Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2002;91:806-813
Published online before print October 3, 2002, doi: 10.1161/01.RES.0000040397.23817.E5
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
91/9/806    most recent
01.RES.0000040397.23817.E5v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Saura, M.
Right arrow Articles by Lowenstein, C. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Saura, M.
Right arrow Articles by Lowenstein, C. J.
Related Collections
Right arrow Cell signalling/signal transduction
Right arrow Endothelium/vascular type/nitric oxide
(Circulation Research. 2002;91:806.)
© 2002 American Heart Association, Inc.

Molecular Medicine

Smad2 Mediates Transforming Growth Factor-ß Induction of Endothelial Nitric Oxide Synthase Expression

Marta Saura, Carlos Zaragoza, Wangsen Cao, Clare Bao, Manuel Rodríguez-Puyol, Diego Rodríguez-Puyol, Charles J. Lowenstein

From the Department of Physiology (M.S., M.R.-P.), Universidad de Alcalá, Madrid, Spain; Unidad de Nefrología (D.R.-P.), Hospital Universitario "Principe de Asturias," Madrid, Spain; Fundacion Centro Nacional de Investigaciones Cardiovasculares (CNIC) (C.Z.), Madrid, Spain; and the Division of Cardiology, Department of Medicine (W.C., C.B., C.J.L.), The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Charles Lowenstein, Division of Cardiology, Department of Medicine (W.C., C.B., C.J.L.), The Johns Hopkins University School of Medicine, Baltimore, MD 21205e-mail: clowenst{at}jhmi.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowRegulation of eNOS Expression
 down arrowTGF-ß and Vascular...
 down arrowTGF-ß Signal...
 down arrowTGF-ß Regulates eNOS
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Transforming growth factor-ß (TGF-ß) increases expression of endothelial nitric oxide synthase (eNOS), although the precise mechanism by which it does so is unclear. We report that Smad2, a transcription factor activated by TGF-ß, mediates TGF-ß induction of eNOS in endothelial cells. TGF-ß induces Smad2 translocation from cytoplasm to nucleus, where it directly interacts with a specific region of the eNOS promoter. Overexpression of Smad2 increases basal levels of eNOS, and further increases TGF-ß stimulation of eNOS expression. Ectopic expression of Smurf, an antagonizer of Smad2, decreases Smad2 expression and blocks TGF-ß induction of eNOS. Because Smad2 can interact with a variety of transcription factors, coactivators, and corepressors, Smad2 may thus act as an integrator of multiple signals in the regulation of eNOS expression.


Key Words: endothelial cell • hypoxia • atherosclerosis


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowRegulation of eNOS Expression
 down arrowTGF-ß and Vascular...
 down arrowTGF-ß Signal...
 down arrowTGF-ß Regulates eNOS
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Although endothelial nitric oxide synthase (eNOS or NOS3) was originally described as a constitutive NOS, subsequent work showed that eNOS expression is regulated by a variety of physiological and pathophysiological factors.1–6 Expression of eNOS is increased by exercise, shear stress, hypoxia, hyperthyroidism, cirrhosis, and endothelial cell proliferation.7–10 Various mediators can also increase eNOS expression, including estrogens, angiotensin II, glucose, oxidized linoleic acid, lysophosphatidylcholine, hydrogen peroxide, and transforming growth factor-ß.1–6 Expression of eNOS is decreased by hypoxia, inflammation, and pulmonary hypertension.11–14 Mediators that decrease eNOS expression include oxidized LDL, lipopolysaccharide, and tumor necrosis factor-{alpha}.


*    Regulation of eNOS Expression
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Regulation of eNOS Expression
down arrowTGF-ß and Vascular...
 down arrowTGF-ß Signal...
 down arrowTGF-ß Regulates eNOS
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The level of eNOS expression is determined by multiple mechanisms, including posttranscriptional regulation15–18 and posttranslational regulation.19–36 Expression of eNOS is also regulated at the transcriptional level. The 5'-flanking region of the eNOS gene lacks a TATA box, but contains elements that mediate constitutive expression of genes in endothelial cells, including Sp1 sites and GATA.37–42 The 5'-flanking region also contains many putative sites for further transcriptional regulation of eNOS. However, only a few of these sites have been formally shown to regulate eNOS transcription, including PEA3 and AP-1 binding sites.43,44 Finally, a region of the eNOS promoter extending from -1269 and -935 mediates TGF-ß induction of eNOS transcription.45


*    TGF-ß and Vascular Pathophysiology
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 *TGF-ß and Vascular...
down arrowTGF-ß Signal...
 down arrowTGF-ß Regulates eNOS
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
TGF-ß belongs to a superfamily of dimeric growth factors, including bone morphogenetic proteins and activins.46–48 TGF-ß regulates diverse biological processes, including cell proliferation, differentiation, and migration. TGF-ß also regulates cellular functions within the cardiovascular system, including angiogenesis, fibrosis, and production of extracellular matrix.


*    TGF-ß Signal Transduction and Smads
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 *TGF-ß Signal...
down arrowTGF-ß Regulates eNOS
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
After its secretion and activation, TGF-ß forms a complex with type I and type II TGF-ß receptors on target cells. Once bound to TGF-ß, the type II TGF-ß receptor phosphorylates the type I receptor, and the type I receptor in turn phosphorylates transcription factors known as Smads. Smad family members mediate TGF-ß signal transduction, and include receptor Smads (Smad2 and Smad3) that associate with and are activated by TGF-ß receptors, common Smads (Smad4) that interact with receptor Smads, and inhibitory Smads (Smad6 and Smad7) that block receptor Smad signaling.46,49–51 The TGF-ß type I receptor phosphorylates Smad2, which then disassociates from the receptor, binds to Smad4, and enters the nucleus. This Smad complex then interacts with other transcription factors, coactivators, and corepressors to modulate gene transcription.52


*    TGF-ß Regulates eNOS
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 up arrowTGF-ß Signal...
 *TGF-ß Regulates eNOS
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
TGF-ß regulates eNOS through pathways that are partially understood.45,53 Harrison and colleagues45 previously showed that TGF-ß regulates eNOS expression in part by activating NF-1 to bind to the eNOS promoter region -1269 and -935. We hypothesized that the Smad signal transduction pathway also plays a role in mediating the effects of TGF-ß on eNOS transcription. We now show that TGF-ß induction of eNOS is mediated in part by Smad2 interacting directly with a specific region of the eNOS promoter.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 up arrowTGF-ß Signal...
 up arrowTGF-ß Regulates eNOS
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Recombinant human TGF-ß was purchased from R&D Systems, Oxon, UK. The human eNOS promoter was a generous gift of Dr Thomas Michel (Brigham and Women’s Hospital, Boston, Mass).39 The Smurf cDNA was a kind gift from Dr X.-H. Feng (Baylor College of Medicine, Houston, Tex).

Cell Culture
Bovine aortic endothelial cells (BAECs) and HUVECs were isolated using methods previously described.9 Human aortic endothelial cells (HAECs) were from Clonetics. Studies were performed on confluent monolayers at passages 2 to 7, made quiescent by serum deprivation with 0% calf serum for 16 hours.

Immunofluorescence
To analyze the cellular localization of Smad2, we cultured BAECs, fixed cells with 4% (v/v) paraformaldehyde, permeabilized them with Triton X-100, incubated them with antibody to Smad2 for 1 hour, washed then 3 times with phosphate buffered saline, and incubated them with antibody to mouse IgG conjugated to FITC (Jackson Immunoresearch). Cells were then photographed with a fluorescent microscope.

Construction of Reporter Plasmids
The 1.6-kb fragment of the eNOS 5'-flanking region was a generous gift from Dr Thomas Michel (Brigham and Women’s Hospital, Boston, Mass). An eNOS promoter reporter plasmid was constructed by inserting the 1.6-kb fragment of the human eNOS promoter upstream of the firefly luciferase gene in the plasmid pGL3, as described previously.9 This promoter was designated -1624 eNOS-Luc. Deletion mutants of the eNOS promoter were generated by polymerase chain reaction (PCR), as described previously.9

eNOS Promoter Activity
Cell transfection was performed using methods modified from those previously reported.9 Photinus luciferase activity was determined using a luminometer and normalized against Renilla activity, using the Dual luciferase reporter kit (Promega), and normalized as relative luciferase light units/mg protein.

Electrophoretic Mobility Shift Assay
Proteins were prepared from nuclear extracts as described previously.54 Nuclear extracts from HAECs were incubated with radiolabeled eNOS promoter fragment at 22°C for 30 minutes. The mixture was then electrophoresed through a nondenaturing polyacrylamide gel. The gel was dried and autoradiographed.

Southwestern Blotting and Immunoblotting
Immunoblotting was performed as described previously.9 For Southwestern blotting, nuclear extracts (50 µg of total protein) from BAECs were electrophoresed through a SDS-10% polyacrylamide gel, transferred to a membrane, denatured with 6 mol/L guanidine, washed, and renatured with decreasing concentrations of guanidine HCl. The membrane was then blocked with 5% dry milk and incubated with [32P]end-labeled fragment of the eNOS promoter. The membrane was then washed and autoradiographed.

Statistical Analysis
Data for promoter activity were analyzed by analysis of variance and Newman-Keuls post hoc testing. Results are expressed as mean±SEM, with a value of P<0.05 considered statistically significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 up arrowTGF-ß Signal...
 up arrowTGF-ß Regulates eNOS
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
TGF-ß Stimulates eNOS Expression
To show that TGF-ß increases eNOS expression in human endothelial cells, we treated HAECs with 2 ng/mL of TGF-ß, and analyzed total RNA by Northern blotting with a radiolabeled eNOS cDNA probe. Densitometry was used to assess relative changes in signal intensity. TGF-ß increases steady-state eNOS mRNA levels after 4 to 8 hours of treatment (Figure 1A). TGF-ß increases steady-state eNOS mRNA levels both in proliferating endothelial cells by about 1.7-fold (Figure 1A, top) and confluent endothelial cells by about 3.6-fold (Figure 1A, bottom). Simultaneous treatment of HAECs with TGF-ß and cyclohexamide (CHX) show that the effect of TGF-ß on eNOS mRNA does not require new protein synthesis (Figure 1B).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. TGF-ß increases eNOS expression. A, TGF-ß increases steady-state levels of eNOS mRNA. Subconfluent (top) and confluent (bottom) HAECs were incubated with 2 ng/mL TGF-ß for 0 to 16 hours. Total RNA was analyzed by Northern blotting with a radiolabeled eNOS probe. B, New protein synthesis is not required for TGF-ß effects on levels of eNOS mRNA. HAECs were incubated for 16 hours with TGF-ß and cyclohexamide (CHX). Total RNA was analyzed by Northern blotting with a radiolabeled eNOS probe. C, TGF-ß increases steady-state eNOS protein levels. HUVECs were incubated with TGF-ß for 0 to 48 hours. Total protein was analyzed by immunoblotting with an antibody to eNOS. These experiments were repeated 3 to 5 times with similar results.

TGF-ß also increases steady-state levels of eNOS protein. Lysates of HUVECs treated with TGF-ß were analyzed by immunoblotting with antibody to eNOS. TGF-ß increases steady-state eNOS protein levels after 8 hours of treatment and produces a maximum level of eNOS protein 16 hours after treatment, with an increase over baseline of approximately 15-fold (Figure 1C).

TGF-ß Transactivates the eNOS Promoter
We next explored the ability of TGF-ß signaling to transactivate the eNOS promoter. Endothelial cells were transfected with a reporter vector, consisting of the 1624 bp of the human eNOS promoter upstream of the AUG start site, driving expression of the Photinus luciferase gene. (Cells were also transfected with a vector constitutively expressing Renilla luciferase, as an internal control for transfection efficiency.) Transfected cells were then treated with vehicle alone or TGF-ß, and the amount of luciferase activity was measured. (Promoter transactivation is calculated as Photinus luciferase/Renilla luciferase/mg cell protein.) The eNOS promoter is activated at basal levels in both BAECs and HUVECs treated with vehicle alone (Figure 2A). TGF-ß increases the transactivation of the eNOS promoter in BAECs and HUVECs (Figure 2A).



View larger version (19K):
[in this window]
[in a new window]
  		Figure 2. TGF-ß transactivates the eNOS promoter. A, TGF-ß transactivates the full-length eNOS promoter. BAECs (left) and HUVECs (right) were transfected with the full-length eNOS promoter, treated with TGF-ß or vehicle, and luciferase activity was measured in cell lysates with a luminometer. B. A region of the eNOS promoter mediates TGF-ß transactivation of the eNOS promoter. BAECs were transfected with deletions of the eNOS promoter-reporter vector, treated with TGF-ß, and luciferase activity in cell lysates was measured. C, TGF-ß transactivates both the wild-type eNOS promoter and a mutant eNOS promoter lacking the NF-1 binding site. HAECs were transfected with the wild-type eNOS promoter-reporter construct (left) or an eNOS promoter lacking an NF-1 binding site (right), treated with TGF-ß or vehicle, and luciferase activity was measured. n=3±SEM with *P<0.005 for TGF-ß vs vehicle.

To determine the region of the eNOS promoter that mediates activation by TGF-ß signaling, we next transfected BAECs with various reporter constructs containing 5' deletions in the eNOS promoter upstream of Photinus luciferase. Transfected cells were then treated with TGF-ß or vehicle, and the amount of luciferase activity was measured. TGF-ß transactivates the -1500 to 0 bp region of the eNOS promoter (Figure 2B). TGF-ß also transactivates the -1300 to 0 and the -1000 to 0 region of the eNOS promoter. However, TGF-ß cannot transactivate the -720 to 0 region of the eNOS promoter. Thus, TGF-ß induction of the eNOS promoter is mediated by the region of the eNOS promoter extending from -1000 to -720 bp.

TGF-ß increases eNOS expression in part by inducing NF-1 to interact with an NF-1 element in the eNOS promoter.45 The human eNOS promoter contains two NF-1 sites, at -1219 to -1191 and at -947 to -935. Deletion of the region from -1300 to -1000 containing the -1219 to -1191 NF-1 site in the human eNOS promoter causes a slight decrease in the responsiveness to TGF-ß, and deletion of the region from -1000 to -720 containing the -947 to -935 NF-1 site abolishes the response to TGF-ß (Figure 2B). However, site-directed mutagenesis of the -947 to -935 NF-1 element of the eNOS promoter has a modest effect on TGF-ß transactivation of the eNOS promoter (Figure 2C).

TGF-ß Activates eNOS Promoter Binding Activity
To further explore the role of the -1000 to -720 bp region of the eNOS promoter in mediating TGF-ß regulation of the eNOS promoter, we performed an electrophoretic mobility shift assay. HAECs were treated with media alone or TGF-ß, nuclear extracts were isolated and incubated with radiolabeled eNOS promoter fragment extending from -1000 to -720, fractionated by SDS-PAGE, and autoradiographed. TGF-ß activates a protein that interacts with the eNOS promoter fragment (Figure 3A).



View larger version (38K):
[in this window]
[in a new window]
  		Figure 3. TGF-ß induces eNOS promoter binding activity. A, Gel-shift assay. HAECs were treated with TGF-ß, and nuclear extracts were incubated with radiolabeled eNOS promoter probe (bp -1000 to -720). Cold eNOS competitor probe or cold NF-1 element probe or antibody to Smad2 or antibody to NF-1 was added to some mixtures (right lanes). Mixture was fractionated by PAGE and autoradiographed. Results are representative of two separate experiments. B, Southwestern assay shows proteins interact with eNOS promoter. BAECs were treated with TGF-ß, and nuclear extracts were collected, fractionated by SDS-PAGE, and transferred to a membrane. Proteins on the membrane were renatured and hybridized with a radiolabeled fragment (-1000 to -720 bp) of the eNOS promoter probe (left). eNOS promoter fragment interacts with a 58-kDa polypeptide. Membranes containing fractionated nuclear extracts were also hybridized with both radiolabeled eNOS promoter probe and 50-fold excess nonlabeled eNOS promoter probe (right). Results are representative of 3 different experiments.

To confirm that the -1000 to -720 bp fragment of the eNOS promoter mediates TGF-ß transactivation of the eNOS promoter, we performed a Southwestern binding assay. BAECs were treated with TGF-ß or vehicle alone, and nuclear extracts were prepared, fractionated by SDS-PAGE, and transferred to a membrane. Proteins on the membrane were renatured by sequential washes in buffer containing decreasing amounts of guanidine. The membrane was then incubated with a radiolabeled -1000 to -720 bp fragment of the eNOS promoter, washed, and autoradiographed. The eNOS promoter -1000 to -720 bp fragment binds to a polypeptide that appears in the nucleus 2 to 6 hours after TGF-ß treatment (Figure 3B, left panel). This binding activity is competed by nonlabeled eNOS promoter fragment (Figure 3B, right panel). Thus TGF-ß leads to the appearance in the nucleus of a polypeptide of approximately 55 kDa that interacts with a specific region of the eNOS promoter.

TGF-ß Activates Smad2 in Endothelial Cells
Because Smad2 is a component of the TGF-ß signaling cascade, we next tested the ability of TGF-ß to activate Smad2 in endothelial cells. One hallmark of Smad2 activation is its translocation to the nucleus. We therefore treated BAECs with TGF-ß, and then analyzed the localization of Smad2 in cells by immunofluorescence with an antibody to Smad2. Smad2 is located in the cytoplasm of resting BAECs, and TGF-ß treatment causes transient translocation of Smad2 into the nucleus (Figure 4A). Smad2 then disappears from the nucleus 4 hours after TGF-ß treatment (not shown). To confirm these results, we performed immunoblotting with antibody to Smad2 of cytosolic and nuclear extracts from BAECs treated with TGF-ß. Smad2 is located in the cytosol but not the nucleus of resting BAECs, and Smad2 disappears from the cytosol and appears in the nucleus of cells within 2 hours of treatment with TGF-ß (Figure 4B). TGF-ß thus induces a translocation of Smad2 from the cytosol into the nucleus of endothelial cells.



View larger version (66K):
[in this window]
[in a new window]
  		Figure 4. TGF-ß activates nuclear translocation of Smad2. A, BAECs were treated with 2 ng/mL TGF-ß for 0 to 2 hours, and the subcellular location of Smad2 was analyzed by immunofluorescence with an antibody to Smad2. B, BAECs were treated with 2 ng/mL TGF-ß for 0 to 2 hours, and nuclear extracts were prepared and analyzed by immunoblotting with an antibody to Smad2. Results are representative of 2 separate experiments.

Smad2 Stimulates Basal eNOS Expression and TGF-ß-Stimulated Expression
We next explored the effect of Smad2 on eNOS expression. We first examined Smad2 expression in control and transfected cells. Smad2 is absent from the nucleus of resting cells, and TGF-ß increases nuclear levels of Smad2 (Figure 5A, left). Transfection of BAECs with a FLAG-Smad2 expression vector increases Smad2 expression. FLAG-Smad2 is present in the nucleus of resting cells transfected with FLAG-Smad2 expression vector, and TGF-ß increases nuclear levels of FLAG-Smad2 (Figure 5A, right).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 5. Smad overexpression transactivates eNOS promoter. A, Immunoblot with antibody to Smad2 of nuclear extracts from BAECs transfected with Smad2 expression vector. B, Luciferase assay of BAECs cotransfected with eNOS promoter-luciferase reporter vector and Smad2 expression vector. n=3±SEM. with *P<0.005 for vehicle vs TGF-ß and **P<0.005 for control vector vs Smad vector. C, Immunoblot of BAECs transfected with Smad2 expression vector, using antibody to eNOS or antibody to ß-tubulin as negative control.

Overexpression of Smad2 increases eNOS promoter transactivation. BAECs were cotransfected with empty vector or with vector-expressing FLAG-Smad2, and also with the eNOS promoter-luciferase reporter vector. Overexpression of Smad2 leads to transactivation of the eNOS promoter even in the absence of TGF-ß (Figure 5B, vehicle-treated Control versus vehicle-treated Smad2). Furthermore, TGF-ß increases the absolute level of transactivation of the eNOS promoter when Smad2 is overexpressed (Figure 5B, TGF-ß-treated Control versus TGF-ß-treated Smads). However, TGF-ß treatment increases eNOS promoter activity over the activity in vehicle-treated cells, by approximately the same percentage in control or Smad2-overexpressing cells.

Finally, we measured the effect of Smad2 overexpression on endogenous eNOS expression. Steady-state protein levels of eNOS are higher in BAECs transfected with a Smad2 expression vector than in nontransfected cells (Figure 5C). TGF-ß treatment increases eNOS protein levels even higher in BAECs overexpressing Smad2 than in control transfected BAECs (Figure 5C). Smad2 overexpression had no effect on steady-state levels of ß-tubulin, demonstrating the specificity of TGF-ß and Smad2 for regulation of eNOS expression (Figure 5C). These data all suggest that Smad2 increases eNOS expression.

Smad2 Interacts With eNOS Promoter
Because Smad2 transactivates the eNOS promoter and increases eNOS expression, we hypothesized that Smad2 interacts directly with the eNOS promoter. We performed an electrophoretic mobility shift assay to test this hypothesis. The addition of antibody to Smad2 blocks the ability of nuclear extracts to interact with the eNOS promoter fragment (Figure 3A). In addition, antibody to NF-1 affects the interaction of nuclear proteins with the eNOS promoter fragment, decreasing the intensity of a lower but not an upper band (Figure 3A).

A Southwestern binding assay confirmed our hypothesis that Smad2 interacts with the eNOS promoter region from -1000 to -720. Nuclear extracts from BAECs were fractionated by SDS-PAGE, transferred to a membrane, renatured, and then hybridized with a radiolabeled eNOS promoter -1000 to -720 bp fragment. Control BAECs do not contain binding activity for the eNOS promoter fragment (Figure 6, lane 1). TGF-ß treatment of BAECs induces a polypeptide in the nucleus with eNOS promoter binding activity (Figure 6, lane 2). However, immunodepletion of these extracts with antibody to Smad2 abolishes this activity (Figure 6, lane 3). Finally, overexpression of Smad2 in TGF-ß-treated BAECs increases this eNOS promoter binding activity (Figure 6, lane 4). Thus, Smad2 (or a complex containing Smad2) interacts directly with the eNOS promoter.



View larger version (26K):
[in this window]
[in a new window]
  		Figure 6. Smad2 interaction with eNOS promoter. A, BAECs were transfected or not with Smad2 expression vector, and then treated or not with TGF-ß. Nuclear extracts were isolated, immunodepleted with antibody to Smad2 or not, fractionated by SDS-PAGE, transferred to a membrane, and renatured. Membrane was hybridized with a radiolabeled eNOS promoter fragment (-1000 to -720) and autoradiographed. B, Duplicate samples were immunoblotted with antibody to Smad2. Results are representative of 3 different experiments.

Smurf Interference With Smad2 Blocks TGF-ß Activation of eNOS Expression
If Smad2 mediates TGF-ß induction of eNOS expression, then interference with Smad2 should block the ability of TGF-ß to activate the eNOS promoter. Smurf-2 modulates TGF-ß signaling by activating the ubiquitination and destruction of Smad2.55 We therefore measured eNOS promoter activity in BAECs overexpressing Smurf-2. BAECs were cotransfected with the eNOS promoter reporter vector and with a vector overexpressing Smurf-2. Smad2 is present in nuclear extracts of control transfected BAECs (Figure 7A). Overexpression of Smurf-2 drastically reduces Smad2 steady-state protein levels, but overexpression of a mutant Smurf-2 has no effect on Smad2 levels (Figure 7A).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Smurf-2 expression decreases Smad2 levels and blocks TGF-ß transactivation of eNOS promoter. A, Smurf-2 decreases Smad2 levels. BAECs were cotransfected with a Smurf-2 expression vector and the eNOS promoter-luciferase reporter vector, and then TGF-ß or vehicle was added. Nuclear extracts were immunoblotted with antibody to Smad2. B, Smurf-2 expression blocks TGF-ß transactivation of eNOS promoter. BAECs were cotransfected as above with a Smurf-2 expression vector and an eNOS promoter-luciferase reporter vector, and then treated with vehicle or TGF-ß treatment. Luciferase activity was measured in cell extracts. Data are representative of 4 different experiments. *P<0.005 for TGF-ß vs vehicle; **P<0.005 for TGF-ß+Smurf-2 vs TGF-ß+control vector.

We then measured the effect of Smurf-2 on eNOS promoter activity. TGF-ß increases eNOS promoter transactivation in control cells, as before (Figure 7B, left). However, TGF-ß has no effect on the eNOS promoter when Smurf-2 is overexpressed (Figure 7B, middle). TGF-ß is able to transactivate the eNOS promoter when a mutant Smurf-2 is expressed (Figure 7B, right). These data support the hypothesis that Smad2 mediates TGF-ß induction of eNOS expression in endothelial cells.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 up arrowTGF-ß Signal...
 up arrowTGF-ß Regulates eNOS
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of this study is that Smad2 mediates TGF-ß regulation of eNOS expression. TGF-ß activates Smad2 in endothelial cells, which translocates from cytoplasm to nucleus, where it interacts with the eNOS 5'-flanking region. Overexpression of Smad2 increases basal eNOS expression, and greatly increases TGF-ß stimulation of eNOS expression. Finally, inhibition of Smad (by overexpression of Smurf) blocks TGF-ß transactivation of the eNOS promoter.

TGF-ß increases eNOS mRNA and protein levels within 4 to 8 hours of treatment (Figure 1). This rapid induction is consistent with the kinetics of phosphorylation and nuclear translocation of Smad2 after TGF-ß binding to the signaling receptor complex (Figure 4). TGF-ß induction of eNOS expression is independent of new protein synthesis (Figure 1B), which is also consistent with a signal transduction pathway involving a presynthesized messenger such as Smad2.

Our data show that Smad2 interacts with the eNOS promoter (Figures 3 and 6). However, there is no Smad consensus binding element within the eNOS promoter. Although consensus Smad binding sites are absent from the eNOS promoter, the DNA binding specificity of Smads can be regulated by various partners that interact with Smads, such as Sp-1 and forkhead activin signal transducer-1.56–58 Despite the lack of a Smad binding element in the eNOS 5'-flanking region, Smad2 may be directed to specific regions of the eNOS promoter by binding partners.

Prior work showed that NF-1 mediates part of the effect of TGF-ß on eNOS expression.45 Harrison and colleagues demonstrated that a region of the bovine eNOS promoter between -1269 to -935 is responsive to TGF-ß. This prior study also showed that NF-1 binds to an NF-1 element from -1034 to -1006 within the bovine eNOS promoter region, and that NF-1 mediates TGF-ß regulation of eNOS. The human eNOS promoter contains two potential NF-1 binding sites: one NF-1 element from -1219 to -1191 (corresponding to the bovine eNOS promoter -1034 to -1006), and a second NF-1 element from -947 to -935. Our data confirm the results of Harrison and colleagues45 that NF-1 plays a role in TGF-ß regulation of eNOS. Our deletion mutation experiments show that the eNOS promoter region -1300 to -1000 containing the NF-1 site studied by Harrison and colleagues (bovine promoter -1034 to -1006, corresponding to human promoter -1219 to -1191) mediates some of the effects of TGF-ß (Figure 2B). Our deletion experiments also show that the region containing the second NF-1 element, -1000 to -720, plays a more significant role in TGF-ß regulation of eNOS (Figure 2B). Gel-shift experiments show that both NF-1 and Smad2 interact with this region of the eNOS promoter (Figure 3A). In addition, our point mutation experiments show that the second NF-1 site between -947 to -935 also mediates some of the effects of TGF-ß (Figure 2C). Differences in the relative contributions of Smad2 and NF-1 to eNOS regulation in these two studies may be due in part to differences in bovine and human eNOS promoter sequences, and bovine and human endothelial cells. Thus, our data confirm the findings of Harrison and colleagues that NF-1 plays a role in TGF-ß regulation of eNOS.

Identification of Smad2 as a regulator of eNOS expression suggests that pathways outside of the TGF-ß signal transduction cascade can regulate eNOS as well. For example, shear stress activates Smad6, an inhibitory Smad, which can bind to Smad2 and prevent its transactivation of promoters.50 Shear stress can also activate MEKK-1, which in turn can activate Smad2 independent of TGF-ß.59 Pathways that modulate the coactivator CREB binding protein (CBP) might also regulate eNOS transcription, because Smad2 interacts with CBP within endothelial cells.52,60 Thus, Smad2 can serve as an integrator of multiple pathways that regulate eNOS expression.

Our findings might explain one aspect of the antiatherogenic properties of TGF-ß. Recent evidence suggests that TGF-ß inhibits atherogenesis in animals and humans.61,62 These antiatherosclerotic effects of TGF-ß may be mediated by multiple mechanisms. However, one possible pathway by which TGF-ß reduces atherosclerosis is by activation of Smad2, followed by an increase in eNOS expression and NO synthesis. Increased NO, produced by higher levels of eNOS induced by Smad2, might in turn inhibit pathways leading to atherosclerosis.


*    Acknowledgments
 
This work was supported by grants from the Spanish Ministry (to M.S.), Spanish Government (to M.R.-P., DGES PM-97-0067; to D.R.-P., CICYT SAF-0054), and IRSIN (to D.R.-P. and M.R.-P.). This research was also supported by an American Heart Association Established Investigator Grant EIG 0140210N, National Institutes of Health grants P01 HL56091, P01 HL65608, NIH R01 HL53615, R01 HL63706, by the Ciccarone Center for the Prevention of Heart Disease, and by the Cora and John H. Davis Foundation (all to C.J.L.).


*    Footnotes
 
This manuscript was sent to Elizabeth G. Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received March 13, 2002; revision received September 6, 2002; accepted September 20, 2002.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowRegulation of eNOS Expression
 up arrowTGF-ß and Vascular...
 up arrowTGF-ß Signal...
 up arrowTGF-ß Regulates eNOS
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Harrison DG, Venema RC, Arnal JF, Inoue N, Ohara Y, Sayegh H, Murphy TJ. The endothelial cell nitric oxide synthase: is it really constitutively expressed? Agents Actions Suppl. 1995; 45: 107–117.[Medline] [Order article via Infotrieve]
  2. Forstermann U, Boissel JP, Kleinert H. Expressional control of the "constitutive" isoforms of nitric oxide synthase (NOS I and NOS III). FASEB J. 1998; 12: 773–790.[Abstract/Free Full Text]
  3. Harrison DG, Sayegh H, Ohara Y, Inoue N, Venema RC. Regulation of expression of the endothelial cell nitric oxide synthase. Clin Exp Pharmacol Physiol. 1996; 23: 251–255.[Medline] [Order article via Infotrieve]
  4. Moncada S. Nitric oxide in the vasculature: physiology and pathophysiology. Ann N Y Acad Sci. 1997; 811: 60–67;discussion 67–69.
  5. Michel T, Feron O. Nitric oxide synthases: which, where, how, and why? J Clin Invest. 1997; 100: 2146–2152.[Medline] [Order article via Infotrieve]
  6. Papapetropoulos A, Rudic RD, Sessa WC. Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res. 1999; 43: 509–520.[Abstract/Free Full Text]
  7. Sessa WC, Pritchard K, Seyedi N, Wang J, Hintze TH. Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression. Circ Res. 1994; 74: 349–353.[Abstract/Free Full Text]
  8. Topper JN, Cai J, Falb D, Gimbrone MA, Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996; 93: 10417–10422.[Abstract/Free Full Text]
  9. Arnet UA, McMillan A, Dinerman JL, Ballermann B, Lowenstein CJ. Regulation of endothelial nitric-oxide synthase during hypoxia. J Biol Chem. 1996; 271: 15069–15073.[Abstract/Free Full Text]
  10. Arnal JF, Yamin J, Dockery S, Harrison DG. Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth. Am J Physiol. 1994; 267: C1381–C1388.[Medline] [Order article via Infotrieve]
  11. Liao JK, Zulueta JJ, Yu FS, Peng HB, Cote CG, Hassoun PM. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest. 1995; 96: 2661–2666.[Medline] [Order article via Infotrieve]
  12. McQuillan LP, Leung GK, Marsden PA, Kostyk SK, Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol. 1994; 267: H1921–H1927.[Medline] [Order article via Infotrieve]
  13. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med. 1995; 333: 214–221.[Abstract/Free Full Text]
  14. Rudic RD, Sessa WC. Nitric oxide in endothelial dysfunction and vascular remodeling: clinical correlates and experimental links. Am J Hum Genet. 1999; 64: 673–677.[CrossRef][Medline] [Order article via Infotrieve]
  15. Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem. 1998; 273: 24266–24271.[Abstract/Free Full Text]
  16. Laufs U, La Fata V, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation. 1998; 97: 1129–1135.[Abstract/Free Full Text]
  17. Yoshizumi M, Perrella MA, Burnett JC Jr, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993; 73: 205–209.[Abstract]
  18. Alonso J, Sanchez de Miguel L, Monton M, Casado S, Lopez-Farre A. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor-{alpha}. Mol Cell Biol. 1997; 17: 5719–5726.[Abstract]
  19. Busconi L, Michel T. Endothelial nitric oxide synthase: N-terminal myristoylation determines subcellular localization. J Biol Chem. 1993; 268: 8410–8413.[Abstract/Free Full Text]
  20. Michel T, Li GK, Busconi L. Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1993; 90: 6252–6256.[Abstract/Free Full Text]
  21. Robinson LJ, Michel T. Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting. Proc Natl Acad Sci U S A. 1995; 92: 11776–11780.[Abstract/Free Full Text]
  22. Robinson LJ, Busconi L, Michel T. Agonist-modulated palmitoylation of endothelial nitric oxide synthase. J Biol Chem. 1995; 270: 995–998.[Abstract/Free Full Text]
  23. Robinson LJ, Ghanouni P, Michel T. Posttranslational modifications of endothelial nitric oxide synthase. Methods Enzymol. 1996; 268: 436–448.[Medline] [Order article via Infotrieve]
  24. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996; 271: 6518–6522.[Abstract/Free Full Text]
  25. Igarashi J, Thatte HS, Prabhakar P, Golan DE, Michel T. Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci U S A. 1999; 96: 12583–12588.[Abstract/Free Full Text]
  26. Gallis B, Corthals GL, Goodlett DR, Ueba H, Kim F, Presnell SR, Figeys D, Harrison DG, Berk BC, Aebersold R, Corson MA. Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. J Biol Chem. 1999; 274: 30101–30108.[Abstract/Free Full Text]
  27. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature. 1999; 399: 601–605.[CrossRef][Medline] [Order article via Infotrieve]
  28. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature. 1999; 399: 597–601.[CrossRef][Medline] [Order article via Infotrieve]
  29. Yeh DC, Duncan JA, Yamashita S, Michel T. Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca2+-calmodulin. J Biol Chem. 1999; 274: 33148–33154.[Abstract/Free Full Text]
  30. Igarashi J, Michel T. Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae: eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction. J Biol Chem. 2000; 275: 32363–32370.[Abstract/Free Full Text]
  31. Dimmeler S, Dernbach E, Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett. 2000; 477: 258–262.[CrossRef][Medline] [Order article via Infotrieve]
  32. Luo Z, Fujio Y, Kureishi Y, Rudic RD, Daumerie G, Fulton D, Sessa WC, Walsh K. Acute modulation of endothelial Akt/PKB activity alters nitric oxide-dependent vasomotor activity in vivo. J Clin Invest. 2000; 106: 493–499.[Medline] [Order article via Infotrieve]
  33. Igarashi J, Bernier SG, Michel T. Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase: differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells. J Biol Chem. 2001; 276: 12420–12426.[Abstract/Free Full Text]
  34. Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem. 2001; 276: 16587–16591.[Abstract/Free Full Text]
  35. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res. 2001; 88: e68–e75.[CrossRef][Medline] [Order article via Infotrieve]
  36. Morales-Ruiz M, Lee MJ, Zollner S, Gratton JP, Scotland R, Shiojima I, Walsh K, Hla T, Sessa WC. Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells. J Biol Chem. 2001; 276: 19672–19677.[Abstract/Free Full Text]
  37. Marsden PA, Heng HH, Scherer SW, Stewart RJ, Hall AV, Shi XM, Tsui LC, Schappert KT. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem. 1993; 268: 17478–17488.[Abstract/Free Full Text]
  38. Venema RC, Nishida K, Alexander RW, Harrison DG, Murphy TJ. Organization of the bovine gene encoding the endothelial nitric oxide synthase. Biochim Biophys Acta. 1994; 1218: 413–420.[Medline] [Order article via Infotrieve]
  39. Robinson LJ, Weremowicz S, Morton CC, Michel T. Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene. Genomics. 1994; 19: 350–357.[CrossRef][Medline] [Order article via Infotrieve]
  40. German Z, Chambliss KL, Pace MC, Arnet UA, Lowenstein CJ, Shaul PW. Molecular basis of cell-specific endothelial nitric-oxide synthase expression in airway epithelium. J Biol Chem. 2000; 275: 8183–8189.[Abstract/Free Full Text]
  41. Tang JL, Zembowicz A, Xu XM, Wu KK. Role of Sp1 in transcriptional activation of human nitric oxide synthase type III gene. Biochem Biophys Res Commun. 1995; 213: 673–680.[CrossRef][Medline] [Order article via Infotrieve]
  42. Zhang R, Min W, Sessa WC. Functional analysis of the human endothelial nitric oxide synthase promoter: Sp1 and GATA factors are necessary for basal transcription in endothelial cells. J Biol Chem. 1995; 270: 15320–15326.[Abstract/Free Full Text]
  43. Cieslik K, Lee CM, Tang JL, Wu KK. Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A. J Biol Chem. 1999; 274: 34669–34675.[Abstract/Free Full Text]
  44. Navarro-Antolin J, Rey-Campos J, Lamas S. Transcriptional induction of endothelial nitric oxide gene by cyclosporine A: a role for activator protein-1. J Biol Chem. 2000; 275: 3075–3080.[Abstract/Free Full Text]
  45. Inoue N, Venema RC, Sayegh HS, Ohara Y, Murphy TJ, Harrison DG. Molecular regulation of the bovine endothelial cell nitric oxide synthase by transforming growth factor-ß1. Arterioscler Thromb Vasc Biol. 1995; 15: 1255–1261.[Abstract/Free Full Text]
  46. Wrana JL. Regulation of Smad activity. Cell. 2000; 100: 189–192.[CrossRef][Medline] [Order article via Infotrieve]
  47. Massague J, TGF-ß signal transduction. Annu Rev Biochem. 1998; 67: 753–791.[CrossRef][Medline] [Order article via Infotrieve]
  48. Massague J, Blain SW, Lo RS. TGFß signaling in growth control, cancer, and heritable disorders. Cell. 2000; 103: 295–309.[CrossRef][Medline] [Order article via Infotrieve]
  49. Massague J, Wotton D. Transcriptional control by the TGF-ß/Smad signaling system. EMBO J. 2000; 19: 1745–1754.[CrossRef][Medline] [Order article via Infotrieve]
  50. Topper JN, Cai J, Qiu Y, Anderson KR, Xu YY, Deeds JD, Feeley R, Gimeno CJ, Woolf EA, Tayber O, Mays GG, Sampson BA, Schoen FJ, Gimbrone MA Jr, Falb D. Vascular MADs: two novel MAD-related genes selectively inducible by flow in human vascular endothelium. Proc Natl Acad Sci U S A. 1997; 94: 9314–9319.[Abstract/Free Full Text]
  51. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Jr, Wrana JL, Falb D, The MAD-related protein Smad7 associates with the TGFß receptor and functions as an antagonist of TGFß signaling. Cell. 1997; 89: 1165–1173.[CrossRef][Medline] [Order article via Infotrieve]
  52. Topper JN, DiChiara MR, Brown JD, Williams AJ, Falb D, Collins T, Gimbrone MA, Jr. CREB binding protein is a required coactivator for Smad-dependent, transforming growth factor ß transcriptional responses in endothelial cells. Proc Natl Acad Sci U S A. 1998; 95: 9506–9511.[Abstract/Free Full Text]
  53. Ying WZ, Sanders PW. Dietary salt enhances glomerular endothelial nitric oxide synthase through TGF-ß1. Am J Physiol. 1998; 275: F18–F24.[Medline] [Order article via Infotrieve]
  54. Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991; 19: 2499.[Free Full Text]
  55. Zhu H, Kavsak P, Abdollah S, Wrana JL, Thomsen GH. A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation. Nature. 1999; 400: 687–693.[CrossRef][Medline] [Order article via Infotrieve]
  56. Pardali K, Kurisaki A, Moren A, ten Dijke P, Kardassis D, Moustakas A. Role of Smad proteins and transcription factor Sp1 in p21(Waf1/Cip1) regulation by transforming growth factor-ß. J Biol Chem. 2000; 275: 29244–56.[Abstract/Free Full Text]
  57. Zhou S, Zawel L, Lengauer C, Kinzler KW, Vogelstein B. Characterization of human FAST-1, a TGF-ß and activin signal transducer. Mol Cell. 1998; 2: 121–127.[CrossRef][Medline] [Order article via Infotrieve]
  58. Labbe E, Silvestri C, Hoodless PA, Wrana JL, Attisano L. Smad2 and Smad3 positively and negatively regulate TGF ß-dependent transcription through the forkhead DNA-binding protein FAST2. Mol Cell. 1998; 2: 109–120.[CrossRef][Medline] [Order article via Infotrieve]
  59. Brown JD, DiChiara MR, Anderson KR, Gimbrone MA Jr, Topper JN. MEKK-1, a component of the stress (stress-activated protein kinase/c-Jun N-terminal kinase) pathway, can selectively activate Smad2-mediated transcriptional activation in endothelial cells. J Biol Chem. 1999; 274: 8797–8805.[Abstract/Free Full Text]
  60. DiChiara MR, Kiely JM, Gimbrone MA Jr, Lee ME, Perrella MA, Topper JN. Inhibition of E-selectin gene expression by transforming growth factor ß in endothelial cells involves coactivator integration of Smad and nuclear factor {kappa}B-mediated signals. J Exp Med. 2000; 192: 695–704.[Abstract/Free Full Text]
  61. McCaffrey TA. TGF-ß and TGF-ß receptors in atherosclerosis. Cytokine Growth Factor Rev. 2000; 11: 103–114.[CrossRef][Medline] [Order article via Infotrieve]
  62. Grainger DJ, Witchell CM, Metcalfe JC. Tamoxifen elevates transforming growth factor-ß and suppresses diet-induced formation of lipid lesions in mouse aorta. Nat Med. 1995; 1: 1067–1073.[CrossRef][Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 BloodHome page
A. Alfranca, J. M. Lopez-Oliva, L. Genis, D. Lopez-Maderuelo, I. Mirones, D. Salvado, A. J. Quesada, A. G. Arroyo, and J. M. Redondo
PGE2 induces angiogenesis via MT1-MMP-mediated activation of the TGF{beta}/Alk5 signaling pathway
Blood, August 15, 2008; 112(4): 1120 - 1128.
[Abstract] [Full Text] [PDF]

Home page
 HypertensionHome page
V. C. Sandrim, A. C.T. Palei, I. F. Metzger, V. A. Gomes, R. C. Cavalli, and J. E. Tanus-Santos
Nitric Oxide Formation Is Inversely Related to Serum Levels of Antiangiogenic Factors Soluble Fms-Like Tyrosine Kinase-1 and Soluble Endogline in Preeclampsia
Hypertension, August 1, 2008; 52(2): 402 - 407.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. Fernandez-Lopez, E. M. Garrido-Martin, F. Sanz-Rodriguez, M. Pericacho, A. Rodriguez-Barbero, N. Eleno, J. M. Lopez-Novoa, A. Duwell, M. A. Vega, C. Bernabeu, et al.
Gene expression fingerprinting for human hereditary hemorrhagic telangiectasia
Hum. Mol. Genet., July 1, 2007; 16(13): 1515 - 1533.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. D. Searles
Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression
Am J Physiol Cell Physiol, November 1, 2006; 291(5): C803 - C816.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Saura, C. Zaragoza, C. Bao, B. Herranz, M. Rodriguez-Puyol, and C. J. Lowenstein
Stat3 Mediates Interelukin-6 Inhibition of Human Endothelial Nitric-oxide Synthase Expression
J. Biol. Chem., October 6, 2006; 281(40): 30057 - 30062.
[Abstract] [Full Text] [PDF]

Home page