Cellular Signalling 27 (2015) 1688–1693
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Coronin 1A depletion protects endothelial cells from TNFα-induced apoptosis by modulating p38β expression and activation Geun-Young Kim, Hanna Kim, Hyun-Joung Lim, Hyun-Young Park ⁎ Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, Cheongju, Republic of Korea
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Article history: Received 29 January 2015 Received in revised form 23 April 2015 Accepted 25 April 2015 Available online 30 April 2015 Keywords: Coronin 1A Tumor necrosis factor α p38β Endothelial cell Apoptosis
a b s t r a c t Coronins are conserved actin-binding proteins that regulate various cellular processes such as migration and endocytosis. Among coronin family members, coronin 1A is highly expressed in hematopoietic lineage cells where it regulates cell homeostasis. However, the expression and function of coronin 1A in endothelial cells have not yet been elucidated. We found that coronin 1A is expressed in the human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell (HBMVEC). In HUVEC depleted of coronin 1A by siRNA transfection, tumor necrosis factor α (TNFα) + cyclohexamide (CHX) treatment resulted in a decrease in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive apoptotic cells. Coronin 1A depletion also resulted in the suppression of caspase 3 and poly(ADP-ribose) polymerase cleavage and a reduction in caspase 3 activity. Next, we examined TNFα-induced activation of several pro- and anti-apoptotic signaling molecules to find the target molecule of coronin 1A and found that p38 phosphorylation was enhanced by TNFα stimulation in coronin 1A-depleted HUVEC. Among the p38 isoforms, the expression of p38β was significantly upregulated after coronin 1A depletion, suggesting that the expression and phosphorylation of antiapoptotic p38β were significantly induced in coronin 1A-depleted HUVEC. Inhibition of p38β upregulation in coronin 1A-depleted HUVEC restored the cleavage of caspase 8 and caspase 3 and induced more apoptosis than in coronin 1A-depleted HUVEC in response to TNFα + CHX. These findings suggest that coronin 1A modulates endothelial cell apoptosis by regulating p38β expression and activation. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Endothelial cell apoptosis is an important element under physiologically normal and pathological conditions [1,2]. Endothelial cells are continuously exposed to pro- and anti-apoptotic stimuli and the fate of endothelial cells is dependent on the balance between these stimuli [3]. Enhanced endothelial cell apoptosis is associated with cardiovascular diseases including atherosclerosis and thrombosis [4,5]. For example, increased endothelial cell apoptosis was observed in atheroprone areas exposed to disturbed flow in the aorta [4]. Among the environmental factors found within the pro-apoptotic regions of atherosclerosis tissue, tumor necrosis factor (TNF)α is the most prominent and is frequently used to study endothelial cell apoptosis because it is the most abundant
Abbreviations: TNFα, tumor necrosis factor α; HUVEC, human umbilical vein endothelial cell; HBMVEC, human brain microvascular endothelial cell; CHX, cyclohexamide; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. ⁎ Corresponding author at: Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, 187, Osongsaengmyeng 2-ro, Osong-eup, Heungdeok-gu, Cheongju-city, Chungcheongbuk-do 361-951, Republic of Korea. Tel.: +82 43 719 8650; fax: +82 43 719 8689. E-mail address: [email protected] (H.-Y. Park).
http://dx.doi.org/10.1016/j.cellsig.2015.04.012 0898-6568/© 2015 Elsevier Inc. All rights reserved.
pro-apoptotic cytokine in disturbed flow areas [6,7] and efficiently induces endothelial cell apoptosis in vitro [4,8,9]. Coronins are highly conserved actin-binding proteins that regulate various cellular processes such as cell motility and phagosome formation [10–12]. The coronin family consists of three subclasses (type I, II and III) [13]. Among type I coronins (1A, 1B and 1C), coronin 1A is highly expressed in hematopoietic lineage cells with little expression in other tissues and regulates cellular homeostasis including cell survival and apoptosis [14–17]. Recently, coronin 1A expression in PC12 cells and the inhibition of neutrite outgrowth by coronin 1A were reported [18], thus extending the role of coronin 1A to other than hematopoietic lineage cells. However, the expression and function of coronin 1A in endothelial cells are still unknown. In this study, we examined the expression of coronin 1A in the human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell (HBMVEC) and studied its function in TNFα-induced apoptosis of HUVEC. 2. Materials and methods 2.1. Cell culture The human umbilical vein endothelial cell (HUVEC) was purchased from Lonza (Walkersville, MD) and cultured in endothelial basal
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medium-2 (EBM-2; Lonza) supplemented with endothelial growth medium-2 (EGM-2) SingleQuots® (Lonza). Vascular endothelial growth factor (VEGF) was provided as one of the components of the EGM-2 SingleQuots®; however, VEGF was not added to the cell culture medium to rule out an effect of VEGF on endothelial cell apoptosis and survival. HUVEC was cultured on 2% gelatin-precoated dishes and used at passages 4 to 9. Human brain microvascular endothelial cell (HBMVEC) was cultured in complete serum containing (CSC) medium (Cell Systems, Kirkland, WA) and used at passages 5–6. Dr. Jin Woong Chung (Dong-A University, Busan, Republic of Korea) kindly provided HBMVEC. 2.2. Sources of materials Antibodies against coronin 1A, α-tubulin and β-actin were purchased from Sigma (St. Louis, MO); anti-cleaved poly-ADP-ribose polymerase (PARP), anti-cleaved caspase 3 and anti-cleaved caspase 8 antibodies were from Cell Signaling Technology (Beverly, MA); antip38α and anti-p38β antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and anti-phospho-p38 (Thr180/Tyr182) antibody was from Cell Signaling Technology or BD Transduction Laboratories (St. Louis, MO). TNFα was purchased from Roche Applied Science (Indianapolis, IN) and cyclohexamide (CHX) was from Sigma. 2.3. siRNA transfection HUVEC was transiently transfected with scrambled, coronin 1A or p38β siRNA using OPTI-MEM (Invitrogen, Carlsbad, CA) and Lipofectamine® RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions. Further experiments were performed after 48 h incubation in complete media. The human coronin 1A siRNA and scrambled siRNA were from Santa Cruz Biotechnology Inc. and human p38β siRNA was from Cell Signaling Technology. 2.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total cellular RNA from either HUVEC or Jurkat cells was extracted using the RNeasy Mini Kit (Qiagen). RNA (500 ng) was reversetranscribed using the RNA to cDNA EcoDryTM Premix (Clontech, Mountain View, CA) and the coronin 1A and glyceraldehyde 3phosphate dehydrogenase (GAPDH) genes were amplified using an amfiEco PCR Premix (GeneDEPOT, Barker, TX). The primer sequences were as follows: for coronin 1A, 5′-CTCCAGCTCCTTCCTCCTCT-3′ (forward) and 5′-GAGACGCGCACATCTTCATA-3′ (reverse); for GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTT GCTGTA-3′ (reverse). The PCR protocol entailed 30 cycles (for coronin 1A) or 25 cycles (for GADPH) of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. 2.5. Western blotting Cells were washed with cold PBS and lysed in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Sigma). Cells were harvested and centrifuged at 13,000 g for 10 min and supernatants were collected. Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of cell lysates was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after heating at 100 °C for 5 min. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and blocked for 1 h at room temperature in 5% nonfat milk (Santa Cruz). Membranes were incubated overnight at 4 °C with appropriate primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. After washing with 0.1% Tween20 containing TBS (Tris-buffered saline), the signals were visualized with ImageQuant LAS4000 mini
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system (GE Healthcare) using either Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore) or Western Blotting Luminol Reagent (Santa Cruz). 2.6. Caspase 3 activity assay The activity of caspase 3 was measured using the CaspACE™ Assay System, Colorimetric (Promega), according to the manufacturer's instructions. Briefly, cells were washed with PBS, resuspended in cell lysis buffer and lysed by repeated freeze-thaw cycles. Then, 20 μg of total protein was incubated with colorimetric substrate (Asp-Glu-ValAsp-p-nitroaniline; DEVD-pNA) to measure caspase 3 activity. Caspase 3 cleaves this substrate to release pNA, which produces a yellow color that can be monitored by a spectrophotometer at 405 nm. 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Cells were seeded onto Lab-TEKTM II Chamber Slides (Nalge Nunc International, Rochester, NY) precoated with 2% gelatin and transfected with the indicated siRNA. After 48 h, cells were exposed to TNFα + CHX for 8 h, washed with cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. To analyze apoptotic cells, the DeadEndTM Fluorometric TUNEL system (Promega) was used according to the manufacturer's instructions. At the final step, slides were mounted in ProLong® Gold antifade reagent with DAPI (Invitrogen) and immediately analyzed under a fluorescence microscopy. The number of TUNEL-positive apoptotic cells was counted from randomly selected five fields. Then, the percentage of TUNEL-positive apoptotic cells was represented relative to total cells counted. 2.8. Immunoprecipitation (IP) assay IP assay was performed as previously described [9,19]. Cells were harvested and lysed in IP lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, 1 mM EDTA, 0.1% SDS and protease inhibitor cocktail) and 200 μg of total protein was immunoprecipitated with specific antibodies or control IgG overnight at 4 °C. Lysates were then incubated with 40 μl of protein G agarose beads (Roche) at 4 °C for 3 h. Immune complexes were collected by centrifugation (2500 rpm for 3 min) and washed five times with IP wash buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1 mM EDTA and protease inhibitor cocktails). The bound proteins were released by heating in 2× SDS gel sample buffer and resolved by SDS-PAGE. 2.9. Statistics The results are presented as the means ± SD. Analyses were performed using Student's t test. Values of P b 0.05 were considered significant. 3. Results 3.1. Expression of coronin 1A in HUVEC Coronin 1A is a leukocyte-specific coronin 1 because it is abundantly expressed in hematopoietic cells [12,14–17,20]. Thus, as a first approach, we examined whether endothelial cells express coronin 1A mRNA by RT-PCR. As shown in Fig. 1A, coronin 1A mRNA was expressed in HUVEC although the expression level was lower than that in the human T cell line, Jurkat. We examined the expression of coronin 1A protein in HUVEC and HBMVEC and found that the coronin 1A is expressed in both endothelial cells (Fig. 1B). We next transfected HUVEC with coronin 1A siRNA and examined the expression of coronin 1A protein by immunoblotting. As shown in Fig. 1C and D, the
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expression of coronin 1A was reduced by 45.9 ± 3.5% in coronin 1A siRNA-transfected HUVEC.
3.2. Depletion of coronin 1A suppresses TNFα-induced HUVEC apoptosis
Fig. 1. Endothelial cell expresses coronin 1A. (A) Total RNA from HUVEC and Jurkat were isolated and the levels of coronin 1A and GADPH were assessed by RT-PCR as described in the Materials and methods. (B) The expression of coronin 1A protein was analyzed in HUVEC and HBMVEC by immunoblotting. (C) The expression of coronin 1A protein in HUVEC transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) was analyzed by immunoblotting. (D) Quantification of coronin 1A protein expression in HUVEC transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM). The intensity of coronin 1A expression was normalized to that of β-actin expression. *P b 0.05 versus scrambled siRNA-transfected HUVEC.
Coronin 1A was reported to regulate the apoptosis of T cells and neutrophils [15,17]. To determine whether coronin 1A is involved in apoptosis of endothelial cells, we stimulated HUVEC with TNFα + cyclohexamide (CHX) [9] after depletion of coronin 1A. First, we examined whether the depletion of coronin 1A affected cell viability using a MTT assay. In scrambled siRNA-transfected groups, TNFα + CHX treatment induced cell death. However, depletion of coronin 1A by siRNA transfection increased cell viability (Supplemental Fig. S1). To show that the increase in cell viability was due to a decrease in cell death caused by apoptosis, we depleted coronin 1A, treated the cells with TNFα + CHX and performed the TUNEL staining. As shown in Fig. 2A and B, the percentage of TUNEL-positive apoptotic cells was significantly decreased after coronin 1A depletion. Next, we examined the cleavage of caspase 3 and PARP, both of which are markers of cell apoptosis [9,21]. In scrambled siRNA-transfected groups, TNFα + CHX treatment induced the cleavage of caspase 3 and PARP. However, the cleavage of these proteins was suppressed when coronin 1A was depleted by siRNA (Fig. 2C). Previously, coronin 1A was suggested to be a substrate of caspase 3 [17]. Consistent with this previous report [17], we observed that the expression of coronin 1A was partially reduced, probably as a result of its cleavage by activated caspase 3 (Fig. 2C). Furthermore, we measured the activity of caspase 3 after cell treatment with TNFα + CHX. As shown in Fig. 2D, TNFα + CHX-induced activation of
Fig. 2. Coronin 1A depletion suppresses TNFα + CHX-induced apoptosis. (A) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml). After 8 h, cells were assayed by TUNEL staining using a DeadEndTM Fluorometric TUNEL system (Promega) as described in the Materials and methods. (B) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were enumerated and expressed as a percentage of total nuclear counts. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. (C) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The cleavage of PARP and caspase 3 was analyzed by immunoblotting. (D) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The activity of caspase 3 was measured by the CaspACE™ Assay System (Promega) as described in the Materials and methods. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX.
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caspase 3 was suppressed by the siRNA-mediated depletion of coronin 1A. These results demonstrate that coronin 1A regulates TNFα + CHX-induced apoptosis of HUVEC. 3.3. Depletion of coronin 1A upregulates p38β and increases its activation in response to TNFα After observing reduced apoptosis of coronin 1A-depleted HUVEC, we hypothesized that coronin 1A might affect the signaling cascade of TNFα-induced apoptosis. To test this hypothesis, we measured the TNFα-induced activation of several pro- and anti-apoptotic molecules including p38, Akt and JNK [9,22–24] after depletion of coronin 1A. TNFα induced the phosphorylation of p38, Akt and JNK (Fig. 3A). However, depletion of coronin 1A increased the phosphorylation of p38 without affecting Akt or JNK activation (Fig. 3A). Among p38 isoforms, p38α induces apoptosis whereas p38β protects against apoptosis [22, 23,25–27]. Interestingly, we found that coronin 1A depletion increased the expression of anti-apoptotic p38β without affecting the expression of pro-apoptotic p38α (Fig. 3A). We further analyzed which isoform of p38 was specifically activated in coronin 1A-depleted HUVEC in response to TNFα stimulation. Isoform-specific antibodies to phosphop38 cannot be made because of the identical phosphorylation sequences of the p38 isoforms [28]. Thus, we immunoprecipitated total protein cell extracts with either p38α or p38β antibody and then analyzed the phosphorylated status of p38 with a phospho-p38 antibody by western blotting. As shown in Fig. 3B, coronin 1A depletion did not affect the expression or phosphorylation of p38α in response to TNFα stimulation. However, coronin 1A depletion increased the expression and subsequent phosphorylation of p38β in response to TNFα stimulation (Fig. 3C). These results suggest that increased p38β expression and its subsequent activation protected coronin 1A-depleted HUVEC from TNFα + CHX-induced apoptosis. 3.4. Increased expression of p38β is critical for the survival of coronin 1Adepleted HUVEC To confirm the protective role of p38β upregulation in coronin 1Adepleted HUVEC, we transfected p38β siRNA into coronin 1A-depleted HUVEC and compared the apoptosis of these cells with that of scrambled siRNA- or only coronin 1A siRNA-transfected HUVEC. Consistent
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with our observations (Figs. 2 and 3), coronin 1A depletion induced the expression of p38β and suppressed TNFα + CHX-induced cleavage of caspase 3 and PARP. However, inhibition of p38β upregulation in coronin 1A-depleted HUVEC completely restored the cleavage of caspase 3 and PARP (Fig. 4A). In addition, we found that coronin 1A depletion decreased the cleavage (activation) of the initiator caspase, caspase 8 and the inhibition of p38β upregulation in coronin 1Adepleted HUVEC restored the cleavage of caspase 8. We measured caspase 3 activity after depletion of both coronin 1A and p38β and found that caspase 3 activity was higher in coronin 1A- and p38βdepleted HUVEC than in coronin 1A-depleted HUVEC (Fig. 4B). Finally, TUNEL staining showed that apoptosis was restored when p38β upregulation was inhibited in coronin 1A-depleted HUVEC (Fig. 4C and D). These results demonstrate that p38β induction is critical for the survival of coronin 1A-depleted HUVEC treated with TNFα + CHX. 4. Discussion The major finding of this study is that coronin 1A is expressed in endothelial cells and regulates TNFα-induced apoptosis. We showed that coronin 1A depletion suppressed TNFα-induced endothelial cell apoptosis and that p38β, which protects endothelial cells from apoptosis [22,23,25,26], was upregulated after coronin 1A depletion and that it suppressed the activation of caspase 8 and caspase 3, suggesting that it played a critical role in the increased survival of coronin 1A-depleted HUVEC. Based on our observations, we propose a new pathway for protection against TNFα-induced endothelial cell apoptosis (Fig. 5). In this model, we suggest that coronin 1A depletion increases cell survival during TNFα-induced apoptosis of HUVEC by positively controlling the expression of p38β and thereby inhibiting the cleavage of caspase 8 and caspase 3. Coronin 1A is a leukocyte-specific coronin and thus has been studied mainly in leukocytes [14–17,20]. Although a recent study showed that coronin 1A inhibits neurite outgrowth in PC12 cells [18], it has remained unclear whether coronin 1A is expressed or functions in endothelial cells. Thus, to our knowledge, the present study is the first to show the expression of coronin 1A and define the role of this factor in endothelial cells. The present study extends our understanding of the role of coronin 1A to cells other than leukocytes and suggests that coronin 1A plays an important role in the regulation of endothelial cell apoptosis.
Fig. 3. Coronin 1A depletion increases the expression and activation of p38β in response to TNFα. (A) HUVEC was transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) for the indicated times. Phosphorylation of p38 at Thr180/Tyr182, phosphorylation of Akt at Ser473, and phosphorylation of JNK at Thr183/Tyr185 were analyzed by immunoblotting. (B, C) HUVEC was transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) for 15 min. Cell lysates were immunoprecipitated with either p38α antibody (B) or p38β antibody (C) and immunoprecipitates were analyzed by immunoblotting with phospho-p38 antibody. The depletion of coronin 1A and expression of p38α (B) and p38β (C) were analyzed in total cell lysates (TCL) by immunoblotting.
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Fig. 4. p38β induction is critical for the survival of coronin 1A-depleted HUVEC. (A) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The cleavage of PARP, caspase 8 and caspase 3 and the expression of p38β and coronin 1A were analyzed by immunoblotting. (B) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The activity of caspase 3 was measured by the CaspACE™ Assay System (Promega) as described in the Materials and methods. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. **P b 0.05 versus coronin 1A siRNA treated with TNFα + CHX. (C) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml). After 8 h, cells were assayed by TUNEL staining using a DeadEndTM Fluorometric TUNEL system (Promega) as described in the Materials and methods. (D) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were enumerated and expressed as a percentage of total nuclear counts. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. **P b 0.05 versus coronin 1A siRNA treated with TNFα + CHX.
Previous studies have shown that coronin 1A is important for cell survival and apoptosis. Coronin 1A-deficient T cells exhibit defects in Ca2+ mobilization and interleukin-2 production resulting in lower survival and more apoptosis than that observed in wild-type T cells [15]. Coronin 1A plays a pro-survival role in neutrophils [17]. In addition, the dimethylformamide-differentiated coronin 1A-overexpressing promyelocytic cell line, PLB985, is subject to less TRAIL-induced apoptosis and reduced activation of caspase-3 and caspase-9 but not caspase-8 [17]. These results are in contrast to those of the present study where
Fig. 5. A model for coronin 1A regulation of endothelial cell survival. Coronin 1A depletion increases the expression and activation of anti-apoptotic p38β after TNFα stimulation, resulting in the suppression of TNFα-induced cleavage of caspase 8, caspase 3 and PARP and an increase in cell survival.
depletion of coronin 1A in HUVEC led to less activation of caspase-8 and caspase-3 resulting in less apoptosis in response to TNFα stimulation. One explanation could be that the role of coronin 1A in apoptosis is cell type dependent or changes depending on the nature of the stimulation. Thus, it may be important to further study the regulatory mechanisms of apoptosis in various cell types exposed to different stimuli. p38 is activated by growth factors, cytokines and environmental stresses and modulates various cellular responses including development, cell growth, differentiation and inflammation [29]. Interestingly, p38 promotes apoptosis in some cell types whereas it increases cell survival in other cell types [23,30–32]. Thus, the function of p38 in apoptosis or survival may depend on cell type and the nature of the stimulus. Four isoforms of p38 MAPK (α, β, γ, δ) have been identified [29]. In particular, although endothelial cells express all isoforms [33], previous studies have shown that p38α induces endothelial cell apoptosis and that p38β protects endothelial cells from apoptosis [22,23,25–27]. Our results showed that both p38α and p38β were activated by TNFα stimulation under normal conditions. Because previous studies have shown that TNFα-activated p38α induces endothelial cell apoptosis [34,35], it is likely that p38α phosphorylation dominates over p38β activation under normal conditions. However, in the coronin 1A-depleted condition, the expression of anti-apoptotic p38β expression was induced. Furthermore, TNFα stimulation of the coronin 1A-depleted cells activated p38β without affecting the expression or activation of pro-apoptotic p38α. The activation of p38β led to a lower level of cleavage of the initiator caspase, caspase 8 and the executioner caspase, caspase 3,
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resulting in a lower level of apoptosis. Conversely, the inhibition of p38β upregulation in coronin 1A-depleted HUVEC restored the activation of caspase 8 and caspase 3 leading to a higher level of apoptosis. Leon Tourian Jr et al previously reported that transfection of p38β siRNA leads to a higher level of caspase 8 and caspase 3 cleavage and apoptosis in response to Fas-mediated apoptotic signaling [31]. However, the present study is the first to show that coronin 1A regulates the expression of p38β, a regulator of the caspase 8-caspase 3-PARP apoptotic pathway, and endothelial cell apoptosis in response to TNFα. Because p38β can induce cell survival via several signaling cascades, we examined the expression of pro-survival molecules that are regulated by p38β including heat shock protein (Hsp) 70 and heme oxygenase1 (HO-1) [22,27]. However, the expression of these proteins was not affected when p38β expression was induced in coronin 1A-depleted HUVEC (Supplemental Fig. S2), suggesting that Hsp70 and HO-1 are not involved in the survival of coronin 1A-depleted HUVEC. 5. Conclusions This study demonstrates that coronin 1A functions in TNFα-induced endothelial cell apoptosis. It is the first to report the involvement of coronin 1A in TNFα-induced apoptosis of endothelial cells via an effect on the p38β-dependent regulatory pathway. Considering the various functions of coronin 1A in leukocytes, it may be worthwhile pursuing this work further by studying the role of coronin 1A in inflammation [17], clearance of infected bacteria [12,36] and vascular diseases such as atherosclerosis [37]. Contributors Geun-Young Kim designed the study, performed experiments and wrote the manuscript. Hanna Kim and Hyun-Joung Lim performed experiments. Hyun-Young Park supervised the project and edited the manuscript. Disclosure The authors disclose no potential conflicts of interest. All authors approved the final article. Acknowledgments This work was supported by a Korean National Institute of Health intramural research grant (2013-NG63003-00). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2015.04.012.
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Coronin 1A depletion protects endothelial cells from TNFα-induced apoptosis by modulating p38β expression and activation Geun-Young Kim, Hanna Kim, Hyun-Joung Lim, Hyun-Young Park ⁎ Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, Cheongju, Republic of Korea
a r t i c l e
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Article history: Received 29 January 2015 Received in revised form 23 April 2015 Accepted 25 April 2015 Available online 30 April 2015 Keywords: Coronin 1A Tumor necrosis factor α p38β Endothelial cell Apoptosis
a b s t r a c t Coronins are conserved actin-binding proteins that regulate various cellular processes such as migration and endocytosis. Among coronin family members, coronin 1A is highly expressed in hematopoietic lineage cells where it regulates cell homeostasis. However, the expression and function of coronin 1A in endothelial cells have not yet been elucidated. We found that coronin 1A is expressed in the human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell (HBMVEC). In HUVEC depleted of coronin 1A by siRNA transfection, tumor necrosis factor α (TNFα) + cyclohexamide (CHX) treatment resulted in a decrease in the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive apoptotic cells. Coronin 1A depletion also resulted in the suppression of caspase 3 and poly(ADP-ribose) polymerase cleavage and a reduction in caspase 3 activity. Next, we examined TNFα-induced activation of several pro- and anti-apoptotic signaling molecules to find the target molecule of coronin 1A and found that p38 phosphorylation was enhanced by TNFα stimulation in coronin 1A-depleted HUVEC. Among the p38 isoforms, the expression of p38β was significantly upregulated after coronin 1A depletion, suggesting that the expression and phosphorylation of antiapoptotic p38β were significantly induced in coronin 1A-depleted HUVEC. Inhibition of p38β upregulation in coronin 1A-depleted HUVEC restored the cleavage of caspase 8 and caspase 3 and induced more apoptosis than in coronin 1A-depleted HUVEC in response to TNFα + CHX. These findings suggest that coronin 1A modulates endothelial cell apoptosis by regulating p38β expression and activation. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Endothelial cell apoptosis is an important element under physiologically normal and pathological conditions [1,2]. Endothelial cells are continuously exposed to pro- and anti-apoptotic stimuli and the fate of endothelial cells is dependent on the balance between these stimuli [3]. Enhanced endothelial cell apoptosis is associated with cardiovascular diseases including atherosclerosis and thrombosis [4,5]. For example, increased endothelial cell apoptosis was observed in atheroprone areas exposed to disturbed flow in the aorta [4]. Among the environmental factors found within the pro-apoptotic regions of atherosclerosis tissue, tumor necrosis factor (TNF)α is the most prominent and is frequently used to study endothelial cell apoptosis because it is the most abundant
Abbreviations: TNFα, tumor necrosis factor α; HUVEC, human umbilical vein endothelial cell; HBMVEC, human brain microvascular endothelial cell; CHX, cyclohexamide; PARP, poly(ADP-ribose) polymerase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling. ⁎ Corresponding author at: Division of Cardiovascular and Rare Disease, Center for Biomedical Sciences, Korea National Institute of Health, 187, Osongsaengmyeng 2-ro, Osong-eup, Heungdeok-gu, Cheongju-city, Chungcheongbuk-do 361-951, Republic of Korea. Tel.: +82 43 719 8650; fax: +82 43 719 8689. E-mail address: [email protected] (H.-Y. Park).
http://dx.doi.org/10.1016/j.cellsig.2015.04.012 0898-6568/© 2015 Elsevier Inc. All rights reserved.
pro-apoptotic cytokine in disturbed flow areas [6,7] and efficiently induces endothelial cell apoptosis in vitro [4,8,9]. Coronins are highly conserved actin-binding proteins that regulate various cellular processes such as cell motility and phagosome formation [10–12]. The coronin family consists of three subclasses (type I, II and III) [13]. Among type I coronins (1A, 1B and 1C), coronin 1A is highly expressed in hematopoietic lineage cells with little expression in other tissues and regulates cellular homeostasis including cell survival and apoptosis [14–17]. Recently, coronin 1A expression in PC12 cells and the inhibition of neutrite outgrowth by coronin 1A were reported [18], thus extending the role of coronin 1A to other than hematopoietic lineage cells. However, the expression and function of coronin 1A in endothelial cells are still unknown. In this study, we examined the expression of coronin 1A in the human umbilical vein endothelial cell (HUVEC) and human brain microvascular endothelial cell (HBMVEC) and studied its function in TNFα-induced apoptosis of HUVEC. 2. Materials and methods 2.1. Cell culture The human umbilical vein endothelial cell (HUVEC) was purchased from Lonza (Walkersville, MD) and cultured in endothelial basal
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medium-2 (EBM-2; Lonza) supplemented with endothelial growth medium-2 (EGM-2) SingleQuots® (Lonza). Vascular endothelial growth factor (VEGF) was provided as one of the components of the EGM-2 SingleQuots®; however, VEGF was not added to the cell culture medium to rule out an effect of VEGF on endothelial cell apoptosis and survival. HUVEC was cultured on 2% gelatin-precoated dishes and used at passages 4 to 9. Human brain microvascular endothelial cell (HBMVEC) was cultured in complete serum containing (CSC) medium (Cell Systems, Kirkland, WA) and used at passages 5–6. Dr. Jin Woong Chung (Dong-A University, Busan, Republic of Korea) kindly provided HBMVEC. 2.2. Sources of materials Antibodies against coronin 1A, α-tubulin and β-actin were purchased from Sigma (St. Louis, MO); anti-cleaved poly-ADP-ribose polymerase (PARP), anti-cleaved caspase 3 and anti-cleaved caspase 8 antibodies were from Cell Signaling Technology (Beverly, MA); antip38α and anti-p38β antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); and anti-phospho-p38 (Thr180/Tyr182) antibody was from Cell Signaling Technology or BD Transduction Laboratories (St. Louis, MO). TNFα was purchased from Roche Applied Science (Indianapolis, IN) and cyclohexamide (CHX) was from Sigma. 2.3. siRNA transfection HUVEC was transiently transfected with scrambled, coronin 1A or p38β siRNA using OPTI-MEM (Invitrogen, Carlsbad, CA) and Lipofectamine® RNAiMAX reagent (Invitrogen) according to the manufacturer's instructions. Further experiments were performed after 48 h incubation in complete media. The human coronin 1A siRNA and scrambled siRNA were from Santa Cruz Biotechnology Inc. and human p38β siRNA was from Cell Signaling Technology. 2.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Total cellular RNA from either HUVEC or Jurkat cells was extracted using the RNeasy Mini Kit (Qiagen). RNA (500 ng) was reversetranscribed using the RNA to cDNA EcoDryTM Premix (Clontech, Mountain View, CA) and the coronin 1A and glyceraldehyde 3phosphate dehydrogenase (GAPDH) genes were amplified using an amfiEco PCR Premix (GeneDEPOT, Barker, TX). The primer sequences were as follows: for coronin 1A, 5′-CTCCAGCTCCTTCCTCCTCT-3′ (forward) and 5′-GAGACGCGCACATCTTCATA-3′ (reverse); for GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-TCCACCACCCTGTT GCTGTA-3′ (reverse). The PCR protocol entailed 30 cycles (for coronin 1A) or 25 cycles (for GADPH) of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s, followed by a final extension at 72 °C for 10 min. 2.5. Western blotting Cells were washed with cold PBS and lysed in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor cocktail (Sigma). Cells were harvested and centrifuged at 13,000 g for 10 min and supernatants were collected. Protein concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of cell lysates was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after heating at 100 °C for 5 min. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) and blocked for 1 h at room temperature in 5% nonfat milk (Santa Cruz). Membranes were incubated overnight at 4 °C with appropriate primary antibodies and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h. After washing with 0.1% Tween20 containing TBS (Tris-buffered saline), the signals were visualized with ImageQuant LAS4000 mini
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system (GE Healthcare) using either Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Millipore) or Western Blotting Luminol Reagent (Santa Cruz). 2.6. Caspase 3 activity assay The activity of caspase 3 was measured using the CaspACE™ Assay System, Colorimetric (Promega), according to the manufacturer's instructions. Briefly, cells were washed with PBS, resuspended in cell lysis buffer and lysed by repeated freeze-thaw cycles. Then, 20 μg of total protein was incubated with colorimetric substrate (Asp-Glu-ValAsp-p-nitroaniline; DEVD-pNA) to measure caspase 3 activity. Caspase 3 cleaves this substrate to release pNA, which produces a yellow color that can be monitored by a spectrophotometer at 405 nm. 2.7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay Cells were seeded onto Lab-TEKTM II Chamber Slides (Nalge Nunc International, Rochester, NY) precoated with 2% gelatin and transfected with the indicated siRNA. After 48 h, cells were exposed to TNFα + CHX for 8 h, washed with cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. To analyze apoptotic cells, the DeadEndTM Fluorometric TUNEL system (Promega) was used according to the manufacturer's instructions. At the final step, slides were mounted in ProLong® Gold antifade reagent with DAPI (Invitrogen) and immediately analyzed under a fluorescence microscopy. The number of TUNEL-positive apoptotic cells was counted from randomly selected five fields. Then, the percentage of TUNEL-positive apoptotic cells was represented relative to total cells counted. 2.8. Immunoprecipitation (IP) assay IP assay was performed as previously described [9,19]. Cells were harvested and lysed in IP lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, 1 mM EDTA, 0.1% SDS and protease inhibitor cocktail) and 200 μg of total protein was immunoprecipitated with specific antibodies or control IgG overnight at 4 °C. Lysates were then incubated with 40 μl of protein G agarose beads (Roche) at 4 °C for 3 h. Immune complexes were collected by centrifugation (2500 rpm for 3 min) and washed five times with IP wash buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1 mM EDTA and protease inhibitor cocktails). The bound proteins were released by heating in 2× SDS gel sample buffer and resolved by SDS-PAGE. 2.9. Statistics The results are presented as the means ± SD. Analyses were performed using Student's t test. Values of P b 0.05 were considered significant. 3. Results 3.1. Expression of coronin 1A in HUVEC Coronin 1A is a leukocyte-specific coronin 1 because it is abundantly expressed in hematopoietic cells [12,14–17,20]. Thus, as a first approach, we examined whether endothelial cells express coronin 1A mRNA by RT-PCR. As shown in Fig. 1A, coronin 1A mRNA was expressed in HUVEC although the expression level was lower than that in the human T cell line, Jurkat. We examined the expression of coronin 1A protein in HUVEC and HBMVEC and found that the coronin 1A is expressed in both endothelial cells (Fig. 1B). We next transfected HUVEC with coronin 1A siRNA and examined the expression of coronin 1A protein by immunoblotting. As shown in Fig. 1C and D, the
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expression of coronin 1A was reduced by 45.9 ± 3.5% in coronin 1A siRNA-transfected HUVEC.
3.2. Depletion of coronin 1A suppresses TNFα-induced HUVEC apoptosis
Fig. 1. Endothelial cell expresses coronin 1A. (A) Total RNA from HUVEC and Jurkat were isolated and the levels of coronin 1A and GADPH were assessed by RT-PCR as described in the Materials and methods. (B) The expression of coronin 1A protein was analyzed in HUVEC and HBMVEC by immunoblotting. (C) The expression of coronin 1A protein in HUVEC transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) was analyzed by immunoblotting. (D) Quantification of coronin 1A protein expression in HUVEC transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM). The intensity of coronin 1A expression was normalized to that of β-actin expression. *P b 0.05 versus scrambled siRNA-transfected HUVEC.
Coronin 1A was reported to regulate the apoptosis of T cells and neutrophils [15,17]. To determine whether coronin 1A is involved in apoptosis of endothelial cells, we stimulated HUVEC with TNFα + cyclohexamide (CHX) [9] after depletion of coronin 1A. First, we examined whether the depletion of coronin 1A affected cell viability using a MTT assay. In scrambled siRNA-transfected groups, TNFα + CHX treatment induced cell death. However, depletion of coronin 1A by siRNA transfection increased cell viability (Supplemental Fig. S1). To show that the increase in cell viability was due to a decrease in cell death caused by apoptosis, we depleted coronin 1A, treated the cells with TNFα + CHX and performed the TUNEL staining. As shown in Fig. 2A and B, the percentage of TUNEL-positive apoptotic cells was significantly decreased after coronin 1A depletion. Next, we examined the cleavage of caspase 3 and PARP, both of which are markers of cell apoptosis [9,21]. In scrambled siRNA-transfected groups, TNFα + CHX treatment induced the cleavage of caspase 3 and PARP. However, the cleavage of these proteins was suppressed when coronin 1A was depleted by siRNA (Fig. 2C). Previously, coronin 1A was suggested to be a substrate of caspase 3 [17]. Consistent with this previous report [17], we observed that the expression of coronin 1A was partially reduced, probably as a result of its cleavage by activated caspase 3 (Fig. 2C). Furthermore, we measured the activity of caspase 3 after cell treatment with TNFα + CHX. As shown in Fig. 2D, TNFα + CHX-induced activation of
Fig. 2. Coronin 1A depletion suppresses TNFα + CHX-induced apoptosis. (A) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml). After 8 h, cells were assayed by TUNEL staining using a DeadEndTM Fluorometric TUNEL system (Promega) as described in the Materials and methods. (B) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were enumerated and expressed as a percentage of total nuclear counts. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. (C) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The cleavage of PARP and caspase 3 was analyzed by immunoblotting. (D) HUVEC was transfected with scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The activity of caspase 3 was measured by the CaspACE™ Assay System (Promega) as described in the Materials and methods. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX.
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caspase 3 was suppressed by the siRNA-mediated depletion of coronin 1A. These results demonstrate that coronin 1A regulates TNFα + CHX-induced apoptosis of HUVEC. 3.3. Depletion of coronin 1A upregulates p38β and increases its activation in response to TNFα After observing reduced apoptosis of coronin 1A-depleted HUVEC, we hypothesized that coronin 1A might affect the signaling cascade of TNFα-induced apoptosis. To test this hypothesis, we measured the TNFα-induced activation of several pro- and anti-apoptotic molecules including p38, Akt and JNK [9,22–24] after depletion of coronin 1A. TNFα induced the phosphorylation of p38, Akt and JNK (Fig. 3A). However, depletion of coronin 1A increased the phosphorylation of p38 without affecting Akt or JNK activation (Fig. 3A). Among p38 isoforms, p38α induces apoptosis whereas p38β protects against apoptosis [22, 23,25–27]. Interestingly, we found that coronin 1A depletion increased the expression of anti-apoptotic p38β without affecting the expression of pro-apoptotic p38α (Fig. 3A). We further analyzed which isoform of p38 was specifically activated in coronin 1A-depleted HUVEC in response to TNFα stimulation. Isoform-specific antibodies to phosphop38 cannot be made because of the identical phosphorylation sequences of the p38 isoforms [28]. Thus, we immunoprecipitated total protein cell extracts with either p38α or p38β antibody and then analyzed the phosphorylated status of p38 with a phospho-p38 antibody by western blotting. As shown in Fig. 3B, coronin 1A depletion did not affect the expression or phosphorylation of p38α in response to TNFα stimulation. However, coronin 1A depletion increased the expression and subsequent phosphorylation of p38β in response to TNFα stimulation (Fig. 3C). These results suggest that increased p38β expression and its subsequent activation protected coronin 1A-depleted HUVEC from TNFα + CHX-induced apoptosis. 3.4. Increased expression of p38β is critical for the survival of coronin 1Adepleted HUVEC To confirm the protective role of p38β upregulation in coronin 1Adepleted HUVEC, we transfected p38β siRNA into coronin 1A-depleted HUVEC and compared the apoptosis of these cells with that of scrambled siRNA- or only coronin 1A siRNA-transfected HUVEC. Consistent
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with our observations (Figs. 2 and 3), coronin 1A depletion induced the expression of p38β and suppressed TNFα + CHX-induced cleavage of caspase 3 and PARP. However, inhibition of p38β upregulation in coronin 1A-depleted HUVEC completely restored the cleavage of caspase 3 and PARP (Fig. 4A). In addition, we found that coronin 1A depletion decreased the cleavage (activation) of the initiator caspase, caspase 8 and the inhibition of p38β upregulation in coronin 1Adepleted HUVEC restored the cleavage of caspase 8. We measured caspase 3 activity after depletion of both coronin 1A and p38β and found that caspase 3 activity was higher in coronin 1A- and p38βdepleted HUVEC than in coronin 1A-depleted HUVEC (Fig. 4B). Finally, TUNEL staining showed that apoptosis was restored when p38β upregulation was inhibited in coronin 1A-depleted HUVEC (Fig. 4C and D). These results demonstrate that p38β induction is critical for the survival of coronin 1A-depleted HUVEC treated with TNFα + CHX. 4. Discussion The major finding of this study is that coronin 1A is expressed in endothelial cells and regulates TNFα-induced apoptosis. We showed that coronin 1A depletion suppressed TNFα-induced endothelial cell apoptosis and that p38β, which protects endothelial cells from apoptosis [22,23,25,26], was upregulated after coronin 1A depletion and that it suppressed the activation of caspase 8 and caspase 3, suggesting that it played a critical role in the increased survival of coronin 1A-depleted HUVEC. Based on our observations, we propose a new pathway for protection against TNFα-induced endothelial cell apoptosis (Fig. 5). In this model, we suggest that coronin 1A depletion increases cell survival during TNFα-induced apoptosis of HUVEC by positively controlling the expression of p38β and thereby inhibiting the cleavage of caspase 8 and caspase 3. Coronin 1A is a leukocyte-specific coronin and thus has been studied mainly in leukocytes [14–17,20]. Although a recent study showed that coronin 1A inhibits neurite outgrowth in PC12 cells [18], it has remained unclear whether coronin 1A is expressed or functions in endothelial cells. Thus, to our knowledge, the present study is the first to show the expression of coronin 1A and define the role of this factor in endothelial cells. The present study extends our understanding of the role of coronin 1A to cells other than leukocytes and suggests that coronin 1A plays an important role in the regulation of endothelial cell apoptosis.
Fig. 3. Coronin 1A depletion increases the expression and activation of p38β in response to TNFα. (A) HUVEC was transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) for the indicated times. Phosphorylation of p38 at Thr180/Tyr182, phosphorylation of Akt at Ser473, and phosphorylation of JNK at Thr183/Tyr185 were analyzed by immunoblotting. (B, C) HUVEC was transfected with either scrambled (10 nM) or coronin 1A siRNA (10 nM) and stimulated with TNFα (5 ng/ml) for 15 min. Cell lysates were immunoprecipitated with either p38α antibody (B) or p38β antibody (C) and immunoprecipitates were analyzed by immunoblotting with phospho-p38 antibody. The depletion of coronin 1A and expression of p38α (B) and p38β (C) were analyzed in total cell lysates (TCL) by immunoblotting.
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Fig. 4. p38β induction is critical for the survival of coronin 1A-depleted HUVEC. (A) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The cleavage of PARP, caspase 8 and caspase 3 and the expression of p38β and coronin 1A were analyzed by immunoblotting. (B) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml) for 6 h. The activity of caspase 3 was measured by the CaspACE™ Assay System (Promega) as described in the Materials and methods. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. **P b 0.05 versus coronin 1A siRNA treated with TNFα + CHX. (C) HUVEC was transfected with scrambled siRNA (10 nM), coronin 1A siRNA (10 nM) or coronin 1A siRNA (10 nM) + p38β siRNA (10 nM) and stimulated with TNFα (5 ng/ml) + CHX (5 μg/ml). After 8 h, cells were assayed by TUNEL staining using a DeadEndTM Fluorometric TUNEL system (Promega) as described in the Materials and methods. (D) Quantification of TUNEL-positive cells. TUNEL-positive apoptotic cells were enumerated and expressed as a percentage of total nuclear counts. *P b 0.05 versus scrambled siRNA treated with TNFα + CHX. **P b 0.05 versus coronin 1A siRNA treated with TNFα + CHX.
Previous studies have shown that coronin 1A is important for cell survival and apoptosis. Coronin 1A-deficient T cells exhibit defects in Ca2+ mobilization and interleukin-2 production resulting in lower survival and more apoptosis than that observed in wild-type T cells [15]. Coronin 1A plays a pro-survival role in neutrophils [17]. In addition, the dimethylformamide-differentiated coronin 1A-overexpressing promyelocytic cell line, PLB985, is subject to less TRAIL-induced apoptosis and reduced activation of caspase-3 and caspase-9 but not caspase-8 [17]. These results are in contrast to those of the present study where
Fig. 5. A model for coronin 1A regulation of endothelial cell survival. Coronin 1A depletion increases the expression and activation of anti-apoptotic p38β after TNFα stimulation, resulting in the suppression of TNFα-induced cleavage of caspase 8, caspase 3 and PARP and an increase in cell survival.
depletion of coronin 1A in HUVEC led to less activation of caspase-8 and caspase-3 resulting in less apoptosis in response to TNFα stimulation. One explanation could be that the role of coronin 1A in apoptosis is cell type dependent or changes depending on the nature of the stimulation. Thus, it may be important to further study the regulatory mechanisms of apoptosis in various cell types exposed to different stimuli. p38 is activated by growth factors, cytokines and environmental stresses and modulates various cellular responses including development, cell growth, differentiation and inflammation [29]. Interestingly, p38 promotes apoptosis in some cell types whereas it increases cell survival in other cell types [23,30–32]. Thus, the function of p38 in apoptosis or survival may depend on cell type and the nature of the stimulus. Four isoforms of p38 MAPK (α, β, γ, δ) have been identified [29]. In particular, although endothelial cells express all isoforms [33], previous studies have shown that p38α induces endothelial cell apoptosis and that p38β protects endothelial cells from apoptosis [22,23,25–27]. Our results showed that both p38α and p38β were activated by TNFα stimulation under normal conditions. Because previous studies have shown that TNFα-activated p38α induces endothelial cell apoptosis [34,35], it is likely that p38α phosphorylation dominates over p38β activation under normal conditions. However, in the coronin 1A-depleted condition, the expression of anti-apoptotic p38β expression was induced. Furthermore, TNFα stimulation of the coronin 1A-depleted cells activated p38β without affecting the expression or activation of pro-apoptotic p38α. The activation of p38β led to a lower level of cleavage of the initiator caspase, caspase 8 and the executioner caspase, caspase 3,
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resulting in a lower level of apoptosis. Conversely, the inhibition of p38β upregulation in coronin 1A-depleted HUVEC restored the activation of caspase 8 and caspase 3 leading to a higher level of apoptosis. Leon Tourian Jr et al previously reported that transfection of p38β siRNA leads to a higher level of caspase 8 and caspase 3 cleavage and apoptosis in response to Fas-mediated apoptotic signaling [31]. However, the present study is the first to show that coronin 1A regulates the expression of p38β, a regulator of the caspase 8-caspase 3-PARP apoptotic pathway, and endothelial cell apoptosis in response to TNFα. Because p38β can induce cell survival via several signaling cascades, we examined the expression of pro-survival molecules that are regulated by p38β including heat shock protein (Hsp) 70 and heme oxygenase1 (HO-1) [22,27]. However, the expression of these proteins was not affected when p38β expression was induced in coronin 1A-depleted HUVEC (Supplemental Fig. S2), suggesting that Hsp70 and HO-1 are not involved in the survival of coronin 1A-depleted HUVEC. 5. Conclusions This study demonstrates that coronin 1A functions in TNFα-induced endothelial cell apoptosis. It is the first to report the involvement of coronin 1A in TNFα-induced apoptosis of endothelial cells via an effect on the p38β-dependent regulatory pathway. Considering the various functions of coronin 1A in leukocytes, it may be worthwhile pursuing this work further by studying the role of coronin 1A in inflammation [17], clearance of infected bacteria [12,36] and vascular diseases such as atherosclerosis [37]. Contributors Geun-Young Kim designed the study, performed experiments and wrote the manuscript. Hanna Kim and Hyun-Joung Lim performed experiments. Hyun-Young Park supervised the project and edited the manuscript. Disclosure The authors disclose no potential conflicts of interest. All authors approved the final article. Acknowledgments This work was supported by a Korean National Institute of Health intramural research grant (2013-NG63003-00). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2015.04.012.
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