzyloxycarbonyl-Val-Ala-Asp(OCH3)-CH2F (z-VAD.fmk) and calpain inhibitor III (MDL28170) were purchased from Sigma (St. Louis, MO, USA). BAPTA/AM was obtained from Molecular Probes, Inc. (Eugene, USA). Caspase-12 polyclonal antibody was obtained from Oncogene Research Products (San Diego, CA, USA). Ethidium bromide was purchased from Wako (Osaka, Japan). BK, TG, and BAPTA/AM were dissolved and stored as stock solutions in 1% dimethyl sulfoxide and diluted to the desired concentrations in solutions indicated for each experiment. All other chemicals were of the best available quality, mostly at analytical grades.

2.6 Statistics
Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed by one-way ANOVA followed by multiple comparisons using Fisher's protected least-significant difference test. Statistical analysis was performed using StatView 5.0 statistic program (Abacus Concepts, Berkley, CA, USA). Statistical significance was assumed at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1 Effects of thapsigargin and bradykinin on cytosolic Ca2+ concentration in ECs
As frequently observed, both TG (5 µM) and BK (10 nM) increased [Ca2+]i in ECs, with F340/F380 ratios from 0.74 ± 0.05 to 5.89 ± 0.52 after 5 min of the treatment with TG, and from 0.73 ± 0.09 to 5.91 ± 0.71 after 1 min of the treatment with BK, respectively (Fig. 1A,B). In addition, in the absence of extracellular Ca2+, both TG and BK deplete internal Ca2+ stores and result in a transient increase in [Ca2+]i (not shown).


Figure 1
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  		Fig. 1 Effects of TG and BK on [Ca2+]i. Ca2+ response to TG (5 µM) (A) and BK (10 nM) (B) in ECs. Data are the average ratio (mean ± SEM) of n=20 cells. Extracellular medium contained 1 mM Ca2+. DNA fragmentation (C) and cleavage of caspase-12 (D) following the treatment with TG and BK for 48 h. Densitometric values are normalized to that of control. Histogram data represent the average (mean ± SEM) of five determinations ({dagger}p<0.05 vs. the level of caspase-12 in the control); lane 1: control, lane 2: TG (5 µM), lane 3: BK (10 nM).

 
3.2 Effects of thapsigargin and bradykinin on ER stress and apoptosis in ECs
ER stress has been shown to induce cleavage of caspase-12, followed by DNA fragmentation [2]. The continuous presence of TG (5 µM) for 48 h was associated with DNA fragmentation (Fig. 1C, lane 2) and cleavage of caspase-12 in ECs (Fig. 1D, lane 2). Caspase-12 expression level was 19.18 ± 19.67% of control level following treatment with TG. However, these effects were not observed with BK (Fig. 1C,D, lane 3). Thus, although increases in [Ca2+]i are observed early in the time courses of treatment with both TG and BK, only TG causes apoptosis and cleavage of caspase-12.

3.3 Effect of BK+BAPTA/AM on apoptosis and ER stress in ECs
BAPTA/AM is widely used as a cytosolic Ca2+ chelator. To confirm this effect, we measured [Ca2+]i following stimulation with BK or TG in ECs pretreated with or without BAPTA/AM. In cells pretreated with BAPTA/AM (100 µM) for 90 min no increases [Ca2+]i were observed following stimulation with either TG or BK, with F340/F380 ratios from 0.59 ± 0.04 to 0.60 ± 0.03 after 8 min of the treatment with TG, and from 0.61 ± 0.03 to 0.61 ± 0.22 after 10 min the treatment with BK, respectively (Fig. 2A,B).


Figure 2
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  		Fig. 2 Effects of BAPTA/AM (100 µM) on TG-and BK-stimulated Ca2+ responses in ECs. Ca2+ responses to TG (5 µM, A) or BK (10 nM, B) in control ECs (open circles) or ECs pretreated for 90 min with 100 µM BAPTA/AM (closed circles). Data are the average ratio (mean ± SEM) of n=20 cells (*p<0.05 vs. control). Extracellular medium contained 1 mM Ca2+. Effect of ER Ca2+ depletion on DNA fragmentation (C) and cleavage of caspase-12 (D) in ECs. Densitometric values are normalized to that of control. Histogram data represent the average (mean ± SEM) of five determinations; (C, D) lane 1: control, lane 2: BK (10 nM), lane 3: BK-stimulated ECs pretreated with BAPTA/AM (100 µM).

 
Having confirmed the Ca2+ chelation effect of BAPTA/AM, we now directly addressed the question of whether increases in [Ca2+]i are necessary for ER stress and apoptosis. In BK-stimulated ECs, pretreatment with BAPTA/AM induced DNA fragmentation (Fig. 2C), but not cleavage of caspase-12 (Fig. 2D), caspase-12 expression being 126.90 ± 32.76% (not significant) and 118.71 ± 64.85% (not significant) of control value following treatment with BK alone and BK plus BAPTA/AM pre-treatment, respectively. Although BK induces Ca2+ release from the ER, this process is compensated for by Ca2+ refilling via the ER Ca2+-ATPase. It is likely that chelation of cytosolic Ca2+ with BAPTA/AM inhibits the refilling process and maintains ER Ca2+ depletion in BK-stimulated ECs.

3.4 Effect of inhibition of calpain on apoptosis and ER stress in TG-stimulated ECs
Calpain is a Ca2+-responsive cytosolic cysteine protease associated with the cleavage of caspase-12 [19]. To examine if the observed effects of TG to cause caspase-12 cleavage are associated with calpain, we used a non-selective caspase inhibitor, z-VAD.fmk (zVAD), and a calpain inhibitor III (CAI), MDL 28170 [19]. Inhibition of either caspase or calpain with zVAD (100 µM) or CAI (120 µM), respectively, prevented the cleavage of caspase-12 stimulated by TG in ECs (Fig. 3C,D). Moreover caspase inhibitor prevented TG-induced DNA fragmentation (Fig. 3A). However, calpain inhibition did not prevent the TG-induced DNA fragmentation as did inhibition of caspase (Fig. 3B).


Figure 3
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  		Fig. 3 Effects of non-selective caspase inhibitor and calpain inhibitor on DNA fragmentation (A, B) and cleavage of caspase-12 (C, D) in TG-stimulated ECs. Densitometric values are normalized to that of control. Histogram data represent the average (mean ± SEM) of five determinations ({dagger}p<0.05 vs. the level of caspase-12 in the control). (A, C) lane 1: control, lane 2: TG (5 µM), lane 3: TG- and zVAD (100 µM)-stimulated ECs. (B, D) lane 1: Control, lane 2: TG (5 µM), lane 3: TG-and CAI (120 µM)-stimulated ECs.

 
3.5 Effect of caspase inhibition on apoptosis caused by ER Ca2+ depletion
To evaluate the apoptosis pathway by ER Ca2+ depletion, we examined the effect of the caspase inhibitor zVAD on DNA fragmentation induced by BK in ECs pretreated with BAPTA/AM. zVAD (100 µM) inhibited the DNA fragmentation in BK-stimulated ECs pretreated with BAPTA/AM (100 µM) (Fig. 4).


Figure 4
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  		Fig. 4 Effects of zVAD.fmk on DNA fragmentation (A) and cleavage of caspase-12 (B) in ECs. Densitometric values are normalized to that of control. Histogram data represent the average (mean ± SEM) of five determinations. (A, B) lane 1: control, lane 2: BK (10 nM), lane 3: BK-stimulated ECs pretreated for 90 min with BAPTA/AM (100 µM), lane 4: BK-and zVAD (100 µM)-stimulated ECs pretreated for 90 min with BAPTA/AM (100 µM).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The ER is the site of assembly of polypeptide chains destined for secretion or routing into various subcellular compartments. The ER is also the site of storing intracellular Ca2+ that is a major second messenger implicated in signal transduction pathways regulating cell cycle, proliferation and apoptosis. Recent studies have indicated that prolonged dysfunction and/or stress in the ER may mediate apoptosis and contribute to the pathogenesis of several diseases, including ischemia-reperfusion injury, Parkinson's Disease, Alzheimer's Disease, and diabetes [20–22]. ER stress-induced apoptosis of ECs has been implicated in the initiation of atherosclerosis [1,23].

In vitro ER stress is caused by a variety of insults, such as inhibitors of protein folding, glycosylation, and trafficking [2,7,24]. Disruption of Ca2+ homeostasis in the ER is also considered important to trigger ER stress and ER stress-induced apoptosis. The concentration of Ca2+ in the ER is maintained higher than that in the cytoplasm since Ca2+ is actively transported into the ER via sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) family of membrane transporters. TG, which inhibits the ER Ca2+-ATPase and disrupts Ca2+ homeostasis, also induces ER stress [2,4,10–12,25]. This agent causes the rise of [Ca2+]i as well as the depletion of stored Ca2+ in the ER. Previous studies demonstrated that the elevation of [Ca2+]i is an important component of the signalling pathway for apoptosis [13–15]. Increased cytosolic Ca2+ can mediate apoptosis through activation of calpains and calcineurin-mediated dephosphorylation of Bad [26,27]. Also Ca2+ may contribute to apoptotic cell recognition and phagocytes by promoting externalization of phosphatidylserine on the plasma membrane [28]. However, it is not known whether the rise of cytosolic Ca2+ concentration or the depletion of stored Ca2+ in the ER is critical to cause apoptosis in ECs.

BK and TG stimulated similar peak increases in [Ca2+]i in ECs (Fig. 1A,B). However in this study we observed that BK increased [Ca2+]i, but failed to cause apoptosis as did TG. These results strongly suggest the important of ER Ca2+ depletion and that the elevation of [Ca2+]i is not sufficient to induce apoptosis in ECs. Although both TG and BK trigger Ca2+ mobilization from the ER in ECs, the former irreversibly depletes the ER Ca2+ content, while the latter does so reversibly. The reversibility of depletion of the ER Ca2+ content would therefore appear to be the simplest explanation for the difference between the effects of these two agents on caspase-12 cleavage and apoptosis. This possibility was tested by use of the cytosolic Ca2+ chelator BAPTA/AM, which keeps the ER Ca2+ content from being refilled after stimulation with BK. Under this condition, DNA fragmentation was observed (Fig. 2C). This finding supports the idea that the ER Ca2+ content rather than the level of cytosolic Ca2+ is an important determinant of the induction of apoptosis in ECs.

It has been suggested that ER stress-dependent apoptotic cell death is caused through the activation of ER-specific caspase-12 [2]. Consistent with these findings, we observed that TG caused DNA fragmentation via cleavage of caspase-12. The inhibition of this enzyme with zVAD, a non-specific caspase inhibitor, prevented the cleavage of caspase-12 and DNA fragmentation in TG-stimulated ECs (Fig. 3). However, ER stress has been shown to induce apoptosis via different pathways [29–32]. For example, in human neuroblastoma SH-SY5Y cells the antiapoptotic protein Bcl-2 inhibits apoptosis induced by TG and the Ca2+ ionophore A23187 [GenBank] , but not that induced by tunicamycin, (an inhibitor of N-glycosylation in ER) and brefeldin A, (an inhibitor of ER-Golgi transport) [29]. Since Bcl-2 has been shown to protect mitochondrial-mediated apoptosis, these results suggest that TG and A23187 [GenBank] may also trigger mitochondria-mediated apoptotic pathways.

In line with these studies, an interesting finding is that depletion of ER Ca2+ content by the combination of BK and BAPTA/AM causes DNA fragmentation but not the cleavage of caspase-12. This appears to argue against the involvement of caspase-12 in the induction of apoptosis by depletion of ER Ca2+ stores in ECs. This position is further supported by the finding that inhibition of calpain with MDL 28170 prevented TG-induced cleavage of caspase-12 but not apoptosis. While only caspase-12 cleavage is prevented by inhibition of calpain, both this and apoptosis are prevented by the non-selective caspase inhibitor zVAD. zVAD can also prevent the cleavage of caspase-8 and -9 [30]. Thus it appears that TG can induce apoptosis in ECs by alternative pathways that may or may not involve caspase-12 and/or other caspase family members (Fig. 5). In support of this observation, a murine cell line lacking expression of caspase-12 is not protected from apoptosis induced by ER stress agents, suggesting that caspase-12 is not always required for the induction of ER stress induced apoptosis [32]. In addition, it has also been demonstrated that TG can cleave caspase-3, -9 in murine myoblast cells [29].


Figure 5
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  		Fig. 5 There are at least two mechanisms inducing apoptosis under ER stress, namely, disruption of protein folding and disruption of ER Ca2+ homeostasis. There are also two signalling pathways leading to apoptosis: caspase-12-dependent and-independent pathways. Disruption of protein folding may induce apoptosis through calpain/caspase-12 dependent pathway. Disruption of ER Ca2+ homeostasis may induce apoptosis through caspase-12-independent pathway.

 
In conclusion, we have demonstrated for the first time that the depletion of ER Ca2+ content can be more crucial rather than the increase of [Ca2+]i to induce apoptosis in ECs, and this pathway involves caspase(s) other than caspase-12. ER stress has been shown to be triggered by the alteration of protein folding as well as the disruption of Ca2+ handling in ER. Since the caspase-12-dependent apoptotic pathway appears to be uninfluenced by the disruption of ER Ca2+ handling, it is possible that this pathway may follow the disruption of protein folding. EC apoptosis leads to EC dysfunction, and increases susceptibility of the vessel wall to atherosclerosis. Therefore, further elucidation of the mechanisms of apoptosis in ECs will contribute to a better understanding of atherosclerosis and the development of relevant treatment modalities.


   Acknowledgements
 
The authors are grateful to Dr. Quang-Kim Tran (University of Missouri-Kansas City) for his helpful insights. This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H. Watanabe and by a grant-in-aid from the Center of Excellence from the Ministry of Education, Culture, Sports, Science, and Technology.


   Notes
 
Time for primary review 32 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

  1. Choy J.C., Granville D.J., Hunt D.W.C., McManus B.M. Endothelial cell apoptosis: biochemical characteristics and potential implication for atherosclerosis. J Mol Cell Cardiol (2001) 33:1673–1690.[CrossRef][Web of Science][Medline]
  2. Nakagawa T., Zhu H., Morishima N., Li E., Xu J., Yuan J., et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyliod-β. Nature (2000) 403:98–103.[CrossRef][Medline]
  3. He H., Lam M., McCormick T.S., Distelhorst C.W. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol (1997) 138:1219–1228.[Abstract/Free Full Text]
  4. Kaneko Y., Tsukamoto A. Thapsigargin-induced persistent intracellular calcium pool depletion and apoptosis in human hepatoma cells. Cancer Lett (1994) 97:147–155.
  5. Rapoport T.A. Transport of Proteins across the endoplasmic reticulum membrane. Science (1992) 258:931–936.[Abstract/Free Full Text]
  6. Sambrook J.F. The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum. Cell (1990) 61:197–199.[CrossRef][Web of Science][Medline]
  7. Kaufman R.J. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational control. Genes Dev (1999) 13:1211–1233.[Free Full Text]
  8. Ferri K.F., Kroemer G. Organelle-specific initiation of cell death pathways. Nat Cell Biol (2001) 3:E255–E263.[CrossRef][Web of Science][Medline]
  9. Cryns V., Yuan J. Proteases to die for. Genes Dev (1998) 12:1551–1570.[Free Full Text]
  10. Thastrup O., Cullen P.J., Drobak B.K., Hanley M.R., Dawson A.P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci U S A (1990) 87:2466–2470.[Abstract/Free Full Text]
  11. Tran Q.K., Ohashi K., Watanabe H. Calcium signaling in endothelial cells. Cardiovasc Res (2000) 48:13–22.[Abstract/Free Full Text]
  12. Yamamoto N., Watanabe H., Kakizawa H., Hirano M., Kobayashi A., Ohno R. A study on thapsigargin-induced calcium ion and cation influx pathways in vascular endothelial cells. Biochim Biophys Acta (1995) 1266:157–162.[Medline]
  13. Caron-Leslie L.M., Cidlowski J.A. Similar actions of glucocorticoids and calcium on the regulation of apoptosis in S49 cells. Mol Endocrinol (1991) 5:1169–1179.[Abstract/Free Full Text]
  14. Kaiser N., Edelman I.S. Calcium dependence of glucocorticoid-induced lymphocytolysis. Proc Natl Acad Sci U S A (1977) 74:638–642.[Abstract/Free Full Text]
  15. McConkey D.J., Nicotera P., Hartzell P., Bellomo G., Wyllie A.H., Orrenius S. Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration. Arch Biochem Biophys (1989) 269:365–370.[CrossRef][Web of Science][Medline]
  16. Watanabe H., Kuhne W., Spahr R., Schwartz P., Piper H.M. Macromolecule permeability of coronary and aortic endothelial monolayers under energy depletion. Am J Physiol (1991) 260:H1344–H1352.[Web of Science][Medline]
  17. Watanabe H., Takahashi R., Zhang X.X., Kakizawa H., Hayashi H., Ohno R. Inhibition of agonist-induced Ca2+ entry in endothelial cells by myosin light-chain kinase inhibitor. Biochem Biophys Res Commun (1996) 225:777–784.[CrossRef][Web of Science][Medline]
  18. Watanabe H., Takahashi R., Zhang X.X., Kakizawa H., Hayashi H., Ohno R. An essential role of myosin light-chain kinase in the regulation of agonist-and fluid flow-stimulated Ca2+ influx in endothelial cells. FASEB J (1998) 12:341–348.[Abstract/Free Full Text]
  19. Nakagawa T., Yuan J. Cross-talk between two cysteine protease families: activation of caspase-12 by calpain in apoptosis. J Cell Biol (2000) 150:887–894.[Abstract/Free Full Text]
  20. Katayama T., Imaizumi K., Sato N., Miyoshi K., Kudo T., Hitomi J., et al. Presenilin-1 mutations downregulate the signaling pathway of the unfolded-protein response. Nat Cell Biol (1999) 1:479–485.[CrossRef][Web of Science][Medline]
  21. Imai Y., Soda M., Inoue H., Hattori N., Mizuno Y., Takahashi R. An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of Parkin. Cell (2001) 105:891–902.[CrossRef][Web of Science][Medline]
  22. Kitamura Y., Nomura Y. Stress proteins and glial functions: possible therapeutic targets for neurodegenerative disorders. Pharmacol Ther (2003) 97:35–53.[CrossRef][Web of Science][Medline]
  23. Takeuchi K., Watanabe H., Tran Q.K., Ignarro L.J., Ohashi K., Hayashi H., et al. Nitric oxide: inhibitory effects on endothelial cell calcium signaling, prostaglandin I2 production and nitric oxide synthase expression. Cardiovasc Res (2004) 62:194–201.[Abstract/Free Full Text]
  24. Mori K. Tripartite management of unfolded proteins in the endoplasmic reticulum. Cell (2000) 101:451–454.[CrossRef][Web of Science][Medline]
  25. Putney J.W. Capacitative calcium entry revisited. Cell Calcium (1990) 11:611–624.[CrossRef][Web of Science][Medline]
  26. Carafoli E., Molinari M. Calpain: a protease in search of a function? Biochem Biophys Res Commun (1998) 247:193–203.[CrossRef][Web of Science][Medline]
  27. Wang H.G., Pathan N., Ethell I.M., Krajewsky S., Yamaguchi Y., Reed J.C., et al. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science (1999) 284:339–343.[Abstract/Free Full Text]
  28. Comfurius P., Senden J.M., Tilly R.H., Schroit A.J., Bevers E.M., Zwaal R.F. Loss of membrane phospholipid asymmetry in platelets and red cells may be associated with calcium-induced shedding of plasma membrane and inhibition of aminophospholipid translocase. Biochim Biophys Acta (1990) 1026:153–160.[Medline]
  29. Kitamura Y., Miyamura A., Inden M., Tsuchiya D., Nakamura K., Taniguchi T., et al. Possible involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in thapsigargin-induced apoptosis in human neuroblastoma SH-SY5Y cells. J Pharmacol Sci (2003) 92:228–236.[CrossRef][Web of Science][Medline]
  30. Jimbo A., Fujita E., Kouroku Y., Ohnishi J., Yonehara S., Momoi T., et al. ER stress induces caspase-8 activation, stimulating cytochrome c release and caspase-9 activation. Exp Cell Res (2003) 283:156–166.[CrossRef][Web of Science][Medline]
  31. Morishima N., Nakanishi K., Takenouchi H., Shibata T., Yukuto Y. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. J Biol Chem (2002) 37:34287–34294.
  32. Obeng E.A., Boise L.H. Caspase-12 and caspase-4 are not required for caspase-dependent endoplasmic reticulum stress-induced apoptosis. J Biol Chem (2005) 280:29578–29587.[Abstract/Free Full Text]

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