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Prepublished online as a Blood First Edition Paper on October 31, 2002; DOI 10.1182/blood-2002-02-0629.
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Blood, 15 March 2003, Vol. 101, No. 6, pp. 2253-2260
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Cellular response to hypoxia involves signaling via Smad
proteins
Hong Zhang,
Hasan O. Akman,
Eric L. P. Smith,
Jin Zhao,
Joanne E. Murphy-Ullrich M. A. Q. Siddiqui, and
Olcay A. Batuman
From the Department of Anatomy and Cell
Biology, the Division of Hematology/Oncology, the Department of
Medicine, the Center for Cardiovascular and Molecular Medicine, and the
Department of Psychiatry, State University of New York Downstate
Medical Center, Brooklyn, NY; and the Department of Pathology, The Cell
Adhesion and Matrix Research Center, University of Alabama at
Birmingham, Birmingham, AL.
 |
Abstract |
The transforming growth factor- (TGF-) family of cytokines
regulates vascular development and inflammatory responses. We have
recently shown that exposure of human umbilical vein endothelial cells
(HUVECs) to hypoxia (1% O2) increases gene expression and bioactivation of TGF-2 and induces its downstream effectors, Smad
proteins (Smads), to associate with DNA. In the present study, we show
that hypoxia-induced TGF-2 gene expression is dependent on
thrombospondin-1-mediated bioactivation of latent TGF-. Blocking TGF-2 but not TGF-1 in hypoxic endothelial cell cultures
inhibited induction of the TGF-2 gene, indicating that an autocrine
mechanism driven by bioactivation of TGF-2 leads to its gene
expression in hypoxic HUVECs. Exposure of HUVECs to hypoxia resulted in
phosphorylation and nuclear transportation of Smad2 and Smad3 proteins
as well as stimulation of transcriptional activities of Smad3 and the transcription factor hypoxia-inducible factor-1 and culminated in
up-regulation of TGF-2 gene expression. Autocrine regulation of
TGF-2 production in hypoxia may involve cross-talk between Smad3 and
HIF-1 signaling pathways, and could be an important mechanism by
which endothelial cells respond to hypoxic stress.
(Blood. 2003;101:2253-2260)
© 2003 by The American Society of Hematology.
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Introduction |
The transforming growth factor- (TGF-) family
of growth factors mediates vascular development and regulates
endothelial responses to mechanical, inflammatory, and hypoxic
stress.1-10 The important role of TGF- in vascular
physiology is indicated by defective vasculogenesis and striking
vascular inflammation leading to death in mice null for TGF-s, their
receptors, or their downstream substrates, the Smad
proteins.3,7,11,12 We recently have shown that exposure of
human umbilical vein endothelial cells (HUVECs) to hypoxia (1%
O2) selectively up-regulates transcription and expression
of TGF-2 by as much as 20-fold and induces Smad2, Smad3, and Smad4
to associate with DNA.9
In vascular endothelium, TGF-2, similar to TGF-1 and TGF-3, is
produced in a latent form in which the bioactive, 25-kDa TGF- dimer (mature TGF-) is noncovalently bound to its propeptide (also known as latency-associated peptide [LAP]) and is unavailable for binding to TGF- membrane receptors.1 An important
physiologic regulator of TGF- bioactivation is thrombospondin-1
(TSP-1), an extracellular matrix protein that is a member of the TSP
family of glycoproteins.13-15 TSP-1, a trimer of
disulfide-linked 180-kDa subunits, is secreted from platelet
-granules, endothelial cells, and vascular smooth muscle cells,
and is deposited in extracellular matrix.16 Binding of
TSP-1 to LAP occurs via amino acid sequence K412RFK415 of TSP-1 and amino acid sequence
L54SKL57 of LAP,15,17 and
potentially induces a conformational change in LAP that allows
interaction of the 25-kDa mature TGF- peptide with its specific
membrane receptors. TSP-1 can activate LAPs associated with both latent
TGF-1 and -2,15 and similarities reported between
TGF-1-null and TSP-1-null animals17,18 suggest that
TSP-1-mediated TGF- bioactivation is physiologically significant.
Mature TGF- can bind to its type I, type II, and type III cell
membrane receptors, the first 2 of which are serine/threonine kinases.19 Once activated by TGF-, the type II receptor
transphosphorylates the type I receptor, which then phosphorylates
Smad2 or Smad3 (receptor-activated Smads [R-Smads]), which in turn
heteromerize with Smad4 (Co-Smad) to translocate to the nucleus. Smad
complexes accumulate in the nucleus, where they regulate gene
transcription by recruiting transcriptional coactivators or inhibitors
to DNA.19 This cross-talk created by the interplay between
Smads and other signaling pathways is largely responsible for the
diverse and context-specific effects of the TGF- family of proteins.
The Smad signaling pathway was recently shown to interact with the
transacting protein complex hypoxia-inducible factor-1 (HIF-1), which
is a well-characterized transcription factor complex that regulates
hypoxia-driven gene expression.20,21 HIF-1 binds DNA as a
heterodimer of 2 basic helix-loop-helix proteins, HIF-1 and the aryl
hydrocarbon receptor nuclear translocator (ARNT, or
HIF-1).22,23 Under normoxic conditions,
HIF-1 is rapidly degraded by E3 ubiquitin ligase
complex,24,25 whereas in hypoxia or in the
presence of transition metals and iron chelators, it is stabilized and
accumulates in the nucleus.25-30 Recent studies have shown
that under normoxic conditions, HIF-1 is targeted for proteasomal
degradation by the ubiquitination complex pVHL, the protein of the von
Hippel-Lindau (VHL) tumor suppressor gene and a component
of an E3 ubiquitin ligase complex. Hydroxylation of HIF-1's proline
residue(s) facilitates binding to VHL, which targets
HIF-1 for degradation by E3 ligase.31,32 Under hypoxic conditions, HIF-1's association with VHL is disrupted,
resulting in inhibition of ubiquitination and proteasomal degradation,
leading to accumulation of HIF-1 in hypoxic cells.29,30
In the present report, we show that exposure of endothelial cells to
hypoxia results in (a) TSP-1-dependent bioactivation of TGF-2; (b)
activation of Smad2 and Smad3; and (c) an additive interplay between
transcriptional activities of Smad3 and HIF-1.
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Materials and methods |
Cell isolation and culture
Primary HUVECs were isolated from segments of normal-term cords
by digestion with collagenase type II, and were pooled and cultured as
previously described.33-35 Cells derived from the same primary culture were used for each set of hypoxic or nonhypoxic culture
conditions in experiments performed in parallel. Each type of
experiment was repeated at least twice, each time with different
primary cell isolates unless otherwise indicated. For exposure to
hypoxia, confluent HUVECs were placed in an incubator (NAPCO 7301;
Precision Scientific, Chicago, IL) at 37°C in humidified 1%
O2/5% CO2/94% N2; for exposure to
nonhypoxic conditions, identically prepared cells were maintained in
standard culture conditions of 37°C in humidified 5%
CO2/95% room air as previously
described.9
Reagents
Recombinant human TGF-2, polyclonal rabbit anti-TGF-2,
monoclonal anti-TGF-1, and isoform-specific controls for rabbit IgG
and mouse IgG were purchased from R&D Systems (Minneapolis, MN).
Monoclonal antibody 133 against stripped TSP-1; peptide LSKL, representing amino terminus TGF-1 LAP amino acid residues 54 to 57;
and scrambled peptide SLLK were prepared as
described.12,14
Assay for bioactive TGF-
Bioactive TGF- levels were determined in HUVEC supernatants
before and after inhibition of TSP-1-mediated TGF- bioactivation. For this purpose, confluent HUVECs, cultured without serum, were exposed to normoxic or hypoxic conditions in the presence or absence of
10 µg/mL anti-TSP-1 monoclonal antibody 133, 28 µM LSKL peptide, 28 µM scrambled peptide SLLK, 10 µg/mL anti-TGF-2 antibody, or 10 µg/mL mouse IgG. After 18 hours, supernatants were collected, and
bioactive TGF- levels were quantitated by the well-established mink
lung epithelial cell (MLEC) bioassay36 within 2 hours of collection. TGF--responsive MLECs stably transfected with the expression construct p800neoLUC containing a truncated plasminogen activator inhibitor-1 promoter fused to the firefly luciferase reporter
gene were kindly supplied by Dr Daniel B. Rifkin (New York University
School of Medicine, New York, NY).37 Cells were maintained
in Dulbecco modified Eagle medium (BioWhittaker, Walkersville, MD)
containing 10% fetal calf serum, 2 mM L-glutamine, and
antibiotics (penicillin G, 100 U/mL; streptomycin, 100 µg/mL; and
geneticin, 250 µg/mL [Gibco BRL, Grand Island, NY]), and
levels of bioactive TGF- in HUVEC supernatants were determined as
previously described.9,36 Briefly, MLECs were detached
with trypsin, washed, plated at 1.6 × 105 cells per well
in a 2-mL volume in 24-well tissue culture plates (Costar, Cambridge,
MA), and allowed to attach for 18 hours at 37°C in 5%
CO2. Medium in the wells was replaced by 2 mL of the following, placed in triplicate wells: control medium, control medium
containing increasing concentrations of recombinant TGF-2 to
generate a standard curve, and 1:10 dilution of conditioned HUVEC
medium to measure bioactive TGF-. Incubations were continued for 18 hours at 37°C, and MLEC lysates from each well were prepared using
reporter lysis buffer (Luciferase Assay System; Promega, Madison, WI).
Luciferase activity was measured as relative light units (RLUs; model
TD-20/20 luminometer, Promega), which were converted to TGF-
activity (picomoles) using the TGF-2 standard curve.36
Total protein content of supernatants was quantitated using Bio-Rad
protein assay reagent (Bio-Rad, Hercules, CA),38 and
TGF-2 levels obtained by the luciferase assay from each well were
normalized to protein. Mean RLUs from triplicate wells (which were
within 5%-7% of one another) were converted to picomoles of TGF-.
This bioassay is sensitive to all 3 isoforms of TGF-, and because
measurement of bioactive TGF-s by enzyme-linked immunosorbent assay
(ELISA) is not yet reliably accomplished by existing commercial kits, isoform specificity of bioactive TGF- in our experiments was
determined by measuring TGF- in supernatants from HUVECs cultured in
the presence or absence of neutralizing anti-TGF-2 antibody as indicated.
RNase protection assay
After exposure to the indicated stimulus, cells were gently
rinsed twice with ice-cold phosphate-buffered saline X1 (PBS) and
scraped quickly into TRIzol (Gibco BRL) for isolation of total RNA
followed by RNase protection analysis using the RiboQuant MultiProbe
RNase protection assay system (PharMingen, San Diego, CA) as previously
described.9 Quantification of mRNA was done from
PhosphorImager (Molecular Dynamics, Sunnyvale, CA) analysis of
radioactive bands using ImageQuant software (Molecular Dynamics). To
control for differences in sample processing, hybridization signals in
each sample were divided by the signal for the ribosomal protein mRNA
(L32), which by independent analysis was found not to change with
exposure to hypoxia (not shown). For determining fold increases in mRNA
levels in hypoxic HUVECs compared with control, the TGF-2/L32 signal
ratio from treated HUVECs was divided by the ratio obtained from
nonhypoxic HUVECs.
Immunoprecipitation and Western blot analysis
Cultures of endothelial cells were exposed to hypoxic or
normoxic conditions for 10 minutes, 30 minutes, 1 hour, or 4 hours, or
were exposed to 100 pM TGF-2 for 30 minutes. At the end of the
culture period, HUVECs were washed twice with ice-cold PBS and lysed
using a Mammalian Cell Lysis Kit (Sigma, St Louis, MO) in the presence
of additional protease inhibitors (Complete Mini Protease Inhibitor
Cocktail Tablets; Roche Diagnostics, Indianapolis, IN). From each cell
lysate, 1 mL (containing 400-500 µg protein) was subjected to
immunoprecipitation with goat anti-Smad2 IgG (10 µg/mL; Santa Cruz
Biotechnology, Santa Cruz, CA) or 10 µg/mL goat anti-Smad3 IgG
(Santa Cruz Biotechnology) for 16 hours at 4°C with mixing, followed
by adsorption to protein G-Sepharose beads (Santa Cruz Biotechnology).
Immunoprecipitates were eluted from beads by boiling, separated by 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),
and transferred to polyvinylidenedifluoride membranes (Immobilon-P;
Millipore, Bedford, MA). Nonspecific binding was blocked by 5% nonfat
milk powder in PBS. Immunoblotting was performed by using 2 µg/mL
each of rabbit antiphosphoserine (Zymed Laboratories, South San
Francisco, CA) or rabbit anti-phospho-Smad2 (Upstate Biotechnology,
Lake Placid, NY), and rabbit anti-Smad2 IgG or rabbit
anti-Smad3 IgG (both from Zymed Laboratories). Horseradish peroxidase-linked donkey anti-rabbit IgG F(ab')2
(Amersham Pharmacia Biotech, Piscataway, NJ) was used as
secondary antibody at 1:4000 dilution. Immunodetection was
carried out by enhanced chemiluminescence (ECL Plus Western blotting
detection system; Amersham Pharmacia Biotech) and autoradiography.
Autoradiograms were scanned and bands were quantified by ImageQuant
software. Fold increases in Smad2 and Smad3 phosphorylation were
calculated by dividing the phospho-Smad2 or phosphoserine values by
their respective total Smad2 or Smad3 values.
Cellular immunofluorescence imaging
For assessing intracellular localization of Smad2 and Smad3,
HUVECs were seeded into gelatin-coated 8-well Permanox chamber slides
(Lab-Tek Chamber Slide System; Nalge Nunc International, Naperville,
IL) in triplicate and exposed to hypoxic or nonhypoxic conditions for
one hour, after which cells were washed with ice-cold PBS, fixed with
4% formaldehyde, permeabilized with 0.5% Triton X-100, and blocked
with 0.4% fetal calf serum. Incubation for 1 hour with primary
antibodies was done using 0.2 µg/mL rabbit anti-human Smad2 IgG
(Zymed Laboratories) or goat anti-human Smad3 IgG (Santa Cruz
Biotechnology) followed by the secondary antibodies, which
were, respectively, fluorescein isothiocyanate-conjugated donkey
anti-goat IgG (3 µg/mL) or rhodamine-conjugated donkey anti-goat
IgG (3 µg/mL; The Jackson Immunoresearch Laboratories, West Grove,
PA) as previously described.39 Negative controls included
secondary antibody alone or replacement of primary antibody with rabbit
IgG or goat IgG. To ensure translocation of Smads from cytoplasm to the
nucleus, cells were also cultured in the presence or absence of 100 pM
recombinant human TGF-2 (R&D Systems) for one hour.
Immunofluorescence microscopy was performed with a Radiance 2000 confocal laser scanning microscope (Bio-Rad, San Francisco, CA)
attached to an Axioskop 2 microscope (Carl Zeiss Microimaging,
Thornwood, NY). Quantitation was performed by scoring 300 HUVECs/slide
as showing predominantly nuclear or cytoplasmic immunofluorescence.
Images of 512 × 512 pixels were obtained and processed using Adobe
Photoshop 6.0 (Adobe Systems, Mountain View, CA).
Plasmid constructs
Plasmid construct pB2-77, containing a deletion mutant of the
human TGF-2 gene promoter ( 77 to +63 base pair [bp]) linked to a
chloramphenicol acetyltransferase (CAT) gene, was generously donated by
Dr S.-J. Kim (National Cancer Institute, Bethesda, MD).40
Expression vectors for Smad3 and Smad4 that contain the FLAG
epitope tag at the amino domain have been described
previously41 and were kindly supplied by Drs Rik Derynck
and Ying Zhang (University of California, San Francisco, CA). The
p3TP-Lux construct (the generous gift of Dr Joan Massagué,
Memorial Sloan-Kettering Cancer Center, New York, NY) is a
well-described artificial promoter construct that was designed to have
maximal responsiveness to TGF-.42 Expression plasmid
encoding human HIF-1 was generously donated by Dr Steven McKnight
(University of Texas Southwestern Medical Center, Dallas,
TX).43 The HIF-1-responsive EPO-luciferase construct (EPO-Lux), which has been previously
described,24,44 is the hypoxia-responsive enhancer from
the human erythropoietin gene (150-bp ApaI/PstI
fragment) and was the generous gift of Dr J. Caro (Jefferson
Medical College, Philadelphia, PA).
Transfection of recombinant plasmids and reporter gene
assays
For transient transfections, plasmids with indicated expression
vectors or corresponding empty constructs were introduced by
electroporation into HepG2 cells or early-passage (second or third)
HUVECs that were 70% confluent, as previously described.9 Plasmid cytomegalovirus -galactosidase (pCMV-gal; 0.5 µg; Promega) was included in each transfection as internal control.
When multiple plasmids were cotransfected, the total amount of DNA was
kept constant by supplementing the samples with empty expression
vectors. To ensure that the same amount of protein was analyzed,
protein concentration of each sample was determined by Bradford assay, and transfection efficiency was determined by -gal activity. CAT/-gal/protein or RLU/-gal/protein ratios for
hypoxic and nonhypoxic transfectants were compared to determine the
fold change in response to hypoxia, as previously
described.9
Quantitative polymerase chain reaction (PCR)
Fluorogenic 5' nuclease quantitative RT-PCR was performed as
described.45 Total RNA was extracted from A549
and human breast carcinoma cell line MDA-MB-468, and reverse
transcription of 2 µg total RNA to cDNA was done with a
RETROscript kit (Ambion, Austin, TX). The primer and probe sequences
for TGF-2, designed with Primer Express 2.0 software (Applied
Biosystems, Foster City, CA), were as follows: forward,
5'-GACCAACCGGCGGAAGA-3'; reverse, 5'-CAGCAATTATCCTGCACATTTCTAA-3';
probe, 6FAMCGTGCTTTGGATGCGGCCTATTGTAMRA. The primers and probes for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from
Applied Biosystems. Agarose gel electrophoresis was used to verify that
the PCR products corresponded to the size predicted for the amplified
fragment. Standard curves of TGF-2 and GAPDH (not shown) were
prepared with first-strand cDNA, and the mean amount of TGF-2
obtained from triplicate samples was normalized to GAPDH RNA for the
same sample.
Statistical analysis
Student t test, 2-tailed, was used to assess
differences between data from hypoxic and normoxic culture conditions.
Means are presented ± 1 SD. Significance level was set at
P  .05.
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Results |
We previously have shown that exposure of HUVECs to hypoxia
stimulates TGF-2 gene expression at a transcriptional level and causes a parallel induction in TGF- production and bioactivation in
these cells.9 Since the TGF- family of proteins can
regulate their own transcription and synthesis,40 it is
possible that hypoxia-driven up-regulation of TGF-2 gene expression
is part of an autocrine pathway triggered by hypoxia-induced
bioactivation of the TGF- ligand. Since transcript and protein
levels of TSP-1 have been shown to be increased in hypoxic endothelial
cells in a time course parallel to hypoxic induction of the TGF-2
gene,46 we tested whether hypoxia-induced bioactivation of
TGF- was TSP-1-dependent.
As shown in Figure 1A, the level of
bioactive TGF- was 15.0-fold ± 1.0-fold higher in supernatants
from hypoxic HUVECs compared with supernatants from nonhypoxic cells
(P < .05). In contrast, exposure of HUVECs to hypoxia in
the presence of either anti-TSP-1 antibody or the peptide LSKL, both
of which are known inhibitors of TGF- activation,14
reduced bioactive TGF- levels in supernatants by 54.0% ± 5.8%
and 60.7% ± 1.3%, respectively (P < .05 for both conditions), compared with the increase observed in control (untreated) hypoxic HUVECs (leftmost bar). Neutralization of TGF-2 by exposure of HUVECs to hypoxia in the presence of specific antibody caused an
equivalent (54% ± 2.6%; P < .05) inhibition in
bioactive TGF- levels compared with supernatants from control
untreated HUVECs, whereas there was no change in supernatants
from HUVECs treated with mouse IgG (P < .05 in both
conditions; Figure 1A). This latter observation is consistent with the
possibility that at least 50% of total bioactive TGF- in
supernatants from hypoxic HUVECs consists of TGF-2. Interestingly,
the increase in bioactive TGF- levels in the presence of control
scrambled peptide SLLK (1.6-fold ± 0.9-fold) was also significant.
However, a corresponding increase in TGF-2 mRNA levels was not
observed in these HUVECs. Whether the effect of SLLK is directed
specifically to bioactivation of TGF- isoforms other than TGF-2
will be determined by measuring bioactive TGF- in supernatants from
HUVECs cultured in the presence of SLLK and neutralizing antibodies to
both TGF-1 and TGF-3 in hypoxic conditions.
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| Figure 1.
Mechanisms responsible for hypoxia-induced activation of
TGF-2 gene expression in HUVECs.
(A) TSP-1-dependent increases in bioactive TGF-2 levels in hypoxic
HUVEC supernatants. Confluent HUVECs, cultured without serum, were
exposed to 20% or 1% O2 in the presence or absence of 10 µg/mL anti-TSP-1 monoclonal antibody 133; 28 µM LSKL peptide,
corresponding to TSP-1-binding amino acid residues 54-57 from TGF-1
LAP; 28 µM scrambled peptide SLLK; 10 µg/mL anti-TGF-2
antibody; or 10 µg/mL mouse IgG. After 18 hours, supernatants were
collected, and bioactive TGF- levels were quantitated by MLEC
bioassay. Results from 3 independent experiments are expressed as fold
change (±SD) in bioactive TGF- levels in hypoxic HUVECs. Values
were derived by dividing the levels obtained from hypoxic supernatants
by each condition's normoxic counterpart (not shown). Asterisks
indicate significant differences (P .05, Student
t, 2-tailed) from the untreated condition (leftmost bar).
(B) TSP-1-dependent increases in TGF-2 mRNA levels in hypoxic
HUVECs. Confluent HUVECs, cultured without serum, were exposed to 20%
or 1% O2 in the presence or absence of 10 µg/mL
anti-TSP-1 monoclonal antibody 133, 28 µM LSKL (inhibitory) peptide,
28 µM SLLK (scrambled) peptide, 10 µg/mL anti-TGF-2 antibody,
10 µg/mL anti-TGF- type II receptor (anti-TGF- RII) antibody,
10 µg/mL mouse IgG, or 10 µg/mL anti-TGF-1 antibody for 18 hours and then subjected to total RNA extraction. For RNase protection
assays, 10 µg total RNA was hybridized to an antisense RNA probe
cocktail containing the templates for TGF-2 and L-32 (ribosomal
protein subunit) genes. Representative results from 1 of 3 independent
experiments are shown in the top panel, in which protected fragments of
TGF-2 and L-32 mRNAs are indicated by arrows. The bottom panel
presents quantification of results from the 3 experiments. TGF-2
signal was normalized to that from L32. Each bar represents the
increase in mRNA levels from hypoxic HUVECs compared with their own
normoxic controls at 18 hours. Asterisks indicate significant
differences (P .05, Student t, 2-tailed)
from the untreated condition (leftmost bar). N indicates
normoxic conditions; H, hypoxic conditions.
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The degree to which hypoxia's effect on bioactivation of LAP might
up-regulate TGF-2 gene expression was examined by inhibiting TSP-1-mediated TGF- activation in hypoxic HUVECs. As shown in Figure 1B, addition of either monoclonal antibody to TSP-1 or the
inhibitory peptide LSKL produced reductions in hypoxia-induced increases in TGF-2 mRNA levels (by 58.9% ± 9.8% and
65.2% ± 5.4%, respectively; P < .05 for both).
Likewise, reducing available receptor ligand by addition of
neutralizing antibody to TGF-2 decreased hypoxia-induced
up-regulation of TGF-2 mRNA levels by 83.0% ± 6.3%
(P < .05). Similar decreases (82.1% ± 3.6%;
P < .05) were observed when the TGF- type II receptor
was blocked by receptor antibody (Figure 1B).
In contrast, neutralization of TGF-1 in cultures by specific
antibody did not affect hypoxic induction of TGF-2 mRNA in HUVECs,
and in fact yielded identical results as treatment with mouse IgG
(Figure 1B), indicating that hypoxia-induced bioactivation of TGF-2,
but not of TGF-1, is responsible for autocrine up-regulation of
TGF-2 gene expression.
The effect of hypoxia on phosphorylation of Smad2 and Smad3
in HUVECs
In order to investigate the downstream effects of hypoxia-induced
stimulation of TGF-2 gene expression in HUVECs, the phosphorylation of Smad2 and Smad3 proteins under hypoxic conditions was undertaken. In
HUVECs exposed to hypoxia for periods of 10 minutes, 30 minutes, 1 hour, and 4 hours, levels of phosphorylated Smad2 increased by 2.1-fold
± 0.3-fold, 3.1-fold ± 0.1-fold, 2.8-fold ± 0.2-fold, and
2.0-fold ± 0.1-fold, respectively, compared with levels in nonhypoxic
HUVECs (P < .05 for all time points; Figure
2). Similarly, the level of
phosphorylated Smad3 in HUVECs exposed to hypoxia for 10 minutes, 30 minutes, 1 hour, and 4 hours was increased by 2.5-fold ± 0.2-fold,
3.1-fold ± 0.5-fold, 2.8-fold ± 0.2-fold, and 2.5-fold
± 0.1-fold, respectively, compared with nonhypoxic HUVECs
(P < .05 for all time points). In contrast, no change was observed in the levels of total Smad2 or Smad3 at any duration of
exposure to hypoxia (Figure 2). The effect of hypoxia-induced Smad2 and
Smad3 phosphorylation in HUVECs was essentially equivalent to that
observed after exposure to 100 pM TGF-2, a natural activator of
Smads (Figure 2B). This finding suggests that TGF-2 and hypoxia may
have comparable effects on Smad activation in vivo as well.
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| Figure 2.
Hypoxia-induced Smad2 and Smad3 phosphorylation in
HUVECs.
HUVECs were grown to confluence and were serum starved overnight,
followed by exposure to 100 pM TGF-2 for 30 minutes or to 1%
O2 (hypoxia) for 10 minutes, 30 minutes, 1 hour, 2 hours,
or 4 hours. As controls, untreated cells were simultaneously analyzed
at the same time points (not shown). (A) Lysates were prepared from
whole cells and quantified. Half were immunoprecipitated
(IP)with goat anti-Smad2 antibody (top panel) and half with
goat anti-Smad3 antibody (bottom panel) overnight at 4°C.
Immunoprecipitates and molecular weight markers (not shown) were
resolved by 10% SDS-polyacrylamide gels. Following electrophoresis and
blotting, the membranes were developed by means of rabbit antibodies
specific for the phosphorylated form of Smad2 (p-Smad2), total Smad2,
phosphoserine (p-Smad3), or total Smad3 followed by
chemiluminescence and autoradiography. Chemiluminescent bands were
quantified using ImageQuant software. Shown are representative results
from 1 of 3 independent experiments. (B) Hypoxia-induced fold increases
in Smad phosphorylation or in total Smad protein were calculated by
dividing the values for p-Smad2, p-Smad3, Smad2, and Smad3
found in hypoxic HUVECs by their respective normoxic control
values. The graph presents mean fold changes (±SD) from 3 independent experiments (*P < .05).
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The nuclear translocation of Smad2 and Smad3 (R-Smads) subsequent to
their hypoxia-induced phosphorylation was studied by immunofluorescence
using specific antisera. Treatment of HUVECs with 100 pM recombinant
TGF-2 under nonhypoxic conditions was used as a positive control. In
unstimulated HUVECs cultured under normoxic conditions, Smad2 (Figure
3A) and Smad3 (Figure 3D) were localized
predominantly in the cytoplasm, with less than 2% of HUVECs
showing nuclear staining for either Smad. The intensity of nuclear
staining for Smad2 and Smad3 in HUVECs exposed to hypoxia (Figure 3C,F)
was similar to that observed in HUVECs exposed to TGF-2 (Figure
3B,E). These data support the notion that the effect of hypoxia on
activation of Smad2 and Smad3 is mediated by TGF-2. This proposition
will be tested in future experiments by determination of nuclear
translocation of Smad2 and Smad3 in hypoxic HUVECs cultured in the
presence of antibody to TGF- isoforms.
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| Figure 3.
Hypoxia-induced Smad2 and Smad3 nuclear translocation in HUVECs.
HUVECs were grown to confluence on gelatin-coated chamber slides, serum
starved overnight, and exposed for 1 hour to 1% O2 or 20%
O2 with or without 100 pM recombinant TGF-2. After cells
were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton
X-100, immunostaining was performed with rabbit anti-Smad2 (10 µg/mL) followed by fluorescein isothiocyanate-conjugated donkey
anti-rabbit IgG (3 µg/mL); or rabbit anti-Smad3 (10 µg/mL),
followed by rhodamine-conjugated donkey anti-rabbit IgG (3 µg/mL).
Immunofluorescence microscopy was performed with a Bio-Rad Radiance
2000 Confocal laser scanning microscope. Immunostaining for Smad2 is
shown (A) in normoxic HUVECs, (B) after treatment with 100 pM TGF-2,
and (C) after exposure to hypoxia; immunostaining for Smad3 is shown
(D) in normoxic HUVECs, (E) after treatment with 100 pM
TGF-2, and (F) after exposure to hypoxia. Original magnification,
× 60.
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Downstream effects of Smad3 activation in hypoxia
Cooperation between transcriptional mechanisms involving Smad3 and
HIF-1 is suggested by their synergistic stimulation of vascular
endothelial growth factor transcription.20 We recently have shown9 that the region spanning 77 to 40 bp
within the 5' TGF-2 promoter, which harbors a Smad binding site and
2 HIF-1 binding sites, is necessary and sufficient to confer hypoxia
responsiveness to the 5' promoter spanning 778 to 40 bp and
containing cis-acting elements that regulate transcription
of the 5.8-kb TGF-2 transcript.40 To explore
downstream effects of hypoxia-induced phosphorylation and nuclear
translocation of Smad3 and its functional relationship to HIF-1,
transient transfection experiments were performed with TGF-2
promoter CAT construct pB2-77, which harbors the 5' TGF-2 promoter
spanning 77 to 63 bp (Figure 4A),
before and after overexpression of Smad3 and HIF-1 in HUVECs. Since
Co-Smad4 is necessary for nuclear translocation of Smad3, a
Smad4-overexpressing vector was also used. As shown in Figure 4B,
activity of this construct was increased by 4.8-fold ± 0.6-fold in
hypoxic conditions compared with that in normoxic conditions
(P < .05). As expected, co-overexpression of Smad3 and
Smad4 augmented the hypoxia-induced activity of pB2-77 by a further
7.4-fold ± 1.3-fold (P < .05) compared with hypoxic
HUVECs, which did not overexpress Smads (Figure 4B). When pB2-77 was
cotransfected with an expression vector coding for HIF-1, there was
a 6.3-fold ± 2.2-fold increase in hypoxia-induced activity of this
promoter construct. The highest activity of pB2-77 in response to
hypoxia was observed after co-overexpression of Smad3, Smad4, and
HIF-1, which resulted in a 14.6-fold ± 2.6-fold increase in
activity of pB2-77 compared with its baseline activity in hypoxic
HUVECs (P < .05). Thus, both Smad3 and HIF-1 are
sufficient to induce hypoxic responsiveness to the TGF-2 promoter,
and, in combination, their effects are additive.
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| Figure 4.
Effect of hypoxia on transcriptional activity of the
TGF-2 promoter.
(A) Schematic structure of recombinant plasmid construct pB2-77
containing the human TGF-2 gene promoter between 77 and +63 bp,
and that is linked to a CAT reporter gene. The transcriptional start
site (arrow) driving the CAT gene and selected protein-binding sites
(Smad, HIF-1) are indicated. (B) HUVECs were transfected with pB2-77,
and cotransfected with indicated Smad and HIF-1 expression vectors
and Rous sarcoma virus (RSV)--gal by electroporation. Each
transfection was divided into 2 plates and exposed to 20% or 1%
O2 for 36 hours. Representative results of 1 of 3 independent CAT assays are shown in the panel on the left. CAT activity
was determined in whole-cell extracts, and its level, given as percent
acetylation, was normalized to protein and to -gal activity in each
experiment. Results are expressed as fold increases (±SD) in CAT
activity in indicated hypoxic conditions compared with that found in
their normoxic counterparts for each transfection. Fold changes
observed in each experimental group are compared with the
hypoxia-induced fold change in HUVECs transfected with the pB2-77-CAT
alone (*P < .05).
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The effect of Smad3 and HIF-1 on hypoxia-induced increases in
TGF-2 mRNA levels
Based on their stimulatory effect on transcription from the
TGF-2 promoter-containing CAT construct, we asked whether either Smads or HIF-1 were necessary and/or sufficient for the occurrence of hypoxia-induced increases in TGF-2 mRNA levels in HUVECs. Because the absence of Smad4 impairs translocation of Smad3 to the nucleus and impedes its functions,9 we examined the
role of Smad3 on hypoxia-induced up-regulation of TGF-2 mRNA levels in Smad4-deficient and Smad4-replete epithelial cell lines. In real-time quantitative PCR experiments not shown here, we found that
overexpression of Smad3 alone did not increase TGF-2 mRNA levels in
hypoxic Smad4-deficient cells. However, co-overexpression of Smad3 and
Smad4 caused approximately 15% higher TGF-2 mRNA levels than were
seen in hypoxic cells overexpressing Smad4 alone (P < .05). We then cotransfected Smad3 as well as Smad4
into Smad4-deficient and control cell lines. As seen in Figure
5, hypoxia had no effect on TGF-2 mRNA
levels in the Smad4-null epithelial breast carcinoma cell line.
However, hypoxia induced a 4.8-fold ± 1.2-fold increase in TGF-2
mRNA in A549 cells, which do express Smad4. As expected, overexpression
of both Smad4 and Smad3 led to a 4.0-fold ± 0.8-fold increase in
hypoxia-induced TGF-2 mRNA levels in MDA-MB-468. Although overlap
between any of the functions of HIF-1 and Smad4 has not been
described, HIF-1 overexpression resulted in a 3.2-fold ± 0.9-fold
increase in TGF-2 mRNA levels in hypoxic MDA-MB-468 cells compared
with untransfected controls (P < .05), indicating that
both Smad3/Smad4 and HIF-1 independently stimulate TGF-2 mRNA
levels in response to hypoxia. Co-overexpression of Smad3, Smad4, and
HIF-1 resulted in an 8.5-fold ± 1.3-fold induction of TGF-2
mRNA levels, mirroring the additive effect of Smads and HIF-1 on
transcriptional activity of the TGF-2 promoter construct p2B-77
(Figure 4B), and suggesting that overexpression of HIF-1 provides at
least partial rescue for Smad4-null cells in hypoxic conditions. In
comparison, under normoxic conditions, TGF-2 mRNA levels were
similar in A549 and MDA-MB-468 cells (Figure 5). When Smad4-deficient
cells were transiently transfected with Smad3/Smad4, there was no
change in TGF- mRNA levels in normoxic conditions. While
overexpression of HIF-1 resulted in a 1.5-fold increase in TGF-2
mRNA levels, co-overexpression of Smad3/Smad4 in addition to HIF-1
resulted in a further increase to 2.5-fold in Smad4-deficient cells
(P < .05 for both; Figure 5). This modest induction of
TGF-2 mRNA levels in normoxia in MDA-MB-468 cells was equivalent to
that seen in control A549 epithelial cells (data not shown). Whether
overexpression of HIF-1 potentiates a signaling pathway downstream
of Smad4 or whether HIF-1 overexpression provides Co-Smad activity
for Smad3 remains to be determined.
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| Figure 5.
Smad3 and HIF-1 are independently capable of inducing
TGF-2 mRNA in response to hypoxia.
Smad4-deficient MDA-MB-468 and Smad4-expressing A549 were
transiently transfected with indicated Smad and HIF-1 expression
vectors and RSV--gal by electroporation. Each transfection was
divided into 2 plates and exposed to either 20% or 1% O2
for 18 hours. TGF-2 mRNA levels were determined by real-time RT-PCR
analysis, using TGF-2-specific TaqMan primers and probe. Amount of
mRNA in each group is normalized to GAPDH. Results are presented as
fold change in TGF-2 mRNA in normoxic ( ) or hypoxic ( )
conditions relative to the levels observed in cell line A549 under
basal normoxic conditions. Bars represent mean ±SD from 3 independent
experiments (*P < .05 for hypoxic and
**P < .05 for normoxic results).
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To explore the effect of Smad3 on HIF-1-mediated transcription in
hypoxic cells, we studied the effect of Smad3 overexpression on
hypoxia-induced transcription from the luciferase construct EPO-Lux, which contains an erythropoietin enhancer highly
responsive to activation by HIF-1 during hypoxia.24,44
Similar to other reports,24,44 activity of
EPO-Lux increased 34.4-fold ± 10.1-fold in hypoxic cells
compared with normoxic controls (Figure
6). Overexpression of Smad3 and Smad4
caused further 2.7-fold ± 0.6-fold and 3.6-fold ± 0.9-fold
increases in EPO-Lux activity, respectively, compared with
hypoxic baseline activity of this plasmid construct
(P < .05 for both). The results are consistent with the
notion that between Smad3 and HIF-1 signaling pathways, there is a
bidirectional stimulatory effect that possibly increases promoter
sensitivity to hypoxia. Whether this effect is due to the interaction
of Smad3 and HIF-1, as suggested by a recent report20
showing their coprecipitation in hypoxic cells, or is via independent
interactions of the proteins with DNA or other transcription factors
remains to be proved. In addition, transcriptional activity of
EPO-Lux was induced by 5-fold after Smad3 and Smad4
overexpression in normoxic conditions (Figure 6). Whether Smads play a
role in the activity of HIF-1 in nonhypoxic conditions remains to be
determined.
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| Figure 6.
Hypoxia-induced increases in transcription from
EPO-Lux are mediated by Smad proteins and HIF-1.
Confluent HepG2 cells were transfected with EPO-Lux, and
cotransfected with indicated Smad and HIF-1 expression vectors, or
empty vector and RSV--gal by electroporation. Each transfection was
divided into 2 plates and exposed to either 20% or 1% O2
for 24 hours. Luciferase activity was determined and results normalized
to RSV--gal activity and protein content of the extracts. Results
are expressed as fold increase in luciferase activity in hypoxic
conditions compared with that found in normoxic conditions for each
transfection. The figure presents the mean (±SD) of 3 independent
experiments (*P < .05).
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Discussion |
In HUVECs exposed to hypoxia, significant up-regulation of
TGF-2 gene expression was found to occur via an autocrine loop dependent upon TSP-1-mediated TGF-2 bioactivation, presented schematically in Figure
7. Furthermore, both
Smad3 and HIF-1 were shown to contribute to hypoxia-induced
transcriptional up-regulation of TGF-2 in endothelial cells. The
effects of proteolysis by plasmin, exposure to reactive oxygen species,
and binding to integrins,47,48 all of which bioactivate
TGF-, on hypoxia-induced elevation of TGF-2 mRNA levels remain to
be elucidated.
The blocking of TSP-1-mediated bioactivation of latent TGF-2 during
exposure to hypoxia inhibited up-regulation of TGF-2 mRNA in HUVECs.
It thus is likely that TGF-2 mediates at least some actions of TSP-1
on hypoxic endothelial cells. Exposure of endothelial cells to hypoxia
results in up-regulation of gene expression of TSP-1, thus inhibiting
neovascularization, in part by its dampening effect on endothelial
response to proangiogenic stimuli,16 and also by its
proapoptotic effects on endothelial cells via downstream signaling from
membrane receptor CD36.49 Inhibition of retinal
angiogenesis by interruption of TSP-1's activation of
TGF-50 suggests that bioactivation of TGF- may be
one mechanism responsible for TSP-1-induced antiangiogenesis. An
antiangiogenic function in vivo was suggested for TGF-2 based on its
inhibitory effect on hepatocyte growth factor-mediated angiogenesis,51 whereas experimental evidence
suggests that TGF-1 has proangiogenic52 and
proapoptotic effects.53,54 Since TGF-2 is only
minimally expressed in vascular endothelium under normoxic
conditions,55,56 the degree to which hypoxic induction of
TGF-2, as distinct from TGF-1 or TGF-3, is associated with
regulation of apoptosis and angiogenesis in endothelium has remained
unclear. The low level of expression of TGF-2 seen in normoxic
conditions in vascular endothelium56 suggests that TGF-2 gene expression and function may be more relevant under hypoxic conditions. Deletion of the TGF-2 gene did not result in the
severe vascular developmental abnormalities seen in deletions of the
genes encoding for Smad5,12 type I TGF-
receptor,10 endoglin,57-59 or
HIF-160 but resulted in perinatal death with cyanosis
and cardiac and pulmonary abnormalities.7 Similar to our
findings of hypoxia-induced Smad activation, the phosphorylation and
subsequent nuclear translocation of Smad proteins were reported to
occur after exposure of HUVECs to laminar shear,61
indicating that the Smad signaling pathway is an important mechanism by
which endothelial cells respond to stress.
Activity of the 5' TGF-2 promoter spanning 778 and +63 bp, which
regulates transcription of the 5.8-kb TGF-2 transcript, is
significantly increased by hypoxia, and within this region, sequences
spanning 77 to 40 bp are necessary and sufficient to mediate the
hypoxia response.9 Our current results show that activity
of the latter hypoxia-responsive promoter region is also increased in
an additive fashion by HIF-1 and Smad3/Smad4, indicating that
HIF-1 and Smad proteins have a potentiating effect on hypoxic
expression of TGF-2. The additive effect of |
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Abstract
Introduction