© 1999 American Heart Association, Inc.
Original Contributions |
Angiotensin II–Induced Transactivation of Epidermal Growth Factor Receptor Regulates Fibronectin and Transforming Growth Factor-ß Synthesis via Transcriptional and Posttranscriptional Mechanisms
From the Department of Medicine II (Y. Moriguchi, H. Matsubara, Y. Mori, S.M, H. Masaki, K.M, Y. Tsutsumi, Y.S, Y. Tanaka, T.I.), Division of Endocrine Hypertension, Metabolism and Nephrology, Kansai Medical University, Moriguchi, Osaka, and Department of Biological Science and Technology (T.N, K.O.), Science University of Tokyo, Chiba, Japan.
Correspondence to Hiroaki Matsubara, Department of Medicine II, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8507, Japan. E-mail: matsubah{at}takii.kmu.ac.jp
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Abstract—The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein–coupled receptors. Here, we report the involvement of EGF-R in Ang II–induced synthesis of fibronectin and transforming growth factor-ß (TGF-ß) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (
5-fold) observed after 12 hours of incubation. Fibronectin
synthesis induced by Ang II or calcium ionophore was completely
abolished by tyrosine kinase inhibitors and intracellular
Ca2+ chelating agents. Ang II–induced fibronectin mRNA was
not affected by protein kinase C inhibitors or protein
kinase C depletion, whereas specific inhibition of EGF-R function by a
dominant negative EGF-R mutant and tyrphostin AG1478 abolished
induction of fibronectin mRNA. We isolated the rat fibronectin gene,
including the 5'-flanking region, and found that the
activator protein-1 (AP-1) binding site present in the
promoter region was responsible for the Ang II responsiveness of this
gene. A gel retardation assay revealed the binding of nuclear protein
to the AP-1 site, which was supershifted with
anti–c-fos and anti–c-jun but not
anti–activating transcription factor (ATF)-2 antibodies.
Conditioned medium from Ang II–treated cells contained TGF-ß
bioactivity, and addition of neutralizing TGF-ß antibody modestly
(46%) inhibited induction of fibronectin. Ang II–induced synthesis of
TGF-ß was also abolished by inhibition of EGF-R function. The effect
of TGF-ß was exerted by stabilizing fibronectin mRNA without
affecting the promoter activity and required de novo protein synthesis.
We concluded that Ang II–induced expression of fibronectin and TGF-ß
is mediated by downstream signaling of EGF-R transactivated by
Ca2+-dependent tyrosine kinase and that Ang II–induced
fibronectin mRNA expression is regulated by 2 different mechanisms,
which are transcriptional control by binding of the
c-fos/c-jun complex to the AP-1 site and
posttranscriptional control by mRNA stabilization due to autocrine or
paracrine effects of TGF-ß. Thus, this study suggests that the
action of Ang II on extracellular matrix formation should be
interpreted in association with the EGF-R signaling cascade.
Key Words: angiotensin II receptor • angiotensin II type 1 receptor • angiotensin II type 2 receptor • angiotensin II • epidermal growth factor receptor
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Cardiac fibroblasts isolated from neonatal rat hearts have abundant high-affinity angiotensin II (Ang II) receptors, which are classified pharmacologically as belonging to the Ang II type 1 receptor (AT1) subtype.1 2 These cells have been used to examine AT1-mediated signaling2 3 4 and extracellular matrix remodeling in heart failure.5 AT1 stimulation was found to stimulate DNA synthesis and cell proliferation1 and also to increase the synthesis of extracellular matrix proteins,4 6 which suggests that cardiac fibroblasts contribute to remodeling of the cardiac interstitium under a variety of physiological and pathological conditions. Ang II, acting via AT1, initiates early biochemical events, including rapid production of diacylglycerol and inositol 1,4,5-triphosphate by phospholipase C–mediated hydrolysis of inositol phospholipids and activation of protein kinase C (PKC).7 8 9 10 Ang II also induces a rapid increase in expression of the growth-associated nuclear proto-oncogenes in a manner similar to that of cellular events mediated by peptide growth factors and stimulates tyrosine phosphorylation of multiple substrates, including p44 and p42 mitogen-activated protein/extracellular signal-regulated kinases (ERKs).11 12 13 14 15 16
Although it was reported that PKC played a dominant role in the Ang
II–induced activation of ERK in vascular smooth muscle cells
(VSMCs)17 18 and in cardiac myocytes,13 other
studies indicated that calcium signaling rather than PKC plays a
critical role in ERK activation in these cells.14 15 16
Certain aspects of signal transduction characteristic of Ang II
stimulation resemble those evoked by growth factors. Activation of
phospholipase C-
, tyrosine kinases, and ERK and expression of
nuclear proto-oncogenes exemplify phenomena common to Ang II and growth
factor signaling.9 12 Recently, cross-talk between G
protein–coupled receptors and growth factor receptors with intrinsic
tyrosine kinase activity was reported. In VSMCs, stimulation of the
AT1 causes transactivation of
platelet-derived growth factor ß receptor19 or
epidermal growth factor receptor (EGF-R),20 resulting in
transmission of AT1-mediated ERK signaling in
association with formation of an adapter protein complex containing the
SH2 domain, such as Grb2 or Shc. Stimulation of Rat-1 cells with
endothelin-1, lysophosphatidic acid, or thrombin induced a rapid
increase in tyrosine phosphorylation of EGF-R and
p185neu, leading to activation of the
ERK.21 Stimulation of Cos-7 cells with Gi- or Gq-coupled
receptors caused phosphorylation of EGF-R associated
with assembly of Shc and Grb2,22 23 and calcium-dependent
EGF-R activation by stimulation of voltage-sensitive calcium channels
was also found in PC12 cells.24 25 Ang II
activated tyrosine phosphorylation of
insulin-like growth factor-1 receptor and insulin receptor substrate-1
in VSMCs.26 In addition, m1 muscarinic acetylcholine
receptor was also shown to transactivate EGF-R and its
downstream signaling, resulting in modulation of potassium channel
function in human embryonic kidney 293 cells.27 Thus,
additional tyrosine kinases that phosphorylate receptor
tyrosine kinases ligand independently appear to contribute in a general
or cell type–specific way to mitogenic signaling mediated
through G protein–coupled receptors. We28 and Eguchi et
al20 recently showed that Ang II–induced ERK activation
and its mitogenic signals are dominantly mediated by
downstream signaling of transactivated EGF-R.
Two thirds of the myocardial cell population is composed of
nonmyocyte cells, the majority of which are
fibroblasts.5 Cardiac fibroblasts are responsible for the
production and deposition of extracellular matrix proteins such
as fibronectin and collagen types 1 and 3.5 6
Fibronectin is an extracellular matrix and plasma protein with various
functions that plays important roles in cardiac remodeling as well as
cell adhesion and migration.29 Although fibronectin
synthesis in cardiac fibroblasts was reported to be induced in response
to Ang II,2 6 the signal transduction mechanism
responsible for this induction remains to be determined. In the
present study, we found that Ang II–induced synthesis of
fibronectin and transforming growth factor-ß (TGF-ß) is regulated
through downstream signaling of EGF-R transactivated by
Ca2+/calmodulin-dependent tyrosine
kinases. Induction of fibronectin mRNA by Ang II was regulated by 2
different mechanisms, which are transcriptional control by binding of
the c-fos/c-jun complex to the
activator protein-1 (AP-1) site and posttranscriptional
control by autocrine or paracrine effects of TGF-ß, which is exerted
by increasing the mRNA stability via de novo protein synthesis and
which upregulates fibronectin mRNA levels by 46%.
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Materials and Methods |
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GF109203X, BAPTA-AM, genistein, ST638, and W7 were purchased from Calbiochem. TMB-8, nifedipine, and calmidazolium chloride were purchased from Calbiochem. A23187, phorbol 12-myristate 13-acetate (PMA), and EGTA were purchased from Sigma. Losartan was kindly provided by DuPont Merck Pharmaceutical. PD123319 was kindly provided by Parke-Davis and Warner-Lambert Co. Fibronectin antibody was purchased from Telios Pharm.
Cell Culture
Cardiac fibroblasts were prepared from ventricles of 1- to
2-day-old Wistar rats and grown as previously
described.30 31 Subcultured fibroblasts from passages 4
and 5, used in this experiment, were >99% positive for
immunostaining with vimentin antibody and were negative
for desmin (for myocytes), smooth muscle 
-actin (for VSMCs), and a
polyclonal antibody against von Willebrand factor (for
endothelial cells) (Sigma). Subconfluent cells were
serum starved for 24 hours and used for the experiments.
Northern Blotting
Total RNA was extracted by guanidinium isothiocyanate–cesium
chloride centrifugation, fractionated on 1%
agarose-formaldehyde gels, and transferred to nylon membranes as
previously reported.30 31 32 33 Blots were then hybridized with
random-primed 32P-labeled cDNA probes consisting
of fibronectin, TGF-ß, and GAPDH as an internal control. Hybridized
signals were measured by scanning densitometry, and fibronectin mRNA
levels were arbitrarily normalized relative to the GAPDH mRNA
levels.
Stable Transfection and Chloramphenicol Acetyltransferase
(CAT) Assay
Cardiac fibroblasts were cotransfected with a 5:1 molar ratio of
the fibronectin promoter-CAT chimeric constructs and pSV2neo DNAs
(15 µg per 10-cm dish) using Lipofectamine Plus reagent
(GIBCO-BRL) according to the manufacturer's instructions followed by
incubation with fresh medium for 48 hours as previously
reported.34 Selection of G418 (500 µg/mL) resistance was
started 48 hours later. After 8 to 12 days, resistance foci appeared,
and pooled foci of stable transformants were subjected to the standard
differential protocol.28 The stable transformants were
serum deprived for 24 hours and then exposed to Ang II (100 nmol/L) for
an additional 12 hours. In each transfection, 5 µg of Rous sarcoma
virus ß-galactosidase was also cotransfected. CAT activity was
normalized for transfection efficiency by ß-galactosidase activity
and for cell density by protein concentration. CAT activities were
determined by a dual-diffusion assay and normalized for cell density by
protein concentration as previously reported.35 36 CAT
activity was expressed relative to the activity of a promoterless CAT
construct.
[35S]Methionine Labeling of Cells and
Immunoprecipitation
Quantification of fibronectin secreted into medium was performed
as previously reported.6 Briefly, fibroblasts were made
quiescent by serum depletion for 12 hours. After 12 hours of incubation
with or without Ang II (0.1 µmol/L), 100 µCi/mL of
[35S] methionine was added for another 12
hours. For immunoprecipitation, aliquots (100 µL) of the medium were
diluted with 900 µL of radioimmunoprecipitation assay buffer (50
mmol/L Tris, 150 mmol/L NaCl, 1% NP-40, 0.5% deoxycholate, and
0.1% SDS) and treated with normal rabbit serum (10 µL) to remove
nonspecific binding, followed by incubation with rabbit antibody to
fibronectin for 16 hours at 4°C. Immune complexes were removed by
protein A–Sepharose beads (Pharmacia LKB) and analyzed by
SDS-PAGE.6
Creation of Mutations in the AP-1 Site
Mutation in the AP-1 site at –453 present in the 5'
flanking region was created by polymerase chain reaction (PCR) overlap
extension mutagenesis as previously reported.36 Briefly, 2
DNA fragments with overlapping ends were first amplified with 2 sets of
primers (A and B, C and D) by using p564-CAT as a template for making
the mutations. Primers A and D were T3 and T7 primers for the pBS
vector, respectively. Primers B and C had the following DNA sequence:
B, 5'-GTGACCGTGGTGGTGTTTGAGAACATTTATCCA-3' (antisense,
nucleotides –453 to –421), and C,
5'-TCAAACACCACCACGGTCAC-3' (sense, nucleotides –439 to
–421). Primer B contained a mutation in the AP-1 site at the 3' end,
and primers B and C were designed to overlap at the 5' end. The 2
respective PCR products were mixed and amplified again with primers
A and D. After confirming the DNA sequence, the PCR product was
subcloned into the 5' end of the CAT gene.
Gel Retardation Assay
The gel retardation assay and preparation of nuclear extract
from cells exposed to Ang II (100 nmol/L) for 45 minutes was performed
as previously reported.36 The final protein concentration
was 0.5 to 1 mg/mL. The oligonucleotides
corresponding to the DNA fragment containing AP-1 and the mutated AP-1
sequence (between nucleotides –438 and –473) or the
ATF/CRE motif (nucleotides –143 to –178) were
synthesized, labeled with [-32P]ATP using T4
kinase (Takara Shuzo), and annealed to make double-strand DNA. Nuclear
extracts were incubated with the probe for 30 minutes at room
temperature in the binding buffer, consisting of (in mmol/L) HEPES
0.5, KCl 60, EDTA 0.1, DTT 0.5, and phenylmethylsulfonyl
fluoride 0.5; 12% glycerol; and 2 µg of poly(dI-dC). The
mixture was loaded on a 6% polyacrylamide gel and
electrophoresed as previously reported.36 Supershift
assays were performed with rabbit polyclonal anti- c-fos IgG
raised against the portion of c-fos containing amino acids
128 to 152 and rabbit anti–c-jun IgG raised against the
portion of c-jun containing amino acids 247 to 263 (Santa
Cruz Biotechnology). Each antibody (1 µg each) was added to the
samples after the initial binding reaction and then incubated for 1
hour at room temperature.
Quantification of Latent and Active TGF-ß
The bioassay for TGF-ß released into medium was performed by
measuring proliferation inhibition of mink lung epithelial cell line
CCL-64 (American Type Culture Collection), as reported
elsewhere.37 Briefly, CCL-64 cells were seeded in 24-well
plates in DMEM containing 10% FCS. After incubation for 24 hours, the
medium was discarded and changed to DMEM containing 1% FCS. After an
additional incubation for 24 hours, control or test samples were added,
and cells were incubated for another 24 hours. Subsequently, 1 µCi/mL
of [3H]thymidine (NEN) was pulsed for 6 hours,
and the amount of [3H]thymidine incorporated
into trichloroacetic acid–precipitable materials was measured by a
liquid scintillation counter. To determine whether cells were producing
latent TGF-ß, latent TGF-ß was converted to the active form by
treating culture supernatant with 10N HCl (final pH, 2.0) for 30
minutes at room temperature, followed by neutralization with equimolar
NaOH. TGF-ß activities were determined on the basis of the standard
curve (TGF-ß concentration versus percentage of inhibition), which
was drawn by using TGF-ß1 (R&D Systems, Inc).
Culture supernatants were serially diluted to fall within the range of
the standard curve. Specificity of the observed effects was proven by
competition of the TGF-ß activity with a purified rabbit
anti–TGF-ß IgG and a control IgG (R&D Systems, Inc).
Membrane Preparation and Binding Assay
The binding assay was performed as previously
reported.6 31 Briefly, membrane fractions were incubated
with 125I-labeled Ang II in the assay
volume for 3 hours at 22°C. Bmax and
Kd values and AT1 and
AT2 densities were calculated by Scatchard
analyses and nonlinear least-squares regression
analysis on the basis of inhibition by CGP42112A.
Measurement of Lactate Dehydrogenase (LDH) Activities
A spectrophotometric enzyme assay (DRI-CHEM slide LDH-P, Fuji
Film) was performed to measure LDH release in the
medium.28 In brief, 10 µL of triplicate medium and
reagent were mixed for 2 minutes at room temperature. Absorbance at 540
nm was determined, and LDH activity (U/L) was automatically calculated
by FD3030 analyzer. One unit of LDH activity is defined
as that amount of enzyme that will catalyze the formation of 1
µmol of NADH per minute.28
Statistical Analysis
The results are expressed as mean±SE. Statistical
analyses were performed with a 1-way ANOVA followed by pairwise
contrasts (control versus conditions) using the Dunnett multiple
comparison test. Data were considered statistically significant at
P<0.05.
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Results |
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Ang II–Induced Fibronectin mRNA Level Is Mediated by AT1 Stimulation
Ang II (100 nmol/L) significantly stimulated fibronectin mRNA accumulation after 6 hours of incubation, reaching a maximal increase after 12 hours, and thereafter maintained a similar level until 24 hours of incubation (Figure 1
). Ang
II–induced fibronectin mRNA levels were increased dose dependently
with a maximal peak at 100 nmol/L (data not shown). Therefore,
subsequent experiments were performed with 100 nmol/L Ang II
stimulation for 12 hours. To determine which Ang II receptor subtypes
mediate fibronectin mRNA accumulation, Ang II receptor densities were
quantified in cardiac fibroblasts, and the effects of the
AT1 antagonist losartan or
the AT2 antagonist PD123319 were
examined. Cardiac fibroblasts (passage 4) expressed only
AT1 (Kd, 0.21±0.01
nmol/L; Bmax, 178±5 fmol/mg protein);
AT2 expression was not detectable in these cells.
Induction of fibronectin mRNA by Ang II was completely blocked by
losartan but not by PD123319 (Figure 1), which
demonstrated that Ang II–induced fibronectin mRNA accumulation is
mainly mediated by AT1, and
AT2 is not involved in this induction mechanism
by Ang II. We previously reported that AT2 is
substantially expressed in primary culture of cardiac fibroblasts from
cardiomyopathic hamsters during heart failure, whereas
AT2 expression is abruptly downregulated with
repeated passages of cells.6 Given that the primary
culture of neonatal rat cardiac fibroblasts also expresses detectable
amounts of AT2, it is likely that downregulation
of AT2 in the cells (passages 4 to 5) used in
this study is due to repeated passages of cells.
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Phorbol Ester–Sensitive PKC Is Not Involved in Ang II–Induced
Fibronectin mRNA Accumulation
Stimulation of AT1 results in generation of
inositol triphosphate and diacylglycerol, which induce the release of
Ca2+ from intracellular stores and PKC
activation, respectively.9 We next examined the effects of
the PKC inhibitors GF109203X and calphostin C on Ang
II–induced fibronectin mRNA levels. Pretreatment with GF109203X
(1 µmol/L) or calphostin C (50 nmol/L) completely inhibited
fibronectin mRNA accumulation in response to 100 nmol/L PMA, whereas no
significant inhibition of Ang II–induced fibronectin mRNA level was
observed (Figure 2). Depletion of phorbol
ester–sensitive PKC by 24-hour incubation with 10 µmol/L of PMA
also did not inhibit Ang II–induced accumulation of fibronectin mRNA
(Figure 2). These results suggest that PKC-mediated pathways do
not play a dominant role in Ang II–induced fibronectin mRNA
accumulation, and that GF109203X-, calphostin C–, and phorbol
ester–sensitive PKC are not involved in this mechanism.
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Ang II–Induced Fibronectin mRNA Accumulation Is Stimulated via
Ca2+/Calmodulin-Dependent Pathway
We have previously shown that exposure of fibroblasts to Ang II
markedly increases intracellular Ca2+ levels, and
this increase was blocked by the intracellular
Ca2+ chelator BAPTA-AM (10 µmol/L) but not
by extracellular Ca2+ chelation by EGTA. These
observations indicated that Ca2+ mobilization
after AT1 stimulation is mainly caused by release
of Ca2+ from intracellular stores.38
As shown in Figure 3, Ang II–induced
fibronectin mRNA levels were completely inhibited by pretreatment with
BAPTA-AM (10 µmol/L) and TMB8 (100 µmol/L), which are
commonly used as intracellular Ca2+ chelators,
but not by pretreatment with EGTA. These Ca2+
chelators did not affect the basal expression level of fibronectin mRNA
(data not shown). In agreement with these results, addition of the
Ca2+ ionophore A23187 also stimulated fibronectin
mRNA accumulation (Figure 3). Elevation of cytosolic
Ca2+ activates a variety of enzymes
through interaction with calmodulin. To examine whether
calmodulin mediates fibronectin mRNA levels in response to
Ang II, fibroblasts were preincubated with the calmodulin
inhibitors W-7 (100 µmol/L) and
calmidazolium (10 µmol/L).These
drugs completely blocked Ang II–induced fibronectin mRNA accumulation
(Figure 3). These calmodulin inhibitors
at the concentrations used in the present study did not affect the
basal level of fibronectin mRNA and did not have any effect on the
PMA-induced increase in fibronectin mRNA level (data not shown), which
suggests that Ang II stimulates fibronectin mRNA accumulation through a
Ca2+/calmodulin-dependent
mechanism.
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Ang II–Induced Fibronectin mRNA Accumulation Is Activated
by Ca2+/Calmodulin-Dependent Protein
Tyrosine Kinases
To determine whether tyrosine kinase activity is required for
Ca2+-dependent fibronectin mRNA accumulation,
cells were pretreated with genistein (100 µmol/L) and ST638
(100 µmol/L), protein kinase inhibitors with a
strong preference for tyrosine-specific kinases,39 40 and
then stimulated by either Ang II or A23187. These
inhibitors completely abolished both Ang II– and
A23187-induced fibronectin mRNA accumulation with no effects on basal
fibronectin mRNA levels (Figure 4). On
the other hand, these tyrosine kinase inhibitors did not
have any effect on PMA-induced increase in fibronectin mRNA level (data
not shown). These findings suggest that protein tyrosine kinases
activated downstream of the
Ca2+/calmodulin pathway are closely
involved in Ang II–induced fibronectin mRNA accumulation.
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We also examined the effects of these kinase inhibitors on cell toxicity at the concentrations used in the above experiments. Although we also measured LDH in culture medium from cells treated with these drugs, no LDH activities were detected. These results suggested that complete inhibition of Ang II– or A23187-induced fibronectin mRNA accumulation by these drugs was not due to nonspecific cytotoxicity.
Ang II–Induced Transactivation of EGF-R Is Involved in Induction
of Fibronectin mRNA
We recently found that in cardiac fibroblasts
Ca2+/calmodulin-dependent tyrosine
kinases activated by AT1 stimulation
cause tyrosine phosphorylation of EGF-R but not
platelet-derived growth factor receptor, which mediates
AT1/Ras/ERK signaling.28 Therefore,
we next examined whether this EGF-R transactivation mechanism is
involved in AT1-mediated fibronectin mRNA
accumulation. Specific inhibition of EGF-R function by the specific
EGF-R antagonist tyrphostin AG147828 20
markedly inhibited both Ang II– and A23187-induced fibronectin mRNA
accumulation (Figure 4
). EGF also dose-dependently stimulated
fibronectin mRNA levels, and this increase was blocked by AG1478
(Figure 4). However, AG1478 did not affect the basal expression
level of fibronectin mRNA (data not shown).
The role of EGF-R in AT1-mediated
fibronectin mRNA accumulation was further analyzed by specific
inhibition of the EGF-R signal. A dominant negative EGF-R mutant
lacking the cytoplasmic domain of human EGF-R (HEGF-R 533del)
was constructed and stably transfected into cardiac fibroblasts. This
mutant inhibited the downstream signaling of rat EGF-R by formation of
signaling-defective heterodimers with the wild-type receptor, and we
have shown that cardiac fibroblasts stably expressing HEGF-R 533del
abolished Ang II– or EGF-induced EGF-R
phosphorylation.28 Using these cloned
cells, we examined Ang II– or EGF-induced effects on fibronectin mRNA
accumulation. In HEGF-R 533del cells, fibronectin mRNA accumulations at
low and higher ligand concentrations (2 and 50 ng/mL EGF) were
completely and partially attenuated, respectively, compared with that
in the control transfected cells, whereas Ang II–induced expression of
fibronectin mRNA was completely blocked (Figure 5A and 5B). We next examined the
involvement of ERK activated downstream of EGF-R on Ang
II–induced fibronectin mRNA levels. Inhibition of ERK activity by ERK
kinase (MEK) inhibitor PD98059 greatly (74%;
P<0.05) inhibited Ang II–induced fibronectin mRNA
accumulation (Figure 5A), which suggests an important role of
ERK activation in induction of fibronectin mRNA after Ang II
stimulation. To test whether PD98059 causes cell injury at the
concentrations used in this study, we measured LDH levels in culture
medium. We did not detect LDH activity in culture medium from Ang
II–stimulated cells pretreated with PD98059 (10 µmol/L), which
indicates that the effect of this drug was not due to induction of
apoptosis or nonspecific cytotoxicity.
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Ang II–Induced Fibronectin mRNA Expression Results in an Increase
in Fibronectin Protein Release
We also examined whether fibronectin mRNA accumulation by Ang II
results in induction at the fibronectin protein level. Fibronectin
protein secreted into the medium was analyzed by
immunoprecipitation using anti-fibronectin antibody as previously
described.6 Immunoprecipitated fibronectin secreted into
medium was increased by 2.8-fold over the control by 0.1 µmol/L
of Ang II. Pretreatment with AG1478 completely abolished Ang
II–induced fibronectin secretion, whereas PD98059 modestly (67%;
P<0.05) inhibited it (Figure 5C). To further confirm
the role of EGF-R transactivation, we tested the effects of Ang II and
EGF on the fibronectin release from cells expressing HEGF-R 533del. As
shown in Figure 5C, fibronectin release induced by Ang II (100
nmol/L) and EGF (2 ng/mL) was completely abolished in HEGF-R 533del
cells. Although we tested the effect of PD123319 on Ang II–stimulated
release of fibronectin, no significant effect was observed in
fibronectin release from cells pretreated with PD123319 (data not
shown).
AP-1 Binding Sequence in the Fibronectin Gene Promoter Confers
Responsiveness to Ang II
To investigate the effects of Ang II on fibronectin gene
transcription, a rat genomic EMBL3 library was screened for the
5'-flanking region of the fibronectin gene with a
32P end–labeled DNA fragment synthesized by PCR
on the basis of the sequence data reported by Patel et
al.41 We isolated the rat fibronectin gene containing 1908
bp of 5'-flanking region from the rat genomic library. The sequence
downstream of position –1080 relative to the transcriptional start
site (+1) was identical to that reported by Patel et al.41
Computer analysis of the sequence revealed the presence of
binding sites for several transcription factors such as AP-1 (TGACGCA
at –453), ATF/CRE (TGACGTCA at –160), AP-2 (CCCCAGGC at –364), and
PEA2 (GACCGCA at –257). To examine the promoter activity of the rat
fibronectin gene in cardiac fibroblasts, we constructed 5' sequential
deletion mutants of the fibronectin promoter using restriction enzyme
sites and fused these mutants to the CAT gene as a reporter (Figure 6A). We established stable
transformants of these chimeric CAT constructs because of the low
efficiency of transient transfection rate into cardiac fibroblasts and
tested Ang II responsiveness of each construct. The longest chimeric
construct (p1908-CAT) had sufficient promoter activity, resulting in
2.8-fold higher CAT activation than the basal activity of the
promoterless construct. When cells transfected with p1908-CAT were
exposed to Ang II, the CAT activity showed an increase of 3.1-fold
relative to that in unstimulated cells, indicating that the Ang
II–responsive element is present in the 1908 bp of 5'-flanking
region. Using stable transformants of 5' sequential deletion mutants,
we found a significant decrease in the basal CAT activity with
deletions between –1908 and –1079, –564 and –414, –202 and –123,
and –123 and +0, which suggests the presence of several positive
cis-regulatory elements (Figure 6A).
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P<0.05 vs the CAT activity of unstimulated cells in
the absence of Ang II. B, Double-stranded
oligonucleotide containing an AP-1 site
(nucleotides –438 to –473) was used as a probe in the gel
shift assay. Nuclear extract was obtained from cells exposed to Ang II
(100 nmol/L) for 45 minutes. AG1478 (250 nmol/L) and PD98059 (10
µmol/L) were added 20 minutes before exposure to Ang II. An unlabeled
oligonucleotide corresponding to
nucleotides –438 to –466 was used as the competitor at
25x and 100x molar excess. AP-1 and mutated AP-1
oligonucleotides were used at 200x molar excess.
Oligonucleotide AP-1 indicates AP-1 sequence
present at –453 (TGACGCA), and mutated
oligonucleotide AP-1 indicates the mutated sequence
TGGATAA. Supershift assays were performed with rabbit polyclonal
anti–c-fos IgG raised against amino acids 128 to 152 of
c-fos and rabbit anti–c-jun IgG raised
against amino acids 247 to 263 of c-jun. Each antibody
was added to the samples after the initial binding reaction and then
incubated for 1 hour at room temperature. C,
Oligonucleotides (–143 to –178) including the ATF/CRE
motif were used as a probe in the gel shift assay. Nuclear extract was
obtained from cells exposed to Ang II (100 nmol/L) or PMA (1
µmol/L) for 45 minutes. The unlabeled ATF/CRE
oligonucleotide corresponding to –143 to –178 was
used as a competitor at 200x molar excess. Cont indicates
control.
Exposure to Ang II of cells stably transfected with p1079-CAT,
p882-CAT, p746-CAT, or p564-CAT induced significant increases
(3-fold) in CAT activity to an extent similar to that in p1908-CAT,
whereas in cells transfected with p414-CAT, p202-CAT, or p123-CAT the
Ang II–induced increases were abolished, which suggests the presence
of an Ang II–responsive element in the promoter region between
nucleotides –564 and –414. Because the AP-1 site
(TGACGCA) was located at nucleotide –453 of the
fibronectin gene and c-fos and c-jun mRNA
expression was upregulated in response to Ang II in cardiac
fibroblasts,28 we tested the involvement of the AP-1
site by site-directed mutation
(TGACGCA
TGGATAA) in this motif of p564-CAT
(termed p564 m-CAT) followed by stable transfection into the cells.
Interestingly, we found that the responsiveness to Ang II was
completely inhibited in cells transfected with p564 m-CAT (Figure 6A), indicating a critical role of the AP-1 site in the Ang
II–induced increase of fibronectin gene transcription. To further
study the role of EGF-R transactivation in Ang II–stimulated
fibronectin promoter activity, we tested the effects of EGF-R
antagonist AG1478 or MEK inhibitor PD98059. As
shown in Figure 5A, treatment of p1908-CAT with AG1478
completely abolished Ang II–stimulated fibronectin promoter activity,
whereas its activity was partially inhibited by PD98059 (64%
inhibition in promoter activity).
c-fos and c-jun Heterodimer Induced
by Ang II Binds to the AP-1 Site of the Fibronectin Gene
Promoter
We have previously shown that c-fos and
c-jun mRNA expression is upregulated in response to Ang II
in cardiac fibroblasts.28 To test whether Ang
II–induced c-fos and c-jun complex binds the
AP-1 binding site of the fibronectin gene promoter to enhance gene
transcription, we performed a gel retardation assay using an
oligonucleotide (nucleotides –438 to
–463), including the AP-1 binding site as a probe. The nuclear extract
from unstimulated control cells contained no protein capable of binding
to the probe, whereas we found a retarded band when the probe was
incubated with nuclear extract from cells stimulated with Ang II
(Figure 6B). An oligonucleotide corresponding to
the AP-1 (TGACGCA) sequence inhibited the binding of nuclear extract,
whereas a mutated oligonucleotide
(TGGATGAA) did not affect binding. Furthermore, protein
that bound to the AP1 oligonucleotide was supershifted
when preincubated with anti–c-fos and
anti–c-jun antibodies, but not anti-ATF2 antibody (Figure 6B). To further study the role of EGF-R transactivation in Ang
II–stimulated gel mobility of AP-1 complex, we tested the effects of
EGF-R antagonist AG1478 or MEK inhibitor
PD98059. As shown in Figure 6B, treatment with AG1478 abolished
Ang II–stimulated binding of AP-1 complex to the AP-1 sequence,
whereas its binding was not completely inhibited by PD98059.
The ATF/CRE motif (TGACGTCA) present at nucleotide
–160 is known to stimulate transcription of several
genes.42 Promoter analyses using CAT reporter
constructs (Figure 6A) revealed that the ATF/CRE motif
present in the promoter region of the fibronectin gene did not
confer Ang II responsiveness. To test whether nuclear extract from Ang
II–treated cells binds the ATF/CRE motif, we performed a gel
retardation assay using an oligonucleotide
(nucleotides –143 to –178) including the ATF/CRE motif.
As shown in Figure 6C, the binding activity of nuclear extract
to the ATF/CRE motif was not enhanced by Ang II treatment, whereas PMA
induces the binding protein specific to the ATF/CRE motif.
TGF-ß Secreted Into Medium After Ang II Treatment Induces
Fibronectin Expression
Because Ang II is known to induce the synthesis and secretion of
TGF-ß, resulting in stimulation of fibronectin expression in
mesangial cells,43 we tested whether this
autocrine or paracrine effect of TGF-ß also occurs in cardiac
fibroblasts. TGF-ß activity was measured using the standard CCL-64
cell bioassay, in which TGF-ß secreted by cells into culture medium
was measured by its ability to inhibit cell growth. As shown in Figure 7A, the mean amount of active
TGF-ß present in 6 control cell cultures was 0.18 ng/mL (open
bars). When stimulated with 100 nmol/L Ang II, a 6.7-fold increase to
1.2 ng/mL was seen. This increased activity was completely abrogated by
neutralizing anti–TGF-ß antibody, but not by nonimmune control
rabbit IgG. Acid treatment of conditioned medium resulted in
significant increases in active TGF-ß in both control and Ang
II–treated cultures. However, acid treatment of control conditioned
medium resulted in 0.8 ng/mL active TGF-ß, whereas active TGF-ß in
Ang II–treated culture medium increased to 2.9 ng/mL (gray bars). This
result indicated that Ang II not only increased the production
of both latent and active TGF-ß but also stimulated the conversion of
latent to active TGF-ß.
|
To study the role of EGF-R transactivation and ERK activation in Ang
II–stimulated release of TGF-ß, we determined Ang II–stimulated
TGF-ß activity using HEGF-R 533del cells or the cells pretreated with
MEK inhibitor PD98059. Ang II–stimulated TGF-ß
activities in the incubation medium were completely inhibited in HEGF-R
533del cells and by treatment with PD98059 (Figure 7A),
indicating that ERK activated downstream of EGF-R is a major
mediator to regulate the release of TGF-ß by Ang II.
To assess whether secreted TGF-ß mediated the effect of Ang II on
fibronectin mRNA accumulation, Ang II–treated cells were coincubated
with neutralizing anti–TGF-ß antibody (100 µg/mL) or control IgG
(100 µg/mL) (Figure 7B). Interestingly, treatment of cultures
with neutralizing antibody significantly (46%; P<0.01)
abolished the induction of fibronectin mRNA expression. Control IgG had
no effect. Addition of a sufficient amount of anti–TGF-ß
neutralizing antibody was confirmed by the finding that the conditioned
medium from cells incubated with Ang II and anti–TGF-ß neutralizing
antibody for 24 hours contained control levels of TGF-ß activity
(0.15 ng/mL) as measured by bioassay. These results suggested that Ang
II–induced fibronectin mRNA synthesis is mediated by autocrine or
paracrine effects of TGF-ß and that this action of TGF-ß is not
sufficient to cause the full induction of fibronectin mRNA.
Ang II Stimulates TGF-ß mRNA Expression Through EGF-R
Transactivation, and TGF-ß Regulates Fibronectin Expression by
Stabilizing Its mRNA With No Effect on Transcriptional
Activity
We next examined the effects of Ang II–induced EGF-R
transactivation on TGF-ß mRNA expression and the mechanism by which
TGF-ß regulates fibronectin synthesis. Addition of Ang II (100
nmol/L) stimulated TGF-ß mRNA accumulation with a time course similar
to that of fibronectin mRNA induction (significant increase at 6 hours
and peak level at 12 hours with a 3-fold increase). Pretreatment with
the EGF-R inhibitor AG1478 completely abolished Ang
II–induced TGF-ß mRNA accumulation (Figure 8A) without affecting the basal
level of TGF-ß mRNA expression (data not shown). To further study the
role of EGF-R transactivation in Ang II–stimulated TGF-ß mRNA
expression, we determined TGF-ß mRNA levels in HEGF-R 533del cells or
the cells pretreated with the MEK inhibitor PD98059. Ang
II–stimulated TGF-ß mRNA accumulations were completely inhibited in
HEGF-R 533del cells and by treatment with PD98059 (Figure 8A),
indicating that ERK activated downstream of EGF-R is a major
mediator to regulate TGF-ß mRNA accumulation by Ang II.
|
indicates Ang II; 
,
TGF-ß, control) represent mean±SE of 6 separate experiments,
and a representative Northern blot is shown.
We also examined the role of TGF-ß in regulating fibronectin gene
expression. TGF-ß (1, 10, and 100 ng/mL) did not significantly
increase the CAT activity of the longest chimeric construct (p1908-CAT)
(data not shown). Therefore, we next tested the effect of TGF-ß on
fibronectin mRNA stability by inhibiting gene transcription with
actinomycin D. In unstimulated control cells, the half-life of
fibronectin mRNA was 14±0.1 hours. When cells were stimulated with Ang
II or TGF-ß, the half -life was significantly (P<0.01)
increased to 23.6±0.1 and 23.2±0.1 hours, respectively, compared with
that in controls (Figure 8B). Moreover, we tested the
involvement of de novo protein synthesis in Ang II– or
TGF-ß–induced stabilization of fibronectin mRNA. Pretreatment of
cells with cycloheximide completely reversed Ang II– or
TGF-ß–induced increases in mRNA stability to the control level
(Figure 8B). These observations suggested that TGF-ß secreted
into the medium by Ang II contributes to the accumulation of
fibronectin mRNA mainly by increasing its mRNA stability rather than
affecting its gene transcription and that this posttranscriptional
effect is exerted via de novo protein synthesis.
|
Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The major new findings of this study were that Ang II–induced synthesis and secretion of fibronectin and TGF-ß are mediated by downstream signaling of EGF-R transactivated by Ca2+/calmodulin-dependent tyrosine kinase, in which the ERK-mediated pathway plays an important role, and that Ang II–induced fibronectin mRNA expression is regulated by 2 different mechanisms, transcriptional control by binding of the c-fos/c-jun complex to the AP-1 site and posttranscriptional control by autocrine and/or paracrine effects of TGF-ß, which is exerted by increasing the mRNA stability and requires de novo protein synthesis.
Fibronectin is important for cell adhesion and cell migration, events that occur in wound healing, organogenesis, and cardiac remodeling, and it is possible that this protein molecule plays an important role in the remodeling of cardiac interstitium secondary to myocardial hypertrophy.44 45 A direct role for Ang II in remodeling is supported by the observation that cultured rat cardiac fibroblasts demonstrate increased fibronectin and collagen types 1 and 3 transcript expression, as well as secretion of new collagen after stimulation with Ang II.2 4 However, little is known about the mechanism by which Ang II stimulates collagenous protein synthesis in cardiac fibroblasts. Recently, we28 and Eguchi et al16 20 clearly showed that in cardiac fibroblasts28 or VSMCs,16 20 Ang II–induced ERK activation and c-fos expression is mainly mediated through downstream signaling of EGF-R transactivated by Ang II in a Ca2+/calmodulin-dependent, PKC-independent manner, and that Ang II as well as the Ca2+ ionophore A23187 induced tyrosine phosphorylation of the EGF-R, which was sufficient to recruit the adaptor proteins that are involved in Ras activation. In this study, we further extended these previous studies and found for the first time that such a Ca2+/calmodulin-dependent transactivation mechanism of EGF-R is operating on both Ang II–induced fibronectin and TGF-ß gene expression.
We also showed that the AP-1 sequence present in the promoter region plays an important role in Ang II responsiveness of the fibronectin gene and that AP-1 complex (containing c-fos/c-jun) is associated with this cognate recognition sequence. A serum response element (SRE) mediates c-fos induction by growth factors, cytokines, and other stimuli that activate ERK.10 46 The c-fos promoter contains SRE, and induction of c-fos expression occurs on formation of a ternary complex factor composed of p62TCF and the serum response factor at the SRE.47 ERK was shown to phosphorylate p62TCF (also known as Elk-1 or SAP-1), resulting in enhanced ternary complex formation.48 Recent studies have shown that Janus kinase/signal transducer(s) and activator(s) of transcription,3 49 Rho-dependent pathways,50 and c-jun N-terminal kinase (JNK)51 also induce c-fos gene expression. As Ang II–induced c-fos expression in cardiac fibroblasts28 or VSMCs20 is completely inhibited by specific inhibition of EGF-R function, it is likely that Ang II–stimulated c-fos expression is mainly regulated by phosphorylated p62TCF after EGF-R–mediated ERK activation. c-jun is 1 of the major components of the transcriptional factor AP-1, which regulates expression of many genes with an AP-1 binding site in their promoter regions.52 The transcriptional activating activity of c-jun is regulated at the posttranslational level by phosphorylation of c-jun. c-jun is phosphorylated at 2 serine residues within N-terminal transactivation domain by JNK.42 JNK was reported to be activated by downstream signaling of EGF via a phosphatidylinositol 3-kinase–dependent mechanism in HeLa cells.53 54 We also found that stimulation of EGF-R is able to stimulate JNK activity in cardiac fibroblasts (S.M., unpublished observation, 1998). In this study, we showed that Ang II–induced transcriptional activity of fibronectin gene and binding of c-fos/c-jun complex to the AP-1 site are completely abolished by specific inhibition of EGF-R function. This finding suggests that in cardiac fibroblasts the transcriptional activating activity of c-jun is enhanced by downstream signaling of EGF-R. Given that inhibition of ERK activity by MEK inhibitor partially inhibited the Ang II–stimulated transcriptional activity of fibronectin gene and binding of the c-fos/c-jun complex, it is likely that the JNK pathway activated downstream of EGF-R is also involved in the transcriptional regulation of fibronectin gene.
ATF-2, a member of the family of CRE binding proteins, is not
activated by agents that increase cAMP. ATF-2 can form a
complex not only with c-jun but also with itself and some
other members of the ATF family, and the complex binds the ATF/CRE
motif to enhance gene transcription.42 Although the
fibronectin gene has an ATF/CRE motif in the 5'-flanking region, the
results from the promoter analyses (Figure 6A) and gel
retardation assays (Figure 6B) indicated that the ATF/CRE motif
is not involved in Ang II responsiveness of this gene. Expression of
the atrial natriuretic peptide,55
endothelin-1,56 and collagenase genes
containing AP-1 sites is regulated by AP-1 activity, whereas the
c-jun promoter and adhesion molecule E-selectin are
upregulated by binding of the ATF-2/c-jun complex to the
ATF/CRE motif.42 This study demonstrated for the
first time that fibronectin is included in the gene family regulated by
the AP-1 complex. Given that collagenase activity is also
stimulated by Ang II,42 it is interesting that both
collagenase and fibronectin genes are transcriptionally
regulated by AP-1 activity. Further studies are needed to define the
interaction between Ang II–induced collagenous protein synthesis and
degradation.
An important function of Ang II in the heart may be stimulation of cardiac fibroblasts to release growth factors. Ang II can directly modulate vascular smooth muscle growth by stimulating release of platelet-derived growth factors, TGF-ß, and basic fibroblast growth factors.57 Primary cultures of neonatal cardiac fibroblasts secrete significant amounts of TGF-ß when stimulated with Ang II,58 which was shown in turn to cause fibrillar collagen synthesis.59 Although similar paracrine or autocrine action of TGF-ß to induce synthesis of fibronectin and collagen proteins was reported in glomerular mesangial cells,43 it remains unclear how TGF-ß regulates the expression of fibronectin. In the present study, we clearly demonstrated that Ang II increases the level of TGF-ß mRNA and production of both latent and active TGF-ß into medium and that this induction is mediated via ERK activated downstream of EGF-R transactivated by Ang II. We also found that TGF-ß did not affect the promoter activity of the fibronectin gene but stimulated fibronectin mRNA accumulation by stabilizing its mRNA metabolism, and that this action requires de novo protein synthesis. TGF-ß has been shown to induce promoter activity of the collagen type 1 gene through a nuclear factor 1 binding site in mouse NIH 3T3 and rat osteosarcoma cells,60 whereas posttranscriptional regulation of collagen type 1 gene has also been reported in lung fibroblasts.61 As there is no nuclear factor site in the 1.9 kb of promoter region of the fibronectin gene, it is plausible that the effect of TGF-ß is exerted at the posttranscriptional level by stabilization of fibronectin mRNA. Eghbali et al59 reported that in cardiac fibroblasts the effect of TGF-ß on collagen type 1 synthesis required de novo synthesis of proteins by examining the effect of cycloheximide and that TGF-ß did not stimulate the induction of proto-oncogenes c-fos, c-jun, or Egr-1. We also confirmed these findings in our cells (Y.M., unpublished observation, 1998). Thus, this study demonstrated that induction of fibronectin by Ang II is regulated by both transcriptional and posttranscriptional mechanisms, and the latter is due to autocrine or paracrine effects of TGF-ß. Although our study also indicated that the effect of TGF-ß on fibronectin mRNA stabilization required de novo protein synthesis, identification of the proteins produced and the mechanism of stabilization of fibronectin mRNA remain to be determined.
In summary, we demonstrated that the expression of fibronectin as well as TGF-ß, a strong mediator of collagenous protein synthesis, is increased through an AT1-mediated transactivation mechanism of EGF-R induced by a Ca2+-dependent tyrosine kinase. We also showed that ERK activation via EGF-R transactivation is a major mediator to regulate the synthesis and release of fibronectin and TGF-ß, whereas a pathway(s) (probably JNK) other than ERK activated downstream of EGF-R is also partially involved in the transcriptional control of fibronectin gene. Recently, we have shown that a Ca2+-dependent tyrosine kinase, Pyk2/CAKß /RAFTK, is partially involved in this transactivation mechanism by examining the effects of dominant negative Pyk2.38 However, the Pyk2-mediated action alone cannot sufficiently account for transactivation of EGF-R, and another mechanism is likely to be responsible for tyrosine phosphorylation of EGF-R. The direct involvement of the EGF-R in Ang II–induced fibronectin and TGF-ß expression presents a novel paradigm for cross-talk between AT1 and growth factor receptor signaling pathways. Therefore, it is important for us to interpret the effects of Ang II on extracellular matrix formation in association with the signaling cascade regulating cellular proliferation and/or differentiation by growth factors.
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Acknowledgments |
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This study was supported in part by research grants from the Ministry of Education, Science and Culture of Japan; Study Group of Molecular Cardiology; Naito Foundation; Clinical Pharmacology Foundation; Japan Medical Association; Japan Smoking Foundation; and Japan Heart Foundation. S.M. is research fellow of the Japan Society for the Promotion of Science.
Received October 30, 1998; accepted March 4, 1999.
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