Kruppel-like Factor 4 Regulates Endothelial Inflammation
2007; Elsevier BV; Volume: 282; Issue: 18 Linguagem: Inglês
10.1074/jbc.m700078200
ISSN1083-351X
AutoresAnne Hamik, Zhiyong Lin, Ajay Kumar, Mercedes Balcells, Sumita Sinha, Jonathan P. Katz, Mark W. Feinberg, Robert E. Gerszten, Elazer R. Edelman, Mukesh K. Jain,
Tópico(s)Myeloproliferative Neoplasms: Diagnosis and Treatment
ResumoThe vascular endothelium plays a critical role in vascular homeostasis. Inflammatory cytokines and non-laminar blood flow induce endothelial dysfunction and confer a pro-adhesive and pro-thrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factor 4 expression has been documented in endothelial cells, but a function has not been described. In this communication we describe the expression in vitro and in vivo of Kruppel-like factor 4 in human and mouse endothelial cells. Furthermore, we demonstrate that endothelial Kruppel-like factor 4 is induced by pro-inflammatory stimuli and shear stress. Overexpression of Kruppel-like factor 4 induces expression of multiple anti-inflammatory and anti-thrombotic factors including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of Kruppellike factor 4 leads to enhancement of tumor necrosis factor α-induced vascular cell adhesion molecule-1 and tissue factor expression. The functional importance of Kruppel-like factor 4 is verified by demonstrating that Kruppel-like factor 4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. Kruppel-like factor 4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. These data implicate Kruppel-like factor 4 as a novel regulator of endothelial activation in response to pro-inflammatory stimuli. The vascular endothelium plays a critical role in vascular homeostasis. Inflammatory cytokines and non-laminar blood flow induce endothelial dysfunction and confer a pro-adhesive and pro-thrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factor 4 expression has been documented in endothelial cells, but a function has not been described. In this communication we describe the expression in vitro and in vivo of Kruppel-like factor 4 in human and mouse endothelial cells. Furthermore, we demonstrate that endothelial Kruppel-like factor 4 is induced by pro-inflammatory stimuli and shear stress. Overexpression of Kruppel-like factor 4 induces expression of multiple anti-inflammatory and anti-thrombotic factors including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of Kruppellike factor 4 leads to enhancement of tumor necrosis factor α-induced vascular cell adhesion molecule-1 and tissue factor expression. The functional importance of Kruppel-like factor 4 is verified by demonstrating that Kruppel-like factor 4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. Kruppel-like factor 4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. These data implicate Kruppel-like factor 4 as a novel regulator of endothelial activation in response to pro-inflammatory stimuli. A fundamental role of the vascular endothelium is to regulate the biological response to inflammatory stimuli (1DiChiara M.R. Kiely J.M. Gimbrone Jr., M.A. Lee M.E. Perrella M.A. Topper J.N. J. Exp. Med. 2000; 192: 695-704Crossref PubMed Scopus (93) Google Scholar). 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However, the function and targets of endothelial KLF4 have remained completely unknown. In this work we demonstrate for the first time that KLF4 regulates critical aspects of endothelial cell inflammatory and thrombotic function. Cell Culture—Human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells were acquired from Cambrex Bioscience and cultured in endothelial cell basal medium-2 media according to the manufacturer's instructions. Bovine aortic endothelial cells were from Cell Applications and were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin. COS7 were from American Type Culture Collection and cultured in the same medium as bovine aortic endothelial cells. THP-1 cells were from American Type Culture Collection and were cultured in RPMI 1640 with 2 mm l-glutamine and 10% fetal bovine serum. Where indicated, HUVECs were treated with human IL1-β, human TNFα (R&D systems), interferon γ (Pierce), and phorbol myristate acetate (PMA; Sigma-Aldrich) at final concentrations of 2.5 ng/ml, 10 ng/ml, 0.1 ng/ml, and 100 ng/ml, respectively, for 4 h. Tissue Preparation and Immunohistochemistry—C57BL/6 mice were anesthetized, and tissues were harvested, rinsed in phosphate-buffered saline, fixed in 4% paraformaldehyde for 48 h, and imbedded in paraffin, and 5-μm sections were cut. Immunohistochemical analysis of formalin-fixed tissues from mice was performed with standard procedures in the Dana Farber/Harvard Cancer Center Histopathology Core using a polyclonal antibody against KLF4 (Santa Cruz, sc-12538). Human tissue was obtained from cardiac transplantation donors under protocols approved by the Human Investigation Review Committee at the Brigham and Women's Hospital (IRB 1999-P-001348). This tissue was frozen, fixed with acetone, and stained with an anti-KLF4 antibody as previously described (40Katz J.P. Perreault N. Goldstein B.G. Actman L. McNally S.R. Silberg D.G. Furth E.E. Kaestner K.H. Gastroenterology. 2005; 128: 935-945Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), except that a 1:5000 dilution of the antibody was used. Adjacent sections were stained with nonimmune IgG as a negative control. RNA Extraction and RNA Blot Analysis—HUVECs were infected with adenovirus expressing green fluorescence protein (Ad-GFP) or adenovirus expressing (via a bicistronic promoter) both GFP and KLF4 (Ad-K4) for 48 h, exposed to the indicated stimulus, and then harvested for total RNA analysis. Total RNA was isolated from cultured cells with Trizol (Invitrogen) as described by the manufacturer. Human lung messenger RNA (mRNA) was purchased from Stratagene. RNA was fractionated on a 1.3% formaldehyde-agarose gel and transferred to nitrocellulose membranes. The membranes were hybridized with 32P-labeled, random-primed cDNA probes, washed, and exposed as described previously (41Feinberg M.W. Jain M.K. Werner F. Sibinga N.E. Wiesel P. Wang H. Topper J.N. Perrella M.A. Lee M.E. J. Biol. Chem. 2000; 275: 25766-25773Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Probes used for Northern analysis were derived as follows. TM, tissue plasminogen activator, VCAM-1, TF, and KLF4 cDNA were generated by reverse transcription-PCR. eNOS cDNA was a kind gift from Dr. J. K. Liao (Harvard Medical School, Boston, MA). Quantitative PCR—Total RNA (2-5 μg) was reverse-transcribed using 1× reverse transcriptase buffer, MgCl2 (2.2 mm), dNTP (2.0 mm), RNasin (0.2 units/μl), and oligo-dT primers (0.5 mm)in20-μl reactions. Reactions were incubated at 70 °C for 2 min and put on ice. Moloney murine leukemia virus reverse transcriptase (0.3 units/μl) was then added, and cDNA was prepared using a 2-step cycle, 48 °C for 1 h and 94 °C for 5 min. Reverse transcription reagents were purchased from Amersham Biosciences. The resulting cDNA was diluted to 100 μl and used in subsequent real time PCR reactions. Real time fluorescence detection was carried out using a Stratagene Mx3005P real-time PCR system. Reactions were carried out in micro 96-well reaction plates using 18 μl of Brilliant Sybr Green Master Mix (Stratagene), forward and reverse primers (0.2 μm each, Invitrogen), and cDNA (2 μl) in a final PCR reaction volume of 20 μl. Amplification parameters were denaturation at 94 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 62 °C for 60 s (except for TM where extension temperature was 57 °C). Samples were analyzed in duplicate, and glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. -Fold induction was calculated after normalization to glyceraldehyde-3-phosphate dehydrogenase using the MX3005P software. Dissociation curves indicated that a single amplification product was made in each reaction. Amplification products using Syber green detection were initially checked by electrophoresis on ethidium bromide-stained agarose gels. The estimated size of the amplified products matched the calculated size for transcript by visual inspection. Primer pairs used for real timer reverse transcription-PCR were as follows: KLF4 (NM_004235) forward primer (5′-3′) ACCAGGCACTACCGTAAACACA, reverse primer (5′-3′) GGTCCGACCTGGAAAATGCT (42Martinez J.M. Afshari C.A. Bushel P.R. Masuda A. Takahashi T. Walker N.J. Toxicol. Sci. 2002; 69: 409-423Crossref PubMed Scopus (106) Google Scholar); KLF2 (NM_016270) forward primer TGCGGCAAGACCTACACCAAGAGT, reverse primer TGCGGCAAGACCTACACCAAGAGT (designed using PrimerSelect, DNASTAR); glyceraldehyde-3-phosphate dehydrogenase (NM_002046) forward primer GCCATCAATGACCCCTTCATT, reverse primer TCTCGCTCCTGGAAGATGG (43Yamamoto J. Ikeda Y. Iguchi H. Fujino T. Tanaka T. Asaba H. Iwasaki S. Ioka R.X. Kaneko I.W. Magoori K. Takahashi S. Mori T. Sakaue H. Kodama T. Yanagisawa M. Yamamoto T.T. Ito S. Sakai J. J. Biol. Chem. 2004; 279: 16954-16962Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar); eNOS (NM_000603) forward primer CTCATGGGCACGGTGATG, reverse primer ACCACGTCATACTCATCCATACAC (44Sonveaux P. Brouet A. Havaux X. Gregoire V. Dessy C. Balligand J.L. Feron O. Cancer Res. 2003; 63: 1012-1019PubMed Google Scholar); TM (NM_000361) forward primer GCCTTAATCAGGTCCTCA, reverse primer TCATGAACTGGATGGGGT (45Cenni E. Ciapetti G. Granchi D. Savarino L. Corradini A. Vancini M. Di L.A. Biomaterials. 2002; 23: 2159-2165Crossref PubMed Scopus (1) Google Scholar). Western Analysis—HUVECs were infected with Ad-GFP or Ad-K4 for 48 h, exposed to the indicated stimulus, and then harvested for total protein analysis. Cellular protein was extracted in radioimmune precipitation assay buffer (Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with the Complete protease inhibitor mixture (Roche Applied Science), and Western blot analyses were performed using the indicated antibodies as previously described (22SenBanerjee S. Lin Z. Atkins G.B. Greif D.M. Rao R.M. Kumar A. Feinberg M.W. Chen Z. Simon D.I. Luscinskas F.W. Michel T.M. Gimbrone Jr., M.A. Garcia-Cardena G. Jain M.K. J. Exp. Med. 2004; 199: 1305-1315Crossref PubMed Scopus (566) Google Scholar). Antibodies recognizing p65, thrombomodulin, VCAM-1, and plasminogen activator inhibitor-1 (PAI-1) were from Santa Cruz Biotechnology, TF antibody was from American Diagnostica, eNOS antibody was from BD Biosciences, anti-KLF4 from CeMines, and anti-tubulin antibody was from Sigma-Aldrich. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Amersham Biosciences. For Western analysis for KLF4-knockdown studies, 150 μg of total cell protein was loaded per lane. Adenoviral and Retroviral Infections—Adenoviral constructs for KLF4 or the "empty virus" control both encode GFP and were generated by the Harvard Gene Therapy Group. The GFP and KLF4 are expressed as separate proteins driven by a bidirectional cytomegalovirus promoter. For adenoviral infection of HUVECs, cells were seeded at 2 × 106/10-cm2 dish, infected with the adenoviral vectors at 15-20 multiplicity of infection, and incubated for 48 h. Transduction efficiencies were typically >85% as measured by GFP positivity and fluorescence-activated cell sorter analyses. For retroviral small interfering RNA studies, the sequence GGACGGCTGTGGATGGAAA (32Rowland B.D. Bernards R. Peeper D.S. Nat. Cell Biol. 2005; 7: 1074-1082Crossref PubMed Scopus (494) Google Scholar) was cloned into the retroviral vector pSUPER.retro (oligo engine), and retroviruses containing the small interfering RNA hairpin and a puromycin resistance gene were generated using the Phoenix packaging cells according to Dr. Nolan's online protocol. For retroviral infection of target cells, retroviral supernatant and culture medium (10% fetal calf serum/Dulbecco's modified Eagle's medium plus 8 μg/ml Polybrene (Specialty Media)) were mixed at a 1:1 ratio and added to pre-confluent cells. Infected cells were selected with 3 μg/ml puromycin. A negative control oligonucleotide (gift from B. Spiegelman) was processed in a similar manner. Laminar Shear Stress Experiments—HUVECs were seeded onto the inner surface of sterilized fibronectin-coated gaspermeable Silastic laboratory tubes by axially rotating the tubes at 10 rotations per hour at 37 °C for 24 h as previously described (46Balcells M. Fernandez Suarez M. Vazquez M. Edelman E.R. J. Cell. Physiol. 2005; 204: 329-335Crossref PubMed Scopus (44) Google Scholar). Closed loops of tubing were created using the section with the confluent HUVEC monolayer and placed in a perfusion bioreactor at 37 °C under 5% CO2. Cells were exposed to static conditions or shear stress of 2, 5, or 20 dynes/cm2 for 24 h before harvest by trypsinization (46Balcells M. Fernandez Suarez M. Vazquez M. Edelman E.R. J. Cell. Physiol. 2005; 204: 329-335Crossref PubMed Scopus (44) Google Scholar). Precipitated cells were resuspended in Trizol for total RNA isolation as described above. cDNA was prepared from RNA using standard techniques. Multiplex Sandwich Enzyme-linked Immunosorbent Assay—HUVECs were infected with Ad-GFP or Ad-K4 for 48 h and treated with TNFα (10 ng/ml) for 5 h, and supernatants were collected for analysis of secreted factors. Analysis was performed with the SearchLight Proteome Arrays multiplex sandwich enzyme-linked immunosorbent assay (Pierce). Each sample was evaluated in duplicate from three independent experiments. Thrombomodulin and Tissue Factor Activity Assays—TM (luminal surface) activity was measured in HUVECs infected with Ad-GFP or Ad-K4 using a chromogenic assay (47Cadroy Y. Diquelou A. Dupouy D. Bossavy J.P. Sakariassen K.S. Sie P. Boneu B. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 520-527Crossref PubMed Scopus (35) Google Scholar, 48Calnek D.S. Grinnell B.W. Exp. Cell Res. 1998; 238: 294-298Crossref PubMed Scopus (63) Google Scholar). Endothelial TM enzymatic activity was measured by the production of activated protein C from protein C. Briefly, HUVECs were washed with cold TBS (50 mmol/liter Tris-HCl, 120 mmol/liter NaCl, 2.7 mmol/liter KCl, and 3 mg/ml bovine serum albumin) three times and then immediately incubated with 150 nmol/liter human protein C (Enzyme Research Laboratories) and 0.5 units/ml thrombin for 1 h at 37°C. The reactions were quenched by adding hirudin (Sigma-Aldrich), and activated protein C activity was measured using a chromogenic substrate (S2366, Chromogenix). The reaction was stopped after 10 min by the addition of acetic acid, and the amidolytic activity of activated protein C generated was read at 405 nm with a spectrophotometer. The TM activity was expressed in arbitrary units using reference curves determined with purified human activated protein C (Enzyme Research Laboratories). For TF activity assays, uninfected and Ad-GFP- and Ad-K4-infected HUVECs were incubated in the presence or absence of 10 ng/ml of TNFα for an additional 5 h. The cells were assayed for total cell-associated TF activity per the manufacturer's instructions using the Actichrome TF kit (American Diagnostica). eNOS Activity Assays—eNOS enzyme activity was measured by monitoring the conversion of l-[3H]arginine to l-[3H]citrulline in labeled cells as described previously (49Greif D.M. Kou R. Michel T. Biochemistry. 2002; 41: 15845-15853Crossref PubMed Scopus (93) Google Scholar). Briefly, HUVECs were incubated in Hepes buffer (25 mm Hepes, pH 7.3, 109 mm NaCl, 5.4 mm KCl, 0.9 mm CaCl2, 1 mm MgSO4, and 25 mm glucose) for 1 h at 37 °C and then labeled with l-[3H]arginine (10Ci/ml) and stimulated with 1 m calcium ionophore A23187 for 10 min at 37 °C. Immediately thereafter, cells were washed 2 times with ice-cold phosphate-buffered saline containing 5 mm EDTA and 5 mm l-arginine, scraped into 2 ml of stop buffer (20 mm sodium acetate, pH 5.5, 1 mm l-citrulline, 2 mm EDTA, and 2 mm EGTA), and sonicated. Aliquots of these lysates were withdrawn to determine total cellular protein abundance and 3H incorporation. l-[3H]Citrulline was isolated from the remaining lysate by anion exchange chromatography with AG 50W-X8 resin (Bio-Rad) and quantitated by liquid scintillation counting. Clotting Assay—HUVEC were plated in 96-well dishes and infected with Ad-GFP or Ad-K4 for 48 h, then incubated for another 5 h in the absence or presence of TNFα. Cells were then rinsed twice with warm phosphate-buffered saline and 100 μl of 37 °C human plasma (Sigma-Aldrich) added to each well. Immediately thereafter, 100 μl of 25 mm CaCl2 was added, and plates were placed in a Vmax kinetic plate reader (Molecular Devices) and read at 405 nm every 20 s for 30 min (50Rose S.L. Babensee J.E. J. Biomed. Mater. Res. A. 2005; 72: 269-278Crossref PubMed Scopus (13) Google Scholar). Fibrin clot formation is indicated when a maximum absorbance is reached. Clotting times are reported at half-maximal absorbance. Adhesion Assays under Flow—HUVEC monolayers were grown to confluency on fibronectin-coated coverslips, infected with Ad-GFP or Ad-K4 for 48 h, then treated for a further 5 h in the presence or absence of 10 ng/ml TNFα. A parallel plate laminar flow chamber (Immunetics, Cambridge, MA) was used to perfuse the monolayer with perfusion medium containing 106 cells/ml THP-1 at an estimated shear stress of 2.0 dynes/cm2 as described previously (51Gerszten R.E. Friedrich E.B. Matsui T. Hung R.R. Li L. Force T. Rosenzweig A. J. Biol. Chem. 2001; 276: 26846-26851Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Monocyte adhesion (>3s) was quantified for three high-powered fields per coverslip. The entire period of perfusion was recorded on videotape. HUVEC Immunostaining—HUVEC monolayers (prepared in parallel with those used for adhesion assays) were released from coverslips with Hepes-buffered solution containing EDTA and EGTA. Staining for VCAM-1, E-selectin, and intercellular cell adhesion molecule-1 was performed as described previously (52Gerszten R.E. Luscinskas F.W. Ding H.T. Dichek D.A. Stoolman L.M. Gimbrone Jr., M.A. Rosenzweig A. Circ. Res. 1996; 79: 1205-1215Crossref PubMed Scopus (59) Google Scholar). Transient Transfections—HUVECs, bovine aortic endothelial cells, or COS7 were plated at a density of 5 × 104/well in 12-well plates 1 day before transfection. Transient transfection studies were performed using FuGENE™ 6 reagent (Roche Applied Science) according to the manufacturer's instructions. A total of 1 μg of plasmid DNA was used per well. Cells were harvested 48 h after transfection at which time they were nearly confluent. In some experiments cells were treated with human TNFα (10 ng/ml) for 5 h before harvest. Luciferase activity was normalized to total cell protein. All transfections were performed in triplicate (n = 9). The TM promoter deletion constructs were made as previously described (25Lin Z. Kumar A. SenBanerjee S. Staniszewski K. Parmar K. Vaughan D.E. Gimbrone Jr., M.A. Balasubramanian V. Garcia-Cardena G. Jain M.K. Circ. Res. 2005; 96: 48-57Crossref PubMed Google Scholar). The VCAM-1 promoter construct was a gift from W. C. Aird (Beth Israel Deaconess Medical Center, Boston, MA). The eNOS promoter was provided by C. J. Lowenstein (The Johns Hopk
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