Angiopoietin-1 Induces Krüppel-like Factor 2 Expression through a Phosphoinositide 3-Kinase/AKT-dependent Activation of Myocyte Enhancer Factor 2

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses. Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses. Angiopoietin-1 (Ang1) 2The abbreviations used are: Ang1, angiopoietin-1; KLF2, Krüppel-like factor 2; COMP, cartilage oligomeric matrix protein; MEF2, myocyte enhancer factor 2; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2; VCAM-1, vascular cell adhesion molecule-1; GFP, green fluorescent protein; GST, glutathione S-transferase; HUVEC, human umbilical vein endothelial cell; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; PCAF, p300/CBP-associated factor; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; BSA, bovine serum albumin; siRNA, small interfering RNA; MOPS, 4-morpholinepropanesulfonic acid; wt, wild type; mut, mutant; Luc, luciferase; CA, constitutive active. is a ligand for endothelium-specific receptor tyrosine kinase Tie-2. Gene-targeting analyses of either Ang1 or Tie2 in mice reveal an essential role of Ang1/Tie2 signaling in developmental vascular formation (1Dumont D.J. Gradwohl G. Fong G.H. Puri M.C. Gertsenstein M. Auerbach A. Breitman M.L. Genes Dev. 1994; 8: 1897-1909Crossref PubMed Scopus (818) Google Scholar, 2Suri C. Jones P.F. Patan S. Bartunkova S. Maisonpierre P.C. Davis S. Sato T.N. Yancopoulos G.D. Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2394) Google Scholar, 3Sato T.N. Tozawa Y. Deutsch U. Wolburg-Buchholz K. Fujiwara Y. Gendron-Maguire M. Gridley T. Wolburg H. Risau W. Qin Y. Nature. 1995; 376: 70-74Crossref PubMed Scopus (1508) Google Scholar). In adult vasculature, Ang1/Tie2 signal maintains quiescence of mature blood vessels by enhancing vascular integrity and endothelial survival (4Wong A.L. Haroon Z.A. Werner S. Dewhirst M.W. Greenberg C.S. Peters K.G. Circ. Res. 1997; 81: 567-574Crossref PubMed Scopus (363) Google Scholar, 5Peters K.G. Kontos C.D. 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Trans-associated Tie2 bridged by Ang1 and extracellular matrix-anchored Tie2 by Ang1 induce distinct signaling pathways preferable for vascular quiescence and angiogenesis via AKT and extracellular signal-regulated kinase (ERK) 1/2, respectively. By performing DNA microarray analysis, we also revealed that a distinct set of genes is regulated by Ang1 in the presence or absence of cell-cell contacts. Among them, Krüppel-like factor 2 (KLF2) was selectively induced by Ang1 in the presence of cell-cell contacts (10Fukuhara S. Sako K. Minami T. Noda K. Kim H.Z. Kodama T. Shibuya M. Takakura N. Koh G.Y. Mochizuki N. Nat. Cell Biol. 2008; 10: 513-526Crossref PubMed Scopus (286) Google Scholar). KLF2 is a zinc finger family of transcription factor functioning in both vascular smooth muscle cells and endothelial cells and is, therefore, essential in developmental vascular formation (12Kuo C.T. Veselits M.L. Barton K.P. Lu M.M. Clendenin C. Leiden J.M. 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Moreover, we revealed that Ang1-induced signaling functionally competes with VEGF-induced inflammatory responses. Reagents, Antibodies, siRNAs, and Recombinant Protein—Ang1 and cartilage oligomeric matrix protein (COMP)-Ang1 were prepared as described before (34Cho C.H. Kammerer R.A. Lee H.J. Steinmetz M.O. Ryu Y.S. Lee S.H. Yasunaga K. Kim K.T. Kim I. Choi H.H. Kim W. Kim S.H. Park S.K. Lee G.M. Koh G.Y. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5547-5552Crossref PubMed Scopus (220) Google Scholar). VEGF was purchased from R&D Systems. Wortmannin and AKT inhibitor IV were obtained from Calbiochem. We generated anti-KLF2 monoclonal antibody against amino acids 2–34 of human KLF2. Anti-green fluorescent protein (GFP) antibody was prepared as described before (35Sakurai A. Fukuhara S. Yamagishi A. Sako K. Kamioka Y. Masuda M. Nakaoka Y. Mochizuki N. Mol. Biol. Cell. 2006; 17: 966-976Crossref PubMed Scopus (127) Google Scholar). Other antibodies were purchased as follows: anti-tubulin, anti-ERK5, anti-FLAG (M2), and anti-β-actin from Sigma-Aldrich; anti-MEF2 from Santa Cruz Biotechnology; anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, and anti-ERK1/2 from Cell Signaling Technology; horseradish peroxidase-coupled sheep anti-mouse and anti-rabbit IgG from GE Healthcare Life Sciences; and Alexa Fluor 488-labeled secondary antibody from Molecular Probes. Stealth siRNAs targeting the genes indicated below were purchased from Invitrogen: human KLF2 (HSS145585, HSS145587), human ERK5 (HSS140815), human AKT1 (validated stealth RNA interference: 12935-001), human AKT2 (validated stealth RNA interference: 12937-40), human MEF2A (HSS106435, HSS106436), human MEF2C (HSS106438, HSS106439), and human MEF2D (HSS106441, HSS106442). Glutathione S-transferase (GST) fusion protein containing transactivating domain of MEF2C (GST-MEF2C) was prepared as described before (36Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar). Plasmids and Adenoviruses—A luciferase reporter plasmid containing the proximal 221-bp region of KLF2 promoter (KLF2wt-Luc) was kindly provided by M. K. Jain (Case Western Reserve University). The MEF2-binding site of the KLF2wt-Luc plasmid was mutated using the QuikChange site-directed mutagenesis kit (Stratagene) using the KLF2wt-Luc vector as a template. Expression plasmids encoding MEF2C and constitutive active mutants of AKT (pCEFL-myrAKT) and PI3K-γ (pcDNA3-PI3Kγ-CAAX) and a luciferase reporter plasmid containing a single MEF2-binding site (pGL3-MEF2) have already been described (36Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar, 37Coso O.A. Montaner S. Fromm C. Lacal J.C. Prywes R. Teramoto H. Gutkind J.S. J. Biol. Chem. 1997; 272: 20691-20697Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 38Murga C. Fukuhara S. Gutkind J.S. J. Biol. Chem. 2000; 275: 12069-12073Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). A cDNA encoding full-length MEF2C amplified by PCR using pCEFL-GST-MEF2C as a template was inserted into p3xFLAG CMV10 vector (Sigma-Aldrich) or cloned into pCMV-DBD vector (Stratagene) to construct the plasmid expressing a Gal4 DNA-binding domain (DBD)-MEF2C fusion protein (Gal4/MEF2C). Plasmids encoding Gal4/MEF2C mutant proteins (Thr-293, Thr-300, Ser-387 (phosphorylation sites by ERK5 and p38), and Thr-404 (potential phosphorylation site by AKT) were replaced with Ala, and 6 Lys residues (Lys-116, Lys-119, Lys-234, Lys-239, Lys-252, Lys-264; acetylation sites by p300) replaced with Arg were generated using the QuikChange site-directed mutagenesis kit. An expression vector encoding FLAG-tagged HDAC5 was generously obtained from C. Grozinger (Harvard University). Other vectors are purchased as follows: pRL-SV40 and pRL-TK from Promega Corp.; pEGFP-C1 from Clontech; and pFR from Stratagene. Recombinant adenovirus vectors encoding GFP and constitutively active form of AKT were kindly provided by H. Kurose (Kyushu University) and Y. Fujio (Osaka University), respectively. Cell Culture, Transfection, siRNA-mediated Protein Knockdown, and Adenovirus Infection—Human umbilical vein endothelial cells (HUVECs) were cultured as described previously (10Fukuhara S. Sako K. Minami T. Noda K. Kim H.Z. Kodama T. Shibuya M. Takakura N. Koh G.Y. Mochizuki N. Nat. Cell Biol. 2008; 10: 513-526Crossref PubMed Scopus (286) Google Scholar) and used for experiments before passage 7. HUVECs were placed on collagen-coated plates at a density of 2,000 cells/cm2 and 40,000 cells/cm2 and cultured overnight to obtain sparse and confluent cell cultures, respectively. U937 cells, a human monocyte-like cell line, were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. HUVECs were transfected using Lipofectamine 2000 reagent (Invitrogen) and Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions. For siRNA-mediated gene silencing, HUVECs were transfected with siRNA duplexes using Lipofectamine RNAiMAX reagent (Invitrogen) and used for the experiments 48–72 h after transfection. HUVECs were infected with adenovirus vectors at the appropriate multiplicity of infection. Forty-eight h after infection, the cells were used for experiments. Real-time Reverse Transcription-PCR—Sparse and confluent HUVECs placed on collagen-coated plates were starved in medium 199 containing 1% BSA for 6 h and stimulated with COMP-Ang1 as described in the figure legends. The cells were stimulated in the presence of 30 μm wortmannin or 8 μm AKT inhibitor IV. To examine the effect of COMP-Ang1 on VEGF-induced VCAM-1 expression, HUVECs starved in medium 199 containing 0.5% BSA for 5 h were prestimulated with or without COMP-Ang1 for 1 h and subsequently challenged with VEGF for 3 h. After the stimulation, total RNA was purified using TRIzol (Invitrogen). Quantitative real-time reverse transcription (RT)-PCR was carried out using QuantiFast SYBR Green RT-PCR kit (Qiagen) as described before (10Fukuhara S. Sako K. Minami T. Noda K. Kim H.Z. Kodama T. Shibuya M. Takakura N. Koh G.Y. Mochizuki N. Nat. Cell Biol. 2008; 10: 513-526Crossref PubMed Scopus (286) Google Scholar). For each reaction, 100 ng of total RNA was transcribed for 10 min at 50 °C followed by a denaturing step at 95 °C for 5 min and 40 cycles of 10 s at 95 °C and 30 s at 60 °C. Fluorescence data were collected and analyzed using Mastercycler ep realplex (Eppendorf). The primers used for amplification were as follows: for human KLF2, 5′-CTACACCAAGAGTTCGCATCTG-3′ and 5′-CCGTGTGCTTTCGGTAGTG-3′; for human VCAM1, 5′-CAAATCCTTGATACTGCTCATC-3′ and 5′-TTGACTTCTTGCTCACAGC-3′; forglyceraldehyde-3-phosphatedehydrogenase (GAPDH), 5′-ATGGGGAAGGTGAAGGTCG-3′ and 5′-GGGGTCATTGATGGCAACAATA-3′. For normalization, expression of human GAPDH was determined in parallel as an endogenous control. Detection of KLF2 Protein Expression—To examine the KLF2 protein expression induced by COMP-Ang1, confluent HUVECs plated on a collagen-coated dish were starved in Humedia-EB2 medium (Kurabo) containing 0.5% fetal calf serum for 12 h and stimulated with 400 ng/ml COMP-Ang1 for the periods as indicated in the figure legends. After the stimulation, the cells were washed once with ice-cold phosphate-buffered saline, harvested by scraping, and pelleted by centrifugation at 4,000 × g for 10 min at 4 °C. The cell pellets were then lysed at 4 °C in radioimmune precipitation buffer containing 50 mm Tris-HCl at pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1× protease inhibitor mixture. The cell lysates were subjected to SDS-PAGE and Western blot analysis as described previously (39Fukuhara S. Sakurai A. Sano H. Yamagishi A. Somekawa S. Takakura N. Saito Y. Kangawa K. Mochizuki N. Mol. Cell. Biol. 2005; 25: 136-146Crossref PubMed Scopus (352) Google Scholar). Detection of ERK5, AKT, and ERK1/2 Activities—Confluent and sparse HUVECs plated on collagen-coated dish were starved in medium 199 containing 1% BSA for 6 h and stimulated with 400 ng/ml COMP-Ang1 for 15 min. The cells were then lysed at 4 °C in lysis buffer containing 25 mm HEPES at pH 7.5, 0.3 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm dithiothreitol, 20 mm β-glycerophosphate, 1 mm sodium vanadate, 1% Triton X-100, and 1× protease inhibitor mixture. To measure the ERK5 activity, in vitro kinase assay was performed as described previously (40Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Briefly, endogenous ERK5 was immunoprecipitated from aliquots of cell lysate with anti-ERK5 antibody at 4 °C for 3 h, and the immunocomplexes were recovered with protein G-Sepharose beads (GE Healthcare Life Sciences). Beads were washed three times with phosphate-buffered saline containing 1% Nonidet P-40 and 2 mm sodium vanadate, once with washing buffer containing 100 mm Tris at pH 7.5 and 0.5 m LiCl and once with kinase reaction buffer containing 12.5 mm MOPS at pH 7.5, 12.5 mm β-glycerophosphate, 7.5 mm MgCl2, 0.5 mm EGTA, 0.5 mm sodium vanadate, and 0.5 mm sodium fluoride. Samples were then resuspended in 15 μl of kinase reaction buffer containing 3 μg of GST-MEF2C, 1 μCi of [γ-32P]ATP, and 20 μm cold ATP and incubated at 37 °C for 90 min. 32P-labeled substrates were separated by SDS-PAGE and detected by autoradiography. To evaluate the phosphorylation of AKT and ERK1/2, aliquots of cell lysate were subjected to Western blot analysis with anti-phospho-AKT and anti-phospho-ERK1/2 antibodies, respectively. The total contents of ERK5, AKT, and ERK1/2 in each cell lysate were also assayed in a parallel run using corresponding antibodies. Luciferase Reporter Assay—Luciferase reporter assay was carried out as described before (36Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar, 40Fukuhara S. Marinissen M.J. Chiariello M. Gutkind J.S. J. Biol. Chem. 2000; 275: 21730-21736Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Confluent HUVECs plated on a collagen-coated 12-well plate were transfected with different expression vectors, together with reporter plasmids as described in the figure legends. The total amount of plasmid DNA was adjusted with empty vector. To examine the effect of COMP-Ang1, the cells were starved and stimulated as described in the figure legends. The cells were lysed using passive lysis buffer (Promega), and luciferase activities in cell extract were determined using a Dual-Luciferase assay system (Promega). Detection of Subcellular Localization of FLAG-tagged HDAC5—Confluent HUVECs plated on a collagen-coated glass base dish were transfected with the plasmid encoding FLAG-tagged histone deacetylase (HDAC) 5. Twenty-four h after the transfection, the cells were starved in medium 199 containing 0.5% BSA for 6 h and subsequently stimulated with vehicle, COMP-Ang1, or VEGF for 3 h. After the stimulation, the cells were fixed and stained with anti-FLAG antibody as described before (10Fukuhara S. Sako K. Minami T. Noda K. Kim H.Z. Kodama T. Shibuya M. Takakura N. Koh G.Y. Mochizuki N. Nat. Cell Biol. 2008; 10: 513-526Crossref PubMed Scopus (286) Google Scholar). Protein reacting with antibody was visualized with Alexa Fluor 488-conjugated secondary antibody. Alexa Fluor 488 and phase contrast images were recorded with an Olympus IX-81 inverted fluorescence microscope. The number of cells expressing FLAG-tagged HDAC5 in the cytoplasm among FLAG-tagged HADC5-expressing cells was counted. Nuclear export of HDAC5 was determined by the HDAC5 in the cytoplasm instead of the nucleus. At least 100 cells were scored for each experiment. Monocyte Adhesion Assay—HUVECs transfected with control or KLF2 siRNA were placed on collagen-coated 24-well plates at the density of 40,000 cells/cm2, cultured overnight, and starved in medium 199 containing 1% BSA for 2 h. The cells were prestimulated with 400 ng/ml COMP-Ang1 for 1 h and challenged with vehicle or 50 ng/ml VEGF in the presence or absence of COMP-Ang1 for 4 h. U937 cells labeled with green fluorescent dye PKH67 (Sigma-Aldrich) were added to the confluent HUVEC monolayers (8 × 105 cells/well) and incubated in RPMI 1640 containing 1% fetal bovine serum for 90 min. The cells were then washed three times with prewarmed Hank's buffered salt solution (Invitrogen) and fixed with 2% formaldehyde. Phase contrast and PKH67 fluorescent images were recorded with an Olympus IX-81 inverted fluorescence microscope. The adherent U937 cells were quantified by measuring fluorescent intensity at five randomly selected fields in each well using MetaMorph 6.1 software (Molecular Devices Corp.). Statistical Analysis—The values are expressed as means ± S.D. Statistical significance was determined using one-way analysis of variance, two-way analysis of variance, or unpaired t test. p values < 0.05 were considered statistically significant. Ang1 Induces KLF2 Expression in Confluent but Not Sparse Cultures of HUVECs—To first examine the KLF2 expression downstream of trans-associated Tie2 at cell-cell contacts and cell-substratum contact-anchored Tie2 in response to Ang1, HUVECs were stimulated with COMP-Ang1, a potent Ang1 variant, under either confluent or sparse culture condition. COMP-Ang1 concentration-dependently induced KLF2 mRNA expression in confluent HUVECs but not in the sparse cells (Fig. 1A). Consistently, KLF2 protein expression was up-regulated upon stimulation with COMP-Ang1 (Fig. 1C). KLF2 mRNA and protein expression by COMP-Ang1 peaked at 1 h after t