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Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells
FEBS Letters 584 (2010) 3131–3136
journal homepage: www.FEBSLetters.org
Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells Monica Lee, Katherine C. Spokes, William C. Aird, Md. Ruhul Abid * The Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, United States
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Article history: Received 2 April 2010 Revised 5 May 2010 Accepted 24 May 2010 Available online 2 June 2010 Edited by Lukas Huber Keywords: NADPH oxidase Ca2+ Reactive oxygen species Signal transduction Endothelial cell
a b s t r a c t The aim of the present study is to determine the role of intracellular Ca2+ in VEGF signaling. We demonstrate that reduction in Ca2+ by chelating compound BAPTA-AM or by IP3-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca2+. These results suggest an essential role for Ca2+ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Vascular endothelial growth factor (VEGF) is a potent endothelial cell (EC)-specific mitogen and chemotactic factor that is involved in wound repair, angiogenesis of ischemic tissue, tumor growth, microvascular permeability, vascular protection, and hemostasis [1,2]. In addition to its mitogenic and chemotactic properties, VEGF promotes EC survival [3–5]. The VEGF family of proteins binds to three major receptor-type tyrosine kinases, Flt-1 (VEGF receptor1), KDR/Flk-1 (VEGF receptor-2), and VEGFR-3 [6,7]. Upon binding to its receptor(s), VEGF activates a number of different intracellular signaling pathways, including phospholipase Cc, protein kinase C, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), nonreceptor tyrosine kinase c-Src, and phosphatidyl inositol 3-kinase (PI3K)/Akt/protein kinase B in ECs. Recently, we have demonstrated that selective VEGF signaling pathway c-Src-PI3K-Akt but not ERK1/2 is dependent on NADPH oxidase-derived reactive oxygen species (ROS) [8]. Although ROS have traditionally been considered harmful for the endothelium, recent findings suggest that ROS play critical roles in signal transduction in ECs. Specifically, we have demonstrated that VEGF induces the activity of NADPH oxidase [9,10], a multi-subunit
* Corresponding author. Address: The Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, RN-232, 330 Brookline Avenue, Boston, MA 02215, United States. Fax: +1 617 667 2913. E-mail address: [email protected] (M.R. Abid).
enzyme complex consists of two membrane-bound components, gp91phox (also known as Nox2) and p22phox, and several cytosolic regulatory subunits, including p47phox, p67phox, and the small GTPase Rac (Rac1 or Rac2). We have also shown that NADPH oxidase is a major source of endothelial ROS and that NADPH oxidase-derived reactive oxygen species (ROS) are, in turn, required for VEGF-mediated induction of cell proliferation, migration and gene expression [8–11]. Subsequent studies have confirmed the importance of ROS in VEGF signal transduction [12,13]. Calcium acts as a second messenger and plays important role in many cellular functions including survival and proliferation. VEGF requires Ca2+ for EC proliferation and plays a critical role in the regulation of intracellular ionized calcium (Ca2+) concentration [14]. VEGF induces influx of Ca2+ from the extracellular space in response to store depletion caused by activation of the transmembrane store-operated channels/capacitative Ca2+ entry channels (SOC/CCE) in ECs [15,16]. VEGF also regulates PLCc-(PIP2)-IP3-mediated activation of inositol 1,4,5-trisphosphate receptor (IP3R) resulting in the release of Ca2+ from the endoplasmic reticulum (ER) into the cytosol [15,17]. Furthermore, inhibition of VEGFR2, Src and IP3 was shown to eliminate calcium and membrane potential transients in HUVEC [16]. The present study is aimed to elucidate the role of Ca2+ in VEGF signaling in ECs and to examine the interrelationship, if any, between ROS and Ca2+ in VEGF signal transduction. Here, we report an interesting finding that VEGFmediated activation of redox-sensitive c-Src-PI3K-Akt but not
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.05.053
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redox-insensitive ERK1/2 is dependent on intracellular Ca2+. We also demonstrate that an increase in intracellular Ca2+ can compensate for the effects of reduction in NADPH oxidase activity on VEGF-induced activation of c-Src-PI3K-Akt and cell proliferation in HCAEC. 2. Materials and methods 2.1. Cell culture and reagents Human coronary artery endothelial cells (HCAEC) were obtained from Clonetics and grown in Endothelial Growth Medium-2-MV (EGM-2-MV) BulletKit (Clonetics, San Diego, CA) at 37 °C and 5% CO2. Endothelial cells from passage 3 to 6 were used for all experiments. Cells were serum starved in 0.5% fetal bovine serum (FBS) for 16 h prior to treatment with 50 ng/ml human VEGF-A165 (PeproTech Inc, Rocky Hill, NJ). Where indicated, cells were pre-incubated for 15 min with cell-permeable calcium chelator BAPTA-AM 5 lmol/l, calcium ionophore A23187 1 lmol/l, store-depletionmediated enhancer of intracellular calcium thapsigargin 1 lmol/l, or inhibitor of IP3-mediated release of calcium 2-Aminoethyl diphenyl borate (2-APB) 5 lmol/l (Calbiochem, San Diego, CA). XTT assay kit was from Roche Applied Science (Indianapolis, IN).
2.3. Western blotting HCAEC were harvested for total protein and Western blots were carried out as previously described [18]. Phospho-specific antibodies against Y416-c-Src, Ser-473 Akt, focal adhesion kinase (FAK), PLCc-1, and ERK1/2 were purchased from Cell Signaling (Beverly, MA). Anti-b-actin antibodies were from Sigma. Western blots were performed in triplicates using cell extracts prepared from three independent experiments. 2.4. Cell proliferation assay XTT assay was employed to determine cell proliferation in HCAEC as described [19]. This assay is based on the conversion of the yellow tetrazolium salt XTT into an orange, water-soluble dye, formazan, by metabolically active cells. Scram-si and sip47phox-transfected HCAEC were serum-strarved for 12 h, pre-treated with vehicle (DMSO) or calcium ionophore A23187 for 15 min and then treated with VEGF for 20 h. HCAEC were then incubated with XTT for 4 h. Formation of formazan was directly quantitated in 96-well plates with an enzyme-linked immunosorbent assay reader at A490–655 nm (model 680; Bio-Rad). Trypan blue exclusion method was also used to count the number of viable cells where indicated as described previously [10,19].
2.2. siRNA transfection 2.5. Rac1 activity assay HCAEC were grown to 70–80% confluence in 6-cm plates and transfected with 100 nM ON-TARGETplus si-RNA against p47phox (Dharmacon, Lafayette, CO) or scrambled siRNA (Scram-si) in Opti-MEM containing 10 lg/ml Lipofectin (Invitrogen, Carlsbad, CA) for 5 h. The cells were then incubated in EGM-2-MV medium for 24 h and serum starved in endothelial basal medium (EBM-2; Clonetics) containing 0.2% serum for 16 h prior to VEGF treatment.
HCAEC pre-treated with or without A23187 for 5 min followed by VEGF treatment for 1 min were lysed in ice-cold lysis buffer (25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM NaF and 10% glycerol). Anti-active rac1-GTP antibody (New East Biosciences) and protein A/G agarose were used to pull down GTP-bound Rac1 from
Fig. 1. VEGF-induced activation of c-Src, FAK and Akt, but not ERK1/2 requires intracellular ionized calcium. (A) Protein extracts from HCAEC were subject to Western blots as described in Section 2. Serum-starved HCAEC were pre-treated with 5 lmol/l BAPTA-AM for 15 min and treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the antibodies shown. Anti-b-actin antibody was used as loading control. Blots shown are representative of three independent experiments. Lower two panels: bar graph shows quantitative densitometric analysis of Akt phosphorylation at 15 min time points and ERK1/2 phosphorylation at 5 min time points in three independent Western blots using NIH J image (-fold change expressed in mean ± S.E.M.). *P < 0.05, VEGF-treated vs. untreated control; P < 0.05, vehicle-pretreated followed by VEGF treatment vs. BAPTA-AM-pre-treated followed by VEGF treatment. (B) Same as in (A) except, in addition to BAPTA-AM, HACEC was pretreated with 1 lmol/l A23187 for 15 min and probed for phosphorylation of c-Src, FAK, Akt and ERK1/2 at indicated time points. Bar graph shows quantitative analysis of c-Src and FAK phosphorylation at 5 min in three independent Western blots. *P < 0.05, VEGF-treated vs. untreated control.
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the cell lysates followed by immunoblotting using anti-Rac1 antibody as per manufacturer’s protocol.
lates VEGF activation of c-Src-PI3K-Akt but not ERK1/2 signaling in HCAEC.
2.6. Statistical analysis
3.2. Increase in Ca2+ selectively activates PI3K-Akt signaling but not ERK1/2
All values are presented as mean ± S.E.M. where appropriate. Statistical significance between two groups was determined by use of a paired t-test, and values of P < 0.05 were considered significant. 3. Results 3.1. Reduction in intracellular Ca2+ inhibits VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 signaling pathway We wanted to examine the role of intracellular Ca2+ in VEGF signal transduction in ECs. Reduction in intracellular Ca2+ levels by cell-permeable chelating agent BAPTA-AM inhibited VEGF-mediated phosphorylation of Akt but not ERK1/2 in HCAEC (Fig. 1), suggesting that selective activation of some but not all VEGF signaling pathways requires intracellular Ca2+. Decrease in intracellular Ca2+ by 2-APB, an inhibitor of IP3-mediated Ca2+ release, produced similar results (Fig. 2). Previously, we have reported that phosphorylation of c-Src by VEGF was essential for downstream activation of PI3K-Akt [8]. Therefore, we wanted to examine whether phosphorylation of c-Src by VEGF was also dependent on intracellular Ca2+. Fig. 1B shows that BAPTA-AM blocked c-Src and FAK phosphorylation by VEGF, suggesting that intracellular Ca2+ selectively modu-
Fig. 2. Increase in intracellular Ca2+ induces Akt but not ERK1/2 phosphorylation in HCAEC, and inhibition of IP3-mediated intracellular release of Ca2+ inhibits Akt activation but not ERK1/2. (A) Protein extracts from HCAEC pre-treated with either calcium ionophore (A23187) or inhibitor of IP3-mediated calcium increase (2-APB) for 15 min, and treated with VEGF for times indicated were subject to Western blots. Membranes were immunoblotted with anti-phospho-Akt antibody, and then stripped and re-probed for phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B and C) Quantitative analyses of Akt (B) and ERK1/2 phosphorylation (C) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, vehicle-pretreated followed by VEGF treatment vs. A23187- or 2-APB-pre-treated followed by VEGF treatment; and #P < 0.05, vehicle-pre-treated control vs. A23187pre-treated control.
Next, we wanted to examine the effects of increase in Ca2+ on VEGF signaling. Increase in Ca2+ by A23187 increased basal and VEGF-induced phosphorylation of Akt but not ERK1/2 in HCAEC (Fig. 2) suggesting that Ca2+ plays an important role in PI3K-Akt activation. Similar results were obtained using thapsigargin-induced increase in intracellular Ca2+ (Fig. 3). 3.3. Increase in Ca2+ compensates for the effects of reduction in NADPH oxidase activity on VEGF-induced c-Src-PI3K-Akt signaling Previously, we have shown that VEGF-mediated activation of c-Src-PI3K-Akt, but not ERK1/2, is dependent on NADPH oxidasederived reactive oxygen species (ROS) [8] (also see Figs. 3 and 4). Together with the data presented in the current study, we hypothesized that Ca2+ lies downstream of NADPH oxidase-derived ROS in VEGF-mediated activation of c-Src-PI3K-Akt in ECs. To test this, we next asked whether increase in Ca2+ can compensate for a lack of NADPH oxidase activity in redox-sensitive VEGF signaling in ECs. In NADPH oxidase-knockdown HCAEC (by si-p47phox),
Fig. 3. Inhibition of NADPH oxidase blocks VEGF-mediated activation of Akt but not ERK1/2, and thapsigargin (Tg)-mediated store-depletion-induced increase in Ca2+ induces Akt but not ERK1/2 phosphorylation. (A) Protein extracts from HCAEC transfected with scrambled siRNA (Scram-si) and pre-treated vehicle (DMSO) for 15 min, HCAEC transfected with siRNA against p47phox (si-p47phox), or pre-treated with thapsigargin for 15 min, and then treated with VEGF for times indicated were subject to Western blots. Membranes were immunoblotted with anti-phospho-Akt antibody, and then stripped and re-probed for phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B and C) Quantitative analyses of Akt (B) and ERK1/2 phosphorylation (C) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, scram-si treated with VEGF vs. si-p47phox-transfected treated with VEGF; and #P < 0.05, vehicle-pre-treated control vs. thapsigargin (Tg)-pretreated control.
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Fig. 4. (A) Excess of intracellular Ca2+ can reverse the effects of NADPH oxidase inhibition on c-Src and Akt activation in HCAEC. (A) Protein extracts from HCAEC transfected with scrambled siRNA (Scram-si) and pre-treated with vehicle (DMSO) or A23187 for 15 min were treated with VEGF for times indicated. Western blots were performed using anti-phospho-c-Src antibody, and then the membranes were stripped and re-probed sequentially for phospho-Akt and phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B) HCAEC transfected with siRNA against p47phox (si-p47phox) and pre-treated with vehicle or A23187 for 15 min and then treated with VEGF were subject to Western blot analyses as in (A). (C–E) Quantitative analyses of c-Src (C), Akt (D) and ERK1/2 phosphorylation (E) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, scram-si plus vehicle treated with VEGF vs. si-p47phox-transfected treated with VEGF (C and D), or scram-si plus vehicle treated with VEGF vs. A23187 pre-treated HCAEC treated with VEGF (D); and #P < 0.05, vehicle-pre-treated control vs. A23187pre-treated control.
VEGF-induced phosphorylation of c-Src and Akt but not ERK1/2 was inhibited (Figs. 3 and 4B). Interestingly, increase in intracellular Ca2+ by A23187 reversed the inhibitory effects of NADPH oxidase inhibition on VEGF-induced phosphorylation of c-Src and Akt (Fig. 4B–E), suggesting that VEGF-induced redox-dependent activation of c-Src-PI3K-Akt occurs through increase in intracellular Ca2+. 3.4. Increase in Ca2+ can reverse the inhibitory effects of NADPH oxidase inhibition on VEGF-induced cell proliferation We and others have previously shown that inhibition of NADPH oxidase-derived ROS blocked VEGF-induced proliferation of ECs including HCAEC [9,12,13]. Here, we wanted to examine whether increase in Ca2+ can compensate for the effects of reduction of NADPH oxidase-derived ROS on VEGF-mediated cell proliferation. Pre-treatment of HCAEC with A23187 reversed the inhibition of VEGF-induced cell proliferation in si-p47phox-transfected HCAEC (Fig. 5A), suggesting that increase in Ca2+ can abrogate the inhibi-
tory effects of NADPH oxidase-knockdown in HCAEC. Cell counting assay using trypan blue exclusion method produced similar results (data not shown). 4. Discussion This study demonstrates that intracellular Ca2+ is required for VEGF-mediated selective activation of c-Src-PI3K-Akt but not ERK1/2. We have previously shown that the same pathway, cSrc-PI3K-Akt but not ERK1/2, is also dependent on NADPH oxidase-derived ROS in ECs [8]. Interestingly, the data presented in the current study demonstrate that an excess of Ca2+ can reverse the effects of NADPH oxidase inhibition on VEGF induction of cSrc-PI3K-Akt signaling and cell proliferation in HCAEC. Together with the effects of Ca2+ on cell proliferation in NADPH oxidase knockdown ECs, the results suggest that intracellular Ca2+ plays a positive role in the redox-sensitive activation of c-Src-PI3K-Akt. The results also suggest that Ca2+ plausibly lies downstream of NADPH oxidease-derived ROS in VEGF-c-Src-PI3K-Akt axis.
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Fig. 5. Ca2+ can reverse the inhibitory effects of NADPH oxidase inhibition on HCAEC proliferation. XTT assay was performed on control (Scram-si) and NADPH oxidase knockdown (si-p47phox) HCAEC that were pre-treated with or without A23187 and treated with VEGF as described in Section 2. The data represents mean ± S.E.M. of fold change using three independent experiments. *P < 0.05, VEGF treated vs. control; P < 0.05, VEGF-treated si-p47phox-transfected vs. VEGF-treated Scram-si-transfected HCAEC. (B) Increase in Ca2+ activates Rac1. Activated GTP-Rac1 was pulled down from HCAEC lysates that were pretreated with or without 1 lmol/l A23187 for 5 min and treated with or without VEGF (50 ng/ml) for 1 min as indicated (upper panel). Lower panel shows Western blot using corresponding total cell lysates and anti-Rac1 antibody. (C) Proposed model for selective requirement for intracellular Ca2+ in NADPH oxidase-dependent activation of c-Src-PI3K-Akt by VEGF in ECs. VEGF-induced activation of PLCc-1ERK1/2 appears to be insensitive to endothelial Ca2+ levels. The colored box indicates Ca2+-dependent VEGF signaling pathways. The ‘‘?” mark indicates as yet unknown Ca2+sensitive signaling intermediate(s) that modulates VEGF induction of redox-sensitive c-Src-PI3K-Akt signaling pathway.
Our data strongly suggest that NADPH oxidase-derived ROS integrate intracellular Ca2+ into the redox-sensitive component of the VEGF signaling pathway. However, the precise mechanism of Ca2+ integration into the VEGF-redox pathway is not known at present. A recent study reported that VEGF-induced activation of calcium/ calmodulin-dependent protein kinase kinase b (CaMKKb) resulted in the activation of AMP-activated protein kinase (AMPK)-Rac1Akt pathway in BAEC [20]. Thus, it is formally possible that Rac1dependent NADPH oxidase-derived ROS production may depend on Ca2+ in ECs (pre-NADPH oxidase role of Ca2+). Our findings that increase in Ca2+ results in Rac1 activation in HCAEC (Fig. 5B) support this notion. Alternatively, VEGF activation of c-Src-PI3K-Akt may depend on Ca2+-sensitive upstream protein kinase(s) yet to be indentified (post-NADPH oxidase role). Since the present study demonstrates that an increase in Ca2+ reversed the effects of NADPH oxidase knockdown on VEGF signaling and cell proliferation, a major post-NADPH oxidase role for Ca2+ would be more appropriate. These questions are currently being addressed in our laboratory. It is evident from the above discussion that a link exists between VEGF signaling and NADPH oxidase activation in ECs as part of an integral circuit that incorporates additional signaling input from intracellular Ca2+ (summarized in Fig. 5C). However, the permissive role of Ca2+ on c-Src-PI3K-Akt signaling is very likely mediated at post-VEGF receptor levels. The findings that activation of VEGFR2 is independent of Ca2+ (data not shown) and redox [8] support this notion. In addition, redox- [8] and Ca2+-independent activation of PLCc-1 by VEGF (data not shown) also supports a post-VEGF receptor role for Ca2+ in c-Src-PI3K-Akt activation. It is interesting to note that although Ca2+ inhibition did not negatively affect VEGF-induced activation of ERK1/2, we observed a shift in the temporal pattern and intensity of ERK1/2 activation (earliest time point for ERK1/2 activation shifted from 1 min to 5 min) in BAPTA-AM treated ECs (Fig. 1A). The precise mechanism for this is not known at present. However, it is plausible that, due to inactivation of PI3K-Akt, PLCc-1-ERK1/2 signaling intermediates may
have greater access to PIP2, a common but limited substrate for both PI3K and PLCc-1 in ECs [21]. Further studies are required to elucidate the mechanism. In summary, we demonstrate that VEGF-mediated activation of redox-sensitive c-Src-PI3K-Akt but not redox-insensitive ERK1/2 signaling pathway is dependent on intracellular Ca2+, and that increase in Ca2+ can compensate for the effects of loss of NADPH oxidase activity on the redox-sensitive arm of VEGF signaling and cell proliferation in HCAEC. This is the first report to demonstrate that Ca2+ is an integral part of the redox-dependent selective VEGF signaling pathways, c-Src-PI3K-Akt but not ERK1/2, in ECs. The findings that two intracellular second messengers, Ca2+ and ROS, lie in the same signaling axis in ECs may have important therapeutic significance in vascular disease state. It is worthwhile to note that changes in endothelial Ca2+ may affect the balance of other intracellular ions including phosphate, which may in turn modulate redox-sensitive signaling cascade in HCAEC. Future studies to elucidate the molecular mechanisms by which Ca2+ and ROS coordinate signal propagation in the endothelium should provide important new insights into the endothelial functions in health and disease. Acknowledgements This work was supported in part by AHA Grants SDG 0453284N and AHA 10GRNT3640011, Department of Medicine, BIDMC, and NIH Grant R01HL077348. References [1] Ferrara, N. (2001) Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. Cell Physiol. 280, C1358–C1366. [2] Dvorak, H.F., Brown, L.F., Detmar, M. and Dvorak, A.M. (1995) Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Pathol. 146, 1029–1039. [3] Gerber, H.P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B.A., Dixit, V. and Ferrara, N. (1998) Vascular endothelial growth factor regulates endothelial cell
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Intracellular Ca2+ can compensate for the lack of NADPH oxidase-derived ROS in endothelial cells Monica Lee, Katherine C. Spokes, William C. Aird, Md. Ruhul Abid * The Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02215, United States
a r t i c l e
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Article history: Received 2 April 2010 Revised 5 May 2010 Accepted 24 May 2010 Available online 2 June 2010 Edited by Lukas Huber Keywords: NADPH oxidase Ca2+ Reactive oxygen species Signal transduction Endothelial cell
a b s t r a c t The aim of the present study is to determine the role of intracellular Ca2+ in VEGF signaling. We demonstrate that reduction in Ca2+ by chelating compound BAPTA-AM or by IP3-endoplasmic reticulum blocker 2-APB selectively inhibited VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 in human coronary artery endothelial cells (HCAEC). We also show that the selective inhibitory effects of NADPH oxidase knockdown on VEGF-mediated activation of c-Src-PI3K-Akt signaling and cell proliferation in HCAEC can be reversed by increase in intracellular Ca2+. These results suggest an essential role for Ca2+ in redox-dependent selective activation of c-Src-PI3K-Akt and endothelial cell proliferation. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Vascular endothelial growth factor (VEGF) is a potent endothelial cell (EC)-specific mitogen and chemotactic factor that is involved in wound repair, angiogenesis of ischemic tissue, tumor growth, microvascular permeability, vascular protection, and hemostasis [1,2]. In addition to its mitogenic and chemotactic properties, VEGF promotes EC survival [3–5]. The VEGF family of proteins binds to three major receptor-type tyrosine kinases, Flt-1 (VEGF receptor1), KDR/Flk-1 (VEGF receptor-2), and VEGFR-3 [6,7]. Upon binding to its receptor(s), VEGF activates a number of different intracellular signaling pathways, including phospholipase Cc, protein kinase C, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), nonreceptor tyrosine kinase c-Src, and phosphatidyl inositol 3-kinase (PI3K)/Akt/protein kinase B in ECs. Recently, we have demonstrated that selective VEGF signaling pathway c-Src-PI3K-Akt but not ERK1/2 is dependent on NADPH oxidase-derived reactive oxygen species (ROS) [8]. Although ROS have traditionally been considered harmful for the endothelium, recent findings suggest that ROS play critical roles in signal transduction in ECs. Specifically, we have demonstrated that VEGF induces the activity of NADPH oxidase [9,10], a multi-subunit
* Corresponding author. Address: The Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, RN-232, 330 Brookline Avenue, Boston, MA 02215, United States. Fax: +1 617 667 2913. E-mail address: [email protected] (M.R. Abid).
enzyme complex consists of two membrane-bound components, gp91phox (also known as Nox2) and p22phox, and several cytosolic regulatory subunits, including p47phox, p67phox, and the small GTPase Rac (Rac1 or Rac2). We have also shown that NADPH oxidase is a major source of endothelial ROS and that NADPH oxidase-derived reactive oxygen species (ROS) are, in turn, required for VEGF-mediated induction of cell proliferation, migration and gene expression [8–11]. Subsequent studies have confirmed the importance of ROS in VEGF signal transduction [12,13]. Calcium acts as a second messenger and plays important role in many cellular functions including survival and proliferation. VEGF requires Ca2+ for EC proliferation and plays a critical role in the regulation of intracellular ionized calcium (Ca2+) concentration [14]. VEGF induces influx of Ca2+ from the extracellular space in response to store depletion caused by activation of the transmembrane store-operated channels/capacitative Ca2+ entry channels (SOC/CCE) in ECs [15,16]. VEGF also regulates PLCc-(PIP2)-IP3-mediated activation of inositol 1,4,5-trisphosphate receptor (IP3R) resulting in the release of Ca2+ from the endoplasmic reticulum (ER) into the cytosol [15,17]. Furthermore, inhibition of VEGFR2, Src and IP3 was shown to eliminate calcium and membrane potential transients in HUVEC [16]. The present study is aimed to elucidate the role of Ca2+ in VEGF signaling in ECs and to examine the interrelationship, if any, between ROS and Ca2+ in VEGF signal transduction. Here, we report an interesting finding that VEGFmediated activation of redox-sensitive c-Src-PI3K-Akt but not
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redox-insensitive ERK1/2 is dependent on intracellular Ca2+. We also demonstrate that an increase in intracellular Ca2+ can compensate for the effects of reduction in NADPH oxidase activity on VEGF-induced activation of c-Src-PI3K-Akt and cell proliferation in HCAEC. 2. Materials and methods 2.1. Cell culture and reagents Human coronary artery endothelial cells (HCAEC) were obtained from Clonetics and grown in Endothelial Growth Medium-2-MV (EGM-2-MV) BulletKit (Clonetics, San Diego, CA) at 37 °C and 5% CO2. Endothelial cells from passage 3 to 6 were used for all experiments. Cells were serum starved in 0.5% fetal bovine serum (FBS) for 16 h prior to treatment with 50 ng/ml human VEGF-A165 (PeproTech Inc, Rocky Hill, NJ). Where indicated, cells were pre-incubated for 15 min with cell-permeable calcium chelator BAPTA-AM 5 lmol/l, calcium ionophore A23187 1 lmol/l, store-depletionmediated enhancer of intracellular calcium thapsigargin 1 lmol/l, or inhibitor of IP3-mediated release of calcium 2-Aminoethyl diphenyl borate (2-APB) 5 lmol/l (Calbiochem, San Diego, CA). XTT assay kit was from Roche Applied Science (Indianapolis, IN).
2.3. Western blotting HCAEC were harvested for total protein and Western blots were carried out as previously described [18]. Phospho-specific antibodies against Y416-c-Src, Ser-473 Akt, focal adhesion kinase (FAK), PLCc-1, and ERK1/2 were purchased from Cell Signaling (Beverly, MA). Anti-b-actin antibodies were from Sigma. Western blots were performed in triplicates using cell extracts prepared from three independent experiments. 2.4. Cell proliferation assay XTT assay was employed to determine cell proliferation in HCAEC as described [19]. This assay is based on the conversion of the yellow tetrazolium salt XTT into an orange, water-soluble dye, formazan, by metabolically active cells. Scram-si and sip47phox-transfected HCAEC were serum-strarved for 12 h, pre-treated with vehicle (DMSO) or calcium ionophore A23187 for 15 min and then treated with VEGF for 20 h. HCAEC were then incubated with XTT for 4 h. Formation of formazan was directly quantitated in 96-well plates with an enzyme-linked immunosorbent assay reader at A490–655 nm (model 680; Bio-Rad). Trypan blue exclusion method was also used to count the number of viable cells where indicated as described previously [10,19].
2.2. siRNA transfection 2.5. Rac1 activity assay HCAEC were grown to 70–80% confluence in 6-cm plates and transfected with 100 nM ON-TARGETplus si-RNA against p47phox (Dharmacon, Lafayette, CO) or scrambled siRNA (Scram-si) in Opti-MEM containing 10 lg/ml Lipofectin (Invitrogen, Carlsbad, CA) for 5 h. The cells were then incubated in EGM-2-MV medium for 24 h and serum starved in endothelial basal medium (EBM-2; Clonetics) containing 0.2% serum for 16 h prior to VEGF treatment.
HCAEC pre-treated with or without A23187 for 5 min followed by VEGF treatment for 1 min were lysed in ice-cold lysis buffer (25 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM NaF and 10% glycerol). Anti-active rac1-GTP antibody (New East Biosciences) and protein A/G agarose were used to pull down GTP-bound Rac1 from
Fig. 1. VEGF-induced activation of c-Src, FAK and Akt, but not ERK1/2 requires intracellular ionized calcium. (A) Protein extracts from HCAEC were subject to Western blots as described in Section 2. Serum-starved HCAEC were pre-treated with 5 lmol/l BAPTA-AM for 15 min and treated with VEGF (50 ng/ml) for the times indicated. Membranes were sequentially blotted, stripped and re-probed with the antibodies shown. Anti-b-actin antibody was used as loading control. Blots shown are representative of three independent experiments. Lower two panels: bar graph shows quantitative densitometric analysis of Akt phosphorylation at 15 min time points and ERK1/2 phosphorylation at 5 min time points in three independent Western blots using NIH J image (-fold change expressed in mean ± S.E.M.). *P < 0.05, VEGF-treated vs. untreated control; P < 0.05, vehicle-pretreated followed by VEGF treatment vs. BAPTA-AM-pre-treated followed by VEGF treatment. (B) Same as in (A) except, in addition to BAPTA-AM, HACEC was pretreated with 1 lmol/l A23187 for 15 min and probed for phosphorylation of c-Src, FAK, Akt and ERK1/2 at indicated time points. Bar graph shows quantitative analysis of c-Src and FAK phosphorylation at 5 min in three independent Western blots. *P < 0.05, VEGF-treated vs. untreated control.
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the cell lysates followed by immunoblotting using anti-Rac1 antibody as per manufacturer’s protocol.
lates VEGF activation of c-Src-PI3K-Akt but not ERK1/2 signaling in HCAEC.
2.6. Statistical analysis
3.2. Increase in Ca2+ selectively activates PI3K-Akt signaling but not ERK1/2
All values are presented as mean ± S.E.M. where appropriate. Statistical significance between two groups was determined by use of a paired t-test, and values of P < 0.05 were considered significant. 3. Results 3.1. Reduction in intracellular Ca2+ inhibits VEGF-induced activation of c-Src-PI3K-Akt but not ERK1/2 signaling pathway We wanted to examine the role of intracellular Ca2+ in VEGF signal transduction in ECs. Reduction in intracellular Ca2+ levels by cell-permeable chelating agent BAPTA-AM inhibited VEGF-mediated phosphorylation of Akt but not ERK1/2 in HCAEC (Fig. 1), suggesting that selective activation of some but not all VEGF signaling pathways requires intracellular Ca2+. Decrease in intracellular Ca2+ by 2-APB, an inhibitor of IP3-mediated Ca2+ release, produced similar results (Fig. 2). Previously, we have reported that phosphorylation of c-Src by VEGF was essential for downstream activation of PI3K-Akt [8]. Therefore, we wanted to examine whether phosphorylation of c-Src by VEGF was also dependent on intracellular Ca2+. Fig. 1B shows that BAPTA-AM blocked c-Src and FAK phosphorylation by VEGF, suggesting that intracellular Ca2+ selectively modu-
Fig. 2. Increase in intracellular Ca2+ induces Akt but not ERK1/2 phosphorylation in HCAEC, and inhibition of IP3-mediated intracellular release of Ca2+ inhibits Akt activation but not ERK1/2. (A) Protein extracts from HCAEC pre-treated with either calcium ionophore (A23187) or inhibitor of IP3-mediated calcium increase (2-APB) for 15 min, and treated with VEGF for times indicated were subject to Western blots. Membranes were immunoblotted with anti-phospho-Akt antibody, and then stripped and re-probed for phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B and C) Quantitative analyses of Akt (B) and ERK1/2 phosphorylation (C) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, vehicle-pretreated followed by VEGF treatment vs. A23187- or 2-APB-pre-treated followed by VEGF treatment; and #P < 0.05, vehicle-pre-treated control vs. A23187pre-treated control.
Next, we wanted to examine the effects of increase in Ca2+ on VEGF signaling. Increase in Ca2+ by A23187 increased basal and VEGF-induced phosphorylation of Akt but not ERK1/2 in HCAEC (Fig. 2) suggesting that Ca2+ plays an important role in PI3K-Akt activation. Similar results were obtained using thapsigargin-induced increase in intracellular Ca2+ (Fig. 3). 3.3. Increase in Ca2+ compensates for the effects of reduction in NADPH oxidase activity on VEGF-induced c-Src-PI3K-Akt signaling Previously, we have shown that VEGF-mediated activation of c-Src-PI3K-Akt, but not ERK1/2, is dependent on NADPH oxidasederived reactive oxygen species (ROS) [8] (also see Figs. 3 and 4). Together with the data presented in the current study, we hypothesized that Ca2+ lies downstream of NADPH oxidase-derived ROS in VEGF-mediated activation of c-Src-PI3K-Akt in ECs. To test this, we next asked whether increase in Ca2+ can compensate for a lack of NADPH oxidase activity in redox-sensitive VEGF signaling in ECs. In NADPH oxidase-knockdown HCAEC (by si-p47phox),
Fig. 3. Inhibition of NADPH oxidase blocks VEGF-mediated activation of Akt but not ERK1/2, and thapsigargin (Tg)-mediated store-depletion-induced increase in Ca2+ induces Akt but not ERK1/2 phosphorylation. (A) Protein extracts from HCAEC transfected with scrambled siRNA (Scram-si) and pre-treated vehicle (DMSO) for 15 min, HCAEC transfected with siRNA against p47phox (si-p47phox), or pre-treated with thapsigargin for 15 min, and then treated with VEGF for times indicated were subject to Western blots. Membranes were immunoblotted with anti-phospho-Akt antibody, and then stripped and re-probed for phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B and C) Quantitative analyses of Akt (B) and ERK1/2 phosphorylation (C) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, scram-si treated with VEGF vs. si-p47phox-transfected treated with VEGF; and #P < 0.05, vehicle-pre-treated control vs. thapsigargin (Tg)-pretreated control.
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Fig. 4. (A) Excess of intracellular Ca2+ can reverse the effects of NADPH oxidase inhibition on c-Src and Akt activation in HCAEC. (A) Protein extracts from HCAEC transfected with scrambled siRNA (Scram-si) and pre-treated with vehicle (DMSO) or A23187 for 15 min were treated with VEGF for times indicated. Western blots were performed using anti-phospho-c-Src antibody, and then the membranes were stripped and re-probed sequentially for phospho-Akt and phospho-ERK1/2. b-Actin was used as loading control. Blots shown are representative of three independent experiments. (B) HCAEC transfected with siRNA against p47phox (si-p47phox) and pre-treated with vehicle or A23187 for 15 min and then treated with VEGF were subject to Western blot analyses as in (A). (C–E) Quantitative analyses of c-Src (C), Akt (D) and ERK1/2 phosphorylation (E) were carried out as described in Fig. 1. *P < 0.05, VEGF-treated relative to untreated control; P < 0.05, scram-si plus vehicle treated with VEGF vs. si-p47phox-transfected treated with VEGF (C and D), or scram-si plus vehicle treated with VEGF vs. A23187 pre-treated HCAEC treated with VEGF (D); and #P < 0.05, vehicle-pre-treated control vs. A23187pre-treated control.
VEGF-induced phosphorylation of c-Src and Akt but not ERK1/2 was inhibited (Figs. 3 and 4B). Interestingly, increase in intracellular Ca2+ by A23187 reversed the inhibitory effects of NADPH oxidase inhibition on VEGF-induced phosphorylation of c-Src and Akt (Fig. 4B–E), suggesting that VEGF-induced redox-dependent activation of c-Src-PI3K-Akt occurs through increase in intracellular Ca2+. 3.4. Increase in Ca2+ can reverse the inhibitory effects of NADPH oxidase inhibition on VEGF-induced cell proliferation We and others have previously shown that inhibition of NADPH oxidase-derived ROS blocked VEGF-induced proliferation of ECs including HCAEC [9,12,13]. Here, we wanted to examine whether increase in Ca2+ can compensate for the effects of reduction of NADPH oxidase-derived ROS on VEGF-mediated cell proliferation. Pre-treatment of HCAEC with A23187 reversed the inhibition of VEGF-induced cell proliferation in si-p47phox-transfected HCAEC (Fig. 5A), suggesting that increase in Ca2+ can abrogate the inhibi-
tory effects of NADPH oxidase-knockdown in HCAEC. Cell counting assay using trypan blue exclusion method produced similar results (data not shown). 4. Discussion This study demonstrates that intracellular Ca2+ is required for VEGF-mediated selective activation of c-Src-PI3K-Akt but not ERK1/2. We have previously shown that the same pathway, cSrc-PI3K-Akt but not ERK1/2, is also dependent on NADPH oxidase-derived ROS in ECs [8]. Interestingly, the data presented in the current study demonstrate that an excess of Ca2+ can reverse the effects of NADPH oxidase inhibition on VEGF induction of cSrc-PI3K-Akt signaling and cell proliferation in HCAEC. Together with the effects of Ca2+ on cell proliferation in NADPH oxidase knockdown ECs, the results suggest that intracellular Ca2+ plays a positive role in the redox-sensitive activation of c-Src-PI3K-Akt. The results also suggest that Ca2+ plausibly lies downstream of NADPH oxidease-derived ROS in VEGF-c-Src-PI3K-Akt axis.
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Fig. 5. Ca2+ can reverse the inhibitory effects of NADPH oxidase inhibition on HCAEC proliferation. XTT assay was performed on control (Scram-si) and NADPH oxidase knockdown (si-p47phox) HCAEC that were pre-treated with or without A23187 and treated with VEGF as described in Section 2. The data represents mean ± S.E.M. of fold change using three independent experiments. *P < 0.05, VEGF treated vs. control; P < 0.05, VEGF-treated si-p47phox-transfected vs. VEGF-treated Scram-si-transfected HCAEC. (B) Increase in Ca2+ activates Rac1. Activated GTP-Rac1 was pulled down from HCAEC lysates that were pretreated with or without 1 lmol/l A23187 for 5 min and treated with or without VEGF (50 ng/ml) for 1 min as indicated (upper panel). Lower panel shows Western blot using corresponding total cell lysates and anti-Rac1 antibody. (C) Proposed model for selective requirement for intracellular Ca2+ in NADPH oxidase-dependent activation of c-Src-PI3K-Akt by VEGF in ECs. VEGF-induced activation of PLCc-1ERK1/2 appears to be insensitive to endothelial Ca2+ levels. The colored box indicates Ca2+-dependent VEGF signaling pathways. The ‘‘?” mark indicates as yet unknown Ca2+sensitive signaling intermediate(s) that modulates VEGF induction of redox-sensitive c-Src-PI3K-Akt signaling pathway.
Our data strongly suggest that NADPH oxidase-derived ROS integrate intracellular Ca2+ into the redox-sensitive component of the VEGF signaling pathway. However, the precise mechanism of Ca2+ integration into the VEGF-redox pathway is not known at present. A recent study reported that VEGF-induced activation of calcium/ calmodulin-dependent protein kinase kinase b (CaMKKb) resulted in the activation of AMP-activated protein kinase (AMPK)-Rac1Akt pathway in BAEC [20]. Thus, it is formally possible that Rac1dependent NADPH oxidase-derived ROS production may depend on Ca2+ in ECs (pre-NADPH oxidase role of Ca2+). Our findings that increase in Ca2+ results in Rac1 activation in HCAEC (Fig. 5B) support this notion. Alternatively, VEGF activation of c-Src-PI3K-Akt may depend on Ca2+-sensitive upstream protein kinase(s) yet to be indentified (post-NADPH oxidase role). Since the present study demonstrates that an increase in Ca2+ reversed the effects of NADPH oxidase knockdown on VEGF signaling and cell proliferation, a major post-NADPH oxidase role for Ca2+ would be more appropriate. These questions are currently being addressed in our laboratory. It is evident from the above discussion that a link exists between VEGF signaling and NADPH oxidase activation in ECs as part of an integral circuit that incorporates additional signaling input from intracellular Ca2+ (summarized in Fig. 5C). However, the permissive role of Ca2+ on c-Src-PI3K-Akt signaling is very likely mediated at post-VEGF receptor levels. The findings that activation of VEGFR2 is independent of Ca2+ (data not shown) and redox [8] support this notion. In addition, redox- [8] and Ca2+-independent activation of PLCc-1 by VEGF (data not shown) also supports a post-VEGF receptor role for Ca2+ in c-Src-PI3K-Akt activation. It is interesting to note that although Ca2+ inhibition did not negatively affect VEGF-induced activation of ERK1/2, we observed a shift in the temporal pattern and intensity of ERK1/2 activation (earliest time point for ERK1/2 activation shifted from 1 min to 5 min) in BAPTA-AM treated ECs (Fig. 1A). The precise mechanism for this is not known at present. However, it is plausible that, due to inactivation of PI3K-Akt, PLCc-1-ERK1/2 signaling intermediates may
have greater access to PIP2, a common but limited substrate for both PI3K and PLCc-1 in ECs [21]. Further studies are required to elucidate the mechanism. In summary, we demonstrate that VEGF-mediated activation of redox-sensitive c-Src-PI3K-Akt but not redox-insensitive ERK1/2 signaling pathway is dependent on intracellular Ca2+, and that increase in Ca2+ can compensate for the effects of loss of NADPH oxidase activity on the redox-sensitive arm of VEGF signaling and cell proliferation in HCAEC. This is the first report to demonstrate that Ca2+ is an integral part of the redox-dependent selective VEGF signaling pathways, c-Src-PI3K-Akt but not ERK1/2, in ECs. The findings that two intracellular second messengers, Ca2+ and ROS, lie in the same signaling axis in ECs may have important therapeutic significance in vascular disease state. It is worthwhile to note that changes in endothelial Ca2+ may affect the balance of other intracellular ions including phosphate, which may in turn modulate redox-sensitive signaling cascade in HCAEC. Future studies to elucidate the molecular mechanisms by which Ca2+ and ROS coordinate signal propagation in the endothelium should provide important new insights into the endothelial functions in health and disease. Acknowledgements This work was supported in part by AHA Grants SDG 0453284N and AHA 10GRNT3640011, Department of Medicine, BIDMC, and NIH Grant R01HL077348. References [1] Ferrara, N. (2001) Role of vascular endothelial growth factor in regulation of physiological angiogenesis. Am. J. Physiol. Cell Physiol. 280, C1358–C1366. [2] Dvorak, H.F., Brown, L.F., Detmar, M. and Dvorak, A.M. (1995) Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Pathol. 146, 1029–1039. [3] Gerber, H.P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B.A., Dixit, V. and Ferrara, N. (1998) Vascular endothelial growth factor regulates endothelial cell
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