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Blood, 1 December 2001, Vol. 98, No. 12, pp. 3324-3331
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Response to hypoxia involves transforming growth factor- 2 and
Smad proteins in human endothelial cells
Hasan O. Akman,
Hong Zhang,
M. A. Q. Siddiqui,
William Solomon,
Eric L. P. Smith, and
Olcay A. Batuman
From the Division of Hematology/Oncology, Department of
Medicine, Center for Cardiovascular and Molecular Medicine, Department
of Psychiatry, State University of New York Downstate Medical Center,
Brooklyn, NY.
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Abstract |
Oxygen deprivation (hypoxia) is a consistent component of ischemia
that induces an inflammatory and prothrombotic response in the
endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O2) increases
messenger RNA and protein levels of transforming growth factor- 2
(TGF-2), a cytokine with potent regulatory effects on vascular
inflammatory responses. Messenger RNA levels of the TGF-2 type II
membrane receptor, which is a serine threonine kinase, also increased. The stimulatory effect of hypoxia was found to occur at the level of
transcription of the TGF-2 gene and involves Smad proteins, a class
of intracellular signaling proteins that mediates the downstream
effects of TGF- receptors. Transient transfection studies showed
that the region spanning  77 and 40 base pairs within the TGF-2
promoter (harboring a Smad-binding "CAGA box") is activated in
hypoxic cells compared with nonhypoxic controls (P < .01). Hypoxia also stimulated transcription from
another promoter, 3TP-Lux, a reporter construct responsive to Smads and TGF-. In addition, specific binding to a Smad-binding
oligonucleotide was observed with nuclear extracts from hypoxic
endothelial cells but not from nonhypoxic cells. It is concluded that
Smad proteins, which can regulate endothelial responses to mechanical
and inflammatory stress, also may play an important role in vascular
responses to hypoxia and ischemia.
(Blood. 2001;98:3324-3331)
© 2001 by The American Society of Hematology.
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Introduction |
One of the earliest responses of endothelium to
hypoxia is inflammation, and the balance between positive and negative
regulators of this inflammatory response is a determinant of
endothelial adaptation to hypoxia. Proinflammatory endothelial
responses induced by hypoxia involve production of cytokines,
chemokines,1-3 tissue factor,4 and
plasminogen activator inhibitor-1 (PAI-1).5 Recent studies
indicate that activation of the transcription factor early growth
response-1 (Egr-1) by hypoxia is responsible for the procoagulant
profile and vascular remodeling that ultimately result in the tissue
damage associated with ischemia.4 Adaptive responses to
hypoxia are also regulated at a transcriptional level, which is best
exemplified by activation of the transacting protein hypoxia-inducible
factor-1 (HIF-1), which stimulates differential expression of specific
genes and results in increases in glucose uptake, glycolytic enzyme
production, and angiogenesis.6,7
The transforming growth factor- (TGF-) family of growth factors
has an especially critical role in modulation of vascular inflammatory
responses and remodeling,8-10 and up-regulation of TGF-
production is an invariant response to vascular injury, indicating that
regulation of gene expression of the TGF- proteins by quiescent and
injured endothelium is likely to be a critical factor affecting the
course of vascular inflammation. This hypothesis is supported by
demonstration of defective vasculogenesis and increased and ultimately
lethal vascular inflammation in mice null for
TGF-s,11,12 their membrane receptors,13,14
or their downstream substrates, the Smad proteins.15,16
Three mammalian isoforms of TGF- (TGF-1, TGF-2,
TGF-3)11,12,17,18 are secreted by endothelial cells in a
latent form in which latency-associated peptide (LAP) and its 25 kd
carboxy-terminal dimer, which is the mature TGF-,19,20
remain noncovalently bound. In vivo release of the mature dimer from
LAP occurs via its degradation or conformational
change19,21 and allows binding of the mature (bioactive)
peptide to its specific membrane receptors TGF-RI, TGF-RII, and
TGF-RIII, of which types I and II are serine threonine
kinases.22-24 Binding of the 25 kd TGF- dimer to the
high-affinity type II receptor results in recruitment and activation of
the type I receptor by phosphorylation at its cytoplasmic domain by the
type II receptor kinase. Activated TGF-R1 phosphorylates intracellular Smad proteins, which propagate downstream signals of
TGF-s.25 Phosphorylation of Smad2 and Smad3
(receptor-activated Smads) causes their dissociation from TGF-R1 and
stimulates their assembly with Smad4 (co-Smad)22,26; this
complex then translocates and accumulates in the nucleus.
Heterodimeric Smad complexes can bind DNA in either orientation of
a 5'-CAGA-' consensus sequence (Smad-binding element
[SBE]27-29) and can directly regulate transcription by recruiting coactivators or inhibitors of
transcription.30
Recent studies of human endothelial cells and in vivo animal studies
have shown that Smad proteins regulate endothelial responses to
mechanical stress such as shear31,32 and to inflammatory stress such as exposure to bacterial lipopolysaccaride.33
It seemed reasonable, therefore, to test the hypothesis that TGF-s and their downstream Smad signaling pathway can be activated by hypoxic
stress in vascular endothelium in humans. In this report, we show that
hypoxia selectively up-regulates the TGF-2 isoform by as much as
20-fold in endothelial cells in a time-dependent fashion. Furthermore,
we show for the first time that hypoxia activates Smad-mediated gene
transcription and Smad-DNA association in human endothelial cells.
These data indicate a new function for Smads in endothelial cells as
one of the signaling pathways activated by hypoxia, one that is capable
of modulating inflammatory consequences of ischemia.
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Materials and methods |
Cell cultures
Primary human umbilical vein endothelial cells (HUVECs) were
isolated and propagated from pooled primary cultures of umbilical veins
by digestion with collagenase type II and were maintained as described
by Gimbrone.34 Briefly, HUVECs were grown on gelatin (2%)-coated tissue culture flasks (Costar, Cambridge, MA) in
endothelial cell growth medium that consisted of medium 199 (Gibco,
Grand Island, NY) supplemented with endothelial cell growth supplement (50 µg/mL; Calbiochem, San Diego, CA), 20% fetal bovine serum, antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin), and heparin (50 µg/mL, porcine intestinal; Sigma, St Louis, MO). Cells were passaged by trypsin-ethylenediaminetetraacetic acid (EDTA) treatment, split 1:4, fed every 1 to 3 days, and used at second or
third passage, when nearly confluent (70%). Cytoplasmic von Willebrand
factor by immunocytochemistry and nonoverlapping cobblestone morphology
verified endothelial cells. For exposure to hypoxia, cells were
incubated under an atmosphere of 1% O2, 5%
CO2, and 94% N2 at 37°C in a NAPCO 7301 incubator (Precision Scientific, Chicago, IL). Previous experiments in
our laboratory have shown that during exposure to hypoxia, the pH of
the medium remains unchanged, pO2 in the medium is 10 to 16 mm Hg, and viability is more than 95% as measured by trypan blue dye
exclusion and determination of release of lactate dehydrogenase.
Experiments were performed in parallel on normoxic and hypoxic cells
that were exposed for the times indicated to hypoxia or recombinant TGF-2 (R&D Systems, Minneapolis, MN). HUVEC viability assessed after
each treatment was consistently more than 95%. HUVEC culture reagent
 -amanitin was obtained from Sigma.
RNase protection assay
Total RNA was prepared from treated or control HUVECs using
TRIzol (Gibco) and quantified as previously described.35
Ribonuclease (RNase) protection analysis was done using the RiboQuant
Multi-Probe RNase protection assay system (PharMingen, San Diego, CA).
In this assay, high-specific-activity RNA probes are synthesized from
a mixture of DNA templates of distinct lengths that correspond to a
sequence in a distinct messenger RNA (mRNA) species. Template sets
hCK-3 and hCR-4 (PharMingen) were used; hCK-3 contains templates for
tumor necrosis factor- (TNF-), TNF-, lymphotoxin-,
interferon-, interferon- , TGF-1, TGF-2, TGF-3, and the
ubiquitously expressed genes for ribosomal protein L32 and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hCR-4 contains
templates for type I interleukin-1 receptor (IL-1RI), type II IL-1
receptor (IL-1RII), p55 type I TNF- receptor (TNF-RI), p75 type
II TNF- receptor (TNF-RII), IL-6 receptor (IL-6R),
glycoprotein 130, TGF-RI, TGF-RII, L32, and GAPDH. T7
polymerase-directed antisense RNA probe synthesis, hybridization to
total RNA, RNase treatments, and gel resolution of protected probes
were done exactly as described by the manufacturer. Briefly, probe was
synthesized by labeling the template with [-32P]UTP
(New England Nuclear, Boston, MA) using T7 RNA polymerase. A total of
6 × 105 cpm of labeled probe in hybridization solution
was added to 10 µg total RNA, and the samples were overlaid with
mineral oil before brief heating to 90°C; hybridization continued for
16 hours at 56°C. After hybridization, the samples were digested for
45 minutes at 30°C with a mixture of RNase A and RNase T1 (as
recommended in the kit), treated with proteinase K, extracted with
phenol/chloroform/isoamyl alcohol (50:50:0.5), precipitated with
ethanol, and resolved on 5% denaturing polyacrylamide sequencing gels.
Radioactive fragments were detected by autoradiography and also by
PhosphorImager (Molecular Dynamics, Sunnyvale, CA), from which
quantification of mRNA was done by a volume integration protocol using
ImageQuant software (Molecular Dynamics). To control for differences in
sample processing, hybridization signals in each sample were divided by
the signal for 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
cytokine/L32 signal ratio from hypoxic HUVECs was divided by the
cytokine/L32 ratio obtained from normoxic HUVECs.
TGF- assay
TGF--responsive mink lung epithelial cells (MLECs) stably
transfected with the expression construct p800neoLUC containing a
truncated PAI-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). Cells were maintained in Dulbecco modified
Eagle medium containing 10% fetal calf serum, 2 mM L-glutamine, and
antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin, 250 µg/mL geneticin; Gibco). To assay bioactive and total TGF-2 levels
in HUVEC supernatants, 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), and were 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; 1:10 dilution of conditioned
HUVEC medium to measure bioactive TGF-; and 1:10 dilution of
conditioned HUVEC medium activated for 10 minutes by heating at 80°C
to measure total 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.20
Total protein content of supernatants was quantitated using Bio-Rad
protein assay reagent (Bio-Rad, Hercules, CA),36 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-2.
This assay is sensitive to all 3 isoforms of TGF-; therefore, to
specifically quantitate total TGF-2 in hypoxic and nonhypoxic
supernatants, aliquots of the identical supernatants used in MLEC assay
were acid-activated and subjected to enzyme-linked immunosorbent assay
(ELISA) (Quantikine, R&D Systems) as previously
described.37 Results (not shown) confirmed that TGF-2
levels were increased by 2-fold in hypoxic supernatants. Because
measurement of bioactive TGF-s by ELISA is not yet reliably accomplished by existing commercial kits, this assay can only confirm
the increase in total TGF-2 determined by the MLEC assay.
Nuclear run-on assay for TGF-2
The rate of transcription of TGF-2 in HUVECs was determined
as described by Greenberg and Ziff38 in nuclei prepared
from HUVECs cultured in either normoxic or hypoxic culture conditions. Briefly, a suspension of 6 × 109 nuclei in 200 µL
glycerol (40%) in 50 mM Tris-HCl buffer (pH 8.3, with 5 mM
MgCl2 and 0.1 mM EDTA) was mixed with 2 × reaction buffer
(10 mM Tris-HCl [pH 8]; 5 mM MgCl2; 0.3 M KCl; 5 mM
dithiothreitol; 1 mM each ATP, CTP, GTP; and 100 µCi [3.7 × 106 Bq] [32P]UTP [1.1 × 1014 Bq, 3000 Ci/mM; PerkinElmer Life
Sciences, Boston, MA]). After incubation for 30 minutes at
30°C, [32P]-labeled RNA was extracted twice with
phenol/chloroform/isoamyl alcohol (25:24:1) and 50 µg transfer RNA,
and 10% trichloracetic acid (TCA) were added. The mixture was filtered
through a 0.45-µm Millipore filter (Millipore, Bedford, MA).
Nuclear RNA from the filter was released by treatment with 20 mM HEPES
buffer (pH 7.5, with 5 mM MgCl2 and 1 mM CaCl2)
and deoxyribonuclease for 30 minutes at 37°C, addition of 45 µL of
0.5 M EDTA and 68 µL of 20% SDS, and incubation at 65°C for 10 minutes. Nuclear RNA was digested with proteinase K, extracted with
phenol/chloroform/isoamyl alcohol, and ethanol-precipitated at 20°C
overnight. TGF-2, Glut-1, and GAPDH complementary DNA probes have
been described.39-41 RNA was hybridized for 36 hours at
65°C to linearized plasmid DNA (TGF-2, Glut-1, GAPDH) immobilized
on nitrocellulose membranes.41 Filters were washed in SSC
(2 ×) at 65°C 3 times for 30 minutes each, incubated at 37°C with
80 µg ribonuclease A, washed again, air dried, and exposed to DuPont
reflection film (DuPont Pharmaceuticals, Wilmington, DE), with an
intensifying screen, at 80°C, and also by exposure to
PhosphorImager. For quantification of TGF-2 transcripts and
determination of the fold increase in transcript levels in hypoxic
HUVECs compared with control, the TGF-2/GAPDH signal ratio from
hypoxic HUVECs was divided by the TGF-2/GAPDH signal obtained from
normoxic HUVECs.
Plasmid constructs
Plasmid constructs containing deletion mutants of the human
TGF-2 gene promoter linked to a chloramphenicol acetyl transferase (CAT) gene were generously donated by Dr S.-J. Kim.42
TGF-2/CAT constructs pB2-778, pB2-257, and pB2-40 were generated by
polymerase chain reaction using genomic DNA containing the 5'-flanking
DNA and the first exon of the TGF-2 gene as template with different 5'-specific oligonucleotide primers, spanning from 778 to 40, and a
common 3'-specific oligonucleotide primer at +63 relative to the
transcription initiation site; amplified DNAs were cloned into the
promoterless pGEM4/SV0CAT vector as described.42 The p3TP-Lux construct (a generous gift from Dr Joan Massagué,
Memorial Sloan-Kettering Cancer Center, New York, NY) is a well
described and widely used artificial promoter construct that was
designed to have maximal responsiveness to TGF-.23 This
plasmid has 3 AP-1 sites corresponding to the collagenase promoter,
concatemerized 5' to a 400-nucleotide region of the PAI-1 promoter,
followed by 70 base pairs (bp) of the adenovirus E4
promoter.43 Activity of this construct is increased
significantly by TGF- as well as by combined overexpression of Smad3
and Smad4. Smad4 is required for this transcriptional activity because
3TP-Lux is inactive in Smad4-null cells and is rescued by Smad4
expression.23 Expression vectors for Smad3 and Smad4 that
contain the FLAG epitope tag at the amino domain have been described
previously25 and were generously supplied by Drs Rik
Derynck and Ying Zhang (University of California, San Francisco, CA).
Transfection of recombinant plasmids and reporter gene
assays
For transient transfections, plasmids containing 5' TGF-2
promoter sequences and p3TP-Lux containing the luciferase reporter gene, with indicated expression vectors or corresponding empty constructs, were introduced by electroporation into early passage (second or third) HUVECs that were 70% confluent, as previously described.35 Briefly, 6 × 106 HUVECs, HepG2
(HB-8065, American Type Culture Collection [ATCC], Manassas,
VA), A549 (CCL-185, ATCC), and MDA-MB-468 (HTB-132, ATCC) were
resuspended in 600 µL respective growth media and reacted with 30 µg reporter plasmid DNA along with the indicated Smad expression
plasmids or corresponding empty constructs (14 µg) and 0.5 µg
plasmid cytomegalovirus -galactosidase (pCMV-gal; Promega)
(included in each transfection as internal control) for 10 minutes at
4°C. When multiple plasmids were cotransfected (eg, reporter and
expression plasmids), the total amount of DNA was kept constant by
supplementing the samples with empty expression vectors.
Electroporation was performed using 220 V and 960 microfarads in a
Bio-Rad Gene Pulser Electroporation System. Transfected cells were
divided into 2 equal aliquots and cultured in 60-mm plates in
endothelial cell growth medium for 6 hours, after which one of each
pair of plates was placed in hypoxic conditions while the duplicate
plate remained in nonhypoxic culture conditions for indicated periods.
Adherent cells were washed with phosphate-buffered saline 36 hours
after transfection, dislodged from plates using a rubber policeman, and
the CAT assay performed at assay conditions predetermined to be within
the linear range for CAT activities of the samples (data not shown).
Luciferase activity was determined using the Luciferase Assay System
(Promega). To ensure that the same amount of protein was analyzed, the
protein concentration of each sample was determined according to the
Bradford assay,36 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 induction by hypoxia. The displayed
error bars in Figures 5 and 6, which are each representative of at
least 3 independent experiments, represent ± 1 SD of the mean.
Electrophoretic gel mobility shift assays
Fresh medium with or without TGF-2 (12.5 ng/mL) was added to
70% confluent HUVECs, and half of the duplicate flasks were placed in
hypoxic conditions. At the end of the indicated incubation periods,
nuclear extracts were prepared from hypoxic and nonhypoxic HUVECs as
described by Andrews and Faller.44 Protein concentrations were determined by the Bio-Rad assay with bovine serum albumin standards; final protein concentration of nuclear extracts was usually
between 5 and 10 mg/mL. Aliquots were stored at 70°C until use. A
double-stranded oligonucleotide corresponding to the 26-bp
sequence29 5'-AGTATGTCT-AGACTGA-3'
harboring the underlined consensus palindromic SBE was used as probe.
The SBE probe was end-labeled with [-32P]dATP using
the Klenow fragment of DNA polymerase I for gel shift studies, and 50 to 100 pg (1 × 104-2 × 104 cpm) was
incubated with 7 µg nuclear extract and 2 to 8 µg double-stranded poly(dIdC) in 20 µL of 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM
MgCl2,1 mM EDTA, 1 mM dithiothreitol, and 5% wt/vol
glycerol for 30 minutes at 4°C after preincubation of all components
except probe for 15 minutes. For competition experiments, 20 to 500 molar excess of the indicated unlabeled oligonucleotide was added to
the binding reaction mixture 5 minutes prior to probe addition. The
following cold oligonucleotides were used for competition for nuclear
extract binding to the probe: an 18 bp sequence corresponding to
nucleotides 1 to 18 of the erythropoietin gene hypoxia-responsive
enhancer45 5'-GCCCTACGTGCTGTCTCA-3' harboring
the HIF-1-binding core sequence (in italics); a 39 bp sequence
corresponding to 77 to 40 bp of the TGF-2 promoter with respect
to transcriptional start site 5'-CCTAGCACGTCACTTTGTTGAAGGCAGACACGTGGTTCA-3'
harboring an underlined CAGA box and an HIF-1-binding core
sequence46; and SBE. For supershift assays, each antibody
(rabbit antihuman Smad2, anti-Smad3 [Zymed, South San Francisco, CA],
rabbit anti-Smad4 [Upstate Biotechnology, Lake Placid, NY], or rabbit
immunoglobulin G) was added to the binding reactions 15 minutes prior
to the probes and incubated for another 30 minutes. After
preelectrophoresis for 1 hour, 4% polyacrylamide gel electrophoresis
was performed in 0.5 × Tris-borate buffer/1% glycerol without EDTA
at 180 V for 2 hours at 4°C. The gel was dried, autoradiographed, and
exposed to a PhosphorImager screen for 18 hours and analyzed using
ImageQuant software.
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Results |
Effect of hypoxia on TGF- mRNA and protein levels in
HUVECs
As shown in Figure 1A, in nonhypoxic
HUVECs, TGF-1 mRNA levels were greater than mRNA levels of TGF-2
or TGF-3. The strongest effect of hypoxia was on TGF-2 mRNA
levels, which increased by 4-fold after 4 hours of exposure to hypoxia
compared with untreated HUVECs, followed by 10-fold and 25-fold
increases at 24 hours and 48 hours, respectively (Figure 1A,B). At each
time point, mRNA from nonhypoxic control HUVECs was also analyzed, and
the TGF- mRNA profile from these cultures remained unchanged (data not shown). Viability of endothelial cells at 24 hours and 48 hours, as
disclosed by trypan blue and lactate dehydrogenase release, was more
than 95%. A strong effect of hypoxia was observed on TGF-
bioactivation (Figure 1C). The level of bioactive TGF-, which was
assayed in supernatants prior to activation of latent TGF-, was 12 times higher in supernatants from hypoxic HUVECs compared with normoxic
control supernatants. Thus, 64% of TGF- in supernatants from
hypoxic HUVECs was bioactive, while only 14% of total TGF- was
bioactive in the nonhypoxic supernatants20 (Figure 1C). The
level of total TGF- (which is determined after activation of latent
TGF-) produced by HUVECs after 48 hours of exposure to hypoxia was
increased by 2.6-fold compared with nonhypoxic cells
(P < .05) both by the MLEC assay (Figure 1C) and by an
ELISA that specifically measures TGF-2 (data not shown). As seen in
Figure 2, nonhypoxic HUVECs have readily
discernable levels of both TGF-RI and TGF-RII mRNA. While hypoxia
did not alter mRNA levels of TGF-RI, there was a modest but
consistent 1.8- to 2.5-fold increase in levels of the serine threonine
kinase TGF-RII in hypoxic HUVECs (P < .05) after
correction for L32 expression (data not shown), suggesting that in
these cells hypoxia could activate TGF- signaling as well.
Up-regulation of vascular endothelial growth factor in this experiment
(Figure 2) confirms hypoxic conditions.47
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| Figure 1.
Time course of hypoxia's effect on TGF- mRNA levels
in HUVECs.
(A) RNase protection analysis of HUVEC mRNA is shown. Total
RNA was extracted from HUVECs after exposure to 20%
O2 or 1% O2 at indicated times; 0 hours
represents the normoxic sample. Each lane contained 10 µg total RNA
hybridized to an antisense RNA probe cocktail that contained the
templates for genes, protected fragments of which were separated on a
5% DNA sequencing gel and are indicated by arrows. (B) The graph
represents the quantification results from 4 independent
experiments. TGF-2 signal was normalized to that from L32 (mRNA for
ribosomal protein subunit); 0 hours is the mean of mRNA levels from
control normoxic HUVECs. Each subsequent bar represents mRNA levels
compared with its own normoxic control (not shown) at indicated hours.
*P < .05. (C) TGF- protein in supernatants from
hypoxic and nonhypoxic HUVECs cultured for 48 hours was determined
before and after heat activation to quantitate bioactive and total
TGF- levels, respectively, by MLEC bioassay. Results are from 4 independent experiments.
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| Figure 2.
Effect of hypoxia on serine threonine kinase
TGF- receptor mRNA levels.
RNase protection analysis of mRNA extracted from HUVECs after exposure
to 20% O2 or 1% O2 at indicated times.
Control represents mRNA from normoxic HUVECs. Each lane contained 10 µg total RNA hybridized to the antisense RNA probe cocktail. Results
are from a single gel in which lanes are separated for display
purposes.
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Level of hypoxia-induced TGF-2 gene expression in
HUVECs
As suggested by Semenza,6,48 several adaptive
responses to hypoxia involve changes in gene expression that occur at
the level of transcription. To examine whether the hypoxia-induced stimulation of TGF-2 mRNA resulted from an up-regulation of its transcriptional activation, we used in vitro transcription assays performed with nuclei isolated from HUVECs exposed to 20% or 1% O2 for 24 hours. The results from 1 of 2 experiments that
yielded identical results are shown in Figure
3 and demonstrate that hypoxia increased
transcription of TGF-2 by 15-fold after normalization to GAPDH (data
not shown). Similarly, transcription of the hypoxia-responsive glucose
transporter-1 (Glut-1)49 gene was also increased under hypoxic culture conditions, whereas transcription of the housekeeping gene GAPDH was largely unchanged. Furthermore, treatment with -amanitin to block type II RNA polymerase activity exhibited very
similar decay curves in hypoxic and normoxic HUVECs (Figure 4B), indicating that hypoxia's effect on
TGF-2 gene expression occurred largely at the transcriptional
level.
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| Figure 3.
Effect of hypoxia on transcriptional activity of
TGF-2 gene in HUVECs.
Transcriptional analysis of TGF-2, Glut-1, and GAPDH genes by
nuclear run-on assay; 1 of 2 representative experiments is shown. (A) 5 µg of each of the plasmids indicated on the right bound to nylon
membrane were hybridized with [32P]-labeled run-on
transcripts from 6 × 109 nuclei isolated from HUVECs
cultured for 24 hours in normoxia (20% O2) or hypoxia (1%
O2). pUC19, the plasmid vector without insert, was used to
estimate the background level. Radioactive bands were detected by
autoradiography and also by PhosphorImager, from which transcripts were
quantitated. (B) Fold increase in transcript levels in hypoxic HUVECs
compared with normoxic controls was done by dividing the TGF-2/GAPDH
signal from hypoxic HUVECs by the TGF-2/GAPDH signal obtained from
normoxic HUVECs, which is shown as 1. The mean of results from 2 independent experiments is shown.
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| Figure 4.
Effect of hypoxia on TGF-2 message stability.
HUVECs subjected to normoxic conditions, C, or to 24 hours of hypoxia
were treated with 1 µg/mL -amanitin for the times indicated. (A)
TGF-2 message obtained with RNase protection analysis in HUVECs
subjected to hypoxia for 24 hours and subsequently treated with
-amanitin for the times indicated. In parallel cultures, normoxic
HUVECs were also cultured with and without -amanitin (not shown
except for the 0-hour control loaded in the first lane). (B) Semilog
plot of TGF-2 transcript half-life in the presence of hypoxic or
normoxic conditions. Data were quantitated by PhosphorImager and the
results plotted with linear regression; data from 1 of the 2 representative experiments are shown.
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To further study hypoxia-induced TGF-2 transcriptional activation,
we performed transient transfection assays using CAT reporter constructs containing the 5' TGF-2 promoter sequences. First, activity of the CAT reporter construct pB2-778, which contains cis-acting elements (Figure
5A) that regulate transcription of the
5.8 kilobase TGF-2 transcript,42 was determined. As
shown in Figure 5B, activity of this construct was increased by 3-fold in response to hypoxia, and further studies using deletion mutants of
this construct showed that the promoter sequence within pB2-77 is
necessary and sufficient to mediate the 4.8 ± 1.2-fold increase in
CAT activity obtained in hypoxic cells compared with nonhypoxic controls. A hypoxia-induced 4.8-fold increase in transcription from the
TGF-2 promoter region between 77 and +63 bp compared with
nonhypoxic controls (P < .001) paralleled the increase
seen in the mRNA levels after 24 hours of exposure to hypoxia,
suggesting that the stimulatory mechanism involves changes associated
with transcriptional events.
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| Figure 5.
Effect of hypoxia on transcriptional activity of the
TGF-2 promoter in HUVECs.
(A) Recombinant constructs containing the human TGF-2 gene promoter
between 778 and +63 bp. The transcriptional start site (arrow)
driving the CAT gene and selected protein-binding sites (Smad, HIF-1,
AP-1) are indicated. (B) Transcriptional activity of the TGF-2
promoter in response to hypoxia. HUVECs were transfected with pB2-778
or its deletion constructs pB2-77 or pB2-40; pB2-77 was also
cotransfected with indicated Smad expression vectors and RSV--gal by
electroporation. Each transfection was divided into 2 plates and
exposed to either to 20% or 1% O2 for 36 hours. CAT
activity was determined in whole-cell extracts, and its level, given as
percent acetylation, was normalized to protein (Bradford assay,
Bio-Rad) and to -gal activity in each experiment. Results are
expressed as fold increases in hypoxic CAT activity compared with
normoxic CAT activity in each transfection. The mean ± SD of 4 independent experiments is shown.
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TGF-s are known to be regulated by cis-acting Smad sites.
Because the TGF- promoter region within the hypoxia-responsive construct pB2-77 harbored a Smad-binding site (Figure 5A) and because
endogenous Smad activity is known to be boosted by Smad overexpression,25,50 we tested the hypoxia response of the 77 to +63 bp CAT construct after Smad3 and Smad4 overexpression in
HUVECs. Results show that overexpression of Smad3 and Smad4 resulted in
a further 1.8-fold increase in construct pB2-77 activity in hypoxic
HUVECs (Figure 5B) compared with nonhypoxic controls (P < .01). Baseline expression of constructs under
normoxic conditions and response of pB2-778 or pB2-40 to overexpression
of Smads did not have a significant effect on their CAT activities
(data not shown).
We next examined whether hypoxia stimulated activity of another
promoter construct with known responsiveness to activation by Smad
proteins using a well described and widely used artificial promoter
construct, plasmid p3TP-Lux (Figure 6A),
which is responsive to TGF- as well as to Smad3 and Smad4
cooverexpression even in the absence of ligand.23 As seen
in Figure 6B, hypoxia increased the luciferase activity of 3TP-Lux by
3.5-fold in HUVECs and by 10-fold in HepG2 cells, which are easier to
transfect than HUVECs and thus are less subject to artifacts resulting
from low transfection efficiencies. These results indicate that
endogenous Smads are activated by hypoxia in both cell types. As
expected, addition of TGF-2 to HUVECs or HepG2 cells increased
luciferase activity of 3TP-Lux (Figure 6A,B). To further determine the
role of Smad proteins on hypoxia-induced activity of p3TP-Lux, we
measured its response to hypoxia after transfection into the
Smad4-null, epithelial breast adenocarcinoma cell line
MDA-MB-468.16 In contrast to results obtained in HUVECs
and HepG2, hypoxia-driven luciferase activity of p3TP-Lux was minimal
in MDA-MB-468 (Figure 6C). When these cells were transiently
transfected to express Smad4, there was a 4-fold increase in the
p3TP-Lux activity, indicating that Smad4 is necessary for
hypoxia-induced transcription in this cell line. Hypoxia also modulates
expression of genes regulated by the transcription factor
AP-1,51-53 and Smad3 and Smad4 potentiate transcription
from AP-1,50 suggesting that Smad and AP-1 responses may
both contribute to hypoxia-induced 3TP-Lux activity.
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| Figure 6.
Hypoxia stimulates transcription from 3TP-Lux in HUVECs.
(A) Structural organization of the 3TP-Lux reporter plasmid,
composed of 3 AP-1 binding sites and a TGF--responsive PAI-1
promoter fragment driving a luciferase reporter gene, is shown. (B)
Response of 3TP-Lux is increased by 4-fold in HUVECs and 10-fold in
HepG2 cells after exposure to 1% O2 or TGF-2 (12.5 ng/mL) for 24 hours. Luciferase activity was determined and results
corrected to CMV promoter-derived -gal activity and protein content
of the extracts. Results from 3 independent experiments are shown. (C)
Response of p3TP-Lux to hypoxia or TGF-2 (12.5 ng/mL) is absent in
the Smad4-defective MDA-MB-468 cell line and can be reinstated by Smad4
expression. MDA-MB-468 in parallel with the lung carcinoma cell line
A549 that expresses Smad4 was transfected with p3TP-Lux by
electroporation either in the presence of Smad4 expression vector or
empty vector. Transfected cells were divided into 3 identical culture
dishes and were exposed to 20% O2 with or without TGF-2
(12.5 ng/mL) or were exposed to 1% O2 for 24 hours.
Luciferase activity from 3 independent experiments is shown.
|
|
Nuclear extract binding to the Smad consensus sequence
Smad binding to DNA was studied by comparing gel retardation
patterns of nuclear proteins from HUVECs exposed to hypoxia, normoxia,
or TGF- to DNA sequences that are specific for binding Smad
proteins. As the probe in the electrophoretic gel mobility shift assay,
we used the well-described oligonucleotide SBE which, as shown by x-ray
crystallography, can associate with Smad3 by either or both of its
palindromic Smad-binding sequences.29 As seen in Figure
7A, after exposure to hypoxia a discrete
gel-shifted band is observed that was absent from control nonhypoxic
nuclear extracts. This band was identical in position to the one
observed with nuclear extracts of TGF-2-treated HUVECs and raised
the possibility that hypoxia induced Smad binding to DNA. Binding to
the SBE by nuclear extracts stimulated by hypoxia or by TGF-2 was
competed by an unlabeled 37 bp oligonucleotide corresponding to the
hypoxia-responsive TGF-2 promoter sequences between 77 and 40 bp
and containing a Smad-binding site. However, unlabeled oligonucleotide
corresponding to the HIF-1 binding site of the erythropoietin promoter
did not compete binding of hypoxic nuclear extracts to the
SBE. Results suggest that the shared Smad-binding site between
the SBE and the hypoxia-responsive TGF-2 promoter between 77 and
40 bp can bind nuclear proteins in hypoxic but not normoxic cells and
that binding is independent of HIF-1.
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| Figure 7.
Hypoxia induces sequence-specific DNA binding activity
by Smads.
(A) Electrophoretic gel mobility shift assays were performed using a
32P-labeled SBE probe as described in "Materials and
methods." Nuclear extracts were as follows: control normoxic HUVECs,
C; HUVECs treated for 30 minutes with 12.5 ng/ml TGF-2, T; or HUVECs
exposed to hypoxia (1% O2) for 4 hours, H. Competition
assays: no competition (); 100-fold molar excess of unlabeled SBE
(100 × SBE); 125-fold molar excess of erythropoietin (125 × EPO),
an HIF-1-containing oligonucleotide; 125-fold molar excess of an
oligonucleotide that corresponds to the 77 to 40 bp TGF-2
promoter (125 × TGF-2). (B) Antibody supershifting: nuclear
extracts from HUVECs were preincubated with anti-Smad2, anti-Smad3, or
anti-Smad4 antibodies for 30 minutes prior to the binding reaction.
Constitutive binding is indicated by the unlabeled arrows.
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|
To identify the transcription factors participating in the DNA
association in nuclear extracts from TGF-2 and hypoxia-treated HUVECs, supershift assays were performed with antibodies specific for
Smad2, Smad3, and Smad4 and also with preimmune sera (not shown). As
shown in Figure 7B, antibody to Smad3 and Smad4 supershifted the bands
seen with nuclear extracts from TGF-2- and hypoxia-treated HUVECs,
while preimmune sera did not induce a supershift (data not shown).
Furthermore, antibody to Smad2 did not induce a supershift but
decreased the intensity of the specific association of hypoxia- and
TGF--treated nuclear extracts with the SBE, indicating that Smad2
function may also be activated by hypoxia in endothelial cells. These
results suggest that hypoxia induces Smad-DNA association similar to
that induced by TGF-2 and support the thesis that hypoxia results in
Smad3 and Smad4 activation.
| |
Discussion |
In this report, we show that hypoxia increases TGF-2 production
and its bioactivation in human endothelial cells; furthermore, hypoxia
effectively up-regulates TGF-2 mRNA levels. This effect occurs at
the transcriptional level, as evidenced by nuclear run-on analysis and
reporter gene experiments using the TGF-2 promoter, and is at least
partly mediated by Smad proteins, because their overexpression further
stimulates TGF-2 promoter activation by hypoxia. It is possible that
an autocrine mechanism in which bioactivation of TGF- by hypoxia
results in TGF-R-mediated activation of Smads and results in
TGF-2 transcription; alternatively, Smad activation occurs by a
pathway that is independent of TGF-R. Activation of Smads
by receptor and intracellular kinases other than those within TGF-
pathway have been described.54,55 For example, the
intracellular kinase mitogen-activated protein kinase-1, an upstream
activator of the stress-activated protein kinase/c-Jun N-terminal
kinase, can activate Smads in endothelial cells.54 In view
of the fact that hypoxia activates intracellular kinases p38, p38
mitogen-activated protein kinase, and stress-activated protein
kinase/c-Jun N-terminal kinase,56 it is likely that Smad
protein signaling in hypoxic endothelial cells is regulated by multiple
mechanisms. Responses to hypoxia of negative regulators of the Smad
pathway for example, Smad7, which can regulate endothelial responses
to inflammation and shear23,57are unknown at this time.
Our results show that the HIF-1-binding oligonucleotide cannot compete
association of hypoxic nuclear extracts with SBE. In addition,
exposure to 200 µM hydrogen peroxide, which depletes HIF-1
accumulation in hypoxic cells and inhibits its ability to bind
DNA,58 is associated with a 4-fold increase in TGF-2 mRNA levels in hypoxic HUVECs and HepG2 (data not shown). Existing reports indicate a possible negative regulation of the Smad pathway by
HIF-1, such as reduction of TGF-1 gene expression in fetal skin by
HIF-1 59 and inhibition of HIF-1 causing TGF-3 gene expression and suppression of its downstream effects on
trophoblasts.60 The potential interaction of Smad and HIF
pathways in regulating gene expression in hypoxic endothelium is of
interest. We are currently examining the effect of HIF-1 and its
relative HIF-2/endothelial PAS domain protein
(EPAS)61,62 on activity of the TGF-2 promoter by
mutating its putative HIF-1-binding sites, and after HIF-1 overexpression.
Similar to our results, up-regulation of TGF- bioactivation by
hypoxia has been shown in perivascular glial cells of
retina,63 but the mechanism is unclear. One possibility is
that it occurs by hypoxic stimulation of endothelial expression of
thrombospondin, which activates TGF- by altering conformation of
LAP.64 Thrombospondin transcript and protein are induced
by hypoxia in a parallel time course to hypoxic activation of TGF-2,
with peak thrombospondin mRNA levels observed at 48 hours and protein
levels at 72 hours.65 Interestingly, a hypoxia-induced
increase in steady-state TSP-1 transcript levels is to some extent due
to increases in mRNA stability.65 The possibility also
exists that cellular redox states altered by hypoxia-induced gene
activation and generation of reactive oxygen intermediaries, or changes
in nitric oxide concentration, or inadvertent reoxygenation of HUVECs
during our experiments contribute to bioactivation of
TGF-2 17,66; these possibilities are to be explored.
Hypoxia causes an up-regulation of TGF-2 expression in peritoneal
mesothelial cells67 and down-regulates it in retinal
cells68 and in dermal fibroblasts, where type II receptor
mRNA was also found to be decreased.69 These results reflect the cell-specific nature of regulation of TGF- genes within
different contexts,17 including hypoxia. Recently, Smad2 binding to nuclear coactivator p300/CREB-binding protein was shown to
inactivate nuclear factor- B in endothelial cells in vitro and in
vivo.33 It is possible that hypoxia-mediated changes in
levels of transcriptional coactivators such as P300/CREB-binding protein70 may represent one mechanism responsible for cell- and context-specific actions of TGF-2 in response to hypoxia.
Nonuniform responses in key endothelial functions such as endothelial
cell growth, development, migration, and angiogenesis in response to
TGF- isoforms71,72 indicate that isoform-specific responses are biologically important. Prior to binding to TGF-RII, TGF-2 is anchored by membrane proteins73 or membrane or
matrix proteoglycans, such as type III receptor
(betaglycan),74,75 endoglin,23 biglycan,
decorin, and fibromodulin.51,76 Therefore, hypoxia-induced
regulation of these proteins can differentially modulate local
concentrations of active TGF-2 which, as suggested by
Roberts,17 is pivotal in regulation of its actions.
Our studies do not address the consequences of Smad activation and
TGF-2 production on functions of hypoxic endothelial cells; these
experiments are under way. A recent study has shown that TGF-2 gene
is specifically induced after infection of HUVECs with an adenovirus
gene transfer vector that confers prolonged survival to endothelial
cells even in the absence of serum.77 Furthermore,
exposure of endothelial cells to TGF-2 was shown to counteract the
angiogenic effects of hepatocyte growth factor via inhibition of its
signaling,78 and antibody to TGF-2 was shown to
stimulate DNA synthesis in pericytes.79 Based on these and
previous findings regarding the antiinflammatory role of the TGF-
family of cytokines,75,80-82 we hypothesize that TGF-2 induces an antiproliferative, antiapoptotic, and antiangiogenic signal
to mitigate the inflammatory signals induced by hypoxia and augments an
adaptive response in the endothelium. Similar to other adaptive
responses, TGF-2 response if prolonged or unopposed can result in
progressive fibrosis and vascular thickening.10,83,84 Understanding hypoxia-driven signaling by Smads can thus help elucidate
the context and isoform-specificity of TGF-2 actions in the vascular
system and in the context of endothelial functions including tumor
angiogenesis.15
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Acknowledgments |
We are thankful |
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Abstract
Introduction