Volume 54, Issue 4, Pages 559-572 (May 2014) VEGF Signals through ATF6 and PERK to Promote Endothelial Cell Survival and Angiogenesis in the Absence of ER Stress  Evdoxia Karali, Sofia Bellou, Dimitris Stellas, Apostolos Klinakis, Carol Murphy, Theodore Fotsis  Molecular Cell  Volume 54, Issue 4, Pages 559-572 (May 2014) DOI: 10.1016/j.molcel.2014.03.022 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 VEGF Induces Transcription of HERPUD1 and DNAJB9 Genes in ECs Independently of Accumulation of Unfolded Proteins in the ER Lumen HUVECs cultured in full medium were induced with VEGF (50 ng/ml) (A–E) or FGF2 (10 ng/ml) (C–E) for the indicated time points, and total RNA was isolated. DNAJB9, HERPUD1, and DTR mRNA levels were quantitated by qRT-PCR. (F) HUVECs were infected with the Gluc-IRES-YFP expressing lentivirus, and VEGF or Tm was added 48 hr postinfection. Gluc activity was measured 6 hr later both in the cell-free conditioned medium and in the cell pellet following addition of 2.5 μM coelenterazine. The results are expressed as mean ± SD of four (A–E) or three (F) independent experiments (∗p < 0.05, ∗∗p < 0.001) (see also Figure S1). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 VEGF Leads to Rapid Activation of the ER Stress Mediators IRE1α, ATF6, and PERK HUVECs cultured in full medium were induced with VEGF (50 ng/ml) or Tm (10 μg/ml) for the indicated time points. (A) Total and p-IRE1α were determined by western blot analysis. Quantitative analysis of p-IRE1α levels relative to total IRE1α is presented in the graph. (B and C) mRNA levels of spliced XBP1 were quantitated by qRT-PCR. (D and E) HUVECs were pretreated with lactacystin (10 μM), and following induction ATF6 (90kDa) and its activated 50 kDa form were determined by western blot analysis. Actin levels were assessed as loading control. (F and G) mRNA levels of GRP78 were quantitated by qRT-PCR. (H) Phosphorylated levels of eIF2α were determined by western blot analysis. Tubulin levels were assessed as loading control. Results are expressed as mean ± SD of three (A, F, and G) or four (B and C) independent experiments. (I) Coimmunoprecipitation of endogenous ATF6 and GRP78 proteins. HUVECs cultured in full medium were induced with VEGF (50 ng/ml) for 30 min and 1 hr, Tm (10 μg/ml) or DTT (1 mM). Immunoprecipitation was performed using either an α-GRP78 or rabbit IgG antibody as control. Immunocomplexes were analyzed by SDS-PAGE and immunoblotted with an α-GRP78 antibody to confirm GRP78 immunoprecipitation and with an α-ATF6 antibody to detect the coimmunoprecipitation of ATF6. Whole-cell lysates (5% of the input) were probed with α-ATF6 (90kda) and α-GRP78 antibodies as control, and actin protein levels were assessed as loading control (lower panel) (∗p < 0.05, ∗∗p < 0.001) (see also Figure S2). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 VEGF Activates the UPR Mediators via the PLCγ Pathway (A) A schematic diagram of VEGF signaling cascades and their inhibitors (B) HUVECs cultured in full medium were pretreated with the inhibitors of (1) p38 MAPK (SB203580, 20 μM), (2) MEK (PD98059, 20 μM), (3) PI3K (Wortmannin, 100 μM), or (4) PLCγ (U73122, 10 μM) prior to VEGF (50 ng/ml) stimulation. Following 1 hr of induction, total RNA was isolated and mRNA levels of HERPUD1 were quantitated by qRT-PCR. Results represent the percentage fold induction of HERPUD1 mRNA levels in the presence each inhibitor compared to the untreated control samples, set theoretically as 100% induction. Values represent the mean ± SD of three independent experiments (∗p < 0.05). HUVECs were pretreated with U73122 (10 μM) for 20 min (C–E) or were transfected with PLCγ siRNA (50 nM) (F) and then induced with VEGF (50 ng/ml) for the indicated time points. Actin levels were assessed as loading control (left panels). Quantitative analysis of p-IRE1α levels relative to total IRE1α (C), activated form ATF6 (50 kDa) normalized to total of ATF6 (90 kDa) relative to actin (D), and p-eIF2α levels relative to actin (E and F) are presented. All results are expressed as mean ± SD of three independent experiments (∗p < 0.05, ∗∗p < 0.001) (see also Figure S3). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 VEGF Activation of UPR Mediators via PLCγ Occurs by a Ca2+-Independent Mechanism and Is PKC Pathway Independent HUVECs were pretreated for 20 min with either the IP3R-channel inhibitor 2-APB (75 μM) (A) or the Ca2+ selective chelator BAPTA (20 μM) (E) followed by stimulation with VEGF (50 ng/ml). Fluo-4 NW was used to monitor the cytosolic Ca2+ by confocal microscopy before and after VEGF stimulation (from 0 to 20 min). Representative images at 0 and 130 s are shown in the left panels in (A) and (E) (see also Movie S1 and Movie S2). A time course graph of Fluo-4 mean fluorescence intensity/cell is presented. HUVECs were pretreated for 20 min with either 2-APB (75 μM) (B), BAPTA (20 μM) (F), or staurosporine (50 nM) (G) followed by stimulation with VEGF (50 ng/ml) for the indicated time points. mRNA levels of XBP-1 s were quantitated by qRT-PCR. Results are expressed as mean ± SD of three independent experiments. HUVECs were either pretreated with lactacystin (10 μM) (C and I) or not (D and H) prior to treatment for 20 min with 2-APB (75 μM) (C and D) or staurosporine (50 nM) (I and H) and were stimulated by VEGF (50 ng/ml) for the indicated time points. ATF6 (90 kDa) and its activated 50 kDa form, p-eIF2α, and p-ERK1/2 were determined by western blot analysis. Quantitative analysis of p-IRE1α levels relative to actin, activated form ATF6 (50 kDa) normalized to total of ATF6 (90 kDa) relative to actin, and p-eIF2α levels relative to actin is presented in the graphs (Quantity One). All results are expressed as mean ± SD of three independent experiments (see also Figure S4). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Activation of Both PLCγ and ATF6 and PERK Pathways Is Essential for VEGF-Induced Phosphorylation of AKT and Endothelial Cell Survival (A and B) HUVECs were transfected with siRNA (20 nM) of IRE1α, ATF6, and eIF2α as well as scrambled siRNA (siSCR) (A) or were treated with the PLCγ inhibitor (U73122, 10 μM) or transfected with siRNA of PLCγ (siPLCγ, 50 nM) (B). VEGF (50 ng/ml) was administered for 24 hr, and cells were evaluated for apoptosis by flow cytometry (FACS) using Annexin V-FITC and PI, as described in Experimental Procedures. Each graph represents the percentage of VEGF-induced survival of HUVECs (±VEGF). The percentage is calculated as fold of plus to minus VEGF-induced endothelial cell survival, either treated with U73122 or transfected with the siRNAs, normalized to the control untreated or transfected with siSCR, respectively. Western blot analysis of IRE1α, ATF6, eIF2α, and PLCγ is presented to determine the knockdown efficiency. HUVECs cultured in full medium were pretreated for 20 min with U73122 (10 μM) (see also Figures S5A and S5B) (C) or transfected for 72 hr with 50 nM of PLCγ siRNA (siPLCγ) or siSCR (D) and then were induced with VEGF (50 ng/ml) for 15 and 30 min. p-AKT(Ser473) was determined by western blot analysis. (E and F) HUVECs were transfected for 72 hr with SCR, IRE1α, ATF6, or eIF2α siRNAs and then were induced with VEGF (50 ng/ml) for 15 and 30 min. p-AKT on Ser473 (E) and Thr308 (F) was determined by western blot analysis. Quantitative analysis of p-AKT levels relative to actin is presented in the graphs (Quantity One). (G and H) HUVECs were cultured in full medium, and VEGF (50 ng/ml) or Tm (10 μg/ml) was added. Total RNA was isolated at the indicated time points, and CHOP mRNA levels (G and H) were quantitated by qRT-PCR. Protein levels of CHOP (I) were determined at the indicated time points. Tubulin levels were assessed as loading control. Results are expressed as mean ± SD of five (A and B) or three (C–H) independent experiments (∗p < 0.05, ∗∗p < 0.001, NS, not statistically significant). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 PLCγ Activates the UPR Mediators IRE1α, ATF6, and PERK via Activation of mTORC1 HUVECs cultured in full medium were pretreated for 20 min with an inhibitor of mTORC1 (rapamycin, 20 nM) and were then induced with VEGF (50 ng/ml) for 1 hr. Levels of spliced XBP1 (A) and GPR78 (C) were quantitated by qRT-PCR. Results are expressed as mean ± SD of three independent experiments. HUVECs cultured in full medium either pretreated with 10 μM lactacystin (B) or not (D) were treated for 20 min with rapamycin (20 nM) and induced with VEGF (50 ng/ml) for 30 min and 1 hr. Protein levels of ATF6 (90 kDa) and its activated 50 kDa form (B) and p-eIF2α (D) were determined by western blot analysis. Actin and tubulin levels were assessed as loading control. Quantitative analysis of the western blots is presented in the graphs (quantity one). (E) HUVECs cultured in full medium were pretreated with the inhibitor of PLCγ (U73122, 10 μM) prior to VEGF (50 ng/ml) induction for 15 min. Protein levels of p-rS6 (Ser235/236) and p-p70S6K (Thr389) were determined. (F) HUVECs cultured in full medium were induced or not with VEGF (50 ng/ml) for 10 min, and α-p-mTOR or rabbit IgG control antibody immunoprecipitations were performed. Immunocomplexes were analyzed by SDS-PAGE and immunoblotted with α-p-mTOR antibody to confirm p-mTOR immunoprecipitation (right) and with α-p-PLCγ antibody to detect the coimmunoprecipitation of p-PLCγ (left). The image is representative of three independent experiments (∗p < 0.05, ∗∗p < 0.001) (see also Figure S6). Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Inhibition of PLCγ Pathway as well as Knockdown of ATF6 and eIF2α UPR Mediators Is Essential for VEGF-Induced Angiogenesis In Vivo Matrigel containing PLCγ inhibitor (U73122, 30 μM) (A) or siRNA (1 μM) targeting PLCγ, ATF6, eIF2α, or scrambled (B) in the presence (Ac, Ad, and Be–Bh) or absence (Aa, Ab, and Ba–Bd) of VEGF (250 ng/ml) was injected subcutaneously into male C57 BL/6 mice. Matrigel plugs were removed 7 days after implantation, fixed, sectioned, and H&E stained (see Figures S7Aa–S7Ad and S7Ba–S7Bh). Images are representative of five mice used per condition. Immunohistochemistry with α-CD31 antibody (red) was used for the identification of endothelial cells (Ae, Af, and Bi–Bl), and DAPI (blue) used for nuclei labeling was visualized using confocal microscopy. Scale bar represents 100 μM. (C and D) Statistical analysis presents the percentage of CD31-positive cells per field in each case. Efficiency of knockdown was determined by western blot analysis. Results are expressed as mean ± SD of ten histological tissue sections (∗p < 0.05, ∗∗p < 0.001) (see also Figure S7). (E) A schematic model of the identified VEGF signaling pathway that leads to activation of IRE1α, ATF6, and PERK via PLCγ/mTORC1 pathway and enhancement of endothelial cell survival and angiogenesis. Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 559-572DOI: (10.1016/j.molcel.2014.03.022) Copyright © 2014 Elsevier Inc. Terms and Conditions