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* Division of Hematology-Medical Oncology, Weill Medical College of Cornell University, New York, New York, USA; and
Department of Pharmacological Sciences, University of Milan, Milan, Italy
1Correspondence: Division of Hematology-Medical Oncology, Weill Medical College of Cornell University, 1300 York Ave., 10021 New York, NY, USA. E-mail: babette{at}med.cornell.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: endothelial cells • inflammation • signal transduction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Vascular endothelial cells represent the primary target of many of these atherogenic risk factors (3)
, and endothelial membrane junctional proteins have been implicated in atherosclerotic plaque morphology (4)
.
ß-catenin, a cytoskeletal component that mediates cell-cell interaction, is also involved in regulating expression of genes pertinent to atherosclerosis. In unstimulated adherent cells, ß-catenin is associated with VE-cadherin in complexes mainly at the plasma membrane. If liberated into the cytoplasm, ß-catenin either translocates to the nucleus or becomes incorporated into a cytoplasmic complex with adenomatosis polyposis coli (APC), glycogen synthase kinases-3ß (GSK-3ß), and axin/conductin proteins. This complex favors phosphorylation and subsequent proteolytic degradation of ß-catenin by the ubiquitin-proteasome system (5)
. If GSK-3ß is inactivated by phosphorylation at Ser-9, ß-catenin phosphorylation and degradation are decreased, favoring translocation of ß-catenin to the nucleus. Within the nucleus, ß-catenin associates with the T cell factor/lymphoid enhancer family (TCF/LEF) of transcription factors, leading to activation of several target genes, including cyclin D1 (6)
, c-myc (7)
, and, potentially, cyclooxygenase-2 (COX-2) (8
9
10
11)
.
COX-2, a key enzyme for prostaglandin biosynthesis, is undetectable in most normal tissues but is rapidly induced during inflammatory reactions (12)
and is overexpressed in atherosclerotic lesions (13)
. COX-2 metabolites produced by endothelial cells, namely prostacyclin (PGI2), thromboxane A2 (TXA2), and prostaglandin E2 (PGE2), influence vascular tone, regional blood flow, vascular permeability and remodeling, and angiogenesis. The balance between these autacoids is consequently critical in a variety of pathophysiologic conditions (14)
.
Here, using mouse cardiac endothelial cells (MCEC), we demonstrate that tobacco smoke alone and in cooperation with inflammatory cytokines up-regulates COX-2 expression through nuclear ß-catenin by modulating the EGFR/Akt/GSK-3ß pathway. We then provide evidence that cigarette smoke exposure in vivo increases activation of the Akt/GSK-3ß/ß-catenin pathway and COX-2 expression in vascular tissue and up-regulates serum levels of the inflammatory cytokine interleukin-1ß.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Eight to ten FVB mouse pups (2–5 days old) were used per preparation. After euthanasia, hearts were removed and placed in Hanks’ balanced salt solution (HBSS; Gibco-Brl, Gaithersburg, MD, USA) without Ca2, Mg2, NaCO3, or phenol red and minced. Tissue was collected in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 0.2% collagenase I (Worthington Biochemical, Lakewood, NJ, USA), 0.005% DNase (Sigma, St. Louis, MO, USA), and 5% fetal calf serum (Gemini Bio-Products, West Sacramento, CA, USA) and then incubated at 37°C for 30 min with occasional agitation. After digestion, the tissue was mechanically disrupted and filtered through sterile 70 µm nylon mesh. After being centrifuged and then washed in 10% FCS-DMEM, endothelial cells were isolated on a 25–40% Percoll (Sigma) gradient. Separated endothelial cells were collected and washed twice in 10% FCS-DMEM before suspension in VEC medium [DMEM/F-12 (Gibco) medium containing 5% fetal calf serum and 1% mouse pup serum, 20 U/ml heparin (Sigma), 10 µg/ml endothelial growth supplement (Biomedical Technologies, Stoughton, MA, USA), 1 mmol/l sodium pyruvate, 1% BME vitamins, 1% ITS-X (Invitrogen, Carlsbad, CA, USA), and 1% ECGF (Sigma)] and then plated for 1 h on a sterile plastic plate to eliminate adherent fibroblasts with nonadherent cells then transferred to a 1% gelatin-coated plate and incubated overnight. Unattached cells were rinsed off and discarded, and adherent cells were fed fresh VEC medium. Two days after isolation, cells were incubated overnight with DNA triple flap lentiviral vectors (kind gifts of Dr. Pierre Charneau, Institut Pasteur, Paris, France; ref 16
), encoding hTERT (final concentration 100 ng p24 U/ml) and SV40-T antigen (20 ng p24 U/ml), and then were permitted to proliferate. Immortalized MCEC were isolated by two rounds of immunopurification, first with CD31 antibody-coated beads and second with VE-cadherin antibody-coated beads (17)
. Clones were cultivated in DMEM with 10 mmol/l penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA), 10 mmol/l HEPES (Sigma), and 5% FBS. Individual transduced MCEC were then isolated by two rounds of limiting dilution cloning, producing several hundred proliferating cell lines. Expression of transfected immortalizing genes and purity of derived MCEC were determined. MCEC were uniformly positive to nuclear SV40-T antigen and cytoplasmic hTERT and exhibited a normal endothelial phenotype with contact inhibition as shown by 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate-labeled LDL (DilAcLDL; Biomedical Technologies) uptake and PECAM-1, VE-cadherin, and von Willebrand Factor expression by immunostaining (Supplemental Fig. 1).
Preparation of tobacco smoke extract
2R4F cigarettes were smoked one at a time in a Borgwaldt piston-controlled apparatus (Model RG-1) using the Federal Trade Commission standard protocol that mimics a standardized human smoking pattern (puff duration, 2 s; frequency, 1 puff/min; vol., 35 ml/puff). The smoke was drawn under sterile conditions through premeasured amounts of sterile PBS, pH 7.4. Smoke dissolved in PBS represents a hydrophilic fraction of main stream cigarette smoke. Many hydrophobic and volatile main stream smoke chemicals are lost by this procedure. Tobacco smoke dissolved in PBS was abbreviated as "TS" and was quantitated in puffs/l of PBS, with one cigarette yielding 
8 puffs drawn into a 10 ml vol. The final concentration of TS in the cell culture medium is expressed as puffs/l medium. Aliquots of TS were snap frozen immediately after preparation and kept at –80 deg C until use (18)
. Biological effects were stable for at least 6 months.
Cell incubation and prostaglandin assays
Two different clones of MCEC were used with similar results. Cells at confluence were starved 24 h in fresh medium containing 0.5% FBS and then incubated in DMEM 0.5% FBS with or without TS and/or IL-1ß, as indicated. PGE2 levels in cell culture supernatants were measured using PGE2 enzyme immunoassay (EIA) kits (Cayman, Ann Arbor, MI, USA).
Immunofluorescence studies
MCEC were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.2% Triton X-100 (Sigma) for 10 min at room temperature. After being washed three times with PBS, the cells were blocked 1 h with PBS containing 5% BSA and then incubated at 4°C overnight with primary antibodies to PECAM-1 (1:30; a gift from Dr. William Mueller, Weill Medical College), vonWillebrand Factor (1:200, Zymed Burlingame, CA, USA), VE-cadherin, SV40-T antigen, h-TERT, and ß-catenin (all 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Cells were washed with PBS and incubated for 1 h at room temperature with appropriate second antibodies labeled with Cy2 or Cy3 (1:300, Jackson). For negative controls, the same procedure was performed without adding the primary antibody. Slides were then mounted with mowiol (Calbiochem, San Diego, CA, USA) and examined using a Zeiss LSM 510 confocal microscope.
Protein preparation
For Western blotting of total cellular extracts, MCEC were lysed in 1 mmol/l Tris-HCl, pH 6.8, 4% SDS, 0.5 mmol/l DTT and protease inhibitor cocktail (Complete, Mini; Roche Diagnostics, Indianapolis, IN, USA). For phosphorylated proteins, total cellular extracts were prepared by sonication of cells in lysis buffer containing 100 mmol/l NaCl, 20 mmol/l Tris-HCl pH 7.4, 2.5 mmol/l EDTA, 10 mmol/l NaF, 1 mmol/l Na3VO4, 10 mmol/l sodium pyrophosphate, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail (19)
.
For nuclear extracts, cells were suspended in hypotonic buffer A (10 mmol/l HEPES, pH 7.6, 10 mmol/l KCl, 0.1 mmol/l MgCl2, 0.1 mmol/l DTT, 0.1 mmol/l EDTA, 0.5 mmol/l PMSF, and 1 mmol/l each of pepstatin, aprotinin, and leupeptin for 10 min in ice and vortexed for 10 s. Nuclei were pelleted and washed with buffer A before suspension in buffer C (10 mmol/l HEPES, pH 7.6, 400 mmol/l NaCl, 1.5 mmol/l MgCl2, 0.1 mmol/l EDTA, 0.1 mmol/l DTT, 0.5 mmol/l PMSF, and 1 mmol/l pepstatin, aprotinin, and leupeptin and 5% glycerol) for 30 min in ice. The supernatants containing nuclear proteins were collected by centrifugation at 12,000 g for 20 min (20)
.
Immunoprecipitation
MCEC cells were lysed in immunoprecipitation buffer (62.5 mmol/l Tris-HCl, 100 mmol/l NaCl, 1% Nonidet P-40, 0.1% Tween 20, 1 mmol/l Na3VO4, 1 mmol/l PMSF, pH 8.0, and protease inhibitors) for 30 min on ice. Antibody against VE-cadherin (1 µg; Santa Cruz) was added to protein A-Sepharose beads (Sigma; 20 µl) and incubated with gentle rocking overnight at 4°C. The protein A-Sepharose beads were sedimented by brief centrifugation and washed with immunoprecipitation buffer; 0.3 mg of cell lysate proteins were added to the protein A-Sepharose beads for 2 h at 4°C after which the beads were washed and the pellet then lysed in 20 µl of 4x Laemmli buffer; 20 µl aliquots of immunoprecipitated protein were separated on 7% SDS-PAGE gels and immunoblotted with antibodies to VE-cadherin or ß-catenin (21)
Western blot analysis
The samples were prepared with Laemmli method, and protein was measured with BCA Protein assay (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated by 7% SDS-PAGE gel and transferred to Optitran BA-S 85 nitrocellulose membranes (Schleicher & Schull, Keene, NH, USA). The equivalency of protein loading was confirmed by Ponceau Red staining. The membranes were blocked for 2 h in blocking buffer and thereafter incubated overnight at 4°C with primary antibody against either COX-2 (1:500, BD Transduction Laboratories), ß-actin (1:10 000, Sigma), EGFR, phospho-EGFR, Akt phospho-Akt (Ser-473 phospho-GSK-3ß (Ser-9, phospho-ß-catenin (Ser-33/37/Thr-41; 1:1 000, Cell Signaling), ß-catenin, and SV40-T (1:500, Santa Cruz). Blots were subsequently incubated with horseradish peroxidase-linked goat anti-mouse (1:7 000, Sigma) or goat anti-rabbit (1:4 000, Santa Cruz) IgG and visualized with the enhanced chemiluminescent detection system ECL-plus (PerkinElmer, Wellesley, MA, USA). SV40-T antigen was used as loading control for nuclear extracts for the in vitro study and Ponceau Red staining as loading control for the in vivo study.
RNA extraction and reverse transcriptase-polymerase chain reaction
Total cellular RNA was isolated from cells with TRIzol reagent, and 1 µg of RNA per 20 µl reaction was reverse transcribed using SuperScript II RNase H (Invitrogen). Samples were amplified for 10 min at 25°C, 15 min at 42°C, 5 min at 99°C, and 5 min at 5°C. cDNA (2.5 µl) was then subjected to 25 cycles of polymerase chain reaction (PCR; denaturation at 94°C for 1 min, annealing at 94°C for 20 s; 65°C for 20 s; 72°C for 1 min, extension at 72°C for 10 min) in a reaction mixture (25 µl), containing 2.5 U TaqDNA polymerase (Invitrogen) and 400 nmol/l forward primer and 400 nmol/l reverse primer.
COX-2 primers were 5'-GGTCTGGTGCCTGGTCTGATGATG-3' and 5'-GTCCTTTCAAGGAGAATGGTGC-3', giving rise to a 720-bp PCR product; ß-actin primers were 5'-GGTCACCCACACTGTGCCCAT-3' and 5'-GGATGCCACAGGACTCCATGC-3', giving rise to a 450-bp PCR product. All reactions were performed in a Perkin-Elmer GeneAmp PCR System 2400 thermocycle. PCR products were electrophoresed on a 1% agarose gel with 0.5 mg/l ethidium bromide and photographed under ultraviolet light. The identity of each PCR product was confirmed by DNA sequencing.
Ex vivo experiments
FVB mice 10–12 wk old were anesthetized and perfused with saline, and aortas were isolated and aortic rings were cut and incubated in 0.5% FBS-DMEM with or without TS/IL-1ß for 6 h and then fixed for study.
Immunohistochemistry
Aortas were fixed in 4% paraformaldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA, USA) overnight with gentle shaking at 4°C, washed in PBS, and embedded in OCT (Tissue Tek), and rapidly frozen in isopentane. Sections (0.012 mm) were washed in PBS and incubated with 3% H2O2 for 10 min. After being washed, the sections were incubated 1 h in blocking solution (PBS, 10% horse serum, 0.1% Triton-X100). Primary antibodies were added at a dilution of 1:100 for anti-mouse COX-2 (BD, Transduction Laboratories), for anti-goat PECAM-1, anti-mouse ß-catenin (both 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-rabbit phospho-ß-catenin (1:200, Cell Signaling, Danvers, MA, USA) and incubated overnight at 4°C. Sections were rinsed and incubated 1 h at room temperature with biotinylated anti-mouse, anti-rabbit, or anti-goat antibodies (1:200; Jackson Laboratories, West Grove, PA, USA), followed by the peroxidase-based Vectastain ABC system (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine (Sigma). Methyl green (Sigma) was used as counterstain. For negative controls, the same procedure was performed without adding the primary antibody.
Transfection of siRNA
Approximately, 5 x 104 cells per well of 24-well plates in antibiotic-free media containing 5% FBS were grown to 50–70% confluence. Transfection with small interferring siRNAs (RNA) was performed using transfection reagent and medium (Santa Cruz) to result in a final ß-catenin siRNA duplex or control siRNA (all from Santa Cruz) concentration of 50 nmol/l. After 7 h of incubation, an equal volume of DMEM/5% FBS was added without removing the transfection mixture. The following day, the cells were retransfected with the same protocol. After 18 h, the transfected cells were washed and starved for 24 h in DMEM/0.5% FBS before use.
Permeability studies
The permeability of MCEC cell monolayers was measured using Transwell polycarbonate insert filters (Corning, pore size 3 µm). Briefly, MCEC at confluence were incubated with or without stimuli in DMEM/0.5% FCS without phenol red; FITC-conjugated dextran (40 kDa, Sigma, 2 g/l) was added to the upper chamber. Aliquots (0.2 ml) were removed from the lower chamber at 0, 5, 15, 30, 60, 90, and 120 min and replaced with fresh assay buffer, and the fluorescence that passed through the cell-covered inserts was determined using a fluorescence multiwell plate reader. The volume cleared was plotted against time, and the slopes of the curves were used to calculate the permeability coefficients (Pe) of the endothelial monolayer, as described by Forster et al. (22)
: 1/PS = 1/me – 1/mf and Pe = PS/s, where me and mf are slopes of the curves, PS is permeability surface area product, and s is the surface area of the filter (1 cm2).
Animal studies and in vivo experiments
All animal experimental procedures were approved by the Weill Medical College Institutional Animal Care and Use Committee. ApoE–/– mice aged 9 wk (5 males per group, Jackson Laboratories) were fed standard chow. Mice were secured in fitted polycarbonate chambers and placed into a 12-port nose-only exposure chamber (CH Technologies, Westwood, NJ, USA) for direct inhalation of cigarette smoke. The cigarettes used in this study were 2R4F research cigarettes (Kentucky Tobacco Research Institute, Lexington, KY, USA) smoked at the rate of 1 puff/minute, 2 s per puff. Puff volume was 35 ml. Mice were treated with either room air or tobacco smoke for 1 h per day, 7 days per week for 15 days at a total suspended particulate (TSP) dose of 200 mg/m3/day. At the end of the entire experimental period, animals were deeply anesthetized with isofluorane. After perfusion with saline, the hearts and aortas were isolated en bloc and lysed in a phosphorylation buffer or processed for nuclear extraction. Aortas were also removed and fixed for immunohistochemical study.
Data analysis
Data are shown as mean ± SD and are based on at least four independent experiments. Statistical analysis was performed by Student’s test or ANOVA and the Fisher’s least significant difference (LSD) post test where appropriate. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Treatment with TS 12.8 puffs/l caused time-dependent translocation of ß-catenin to the nucleus within 30 min of treatment (Fig. 1
A) and increased total ß-catenin levels (Fig. 1B
). This effect was TS-concentration dependent (Fig. 1A, C
).
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We also observed that IL-1ß, a major proinflammatory cytokine that increases in smokers and plays a key role in atherosclerosis (1
, 2
, 23
, 24)
, produced ß-catenin translocation to the nucleus after 30 min (Fig. 1A, C
).
It was of interest that a low concentration of TS interacted with IL-1ß to accelerate and to increase nuclear ß-catenin translocation in MCEC. Whereas ß-catenin translocation to the nucleus was observed within 60 min of starting treatment with TS 6.4 puffs/l alone (Fig. 1A, C
) and by 30 min with IL-1ß alone, it occurred within 15 min with the combination of stimuli (Fig. 1A, C
). Moreover, the combined treatment further augmented ß-catenin accumulation in the cells (Fig. 1B
). Our results showed that TS with or without IL-1ß both accelerates and augments accumulation of ß-catenin in nuclei of endothelial cells.
TS and IL-1ß cooperate to induce COX-2 expression in MCEC in a dose- and time-dependent manner
ß-Catenin present in the nucleus can act as a transcription cofactor with the TCF/LEF family of DNA binding protein to modify expression of many genes involved in atherosclerosis such as COX-2, an early response gene (8
9
10
11)
. COX-2 mRNA increased in MCEC after 30 min of treatment with TS 12.8 puffs/l (Fig. 2
A), and COX-2 protein was up-regulated by 1–3 h with further increases by 6 h (Fig. 2B
). Concentrations of TS that alone (up to 6.4 puffs/l) induced only a modest COX-2 expression were more effective in the presence of IL-1ß (Fig. 2C
).
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Exposing MCEC to TS also altered the kinetics of COX-2 induction by IL-1ß. Indeed, exposure to TS alone 6.4 puffs/l barely altered COX-2 mRNA after 60 min and COX-2 protein levels after 6 h, while exposure to IL-1ß alone moderately increased COX-2 (30 min for mRNA and 3–6 h for the protein; Fig. 2A, D
). However, a marked increase in COX-2 mRNA occurred within 15 min in MCEC simultaneously exposed to TS/IL-1ß, with COX-2 protein up-regulated by 60 min, peaking at 3–6 h (Fig. 2A, D
).
A cooperation between TS and IL-1ß was observed also in terms of PGE2 release (Fig. 2E
) while expression of the constitutive enzyme COX-1 was not modified after any treatment (data not shown).
To demonstrate that TS/IL-1ß exposure has similar effects in primary vascular tissue as in vitro in MCEC, we replicated these studies by ex vivo incubation of freshly obtained aortic rings with TS/IL-1ß and observed increased levels of ß-catenin and COX-2 protein localized in the aortic endothelium within 6 h (Fig. 3
).
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TS/IL-1ß-mediated accumulation of ß-catenin in the nucleus modulates COX-2 expression and activity
To determine whether ß-catenin was directly involved in regulating COX-2 expression, MCEC cells were first transfected with ß-catenin siRNA or with a non-specific siRNA and then exposed to different concentrations of TS alone or TS/IL-1ß. Transfection of MCEC with siRNA directed against ß-catenin reduced the levels of ß-catenin by 80% compared with non-specific siRNA but did not affect the levels of ß-actin (Fig. 4
A). COX-2 protein and activity clearly decreased in cells transfected with ß-catenin after treatment compared to cells transfected with non-specific siRNA (Fig. 4A, B
). COX-2 expression and activity induced by IL-1ß alone were only moderately reduced in cells transfected with ß-catenin siRNA. Our results indicate that COX-2 induction by TS alone or TS/IL-1ß is mediated by nuclear accumulation of ß-catenin.
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TS and IL-1ß act to destabilize endothelial junctions and to reduce ß-catenin degradation
ß-Catenin translocation into the nucleus requires not only its release from membrane complexes with VE-cadherin but also decreased degradation in the cytoplasm by the ubiquitin-proteasome pathway-involving GSK-3ß (11)
. We determined the effect of TS exposure on both these steps. We first analyzed the changes in ß-catenin/VE-cadherin complexes in plasma membranes of MCEC monolayers that were exposed to TS with or without IL-1ß. Treatment of MCEC monolayers with TS alone (12.8 puffs/l) or TS/IL-1ß markedly decreased the association between ß-catenin and VE-cadherin concomitant with ß-catenin trafficking from junctions into the cytosol (Fig. 5
A). Disruption of the ß-catenin/VE-cadherin complex increased monolayer permeability, as shown in Fig. 5B
. Permeability increased only slightly with TS alone (20% increase), while it increased strongly (360% increase) with IL-1ß, compared with control. However, exposure to TS/IL-1ß in combination dramatically increased the extent of permeability (689% increase) and accelerated its onset. These effects progressed over 2 h, reaching 50% increase with TS alone, 480% increase with IL-1ß, and 1400% increase with TS/IL-1ß compared with untreated controls.
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We then explored the possibility that exposure of MCEC to TS/IL-1ß modulated ß-catenin degradation by diminishing phosphorylation of ß-catenin on Ser-33/37/Thr-41. TS at a low concentration decreased ß-catenin phosphorylation within 60 min (Fig. 5C
). TS at high concentration and IL-1ß each also reduced phosphorylation of ß-catenin, starting within 15–30 min (Fig. 5C
). However, cells exposed to a low concentration of TS plus IL-1ß for only 5 min already showed decreased ß-catenin phosphorylation (Fig. 5C
). These data suggest that TS cooperated with IL-1ß by destabilizing intercellular endothelial junctions and subsequent increasing endothelial permeability. These events lead to increase of ß-catenin in the nucleus as consequence of its decreased degradation and increased accumulation in the cytosol.
TS/IL-1ß-activated PI3-kinase/Akt/GSK-3ß signaling axis regulates nuclear ß-catenin translocation and COX-2 expression
In unstimulated mammalian cells, GSK-3ß is constitutively active and induces the phosphorylation of soluble ß-catenin, favoring proteosomal degradation of the latter. Phosphorylation of GSK-3ß at Ser-9, which inhibits its kinase activity, results in an increase in intracellular free ß-catenin. We showed that treatment of MCEC with TS 6.4 puffs/l for 60 min or with TS 12.8 puffs/l or IL-1ß alone for 30 min increased phosphorylation of GSK-3ß (Fig. 6
A). Moreover, exposure to a low concentration of TS plus IL-1ß resulted in even more rapid phosphorylation of GSK-3ß, observed within only 15 min (Fig. 6A
). Since TS and/or IL-1ß modulated GSK-3ß activity, we investigated whether the level of GSK-3ß activity regulated both ß-catenin relocalization and COX-2 expression. Inhibition of GSK-3ß activity by its known inhibitor LiCl rapidly induced GSK-3ß phosphorylation and decreased ß-catenin phosphorylation, resulting in increased translocation of ß-catenin to the nucleus (Fig. 6B, C
) and increased COX-2 expression (Fig. 6D
).
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Protein kinase B (PKB/Akt), a serine/threonine kinase located downstream of phosphytidylinosoitol-3-kinase (PI3K), has been demonstrated to phosphorylate GSK-3ß at an inhibitory site. Treatment of MCEC with TS and/or IL-1ß rapidly led to increased Akt phosphorylation at Ser-473 and enhanced its kinase activity. Activation of Akt was observed between 5–30 min, with stimulus-dependent kinetics (Fig. 7
A). To prove that Akt activated by TS and/or IL-1ß induced a signaling cascade that modulates ß-catenin activation and COX-2 expression, we pretreated MCEC with the PI3K inhibitor LY294002 (2.5 µmol/l) for 1 h before stimulation. The rapid induction by TS or TS/IL-1ß of Akt and GSK-3ß phosphorylation, followed by ß-catenin translocation into the nucleus with subsequent COX-2 expression, was almost entirely suppressed by treatment of MCEC with LY294002 (Fig. 7B, C, D
). Moreover, pretreatment with the PI3K inhibitor completely abolished the increase in endothelial monolayer permeability induced by TS/IL-1ß (Fig. 7E
). Taken together, these data support the hypothesis that activation of PI3K by TS/IL-1ß increases endothelial monolayer permeability, induces Akt activation, and promotes GSK-3ß-phosphorylation. The consequent inhibition of GSK-3ß activity contributes to increased levels of nuclear ß-catenin, favoring increased COX-2 expression.
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TS/IL-1ß modulated nuclear ß-catenin translocation and COX-2 expression by EGFR/AKT pathway
We previously have shown in epithelial cells that TS modulated COX-2 expression by direct activation of EGFR (18)
. We therefore tested whether TS-induced EGFR activation in MCEC affected translocation of ß-catenin to the nucleus. TS and/or IL-1ß stimulated phosphorylation of EGFR in MCEC (Fig. 8
A). Importantly, an inhibitor of EGFR tyrosine kinase activity (0.5 µmol/l AG1478) incubated with MCEC for 1 h before exposure to TS and/or IL-1ß, blocked the phosphorylation of Akt and GSK-3ß (Fig. 8B
), nuclear ß-catenin accumulation (Fig. 8B, C
), and COX-2 expression (Fig. 8D
). These results are consistent with a role of the EGFR in inducing ß-catenin translocation to nucleus by TS and demonstrate that activation of the Akt/GSK-3ß pathway is required for this effect.
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Exposure of mice to cigarette smoke increased Akt/GSK-3ß phosphorylation, nuclear ß-catenin accumulation, COX-2 expression, and interleukin-1ß serum levels
The studies presented above indicate that TS alone or in association with IL-1ß induces the expression of COX-2 in cultured endothelial cells via the Akt/GSK-3ß/ß-catenin pathway. To assess the in vivo relevance of these in vitro observations of TS effects, we examined activation of this pathway and COX-2 expression in cardiovascular tissues (CT) of mice exposed to cigarette smoke. When ApoE–/– mice were exposed or sham-exposed to cigarette smoke for 1 h daily for 15 days, body weight was unaffected. Statistically significant increases in Akt (168% increase, P <0.01), GSK-3ß phosphorylation (237% increase, P <0.01) (Fig. 9
A, C), and ß-catenin (288% increase, P<0.05) were associated with enhanced ß-catenin accumulation in the nuclear fraction (Fig. 9B, C
) and increased expression of COX-2 protein (63% increase, P<0.05) in CT of smoke exposed ApoE–/–mice compared with control mice. Immunohistochemical staining confirmed decreased phospho-ß-catenin and increased expression of COX-2 in aortic endothelial cells of smoker mice (Fig. 9D
). Moreover, a significant increase in serum interleukin-1ß (IL-1ß) was detected in smoke-treated mice compared to controls (45% increase, P<0.05) (Fig. 9E
), confirming published data that smoking induces this cytokine (2)
. Taken together, our data suggest thatexposing live animals to cigarette smoke induces activation of the Akt/GSK-3ß pathway, accumulation of nuclear ß-catenin, and up-regulation of COX-2 expression in cardiovascular tissue. In addition, the serum level of IL-1ß increases in smoker mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2
, 8
, 25)
. The ability of cigarette smoke to induce vascular inflammation appears fundamental to the broad effects of smoking on vascular pathophysiology (1)
. Activation of inflammatory genes that may be induced by smoke is necessary for the cascade of events that culminates in erosion of fibrous plaques and rupture of atheromatous plaques, but the mechanisms involved in vascular response to smoke are likely to be quite complex. In the present study, we have explored the mechanisms underlying effects of cigarette smoke on vascular endothelium by focusing on activation of two genes, ß-catenin and COX-2, that play key roles in inflammation and vascular remodeling, crucial events contributing to cardiovascular disease. We hypothesized that cigarette smoke can directly or via cooperation with proinflammatory cytokines induce changes in these genes that affect vascular responses.
We showed, both in vitro and in vivo, that cigarette smoke exposure resulted in enhanced translocation of ß-catenin to the nucleus and up-regulation of COX-2 expression in vascular cells. Up-regulation of nuclear ß-catenin is a novel molecular target of cigarette smoke that has not previously been reported. ß-Catenin, a key membrane component of cell-cell interactions, also acts as a coactivator of TCF/LEF and promotes expression of many genes, such as cyclin D1 (6)
, vascular endothelial growth factor (VEGF) (26)
, and COX-2 (8
9
10
11)
that affect vascular proliferation and function. Here we show that TS-mediated COX-2 induction requires nuclear ß-catenin translocation, as documented by its inhibition after cellular transfection with ß-catenin siRNA. Our findings that ß-catenin activation increased COX-2 expression and activity suggest a previously unrecognized mechanism by which smoke favors atherogenesis. PGE2 generation facilitated by increased COX-2 in turn can increase the expression and activity of many different genes, such as MMP-9 (27)
, angiopoietin (28)
, and c-myc (29)
, which are associated with matrix degradation, remodeling, angiogenesis, and vascular cell proliferation. These processes contribute to the instability of atherosclerotic plaques as well as to a variety of other pathologies, ranging from joint damage in rheumatoid arthritis to tumor cell invasion (27
, 28
, 30)
.
ß-Catenin translocation to the nucleus involves its phosphorylation on serine/threonine and tyrosine residues: decreased serine/threonine phosphorylation of ß-catenin deregulates its turnover, destabilizes the VE-cadherin adhesion complex in the plasma membrane, and facilitates nuclear translocation of ß-catenin and subsequent increases in gene transcription (31)
. Tyrosine phosphorylation of ß-catenin can result in its dissociation from VE-cadherin and loss of association with cytoskeleton, promoting reduced cell-cell adhesion. Biological consequences of disrupting the membrane complexes between VE-cadherin and ß-catenin at endothelial cell contacts include increased vascular permeability (32)
, promoting infiltration of plasma lipids into the subendothelium, the first step of atherogenesis. Increased monocyte penetration into the vascular wall would also be expected but has not been examined here. It has been reported that smoking induced a considerable change in endothelial morphology of the aorta, consisting of the formation of blebs, microvillus-like projections, increased number of plasmalemmal vesicles and an increase in the number of Weibel-Palade bodies in vivo (33)
. Moreover, another report documented a dramatic, yet reversible, contraction of endothelial cells that was observed after treatment with low concentration of cigarette smoke in vitro (34)
. The spatial opening of gaps between endothelial cells (via cell contraction) or endothelial morphological changes led to atherosclerosis-relevant endothelial denudation of the blood vessels and to aggregation of endothelial cells with consequent acceleration of atherosclerosis.
Here we have demonstrated that exposure to TS induces disruption of VE-cadherin/ß-catenin membrane complexes with increase of transendothelial permeability, and diminishes the serine/threonine phosphorylation and degradation of ß-catenin. Thus two different effects of smoking on ß-catenin trafficking appear to promote its accumulation first in the cytoplasm and then in the nucleus. Activation of PI3K signaling pathways results in induction of endothelial monolayer permeability and in inactivation of GSK-3ß via its Akt-mediated phosphorylation. We showed that TS both induced activation of Akt and inactivation of GSK-3ß, effects that implicate this pathway as responsible for the nuclear accumulation of ß-catenin. Moreover, an independent pharmacological inhibitor of PI3K decreased endothelial monolayer permeability and Akt activation mediated by TS, promoted GSK-3ß activation, enhanced phosphorylation and degradation of ß-catenin, and reduced nuclear ß-catenin accumulation and COX-2 expression. Our findings are consistent with a recent study in epithelial cells (35)
, demonstrating that PI3K inhibitors regulated Akt activity induced by two components of smoke. Although in one study transfection of HUVEC with constitutively active Akt did not influence cytosolic ß-catenin levels or ß-catenin/Tcf/LEF activation (36)
, we consider that these differing results may reflect different approaches used to induce Akt activation. Because Wnt signaling results in inhibition of GSK-3ß activity, it might be interesting to test if Wnt signaling is involved in these effects of smoke on MCEC. Importantly, we showed that NS398, a pharmacological inhibitor of COX-2 activity, initially did not alter TS-mediated increased transendothelial permeability but became effective after 30 min of treatment with smoke (Supplemental Fig. 2). These results suggest that endothelial permeability changes in response to TS are initially modulated by PI3K activation, which also induces activation of the Akt/ß-catenin pathways as well as COX-2 expression and activity, while later permeability changes are regulated by COX-2-mediated production of prostaglandins.
Our data also provide evidence for a role of EGFR as a mediator of ß-catenin function in TS-exposed MCEC. We previously showed that TS induces EGFR slow time frame phosphorylation in oral epithelial cells (18)
. Here, we demonstrated for the first time in endothelial cells that a rapid TS-mediated activation of EGFR resulted in the cellular redistribution of ß-catenin, particularly into the nuclear fraction, and up-regulation of COX-2 expression. Correspondingly, inhibition of EGFR activity reduced phosphorylation of AKT and GSK-3ß, decreased nuclear ß-catenin activity, and decreased COX-2 detectable in these cells after treatment with TS. The precise mechanisms underlying TS-mediated activation of EGFR/Akt/GSK-3ß and translocation of ß-catenin remain to be clarified. Reactive oxygen species (ROS) are known to increase EGFR phosphorylation (37)
, so that smoke-derived increases in superoxide may activate EGFR to modulate the ß-catenin pathway. Here, we exclude that unstable/volatile compounds (likely ROS) already present in the TS might be implicated in EGFR phosphorylation. Fresh TS and aged TS (incubated for 18 h at 37°C) both equally induced activation of EGFR. (Supplemental Fig. 3). However, it remains possible that intracellular ROS produced after TS came into contact with the cells was responsible for mediating increased phosphorylation of EGFR.
Our hypothesis that inflammatory conditions can amplify and accelerate vascular dysfunction induced by smoke is confirmed by the data presented here. What is of interest is that low concentrations of TS that alone are minimally effective interact with IL-1ß to induce COX-2 expression in MCEC (similar findings were observed with TNF-
, data not shown). Moreover, low TS concentrations combined with low IL-1ß concentrations increased both the rate and extent of EGFR/Akt/ß-catenin/COX-2 signaling, leading to earlier and greater production of PGE2.
In vivo, the mechanisms by which smoking modulates inflammatory genes in endothelial cells may be even more complex that those we described in our in vitro studies.
One recent report had shown that in ApoE–/– mice exposed daily to secondhand smoke (SHS) experience macrophage infiltration into the aorta by 21 days. The fact that C57 wild-type mice exposed to SHS developed cardiovascular dysfunction later than did ApoE–/– mice (25)
suggests that smoking also interacts with other risk factors to promote atherosclerosis. We deliberately smoke-exposed young (9 wk) ApoE–/– mice (which do not develop atherosclerotic lesions until>15 wk age when fed standard chow) to mainstream smoke for only 15 days to study the early effects of smoking on vascular responses before the appearance of established cardiovascular disease. We observed that up-regulation of phosphorylation of Akt and GSK-3ß, induction of nuclear ß-catenin accumulation and COX-2 expression in cardiovascular tissue, and increased IL-1ß serum levels were all detected in ApoE–/– mice that were killed immediately after the last session of smoke exposure. Even 1 wk of smoke exposure of mice has been shown to increase IL-1ß and TNF- in mouse bronchoalveolar lavage fluids (38)
. We hypothesized that, in vivo, soluble smoke-components present in the blood may interact with as well as increase these (and perhaps other) cytokines (2
, 23
, 24)
to activate the signaling pathways described. As circulating cytokine levels poorly reflect tissue levels, this process could reconcile the difference between the amount of IL-1ß detected in the serum of smoker mice (10 ng/l) and the concentration used in the in vitro study (2 µg/l).
The early vascular expression (15 days) of COX-2 after smoking that we documented compared with the later induction of COX-2 (60 days) with nicotine administration detected by Lau et al. (2)
is not surprising. TS prepared from low nicotine cigarettes (Quest 3) also interacted with IL-1ß to induce Akt phosphorylation and COX-2 expression in MCEC (data not shown). We can speculate that many different chemical components present in cigarette smoke rather than nicotine alone may interact to promote cardiovascular damage.
In conclusion, our in vivo experiments provide evidence that smoking increases Akt activation, nuclear ß-catenin accumulation, and COX-2 expression in cardiovascular tissue associated with decreased GSK-3ß activation and ß-catenin degradation. Complementary in vitro experiments demonstrate that smoke-induced activation of EGFR/Akt/GSK-3ß pathways increases nuclear ß-catenin accumulation and that nuclear ß-catenin was necessary for smoke-mediated modulation of COX-2 expression and activity. Concomitantly, smoke-induced translocation of ß-catenin resulted in enhanced permeability of endothelial monolayers, a process that could enhance lipid infiltration. Interaction between smoke and inflammatory cytokines promoted both of these processes. These data suggest a novel mechanism by which smoking contributes to the initiation and evolution of cardiovascular disease.
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ACKNOWLEDGMENTS |
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Received for publication October 16, 2006. Accepted for publication January 11, 2007.
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P < 0.05 vs. IL-1ß.
