Reagents and Antibodies
Materials were purchased as follows: forskolin (FSK) and latrunculin A (Lat.A) were from Calbiochem (San Diego, CA), Epac-specific activator 007 was from Tocris Bioscience (Bristol, United Kingdom), PKA-specific activator 6-Bnz was from Biolog Life Science Institute (Bremen, Germany), H89 was from Sigma-Aldrich (St. Louis, MO), and Cellmatrix type I-C was from Nitta Gelatin (Osaka, Japan). Antibodies used here were purchased as follows: anti-VE-cadherin was from Santa Cruz Biotechnology (Santa Cruz, CA), BD Biosciences (San Jose, CA), and Cell Signaling Technology (Danvers, MA); anti-α-catenin was from Zymed Laboratories (South San Francisco, CA), anti-β-catenin and anti-p120-catenin were from BD Bioscience, anti-cAMP response element-binding protein (CREB) and anti-phospho-CREB (Ser133) were from Cell Signaling Technology; anti-Rap1 was from Santa Cruz Biotechnology; anti-β-actin and anti-β-tubulin were from Sigma-Aldrich; rhodamine-phalloidin, Alexa 488-labeled goat anti-mouse immunoglobulin G (IgG), Alexa 633-labeled goat anti-mouse IgG, and Alexa 546-labeled goat anti-rabbit IgG were from Invitrogen (Carlsbad, CA); horseradish peroxidase-coupled goat anti-mouse and horseradish peroxidase-coupled goat anti-rabbit IgG were from GE Healthcare (Piscataway, NJ); and horseradish peroxidase-coupled donkey anti-goat IgG was from Santa Cruz Biotechnology.
Cell Culture, Transfection, Small Interfering RNA (siRNA)-mediated Protein Knockdown, and Adenovirus Infection
Human umbilical vein endothelial cells (HUVECs) were purchased from Kurabo (Kurashiki, Japan), maintained as described previously (Fukuhara et al., 2008
), and used for the experiments before passage 9. 293T cells were cultured in DMEM (Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum and antibiotics (100 μg streptomycin/ml and 100 U penicillin/ml). HUVECs and 293T cells were transfected using Lipofectamine 2000 and 293 fectin reagents (Invitrogen), respectively. Stealth siRNAs targeted to human VE-cadherin (HSS101682), human α-catenin (HSS102451 and 5′-UUAUUAGAGGGCCCUUUACUAUUGG-3′), human β-catenin (VHS50819 and VHS50822), and human p120-catenin (HSS102463 and HSS102465) were purchased from Invitrogen. As a control, siRNA duplexes with irrelevant sequences were used. HUVECs were transfected with 20 nM siRNA duplexes using Lipofectamine RNAi MAX reagent (Invitrogen). After incubation for 48 h, the cells were replated, cultured for additional 24 h, and were used for the experiments.
Recombinant adenoviruses encoding Rap1GAP and LacZ were obtained from S. Hattori (The Institute of Medical Science, University of Tokyo, Tokyo, Japan) and M. Matsuda (Research Institute for Microbial Disease, Osaka University, Osaka, Japan), respectively. HUVECs were infected with adenoviruses at the appropriated multiplicities of infection as described in the figure legends.
cDNAs for human VE-cadherin and human platelet/endothelial cell adhesion molecule (PECAM)1 were amplified from human heart cDNAs by reverse transcription-polymerase chain reaction (PCR) and cloned into pEGFP-N1 vector (Clontech, Mountain View, CA) to construct pEGFP-N1-VEC encoding VE-cadherin carboxy-terminally tagged with green fluorescence protein (VEC-GFP) and pEGFP-N1-PECAM1 encoding PECAM1 carboxy-terminally tagged with GFP (PECAM1-GFP), respectively. To generate the plasmid encoding VEC-GFP lacking β-catenin binding domain (VECΔβ-GFP) and that encoding VEC-GFP lacking cytoplasmic domain (VECΔC-GFP), amino acids 1-700 and 1-631 fragments of VE-cadherin were amplified by PCR and subcloned into pEGFP-N1 vector, namely, pEGFP-N1-VECΔβ and pEGFP-N1-VECΔC, respectively. To generate pEGFP-N1-VECΔC-α vector encoding VEC-GFP mutant in which cytoplasmic domain of VE-cadherin is replaced with α-catenin (VECΔC-α-GFP), a cDNA encoding full-length α-catenin was amplified by PCR using an expression vector for α-catenin (a gift from A. Nagafuchi, Kumamoto University, Kumamoto, Japan) as a template and inserted into the site immediately upstream of GFP in pEGFP-N1-VECΔC vector. Similarly, a cDNA encoding amino acids 327–906 fragment of α-catenin was amplified by PCR and inserted into the same site of pEGFP-N1-VECΔC vector to construct the plasmid expressing VECΔC-αΔN-GFP. An siRNA-insensitive version of pEGFP-N1-VECΔC-α plasmid, namely, pEGFP-N1-VECΔC-α-in vector, was generated using QuickChange Site-directed mutagenesis kit (Stratagene, La Jolla, CA). A cDNA fragment encoding PECAM1 lacking cytoplasmic region was amplified by PCR and inserted into pEGFP-N1-vector to generate the pEGFP-N1-PECAM1ΔC plasmid encoding PECAM1-GFP mutant lacking the cytoplasmic region of PECAM1 (PECAM1ΔC- GFP). To construct the pEGFP-N1-PECAM1ΔC-α plasmid encoding PECAM1-GFP mutant in which cytoplasmic region is replaced with α-catenin (PECAM1ΔC-α-GFP), a cDNA encoding full-length α-catenin amplified by PCR was inserted into the site immediately upstream of GFP in pEGFP-N1-PECAM1ΔC vector. Similarly, a cDNA encoding cytoplasmic region of VE-cadherin was also inserted into the same site of pEGFP-N1-PECAM1ΔC vector to construct the pEGFP-N1-PECAM1ΔC-VEC/C plasmid encoding PECAM1-GFP mutant in which cytoplasmic region is replaced with that of VE-cadherin (PECAM1ΔC-VEC/C-GFP).
Fluorescence Recovery after Photobleaching Analysis
HUVECs plated on a 35-mm-diameter collagen-coated glass-base dish (Asahi Techno Glass, Chiba, Japan) were transfected with the expression plasmids encoding VEC-GFP, PECAM1-GFP, and their mutants and cultured for 24 h at confluent cell density. The cells were then starved in medium 199 containing either 1 or 0.1% bovine serum albumin (BSA) for 3 h, and stimulated with vehicle, 10 μM FSK, 0.2 mM 007, and 0.2 mM 6-Bnz for 30 min. Fluorescence recovery after photobleaching (FRAP) experiments were performed on a FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan) with a 60× objective lens, and GFP fluorescence was imaged by the excitation with 473-nm diode laser. All experiments were performed at 37°C with 5% CO2 using a heating chamber (Tokai Hit, Shizuoka, Japan). GFP-positive cells surrounded by GFP-negative cells were selected and subjected to FRAP analysis. GFP fluorescence at the cell–cell contacts was bleached for 5 s using 405-nm diode laser set at full power. To monitor fluorescence recovery, images were acquired every 90 s over a period of 50–60 min using the FluoView version 1.7c software (Olympus). Using Excel software (Microsoft, Redmond, WA), data were corrected for the overall loss in total fluorescence intensity as a result of the imaging scans. The fluorescence intensity of the bleached region over time was normalized with the prebleached fluorescence intensity. Recovery measurements were quantified by fitting normalized fluorescence intensities of bleached areas to a one-phase exponential association by using Prism 5 software (GraphPad Software, San Diego, CA). This program was also used for plotting of the data and statistical analysis.
Monolayer-cultured HUVECs grown on a collagen-coated glass-base dish were starved in medium 199 containing either 0.5 or 0.1% BSA for 3 h and subsequently stimulated with vehicle, 10 μM FSK, 0.2 mM 007, or 0.2 mM 6-Bnz for 30 min. After stimulation, the cells were fixed in phosphate-buffered saline (PBS) containing 2% formaldehyde for 30 min at 4°C, permeabilized with 0.05% Triton X-100 for 30 min at 4°C, and blocked with PBS containing 4% BSA for 1 h at room temperature. The cells were then stained with rhodamine-phalloidin for 20 min and with anti-VE-cadherin, anti-α-catenin, anti-β-catenin, and anti-p120-catenin antibodies for 60 min at room temperature. Protein reacting with antibody was visualized with species-matched Alexa 488-, Alexa 546- or Alexa 633-labeled secondary antibodies. Fluorescence images of GFP, rhodamine, Alexa 488, Alexa 546, and Alexa 633 were recorded with an Olympus IX-81 inverted fluorescence microscope (Olympus) equipped with pE-1 LED excitation system (CoolLED, Andover, United Kingdom) with a cooled charge-coupled device camera CoolSNAP-HQ (Roper Scientific, Trenton, NJ) and appropriate filter sets for GFP, Alexa 488, Alexa 546, and Alexa 633, and with a FluoView FV1000 confocal microscope with 60× and 100× oil immersion objective lens. To quantify the levels of F-actin at cell–cell contacts, fluorescence intensity of rhodamine along the 5-pixel-width lines randomly drawn on the rhodamine images was determined by line intensity scanning using MetaMorph software (Molecular Devices, Sunnyvale, CA). Peak fluorescence intensity at the points across the cell–cell contacts was taken as the value of F-actin at cell–cell contacts. A minimum of 80 contacts were analyzed per experiment, and experiments were repeated three times.
Detection of GTP-bound Form of Rap1 and Phosphorylated CREB
Rap1 activity and phosphorylation of CREB were assessed as described previously (Fukuhara et al., 2005
). In brief, HUVECs starved in medium 199 containing 1% BSA for 6 h were stimulated with vehicle, 10 μM FSK, 0.2 mM 007, or 0.2 mM 6-Bnz for 15 min and lysed at 4°C in a pull-down lysis buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2
, 1% Triton X-100, 1 mM EGTA, 1 mM dithiothreitol, 1 mM Na3
, and 1× protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). GTP-bound Rap1 was collected on the glutathione transferase-tagged Rap1 binding domain of RalGDS precoupled to glutathione-Sepharose beads and subjected to Western blot analysis with anti-Rap1 antibody. Aliquots of total cellular lysates were also subjected to Western blot analysis with anti-Rap1, anti-phospho-CREB, anti-CREB, and anti-β-actin antibodies.
Fluorescence-activated Cell Sorting (FACS) Analysis
Expression levels of VEC-GFP, PECAM1-GFP, and their mutants were analyzed by FACS analysis using FACSAria cell-sorting system (BD Biosciences).
Data are expressed as either mean ± SD or mean ± SE as indicated in figure legends. Statistical significance was determined using Student's t test for paired samples or one-way analysis of variance and nonparametric tests for multiple groups. Data were considered statistically significant if p values <0.05.