Am J Physiol Lung Cell Mol Physiol 300: L740–L752, 2011.
First published January 14, 2011; doi:10.1152/ajplung.00146.2010.
Hypoxia-inducible factor-1␣ mediates TGF-␤-induced PAI-1 production
in alveolar macrophages in pulmonary fibrosis
Manabu Ueno,1 Toshitaka Maeno,1 Miyuki Nomura,1 Kana Aoyagi-Ikeda,1 Hiroki Matsui,1
Kenichiro Hara,1 Toru Tanaka,1 Tatsuya Iso,1,2 Tatsuo Suga,1 and Masahiko Kurabayashi1
1
Department of Medicine and Biological Science, and 2Education and Research Center, Gunma University Graduate School
of Medicine, Maebashi, Japan
Submitted 11 May 2010; accepted in final form 10 January 2011
pulmonary fibrosis; hypoxia inducible factor-1␣; transforming growth
factor-␤; plasminogen activator inhibitor-1
PULMONARY FIBROSIS IS AN INTEGRAL component of many interstitial lung diseases that occur as a consequence of acute or
chronic inflammatory disorders (1). Fibrosis represents a final
common consequence for a large variety of disease processes
in which normal control of tissue repair is compromised and
excess fibrous material accumulates in the tissues (13, 52).
Pathological studies using animal models and human pulmonary fibrosis specimens identified the presence of proliferation
myofibroblasts, extracellular matrix deposition, epithelial cell
apoptosis, alveolar remodeling, and infiltration of inflammatory cells such as macrophages, neutrophils, and lymphocytes
Address for reprint requests and other correspondence: M. Kurabayashi,
Dept. of Medicine and Biological Science, Gunma Univ. Graduate School of
Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan (e-mail:
[email protected]).
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(17, 50). Although the basic mechanisms of pulmonary fibrosis
remain unknown, increased expression of TGF-␤1 is believed
to be a critical mediator of a complex, multi-phase process
during initiation and progression of fibrosis and remodeling
(42, 48). For example, elevated levels of TGF-␤1 mRNA and
TGF-␤ protein were detected in alveolar macrophages, myofibroblasts, and fibroblasts in lung biopsies obtained from
idiopathic pulmonary fibrosis (IPF) patients and rodent bleomycin (BLM)-induced pulmonary fibrosis models (5, 25, 26,
35, 36, 44). In addition, pharmacological and genetic intervention
targeting TGF-␤1 action on fibroblasts, such as administration of
anti-TGF-␤1 neutralizing antibody, dominant negative TGF-␤
receptor, or interferon (IFN)-␤, which is expected to be a potent
inhibitor of fibrotic disorders, ameliorates BLM-induced pulmonary fibrosis (19). Furthermore, enforced expression of bioactive
human TGF-␤1 in the lungs of transgenic mice was shown to
recapitulate several key pathophysiologies observed in fibrotic
lung disorders (25).
TGF-␤1 promotes multiple features associated with fibrosis,
such as fibroblast migration, proliferation, myofibroblast differentiation, and excessive production of extracellular matrix
(ECM) components (44). TGF-␤1 acts through binding to a
transmembrane receptor, which in turn phosphorylates Smad2
and Smad3 (29, 30, 47). These activated Smads then translocate to the nucleus with binding partner Smad4, and this
complex regulates a wide range of target genes that are relevant
to tissue fibrosis through cis-regulatory elements that serve as
binding sequence of Smad proteins. Positive and negative
changes in the expression of several hundred TGF-␤1-responsive genes at least partly depend on the associated partners of
Smads (43).
cDNA microarray-based gene expression profiling data obtained from a BLM-induced pulmonary fibrosis model and
from IPF patients revealed that expression of the genes involved in oxygen transport are the most significantly dysregulated, suggesting an important role for hypoxia-inducible factor-1 (HIF-1) signaling in pulmonary fibrosis (49). HIF-1 is a
basic helix-loop-helix transcription factor composed of HIF-1␣
and HIF-1␤ (40, 41). While under normoxia, HIF-1␣ becomes
hydroxylated at proline residues and degraded in the proteasome; under hypoxia, HIF-1␣ becomes stabilized, translocates
to the nucleus, and induces transcription of target genes.
Recent studies have demonstrated that HIF-1␣ expression is
increased by non-hypoxic stimuli such as TGF-␤1 and PDGF,
inflammatory cytokines (tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, and angiotensin II in vascular smooth muscle
cells (SMC), and by a variety of growth factors including
epidermal growth factor, insulin, and insulin-like growth factor
in hepatocytes (9, 12, 15, 23, 24, 34, 38, 45). However, it
1040-0605/11 Copyright © 2011 the American Physiological Society
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Ueno M, Maeno T, Nomura M, Aoyagi-Ikeda K, Matsui H,
Hara K, Tanaka T, Iso T, Suga T, Kurabayashi M. Hypoxiainducible factor-1␣ mediates TGF-␤-induced PAI-1 production in
alveolar macrophages in pulmonary fibrosis. Am J Physiol Lung
Cell Mol Physiol 300: L740 –L752, 2011. First published January
14, 2011; doi:10.1152/ajplung.00146.2010.—Hypoxia-inducible factor-1␣
(HIF-1␣), a transcription factor that functions as a master regulator of
oxygen homeostasis, has been implicated in fibrinogenesis. Here, we
explore the role of HIF-1␣ in transforming growth factor-␤ (TGF-␤)
signaling by examining the effects of TGF-␤1 on the expression of
plasminogen activator inhibitor-1 (PAI-1). Immunohistochemistry of
lung tissue from a mouse bleomycin (BLM)-induced pulmonary
fibrosis model revealed that expression of HIF-1␣ and PAI-1 was
predominantly induced in alveolar macrophages. Real-time RT-PCR
and ELISA analysis showed that PAI-1 mRNA and activated PAI-1
protein level were strongly induced 7 days after BLM instillation.
Stimulation of cultured mouse alveolar macrophages (MH-S cells)
with TGF-␤1 induced PAI-1 production, which was associated with
HIF-1␣ protein accumulation. This accumulation of HIF-1␣ protein
was inhibited by SB431542 (type I TGF-␤ receptor/ALK receptor
inhibitor) but not by PD98059 (MEK1 inhibitor) and SB203580 (p38
MAP kinase inhibitor). Expression of prolyl-hydroxylase domain
(PHD)-2, which is essential for HIF-1␣ degradation, was inhibited by
TGF-␤1, and this decrease was abolished by SB431542. TGF-␤1
induction of PAI-1 mRNA and its protein expression were significantly attenuated by HIF-1␣ silencing. Transcriptome analysis by
cDNA microarray of MH-S cells after HIF-1␣ silencing uncovered
several pro-fibrotic genes whose regulation by TGF-␤1 required
HIF-1␣, including platelet-derived growth factor-A. Taken together,
these findings expand our concept of the role of HIF-1␣ in pulmonary
fibrosis in mediating the effects of TGF-␤1 on the expression of the
pro-fibrotic genes in activated alveolar macrophages.
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HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
MATERIALS AND METHODS
Antibodies. HIF-1␣, PHD-1, PHD-2, and PHD-3 (Novus Biological, Littleton, CO), PAI-1 (Abcam, Cambridge, MA), Mac3 (BD
Biosciences, Mississauga, ON), Smad3, phospho-Smad3, p44/42,
phospho-p44/42, p38, and phospho-p38 (Cell Signaling Technology,
Beverly, MA).
Mice. Animal experiments were performed according to the guidelines of the Committee of Experimental Animal Research of Gunma
University. Male 8-wk-old C57BL/6 mice were anesthetized and
instilled with BLM (5 mg/kg) intratracheally as an aerosol using a
microsprayer (Penn-Century, Philadelphia, PA). Control mice received saline. Mice were killed 1, 3, 7, 14, and 21 days after BLM
administration.
Tissue processing. The left lungs were ligated and frozen. Tissue
was removed for RT-PCR. The right lungs were inflated by instilling
with 10% formalin at a constant pressure of 25-cm formalin (for 10
min) and fixed for 24 h before paraffin embedding. Serial sections
were prepared for histological analysis.
Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed using a 20-G intravenous catheter inserted into the trachea.
Lungs were lavaged with 0.75 ml of PBS four times and then
centrifuged at 3,000 revolutions/min for 3 min. Supernatant without
cell pellets were used for the PAI-1 ELISA.
Immunohistochemistry. Lung sections were deparaffinized in xylene and rehydrated through graded ethanol washes. HIF-1␣ and mac3
immunostaining was performed using the Vectastatin Elite ABC Kit
(Vector Laboratories, Burlingame, CA). PAI-1 staining was performed using the CSA Kit (Dako, Glostrup, Denmark). Negative
controls were stained by substituting the primary antibody for a
nonspecific antibody.
RNA analysis. Total RNA was extracted using RNAiso Plus (Takara Bio, Kyoto, Japan) according to the manufacturer’s protocol.
Single-stranded cDNAs were synthesized from 1 ␮g of total RNA and
semiquantitative RT-PCR was performed with an RT-PCR kit (Takara Bio). Real-time RT-PCR was performed using SYBER green
(TOYOBO, Osaka, Japan) according to the manufacturer’s protocol.
Each experiment was performed using three samples in each condition. The relative quantities of transcripts were determined using
ImageJ software for Windows. All primer sequences are shown in
Table 1.
Cell culture and stimulation. MH-S cells (derived from mouse
alveolar macrophage) were obtained from ATCC. Primary alveolar
macrophages were obtained from BAL fluid that we collected from
lungs of C57BL/6 mice using the method described in BAL. These
cells were cultured in RPMI1640 medium supplemented with 10%
FBS and 1% penicillin-streptomycin (GIBCO, Gaithersburg, MD) at
37°C in a 5% CO2 atmosphere. Serum-free medium was added for 24
h before 5 ng/ml rhTGF-␤1 (Roche Diagnostics, Manneheim, Germany) was added to the medium and cells were incubated at 37°C.
Alternatively, cells were pretreated for 1 h with SB431542 (Sigma, St.
Louis, MO), PD98509, and SB203580 (Calbiochem, San Diego, CA)
before rhTGF-␤1 (5 ng/ml) was added. For hypoxic stimulation, cells
were incubated in a jar containing Anaero Pack-Anaero (⬍1% O2, 5%
CO2) or Anaero Pack-MicroAero (8% O2, 5% CO2) (Mitsubishi Gas
Chemical, Tokyo, Japan).
siRNA transfection. Transfection of siRNA plasmid was performed
with Lipofectamine RNAiMAX Reagent (Invitrogen Life Sciences,
Carlsbad, CA) according to the manufacturer’s protocol. siRNA
oligonucleotides were purchased from Hayashi Kasei. The target
sequences of HIF-1␣ siRNA, PHD-2 siRNA, and control siRNA are
stated in Table 1. Control siRNA/HIF-1␣ siRNA (20 nM) or 100 nM
control siRNA/PHD-2 siRNA were used for the experiment.
Microarray analysis. cDNA microarray analysis of MH-S cells
transfected siHIF-1␣, or siGFP was performed using an oligo microarray system (Agilent). Total RNA was isolated from MH-S cells using
an RNeasy Mini kit (QIAGEN, Hilden, Germany) 6 h after TGF-␤1
Table 1. Primer sequences used for real-time RT-PCR and siRNA analyses
Test
RT-PCR analyses
Real-time PCR analyses
siRNA analyses
Gene
Forward
Reverse
HIF-1␣
PAI-1
TGF-␤1
Procollagen 1
Procollagen 3
18s
HIF-1␣
PAI-1
PDGF-A
GAPDH
HIF-1␣
PHD-2
GFP (control)
5=-TGCTCATCAGTTGCCACTTC-3=
5=-TGATGGCTCAGAGCAACAAG-3=
5=-ATACGCCTGAGTGGCTGTCT-3=
5=-AGGCTTCAGTGGTTTGGATG-3=
5=-AATGGCTCACCAGGACAAAG-3=
5=-GTTGGTGGAGCGATTTGTCT-3=
5=-GCAGCAGGAATTGGAACATT-3=
5=-TGATGGCTCAGAGCAACAAG-3=
5=-GAGATACCCCGGGAGTTGAT-3=
5=-AACGACCCCTTCATTGAC-3=
5=-CAGUUACGAUUGUGAAGUUAA-3=
5=-AUGCGUGACAUGUAUAUAUUA-3=
5=-GUUCAGCGUGUCCGGCGAGTT-3=
5=-TGGGCCATTTCTGTGTGTAA-3=
5=-GCCAGGGTTGCACTAAACAT-3=
5=-TTCTCTGTGGAGCTGAAGCA-3=
5=-GCAATACCAGGAGCACCATT-3=
5=-ATCCATCTTTGCCATCTTCG-3=
5=-GGCCTCACTAAACCATCCAA-3=
5=-GCATGCTAAATCGGAGGGTA-3=
5=-GCCAGGGTTGCACTAAACAT-3=
5=-ACTTTGGCCACCTTGACACT-3=
5=-TCCACGACATACTCAGCAC-3=
5=-AACUUCACAAUCGUAACUGGU-3=
5=-UAAUAUAUACAUGUCACGCAU-3=
5=-CUCGCCGGACACGCUGAACTT-3=
HIF, hypoxia-inducible factor; PAI, plasminogen activator inhibitor; TGF, transforming growth factor.
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remains unclear whether genes that are relevant to lung function under physiological and pathological conditions are regulated by HIF-1 under normoxia.
Among the numerous genes whose expression is regulated by
TGF-␤ and HIF-1␣, plasminogen activator inhibitor-1 (PAI-1),
the primary inhibitor of plasminogen activators (u-PA, t-PA),
has been proven to play a key role in the development of
pulmonary fibrosis (8, 14). BLM-induced fibrosis is more
severe in transgenic mice overexpressing the PAI-1 gene than
wild-type mice, whereas PAI-1 knockout mice were protected
from fibrosis. Several hypotheses have been proposed to explain how PAI-1 deficiency prevents lung fibrosis, varying
from an enhancement of plasmin-mediated proteolysis of fibrin
to enhanced proteolysis of growth factors and matrix metalloproteinases (MMP) that degrade matrix glycoproteins (27). In
addition, PAI-1 was consistently and dramatically upregulated
in pulmonary fibrosis (33).
The objective of the present study was to determine whether
HIF-1␣ could be implicated in the induction of PAI-1 expression in alveolar macrophages. We demonstrated that TGF-␤
induces PAI-1 expression through the accumulation of HIF-1␣
via Smad3-dependent inhibition of PHD-2 expression. Furthermore, we demonstrate that TGF-␤1 induction of PDGF-A was
significantly diminished by HIF-1␣ knockdown, suggesting the
important role of HIF-1␣ in alveolar macrophages in pulmonary fibrosis.
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Fig. 1. Localization of hypoxia-inducible factor (HIF)-1␣ and plasminogen activator inhibitor (PAI)-1 in bleomycin (BLM)-induced pulmonary fibrosis. C57BL/6
mice were instilled intratracheally with BLM (5 mg/kg) or saline. A and B: the expression of HIF-1␣ and PAI-1 was detected by immunohistochemistry. The
lung sections from control lung and BLM-instilled lung on days 1, 3, 7, 14 and 21, are presented (magnification, ⫻100). C: the expression of HIF-1␣, PAI-1,
and Mac3 on day 7 is shown (magnification, ⫻400).
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ersham Biosciences, Piscataway, NJ). All experiments were repeated
at least three times. The relative quantities of protein products were
determined using ImageJ software for Windows.
ELISA. The concentration of PAI-1 protein in the BAL fluid and
culture medium was measured using the mouse active PAI-1 ELISA
kit (Innovative Research, Novi, MI) and PDGF-AA ELISA kit (R&D
Systems, Minneapolis, MN), according to the manufacturer’s instructions. Each sample was assayed in triplicate (n ⫽ 5).
Statistical analysis. Data are expressed as means ⫾ SD. The
differences were examined for significance using an ANOVA with
Tukey’s procedure post hoc comparison as appropriate, using SPSS
software for Windows.
RESULTS
Expression of HIF-1␣ and PAI-1 in BLM-induced lung injury
and fibrosis. The early phase after BLM administration is
characterized by acute inflammatory reaction: inflammatory
cells such as neutrophils, macrophages, and lymphocytes infiltrate in the lung, and the increase of pro-inflammatory
Fig. 2. Expression of HIF-1␣, PAI-1, and transforming growth factor (TGF)-␤1 in BLM-induced pulmonary inflammation and fibrosis.
A: total RNA (left) was extracted from the left lung
after instillation of BLM or saline. The expression of HIF-1␣, PAI-1, TGF-␤1 and procollagen
was analyzed by RT-PCR. The mRNA levels of
HIF-1␣, PAI-1, and TGF-␤1 (right) were normalized against the levels of 18S mRNA: the
result are arbitrarily indicated as values relative
to the levels in the controls and are the means ⫾
SD of three separate experiments. **Significant
difference compared with controls (n ⫽ 3; P ⬍
0.01). B: bronchoalveolar lavage (BAL) was performed from control lung and BLM-instilled
lung 1, 3, 7, and 14 days after BLM instillation.
Activated-PAI-1 protein levels in BAL fluid
(BALF) were measured by ELISA. Data represent means ⫾ SD from triplicate experiments
(*P ⬍ 0.05; **P ⬍ 0.01). C: total RNA (left)
was extracted from alveolar macrophages after
instillation of BLM or saline. The expression of
HIF-1␣ and PAI-1 was analyzed by RT-PCR.
The mRNA levels of HIF-1␣ and PAI-1 (right)
were normalized against the levels of 18S
mRNA: the results are arbitrarily indicated as
values relative to the levels in the controls and
are means ⫾ SD of three separate experiments.
**Significant difference compared with controls
(P ⬍ 0.01; n ⫽ 3).
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treatment and subjected to DNase treatment according to the manufacturer’s instructions. The quantity and purity of total RNA was
determined by spectrophotometry readings at 260 and 280 nm. The
integrity of intact total RNA was verified using a low RNA fluorescent
linear amplification kit (Agilent). For hybridization, Cy3-labeled
cRNA from siHIF-1␣ and siGFP-transfected cells, respectively, was
combined and hybridized to Whole Mouse Genome Oligo Microarray
44K ⫻4 pack (Agilent), according to the manufacturer’s protocol.
Oligonucleotide microarray slides were scanned using an Agilent
microarray scanner, and the gene expression profiles were analyzed
using Agilent Whole Mouse Genome Oligo Microarray software.
Western blot analysis. Cells were washed twice with PBS, harvested, and then lysed in UTB buffer (8 M urea, 50 mM Tris·HCL, pH
7.5, 150 mM 2-mercaptoethanol and deionized-water) or RIPA buffer
(20 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% Na
deoxycholate, 0.1% SDS, deionized-water and protease inhibitors).
After sonication and removal of debris by centrifugation, 50 ␮g of
protein from each sample were resolved by SDS- PAGE and transferred to nitrocellulose membranes. The membranes were immunoblotted with the indicated antibodies and visualized with ECL (Am-
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HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
days after BLM instillation. The concentration of activated-PAI-1
protein was markedly increased in BAL fluid at 7 days after BLM
instillation by ELISA (Fig. 2B). On day 14, activated PAI-1 level
was decreased and remained at the higher level on day 21. We
also performed reverse zymography to examine the activity of
PAI-1 but failed to show the change in PAI-1 activity. A failure
might be due to the sensitivity problem of the zymogram to detect
an increase in PAI-1 activity. These results indicate that PAI-1
protein expression as detected by immunohistochemistry was
biologically active and suggests a role for PAI-1 in the fibrosing
phase rather than the acute inflammatory phase in this model.
Moreover, we obtained primary alveolar macrophages by BAL
from C57BL/6 mice after BLM injection and examined the
mRNA expression of HIF-1␣ and PAI-1. On day 7 and 14, PAI-1
mRNA expression, but not HIF-1␣, was increased in alveolar
macrophages (Fig. 2C).
Effect of TGF-␤1 on the expression of HIF-1␣ and PAI-1 in
alveolar macrophage. It has been established that a key mediator implicated in BLM-induced pulmonary fibrosis is TGF-␤1
(4, 22). Thus we next examined whether TGF-␤1 was directly
Fig. 3. Effect of TGF-␤1 on the expression of HIF-1␣
and PAI-1 in MH-S cells. Alveolar macrophages
[MH-S cells (A), primary alveolar macrophages (B)]
were treated with TGF-␤1 (5 ng/ml) for up to 24 h (0,
2, 6, 12, 24 h). Then total RNA was extracted from
macrophages. The expression of HIF-1␣ and PAI-1
mRNA was analyzed by real-time PCR. The mRNA
levels of HIF-1␣ and PAI-1 were normalized against
the levels of GAPDH mRNA: the results are arbitrarily
indicated as values relative to the levels in the controls
and are means ⫾ SD of three separate experiments.
**Significant difference compared with controls (P ⬍
0.01; n ⫽ 3). C: serum-starved MH-S cells (left) were
treated with TGF-␤1 (5 ng/ml) for up to 24 h (0, 2, 6,
12, 24 h). Then the whole cell lysates were subject to
Western blot analysis to detect HIF-1␣ protein. The
protein levels of HIF-1␣ (right) were normalized
against the protein levels of ␤-actin: the results are
arbitrarily indicated as values relative to the levels in
the controls and are means ⫾ SD of three separate
experiments. Significant difference compared with
controls (n ⫽ 3): *P ⬍ 0.05; **P ⬍ 0.01. D: activatedPAI-1 protein level in culture media were measured by
ELISA. Data represent means ⫾ SD from triplicate
experiments (*P ⬍ 0.05; **P ⬍ 0.01).
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cytokines (IL-1, TNF-␣) (32, 39). The late phase is characterized by fibrotic reaction: the increase of pro-fibrotic markers
(TGF-␤, PDGF), the proliferation and activation of fibroblasts,
and extracellular matrix deposits (32). To investigate whether
HIF-1␣ was associated with pulmonary fibrosis through the
production of PAI-1, we first examined the localization of
HIF-1␣ and PAI-1 in the BLM-induced pulmonary inflammatory and fibrosis model by immunohistochemistry. As shown
in Fig. 1, A and B, the expression of HIF-1␣ was increased on
day 3 after BLM injection, which was not detected in control
mice. Thereafter, the number of inflammatory cells was significantly increased, and most of the inflammatory cells were HIF-1␣
positive on day 7. The number of PAI-1 positive inflammatory
cells was also significantly increased on day 7. The HIF-1␣ and
PAI-1 positive cells were identified as alveolar macrophages
because those cells were also mac3-positive (Fig. 1C).
Next, we analyzed the change in expression of HIF-1␣, PAI-1,
and TGF-␤1 mRNA by RT-PCR. As shown in Fig. 2A, the
expression of PAI-1 and TGF-␤1 mRNA, but not HIF-1␣ mRNA,
was barely detectable in control lungs and increased at 7 to 21
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induced HIF-1␣ expression was completely abrogated by
SB431542 [type I TGF-␤ receptor/activin-like kinase 5
(ALK5) inhibitor], whereas neither PD98509 (MEK1 inhibitor) nor SB203580 (p38MAP kinase inhibitor) exerted measurable effects on TGF-␤1 induction of HIF-1␣ expression. Moreover, we examined whether SB431542 indeed inhibited ALK5mediated Smad3-phosphorylation by using the antibody specific
for phosphorylated Smad3. As shown in Fig. 4B, TGF-␤1 increased the phosphorylation of Smad3, and SB431542 efficiently
blocked this effect. In a similar way, we examined the effects of
TGF-␤1 on MEK1 and p38 MAPK phosphorylation. However,
TGF-␤1 had no effects on phosphorylated forms of MEK1 (phospho p44/42) levels and phosphorylated form of p38 (phospho
p38) levels (data not shown). These results were consistent with
the data that neither PD98509 (MEK1 inhibitor) nor SB203580
(p38 inhibitor) blocked TGF-␤1-induced HIF-1␣ expression.
HIF-1␣ is subjected to oxygen-dependent prolyl hydroxylation, and among the three prolyl-hydroxylase isoforms, PHD-1,
PHD-2 and PHD-3, that hydroxylate the key proline residues
Fig. 4. Effect of ALK5 receptor inhibitor on
TGF-␤1-induced HIF-1␣ protein expression.
A: serum-starved MH-S cells (left) were pretreated for 1 h with SB431542 (ALK5 receptor
inhibitor), PD98059 (MEK1 inhibitor), and
SB203580 (p38MAP kinase inhibitor), and
then treated with TGF-␤1 (5 ng/ml) or vehicle
for 6 h. Cells were harvested for analysis of
HIF-1␣ protein by Western blot analysis. B
and C: MH-S cells (left) were pretreated for 1
h with the indicated concentration of
SB431542, and then treated by TGF-␤1 (5
ng/ml) or vehicle for 1 h. Cells were harvested
for analysis of phosphorylated Smad3, total
Smad3, HIF-1␣, PHD-1, PHD-2, PHD-3, and
␤-actin by Western blot analysis. A–C: the
protein levels (right) of HIF-1␣, phosphorylated Smad3, total Smad3, and PHD-2 were
normalized against the protein levels of ␤-actin. All experiments were repeated at least
three times. The result are arbitrarily indicated
as values relative to the levels in the controls
and are means ⫾ SD of three separate experiments (*P ⬍ 0.05; **P ⬍ 0.01).
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involved in the upregulation of HIF-1␣ and PAI-1 in MH-S
cells, an established mouse alveolar macrophage cell line, and
primary alveolar macrophages. Exposure of cells to TGF-␤1 (5
ng/ml) clearly increased PAI-1 but not HIF-1␣ mRNA levels
shown by real-time PCR (Fig. 3A). Moreover, in primary
alveolar macrophages, TGF-␤1 stimulation also increased
PAI-1 mRNA expression but not HIF-1␣ (Fig. 3B). Western
blot analysis showed that HIF-1␣ protein was induced by
TGF-␤1 at 2 h after stimulation and reached a maximum at 6 h
(Fig. 3C), suggesting that TGF-␤1 increased HIF-1␣ protein
expression predominantly at the posttranscriptional level. In
addition, we found that the activated-PAI-1 protein concentration was robustly increased by TGF-␤ in culture medium of
MH-S cells by ELISA (Fig. 3D).
Inhibition of TGF-␤1-induced HIF-1␣ protein expression by
SB431542. To determine the intracellular signaling cascade
mediating the induction of HIF-1␣ in response to TGF-␤1, we
examined the effects of protein kinase inhibitors on HIF-1␣
expression in MH-S cells. As shown in Fig. 4A, TGF-␤1-
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HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
MH-S cells transfected with HIF-1␣ siRNA displayed a complete loss of HIF-1␣ protein, whereas transfection of siRNA
targeting GFP did not abrogate HIF-1␣ expression. More
importantly, HIF-1␣ siRNA but not GFP siRNA completely
prevented the induction of HIF-1␣ protein by TGF-␤1. Furthermore, real-time RT-PCR revealed that HIF-1␣ siRNA
significantly blunted the TGF-␤1 induction of PAI-1 mRNA
expression (P ⬍ 0.01) (Fig. 5B). Consistently, MH-S cells
transfected with HIF-1␣ siRNA showed a blunt increase in
activated PAI-1 protein levels in the culture medium in response to TGF-␤1 (0.038 ⫾ 0.001 ng/ml vs. 0.018 ⫾ 0.003
ng/ml, siGFP vs. siHIF1␣; P ⬍ 0.05) (Fig. 5C). These results
demonstrated that TGF-␤1 induces PAI-1 expression largely
through HIF-1␣-dependent mechanisms.
Expression profiling of the of TGF-␤1-inducible genes through
HIF-1␣ in MH-S cells. To examine the role of HIF-1␣ in the
TGF-␤1 induction of gene expression in alveolar macrophages,
we performed a comprehensive gene expression pattern analysis
using a microarray comprised of 45,019 probes. For these studies,
Fig. 5. Effect of HIF-1␣ siRNA on PAI-1 and
PDGF-A expression. A: serum-starved MH-S
cells (left) were transfected with HIF-1␣ siRNA
or GFP siRNA (control) for 24 h and then treated
with TGF-␤1 (5 ng/ml) or vehicle for 6 h. Whole
cell extracts were analyzed for HIF-1␣ protein
expression by Western blot analysis. ␤-Actin
was measured as internal control. The protein
levels of HIF-1␣ (right) were normalized against
the protein levels of ␤-actin. All experiments
were repeated at least three times. Data represent
means ⫾ SD from triplicate experiments (*P ⬍
0.05; **P ⬍ 0.01). B: total RNA of MH-S cells
transfected with HIF-1␣ siRNA or GFP siRNA
was extracted for analysis of PAI-1 mRNA by realtime RT-PCR. The data are shown as means ⫾
SD of three separate experiment. The results are
arbitrarily indicated as values relative to the level
in siGFP (*P ⬍ 0.05; **P ⬍ 0.01). C: culture
media from MH-S cells transfected with either
HIF-1␣ siRNA or GFP siRNA was assayed and
subjected to the analysis of activated-PAI-1 protein level by ELISA. Data represent means ⫾ SD
from triplicate experiments (*P ⬍ 0.05; **P ⬍
0.01). D: total RNA of MH-S cells transfected
with HIF-1␣ siRNA or GFP siRNA was extracted for analysis of PDGF-A mRNA
by real-time RT-PCR. The data are shown as
means ⫾ SD of three separate experiment. The
results are arbitrarily indicated as values relative
to the level in siGFP (*P ⬍ 0.05; **P ⬍ 0.01).
E: culture media of MH-S cells transfected with
either HIF-1␣ siRNA or GFP siRNA was subjected to the analysis of PDGF-AA protein levels
by ELISA. Data represent means ⫾ SD from
triplicate experiments (*P ⬍ 0.05; **P ⬍ 0.01).
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(Pro402 and Pro564) in vitro, PHD-2 plays the critical role in
setting the low steady-state levels of HIF-1␣ in normoxia (2).
In addition, McMahon et al. (31) showed that TGF-␤1 decreased PHD-2 mRNA and protein levels. Thus we examined
whether PHD-2 was involved in TGF-␤1-induced HIF-1␣
protein expression in alveolar macrophage. As shown in Fig.
4C, an induction of HIF-1␣ by TGF-␤1 was accompanied by a
downregulation of PHD-2, and this response was abrogated by
SB431542. These results suggest that TGF-␤1-induced HIF-1␣
accumulation is mediated via ALK5-induced phosphorylation
of Smad3 and a reduction of PHD-2 expression.
Inhibition of TGF-␤1-induced PAI-1 protein expression by
siRNA targeting HIF-1␣. To verify the role of HIF-1␣ in the
TGF-␤1-induced PAI-1 expression, we evaluated the impact of
the specific silencing of HIF-1␣ on PAI-1 expression in MH-S
cells. We transiently transfected MH-S cells with 21-bp siRNA
duplexes corresponding to either HIF-1␣ or irrelevant GFP as
a control, incubated the cells for 24 h, and then stimulated them
with TGF-␤1 (5 ng/ml) for 6 h in normoxia. As shown in Fig. 5A,
L747
HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
MH-S cells were transiently transfected with siRNA targeting
either HIF-1␣ or GFP, and incubated in normoxia in the presence
or absence of TGF-␤1 (5 ng/ml) for 6 h. To simplify our analyses,
we compared the fold-difference of TGF-␤1 induction of each
mRNA expression in MH-S cells. Table 2 lists the genes whose
expression was induced by TGF-␤1 less preferentially in MH-S
cells transfected with HIF-1␣ siRNA than in cells transfected with
GFP siRNA. This analysis identified 58 genes that were upregulated more than twofold by TGF-␤ and abrogated by HIF-1␣
silencing. These genes included PAI-1, growth factors, and
receptors such as PDGF-A and type I TGF-␤ receptor,
transcription factors, and cofactors such as HIF-1␣, Snail,
Id1, Id2, and Id3, anti-peptidase such as Serpine, and multiple genes implicated in carbohydrate and lipid metabolism.
Notably, HIF-1␣ mRNA levels were increased 2.71-fold by
TGF-␤1, and HIF-1␣ silencing markedly inhibited this induction, suggesting the auto-regulation of HIF-1␣ gene
expression in response to TGF-␤1.
Table 2. Transcripts of MH-S cells stimulated with TGF-␤1 after HIF-1␣ interference
Fold Difference
Gene
Symbol
NM_010431
NM_009647
NM_009760
NM_022655
NM_018870
NM_007452
BC062654
NM_018865
NM_007802
NM_133955
NM_011673
NM_008321
NM_011961
NM_011427
NM_178266
NM_019568
NM_009370
NM_009932
NM_016803
NM_007635
NM_008871
HIF1a
Ak3l1
Bnip3
Ireb2
Pgam2
Prdx3
Gnas
Wisp1
Ctsk
Rhou
Ugcg
Id3
Plod2
Snai1
Mbtps2
Cxcl14
Tgfbr1
Col4a2
Chst3
Ccng2
PAI-1
Gene Descryption
siHIF1a/siGFP
siGFP
TGFb1/siGFP
siHIF1a
TGFb1/siGFP
siHIF1a TGFb1/siGFP
TGFb1
Hypoxia inducible factor 1, alpha subunit
Adenylate kinase 3 alpha-like 1
BCL2/adenovirus E1B interacting protein 1
Iron responsive element binding protein 2
Phosphoglycerate mutase 2
Peroxiredoxin 3
Guanine nucleotide binding protein, alpha stimulating
WNT1 inducible signaling pathway protein 1
Cathepsin K
Ras homolog gene family, member U
UDP-glucose ceramide glucosyltransferase
Inhibitor of DNA binding 3
Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2
Snail homolog 1
Membrane-bound transcription factor peptidase, site 2
Chemokine (C-X-C motif) ligand 14
Transforming growth factor, beta receptor 1
Procollagen, type IV, alpha 2
Carbohydrate sulfotransferase 3
Cyclin G2
Plasminogen activator inhibitor 1
0.46
0.34
0.5
0.9
0.57
0.53
3.4
1.1
0.74
0.68
0.62
1.1
0.73
2
0.95
1
0.8
1.8
0.89
1.2
0.97
2.8
2.1
2.6
2.6
1.9
1.6
11
126
2.1
6.3
2.1
30
2.9
30
1.9
21
5
5.3
8
6.2
72
0.64
0.41
0.71
0.73
0.58
0.49
3.6
40
0.69
2.1
0.72
10
1
11
0.69
8.1
1.9
2.1
3.1
2.5
30
0.22
0.2
0.27
0.27
0.3
0.3
0.3
0.31
0.32
0.32
0.33
0.34
0.35
0.36
0.36
0.38
0.38
0.38
0.38
0.4
0.4
0.96
0.93
11.1
1.2
0.6
0.83
0.59
1.2
1.1
0.87
1.4
1.2
1
0.74
0.87
0.7
1.1
1.1
0.81
0.88
1.1
1.1
1.1
0.92
0.72
1.4
0.84
1.2
0.86
0.95
1.5
5.8
1.8
2.3
3.4
1.6
1.9
1.6
7.8
2.8
2
3.7
2.7
10
1.9
1.9
8.5
2.7
6.9
1.9
4.5
2.2
5.7
2.4
2.4
1.5
5.1
3.2
2.3
2
2
2.3
2.4
0.74
0.98
1.5
0.71
0.81
0.7
3.5
1.3
0.9
1.7
1.2
4.5
0.87
0.9
4
1.3
3.3
0.9
2.2
1.1
2.8
1.2
1.2
0.76
2.6
1.6
1.2
0.99
1
1.2
0.41
0.41
0.42
0.42
0.42
0.43
0.44
0.44
0.45
0.45
0.45
0.46
0.45
0.45
0.46
0.46
0.47
0.47
0.47
0.48
0.48
0.48
0.49
0.49
0.49
0.49
0.49
0.49
0.49
0.5
0.5
(Serpine 1) (serine peptidase inhibitor, clade E, member 1)
NM_008808
NM_172665
NM_023119
NM_177798
NM_008826
NM_024454
NM_010699
NM_010495
NM_172823
NM_010107
NM_008712
NM_080844
NM_134050
NM_013509
NM_022801
NM_027208
NM_172784
BC029674
NM_017399
NM_011498
NM_011198
NM_010496
NM_009424
NM_007404
NM_024169
NM_013650
NM_019732
NM_178647
NM_008812
NM_011519
NM_028176
Pdgfa
Pdk1
Eno
1
Frs2
Pfkl
Rab21
Ldha
Id1
Lmln
Efna1
Nos1
Serpinc 1
Rab15
Eno2
Mark3
Bdh2
Lrp11
Flt1
Fabp1
Bhlhb2
Ptgs2
Id2
Traf6
Adam9
Fkbp11
S100a8
Runx3
Cggbp1
Padi2
Sdc1
Cda
Platelet derived growth factor, alpha
Pyruvate dehydrogenase kinase, isoenzyme 1
Enolase 1
Fibroblast growth factor receptor substrate 2
Phosphofructokinase
RAB21, member RAS oncogene family
Lactate dehydrogenase A
Inhibitor of DNA binding 1
Leishmanolysin-like (metallopeptidase M8 family)
Ephrin A1
Nitric oxide synthase 1
Serine peptidase inhibitor, clade C, member 1
RAB15, member RAS oncogene family
Enolase 2
MAP/microtubule affinity-regulating kinase 3
3-Hydroxybutyrate dehydrogenase, type 2
Low-density lipoprotein receptor-related protein 11
FMS-like tyrosine kinase 1
Fatty acid binding protein 1
Basic helix-loop-helix domain containing, class B2
Prostaglandin-endoperoxide synthase 2
Inhibitor of DNA binding 2
Tnf receptor-associated factor 6
A disintegrin and metallopeptidase domain 9
FK506 binding protein 11
S100 calcium binding protein A8
Runt-related transcription factor 3
CGG triplet repeat binding protein 1
Peptidyl arginine deiminase, type II
Syndecan 1
Cytidine deaminase
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GeneBank
Accession No.
L748
HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
oxygen. These results suggest that, although an increase in
PAI-1 production by either TGF-␤1 or hypoxia is mediated by
HIF-1␣ accumulation, the mechanisms by which TGF-␤1 and
hypoxia increase PAI-1 production are different from each
other.
We next examined the cooperative effects of hypoxia and
TGF-␤1 on HIF-1␣ and PAI-1 expression. Compared with
TGF-␤1 stimulation in normoxia, TGF-␤1 and ⬍1% oxygen
remarkably induced HIF-1␣ and PAI-1 production, although
8% oxygen had less additive effect on HIF-1␣ and PAI-1
production (Fig. 7, E and F). These results suggest that there is
cross-talk between the signal transduction pathways induced
by TGF-␤1 and ⬍1% hypoxia resulting in stimulation of
TGF-␤1 gene transcription.
DISCUSSION
Here, we demonstrate that HIF-1␣ is abundantly expressed
in alveolar macrophages in a mouse BLM-induced pulmonary
fibrosis model at the alveolitis stage (day 7) and in interstitial
cells at the fibro-proliferating stage (day 14). Among numerous
genes that are known to be regulated by HIF-1␣, we focus on
the PAI-1 gene because PAI-1 has been documented as a
pro-fibrotic mediator of lung fibrosis both in animal models
and in humans (3, 46). Our immunohistochemistry data
showed predominant expression of PAI-1 and HIF-1␣ in alveolar macrophages in the mouse BLM-induced pulmonary fibrosis model, and an in vitro study using MH-S cells demonstrated that TGF-␤1 induced HIF-1␣ stabilization via ALK5/
Smad3 signaling and PHD2 downregulation in normoxia.
Notably, HIF-1␣ silencing attenuated TGF-␤1 induction of
PAI-1 expression, indicating the crucial role of HIF-1␣ in
mediating TGF-␤1 induction of PAI-1 expression.
One of the major findings in this study is that alveolar
macrophages respond to TGF-␤1 to induce PAI-1. This finding
Fig. 6. Effect of PHD-2 siRNA on TGF-␤1 induced HIF-1␣ expression. Left: serum-starved
MH-S cells were transfected with PHD-2 siRNA
or GFP siRNA (control) for 24 h and then treated
with TGF-␤1 (5 ng/ml) or vehicle for 6 h. Whole
cell extracts were analyzed for PHD-2 and
HIF-1␣ protein expression by Western blot analysis. ␤-Actin was measured as internal control.
Right: the protein levels of PHD-2 and HIF-1␣
were normalized against the protein levels of
␤-actin. All experiments were repeated at least
three times. Data represent means ⫾ SD from
triplicate experiments (*P ⬍ 0.05; **P ⬍ 0.01).
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To validate the microarray analysis, we examined the effects
of TGF-␤1 and HIF-1␣ siRNA silencing on PDGF-A expression. As shown in Fig. 5D, real-time RT-PCR showed that
TGF-␤1 increased PDGF-A mRNA levels ⬃4.2-fold in MH-S
cells transfected with the GFP siRNA, and this induction was
significantly blunted in MH-S cells transfected with HIF-1␣
siRNA. These results were confirmed by ELISA, indicating
that PDGF-A is upregulated by TGF-␤1 by, at least in part,
HIF-1␣-dependent mechanisms (Fig. 5E).
Effect of PHD-2 silensing on HIF-1␣ protein expression. To
investigate the involvement of PHD-2 in the HIF-1␣ induction
by TGF-␤1 stimulation, we transfected MH-S cells with
PHD-2 siRNA or GFP siRNA, incubated the cells for 24 h, and
then stimulated them with TGF-␤ (5 ng/ml) for 6 h in normoxia. As shown in Fig. 6, PHD-2 protein expression was
substantially reduced in PHD-2 siRNA transfected cells compared with GFP siRNA transfected cells. We found that PHD-2
siRNA but not GFP siRNA clearly increased HIF-1␣ protein
levels. In addition, an increase in HIF-1␣ protein levels by
TGF-␤1 was clearly enhanced in MH-S cells transfected with
PHD-2 siRNA. These results suggest that TGF-␤1 induction of
PHD-2 expression is causally linked with a downregulation
of PHD-2 expression by TGF-␤1.
Effect of hypoxia on the expression of HIF-1␣ and PAI-1 in
alveolar macrophage. We examined the effects of hypoxia on
HIF-1␣ and PAI-1 expression in MH-S cells. Western blot
analysis showed that a low oxygen concentration (8%) modestly increased HIF-1␣ expression but had no measurable
effects on PAI-1 production (Fig. 7, A and B). In contrast, an
oxygen concentration of ⬍1% clearly increased steady-state
levels of HIF-1␣ protein and activated PAI-1 production (Fig.
7, C and D). It is interesting to note that the magnitude of the
increase in HIF-1␣ protein levels induced by TGF-␤1 was less
than that induced by ⬍1% oxygen, despite the increased
production of PAI-1 induced by TGF-␤ compared with ⬍1%
HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
L749
expands our concept of the role of activated alveolar macrophages in fibrosis to be more than just the cells that produce a
variety of cytokines, oxidants, and profibrosing products, including TGF-␤1. Few studies have described that alveolar
macrophages respond to TGF-␤1 despite the major advances
being made in understanding its role in pulmonary fibrosis.
Most of the previous studies showed that TGF-␤ exerts its
effects on alveolar epithelial cells or resident fibroblasts to
induce the production of organized alveolar exudates with
proliferating myofibroblasts and synthesis of connective tissue
(11, 21, 35, 51, 53). Our study showed for the first time that
TGF-␤1 induces PAI-1 and PDGF-A production in alveolar
macrophages through ALK5 activation and Smad3 phosphorylation.
In this study, we demonstrated that HIF-1␣ expression was
increased in alveolar macrophages in the BLM model of
AJP-Lung Cell Mol Physiol • VOL
pulmonary fibrosis. These results are consistent with a previous
study using microarray-based mRNA profiling of IPF patients,
which demonstrates an increase in the expression of the genes
that regulate oxygen transport and the hypoxic response (49).
Higgins et al. (16) showed that, in cultured tubular epithelial
cells and in a murine unilateral ureteral obstruction (UUO)
model of tubulointerstitial fibrosis, hypoxic stabilization of
HIF-1␣ in renal epithelial cells promotes interstitial fibrosis via
the induction of EMT. In addition, Madjdpour et al. (28)
described that alveolar hypoxia induced macrophage recruitment and enhanced expression of HIF-1␣ and inflammatory
mediators. Thus it is possible that hypoxia in alveolar macrophages in BLM-induced pulmonary fibrosis is attributable to
the accumulation of HIF-1␣ in those cells. However, this
assumption is unlikely because the alveolar space does not
seem to be hypoxic enough to induce HIF-1␣, given that
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Fig. 7. Effects of hypoxia on HIF-1␣ and
PAI-1 expression. A, C, E: MH-S cells (top)
were incubated either in normoxia or hypoxia
(8% O2, ⬍1% O2) with or without TGF-␤1
stimulation (5 ng/ml, 6 h) for indicated times.
Whole cell extracts were analyzed for HIF-1␣
protein expression by Western blot analysis.
The protein levels of HIF-1␣ (bottom) were
normalized against the protein levels of ␤-actin: the results are indicated as values relative
to the levels in the controls and are means ⫾
SD of three experiments. Significant difference compared with controls (n ⫽ 3): *P ⬍
0.05; **P ⬍ 0.01. B, D, F: culture media of
MH-S cells were exposed to hypoxia (8% O2,
⬍1% O2) with or without TGF-␤1 stimulation
and were subjected to the analysis of activatedPAI-1 protein level by ELISA. Data represent
means ⫾ SD from triplicate experiments
(*P ⬍ 0.05, **P ⬍ 0.01).
L750
HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
AJP-Lung Cell Mol Physiol • VOL
Fig. 8. Schematic diagram of the mechanisms of TGF-␤ and hypoxia induction
of PAI-1 expression in alveolar macrophages. Binding of TGF-␤1 to the type
II receptor (TGF-␤RII) allows this receptor to bind to the type I receptor
(TGF-␤RI) on the cell surface of the alveolar macrophage, resulting in
phosphorylation of the kinase domain of the type I receptor. This in turn
phosphorylates the transcription factors Smad2 or Smad3, which bind to
Smad4, the common Smad, and the resulting complex moves from the
cytoplasm into the nucleus. In the nucleus, the Smad complex binds to the
CAGA box, which serves as Smad binding element (SBE) within the PAI-1
promoter and induces transcription. Our study showed that TGF-␤1 decreases
PHD2 expression via the ALK5/Smad3 pathway. Under conditions where
PHD2 expression is decreased, HIF-1␣ translocates to the nucleus, forms a
complex with the p300/CBP coactivator proteins, and heretodimeraizes with
HIF-1␤. The HIF-1␣/HIF-1␤ dimer recognizes the HRE to induce PAI-1 gene
translocation. Under conditions where PHD2 expression remains high, such as
in normoxia or no TGF-␤1 stimulation, HIF-1␣ is hydroxylated on conserved
prolyl residues and subjected to polyubiquitination by the pVHL complex and
proteasomal degradation.
tosis (41). Our microarray analyses of TGF-␤1 stimulation of
MH-S cells with or without HIF-1␣ silencing revealed that
TGF-1 regulates a cluster of genes relevant to fibrosis through
a HIF-1-dependent mechanism. These include PAI-1, PDGF-A, and
type I TGF-␤ receptor. Previous studies with HIF-1␣ knockout
mice revealed that HIF-1 activity is required not only for the
O2 homeostasis under reduced oxygen conditions but also for
the immune system under normoxic condition (6). Our study
provides clues to understanding macrophage biology in the
regulation of the inflammation and fibrosis in response to the
inflammatory microenvironment in which a variety of cytokines, including TGF-␤1, are locally expressed and input signals into macrophages. Our study also supports the hypothesis
that HIF-1␣ plays an early role in the development of pulmonary fibrosis, which has been proposed by Tzouvelekis et al.
(49). They performed comparative expression profiling and
meta-analysis of the results from different animal models and
IPF patients and found that HIF-1␣ signaling was a statistically
significant deregulated pathway.
In conclusion, our study provides convincing data indicating
the effects of TGF-␤1 on PAI-1 expression in alveolar macrophages mediated via ALK5/Smad3. In addition, this study has
highlighted a crucial role of HIF-1␣ in mediating the effects of
TGF-␤1 on PAI-1 expression. We showed that TGF-␤1 induces
PAI-1 and PDGF-A expression via HIF-1␣-dependent mech300 • MAY 2011 •
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HIF-1␣ stabilization can occur at oxygen tensions lower than
5% O2 (equivalent to ⬃38 Torr) (16). Therefore, we hypothesize that HIF-1␣ accumulates in alveolar macrophages in
normoxia during pulmonary fibrosis at the alveolitis stage
in vivo.
Other than hypoxic activation, HIF-1␣ has been reported to
be activated by growth factors, cytokines, hormones, or nitric
oxide (NO) (9, 10, 12, 15, 18, 38, 45). Signal transduction
pathway investigations demonstrated that HIF-1␣ protein levels are subjected to complex modulation by those stimuli in
normoxia. In tubular cells, HIF-1␣ stabilization by NO and
inflammatory cytokines are reactive oxygen species (ROS)sensitive (37, 38), whereas in HepG2 cells this process is
mediated by phosphatidyl inositol 3-kinase (PI3K)-AKT signaling (45). With regard to insulin and insulin-like growth
factor-1 (IGF-1), and possibly other tyrosine kinase such as
EGF receptor and v-Src, an increase in HIF-1␣ protein levels
is due to an increase in the rate of HIF-1␣ protein synthesis.
This increase is mediated by the PI3K-AKT-FRAP (FKBPrapamycin-associated protein) pathway that phosphorylates
and activates the translational regulatory proteins eIF-4E-binding protein 1 and p70 S6 kinase (p70S6K) (23, 24). In addition,
mitogen-activated protein kinase (MAPK) is required for
HIF-1␣ induction by IGF-1 (9). In this study, we showed that
TGF-␤1 increases HIF-1␣ protein levels through a decrease in
the expression of a key limiting prolyl-4-hydroxylases, PHD-2,
that hydoxylates HIF-1␣ at two proline residues (Pro402 and
Pro564).
Our finding that TGF-␤1 induces PAI-1 expression in a
HIF-1␣-dependent manner merits further discussion. It is noteworthy that magnitude of induction by TGF-␤1 is more predominant than that induced by ⬍1% hypoxia, despite the fact
that the accumulation of HIF-1␣ following TGF-␤1 stimulation
is less than that observed in 1% hypoxia. The most plausible
explanation for this discordance between HIF-1␣ and PAI-1
induction by TGF-␤ may be explained as depicted in Fig. 8.
TGF-␤1 induces PAI-1 gene expression through at least two
distinct mechanisms. In addition to the mechanism identified in
this study, a previous study demonstrated that PAI-1 gene
expression is highly induced by TGF-␤1 through the TGF-␤responsive element, referred to as the CAGA box, to which
Smad3/Smad4 bind (7). In contrast, hypoxic induction of
PAI-1 gene expression is exclusively dependent on the HIF-1/
hypoxia response element (HRE) (20). Thus we assume that
TGF-␤1 induction of PAI-1 gene expression is mediated by
both CAGA box and HRE, whereas hypoxic induction is
mostly or completely mediated by HRE alone.
Stimulation of MH-S cells with TGF-␤1 under hypoxia
(⬍1% O2) robustly increased PAI-1 protein levels, suggesting
that the effects of TGF-␤1 and hypoxia on PAI-1 expression
are synergistic rather than additive. Although the precise mechanisms underlying these results remain unclear, it is intriguing
to speculate that the HIF-1/HRE complex and Smad3/4/
CAGA-box complex directly or indirectly interact and activate
PAI-1 gene transcription. Further study using the PAI-1 promoter mutation constructs containing either the CAGA box or
HRE are required to prove this hypothesis.
To date, over 100 genes are known to be targets of HIF-1␣
under low oxygen concentrations (typically ⬍2% O2). Those
include the genes involved in glucose metabolism, erythropoiesis, angiogenesis, vascular tone, cell proliferation, and apop-
HIF-1␣ MEDIATES TGF-␤1-INDUCED PAI-1 PRODUCTION
anisms in normoxia, and the effects of TGF-␤1 on hypoxic
cells were remarkable. Combined with the microarray data that
identify a set of genes regulated by TGF-␤1 via HIF-1␣ in
MH-S cells, these data have relevance to the pathogenesis of
pulmonary fibrosis given that a number of HIF-1-dependent
genes are expressed in IPF patients. The role of HIF-1␣ in
macrophages in pulmonary fibrosis in vivo needs to be verified
by experiments using alveolar macrophage-specific HIF-1␣
knockout mice.
ACKNOWLEDGMENTS
We are grateful to Yoshiko Nonaka, Miki Matsui, Keiko Arai, and Yukiyo
Tosaka for technical assistance.
GRANTS
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the author(s).
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