Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function

Figures

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  Fig. 1.. VEGF-A-stimulated Tpl2 expression modulates endothelial cell responses. (A) Endothelial cells stimulated with VEGF-A (25 ng/ml) for 2, 4, 6, 8 or 24 h were lysed and subjected to immunoblot analysis. (B) Quantification of VEGF-A-stimulated changes in relative Tpl2 protein levels using immunoblotting data. Error bars indicate ±s.e.m. (n=6). Statistical test=one-way ANOVA; *P<0.05, **P<0.01. (C) Endothelial cells stimulated with VEGF-A (25 ng/ml) for 1.5 or 3 h prior to quantification of relative Tpl2 mRNA levels using qRT-PCR. Error bars indicate ±s.e.m. (n=3). Statistical test=one-way ANOVA; **P<0.01, ****P<0.0001. (D) Immunoblot analysis of control (mock-transfected), scrambled siRNA-treated or Tpl2-depleted endothelial cells (E–H). Control (mock-transfected), scrambled or Tpl2-depleted endothelial cells were stimulated with VEGF-A (25 ng/ml), processed with different assays (see Materials and Methods) and quantified for cellular responses in (E,F) endothelial tubulogenesis, or (G,H) endothelial-leukocyte interactions. Scale bars: 1000 µm. Error bars indicate ±s.e.m. (n=3). In panels F and H, statistical tests=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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  Fig. 2.. Tpl2 is required for VEGF-A-stimulated ATF-2-dependent VCAM-1 expression. (A) Control, scrambled, Tpl2 or ATF-2-specific siRNA-treated endothelial cells were stimulated (25 ng/ml) with either VEGF-A165 or VEGF-A121 (negative control) for 4 or 8 h, prior to immunoblot analysis. (B–E) Quantification of protein levels by immunoblot analyses (see the Materials and Methods) of VEGF-A (25 ng/ml) stimulated control, scrambled, Tpl2-depleted or ATF-2 depleted endothelial cells for protein levels of (B) VCAM-1, (C) Tpl2, (D) ATF-2-pT71 and (E) ATF-2. Error bars indicate ±s.e.m. (n=3). In panels B–E, statistical tests=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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  Fig. 3.. Tpl2-selective kinase inhibitor treatment attenuates ATF-2 phosphorylation and expression levels. (A–F) Analysis of basal endothelial signaling in non-stimulated cells. (A) Immunoblot analysis of endothelial cells treated with Tpl2-selective kinase inhibitor (2 µM) for 0, 1, 2, 4 or 8 h to assess effects on VEGFR2, ATF-2 and Tpl2 status. Quantification of immunoblot data for Tpl2-selective inhibitor time course for (B) ATF-2-pT71, and (C) Tpl2 levels. Error bars indicate ±s.e.m. (n=4). In panels B and C, statistical tests=one-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. (D) Immunoblot analysis of endothelial cell response to Tpl2-selective kinase inhibitor titration (2–50 µM, 4 h). (E,F) Quantification of immunoblot data from Tpl2-selective kinase inhibitor titration on (E) ATF-2-pT71, and (F) ATF-2 levels. Error bars indicate ±s.e.m. (n=3). In panels E and F, statistical tests=one-way ANOVA; *P<0.05, **P<0.01. (G) Endothelial cells were pre-treated with a Tpl2-selective kinase inhibitor (2 µM; 30 min) prior to stimulation with either (25 ng/ml) VEGF-A165 (165) or VEGF-A121 (121, negative control) for 0 or 8 h before lysis and immunoblot analysis. (H) Quantification of…

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  Fig. 4.. Tpl2-selective kinase inhibitor treatment impairs VEGF-A-stimulated endothelial cell tubulogenesis and aortic sprouting. (A) Endothelial cells co-cultured with primary human fibroblasts were stimulated with VEGF-A (25 ng/ml)±Tpl2-selective kinase inhibitor (2 µM). Endothelial cell tubules were fixed and stained for the Tpl2-selective kinase endothelial cell-specific marker and cell adhesion molecule (PECAM-1) for visualization using fluorescence microscopy. (B) Quantification of endothelial tubule length from fluorescence microscopy datasets. Error bars indicate ±s.e.m. (n=3). Statistical test=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001. (C) Endothelial sprouting from mouse aortic slices stimulated with VEGF-A (25 ng/ml)±2 µM Tpl2-selective kinase inhibitor followed by fixation, staining with FITC-conjugated endothelial-specific lectin (see the Materials and Methods) and visualization using fluorescence microscopy. (D) Quantification of endothelial sprout number from mouse aortas upon treatment with VEGF-A±Tpl2, selective kinase inhibitor. Error bars indicate ±s.e.m. (n≥4). Statistical test=two-way ANOVA; ***P<0.001, ****P<0.0001. Scale bars: 1000 µm.

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  Fig. 5.. Tpl2 modulates eNOS phosphorylation and endothelial cell permeability. (A) Immunoblot analysis of control, scrambled or Tpl2-specific siRNA-treated endothelial cells to assess VEGFR2, eNOS, Akt and Tpl2 status. Quantification of immunoblot data for levels of (B) Akt-pS473 and (C) eNOS-pS1177 levels. Error bars indicate ±s.e.m. (n≥3). In panels B and C, statistical tests=one-way ANOVA; **P<0.01. (D–E) Assessment of endothelial cell permeability by measuring trans-endothelial electrical resistance (TEER) on control, scrambled or Tpl2-depleted endothelial cells under (D) basal conditions in serum-free MCDB131 media, or (E) with addition of VEGF-A (25 ng/ml). High TEER values correspond to reduced cell monolayer permeability indicative of more stable cell–cell junctions. Horizontal red dotted and blue dashed lines depict baselines for control/scrambled or Tpl2-depleted cells respectively. Error bars indicate ±s.e.m. (n=3). In panels D and E, statistical tests=two-way ANOVA; *P<0.05, **P<0.01, ***P<0.001.

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  Fig. 6.. Tpl2 modulates endothelial cell adhesion. Endothelial cells were subjected to controls scrambled siRNA duplex treatment (Scr RNAi) or Tpl2-specific siRNA duplex (Tpl2 RNAi) and analyzed by confocal laser microscopy. (A) Endothelial cells subjected to RNAi were fixed and stained for cell-surface adhesion proteins such as VE-cadherin, PVR and PECAM-1. Arrows indicate cells showing increased staining for VE-cadherin and PECAM-1 under conditions of Tpl2 knockdown. Scale bar: 20 µm. (B) Endothelial cells subjected to RNAi were fixed and stained for Nectin 2, PVR and PECAM-1. Arrows indicate cells showing increased staining for F-actin and PECAM-1 upon Tpl2 knockdown.

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  Fig. 7.. Probing the link between Tpl2, Akt activation and ATF-2 status. (A) Immunoblot analysis of endothelial cells titrated with Tpl2-selective kinase inhibitor (2–50 µM, 4 h). Quantification of immunoblot data upon inhibitor titration on activated kinases such as (B) Akt-pS473, (C) ERK1/2-pT202/pY204, (D) p38 MAPK-pT180/pY182 and (E) JNK-pT183/pY185. Error bars indicate ±s.e.m. (n=3). In panels B–E, statistical tests=one-way ANOVA; *P<0.05. (F) Immunoblot analysis of endothelial cells treated with small molecule kinase inhibitors (2 µM, 30 min) specific for ERK1/2 (PD98059), p38 MAPK (SB203580), JNK (SP600125) and Akt (MK2206). (G) Quantification of immunoblot data for relative ATF-2-pT71 levels upon inhibition of different protein kinases. Error bars indicate ±s.e.m. (n=3).

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  Fig. 8.. Schematic showing a role for Tpl2 in endothelial function the cellular response to VEGF-A. Step 1: VEGF-A stimulation promotes increased Tpl2 expression and activation via an unknown transcription factor (TF-X). Step 2: Tpl2 maintains phosphorylation of residue T71 on ATF-2 stabilizes ATF-2 levels. Step 3: VEGF-A-stimulated hyperphosphorylation of residue T71 on ATF-2 stimulates VCAM-1 gene transcription leading to increased VCAM-1 levels and increased endothelial-leukocyte interaction. Step 4: basal Tpl2 activity promotes phosphorylation of residue S473 on Akt, causing Akt activation leading to eNOS-S1177 phosphorylation. Step 5: eNOS phosphorylation and activation leads to NO production and increased endothelial cell permeability. Grey arrows depict undefined mechanism.