From the Division of Cardiology, University Hospital of Geneva,
Switzerland and the Department of Medicine, Emory
University, Atlanta, Georgia 30322
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ABSTRACT |
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Angiotensin II (Ang II) and basic fibroblast
growth factor (bFGF) are important modulators of cell growth under
physiological and pathophysiological conditions. We and others have
previously shown that these growth factors increase insulin-like growth
factor-1 receptor (IGF-1R) number and mRNA in vascular smooth
muscle cells and that this effect is transcriptionally regulated. To
study the mechanisms and the signaling pathways involved, IGF-1R
promoter reporter constructs were transiently transfected in
CHO-AT1 cells that overexpress angiotensin
AT1 receptors. Our findings indicate that Ang II and bFGF
significantly increased IGF-1R promoter activity up to 7- and 3-fold,
respectively. The effect induced by Ang II was mediated via a tyrosine
kinase-dependent mechanism, since tyrphostin A25 largely
inhibited the Ang II-induced increase in promoter activity. In
addition, co-transfection of dominant negative Ras, Raf, and MEK1 or
pretreatment with the MEK inhibitor PD 98059 dose-dependently decreased both the Ang II- and
bFGF-induced increase in IGF-1R transcription and protein expression,
suggesting that the Ras-Raf-mitogen-activated protein kinase kinase
pathway is required for both growth factors. Reactive oxygen species
have been shown to act as second messengers in Ang II-induced
signaling, and activation of the transcription factor NF- The vascular response to injury requires a coordinated interaction
between hemostatic and inflammatory systems and is regulated by
cytokines and growth factors that act locally to regulate cellular proliferation and tissue repair. Among the many growth factors that
have been shown to be implicated in the response to vascular injury,
angiotensin II (Ang II)1 is
of particular interest. It stimulates a variety of physiological responses related to regulation of blood pressure, salt, and fluid homeostasis (1). However, Ang II has also been shown to function, either directly or indirectly, as a growth factor for vascular smooth
muscle cells, cardiac fibroblasts, and cardiac myocytes (2-7). The
array of genes induced by Ang II includes proto-oncogenes such as
c-fos, c-jun, c-myc, and
egr-1 (5, 7-9), genes encoding extracellular matrix
proteins such as collagen, fibronectin, and tenascin (10-12), and
genes for growth factors like transforming growth factor
Accumulating evidence has shown that the insulin-like growth factor-1
receptor (IGF-1R) is a convergence point of the control of cell growth.
Thus, a functional IGF-1R autocrine loop is required for the mitogenic
effects of various growth factors, such as PDGF (19, 20), epidermal
growth factor (20-22), thrombin (23), bFGF (24-26), and Ang II (14).
Furthermore, we and others have demonstrated that PDGF (24-26),
thrombin (27), bFGF (25, 26, 28), and Ang II (25) increase IGF-1R
density on vascular cells. Inhibition of this effect by IGF-1R
antisense phosphorothioate oligonucleotides inhibits the Ang II-induced
cellular growth (29).
Ang II exerts its effects through specific G-protein-coupled receptors,
predominantly through the AT1 receptor subtype. These receptors induce intracellular calcium mobilization; activation of
tyrosine kinases such as p125FAK, p46SHC, and
p54SHC; induction of serine/threonine kinases, including
protein kinase C and mitogen-activated protein kinases (MAPKs) (13,
30-35); and stimulation of the Janus kinase (Jak)/signal transducer
and activator of transcription (STAT) pathway (36). The bFGF receptor, however, belongs to the receptor tyrosine kinase family (37-39) and
couples to a variety of signaling pathways, including phospholipase C- The purpose of the present studies was to localize the Ang II- and the
bFGF-responsive elements in the IGF-1R promoter and define the
signaling cascades whereby these two growth factors increase IGF-1R
gene transcription or protein expression. We show that Ang II and bFGF
positively regulate transcriptional activity of the IGF-1R gene and
that they increase IGF-1R gene expression via common as well as
distinct signaling pathways.
Materials--
Cell culture media and LipofectAMINE were
purchased from Life Technologies (Basel, Switzerland), and human Ang II
was from Sigma. Human recombinant bFGF, PD 98059, genistein, tyrphostin A25, eicosatetrayonic acid (ETYA), SB 203580, and BAPTA/AM were from
Calbiochem. The Dual-LuciferaseTM Reporter Assay was from
Promega (Madison, WI), and salmon sperm DNA was purchased from
Stratagene (La Jolla, CA). The enhanced chemiluminescence reagents and
the horseradish peroxidase-conjugated anti-rabbit immunoglobulin were
from Amersham (Pharmacia Biotech). The polivinylidene fluoride blotting
membranes were from Millipore Corp. (Bedford, MA), and the anti-MAPK
and phosphospecific anti-MAPK (p42/p44) antibodies were purchased from
New England Biolabs (Beverly, MA). The antibody against the Cell Culture--
CHO-AT1 cells (kindly provided by
Dr. E. Clauser, INSERM, Paris) stably overexpressing the Ang II
AT1A receptor (49) were grown in Ham's F-12 medium
supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.75 mg/ml G418 and incubated at 37 °C in a
humidified 5% CO2 atmosphere. The parental cell line
CHO-K1 (ATCC, Rockville, MD) was cultured under the same conditions but without the addition of G418.
Plasmids and Transfections--
The full length promoter of the
IGF-1R ( Western Blot Analysis--
Cultured CHO-AT1 or
CHO-K1 cells were serum-starved for 24 h prior to the addition of
Ang II (100 nM) or bFGF (10 ng/ml) for the times indicated.
In some experiments, cells were incubated with the MAPKK inhibitor PD
98059 (10 µM), the tyrosine kinase inhibitor tyrphostin
(10 µM), the p38 MAPK inhibitor SB 203580 (10 µM), or ETYA (10 µM) 1 h prior to the
addition of Ang II or bFGF. Cells were washed in ice-cold
phosphate-buffered saline and lysed in lysis buffer containing 150 mM NaCl, 10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 5 mM dithiothreitol,
0.1 mM phenylmethylsulfonyl fluoride, 5 mM
Effect of Ang II on IGF-1R Promoter Activity and Localization of
the Ang II-responsive Element--
To measure the effect of Ang II on
IGF-1R gene expression and to localize the Ang II-responsive region,
CHO-AT1 cells were transiently transfected with IGF-1R
promoter constructs containing a luciferase reporter gene under the
control of the proximal promoter region of the IGF-1R gene together
with the Renilla luciformis thymidine kinase expression
vector (pRL-TK) as internal control. Ang II (100 nM)
significantly increased IGF-1R promoter activity between 2.3- and
7-fold depending on the promoter constructs (Fig. 1B). No effect was observed
when the promoterless pOLUC was used (data not shown). The biggest
effect induced by Ang II was seen with the promoter construct
containing 476 base pairs of the 5'-flanking region and 640 base pairs
of the 5'- untranslated region (UTR) (p( Effect of bFGF on IGF-1R Gene Transcription--
The stimulatory
effect induced by bFGF in CHO-AT1 cells ranged between 1.7- and 3.2-fold (Fig. 1C), and the same was found in the
parental cell line CHO-K1 (data not shown). Previous studies have
suggested that the bFGF-response element may be located between nucleotides Effect of Ang II and bFGF on IGF-1R Protein Levels--
To assess
whether Ang II or bFGF increased IGF-1R protein levels, cell lysates of
cells treated with or without the corresponding growth factor were
assayed for IGF-1R protein levels by Western immunoblot. Fifty
micrograms were fractionated on a 7.5% reducing SDS-PAGE. Ang II and
bFGF increased IGF-1R protein expression, with a maximum seen after
24 h of incubation (Fig. 2).
Incubation with Ang II or bFGF for 48 h did not further increase
IGF-1R protein levels (not shown).
Common and Distinct Signaling Pathways of Ang II and bFGF on the
Stimulation of IGF-1R Gene Expression and on Protein Level--
The
Ang II AT1 receptor belongs to the family of
G-protein-coupled seven-transmembrane domain receptors (60), whereas
the bFGF receptor is characterized by a single transmembrane domain that has intrinsic tyrosine kinase activity (37-39). To define signal
transduction pathways by which Ang II and bFGF stimulate transcription
of the IGF-1R, cells were transfected with the indicated promoter
reporter constructs and either pretreated with various inhibitors or
co-transfected with increasing doses of dominant negative expression
constructs prior to the addition of Ang II or bFGF. The
protein-tyrosine kinase inhibitor tyrphostin A25 (10 µM)
decreased the Ang II response by 60-70%, suggesting that protein
tyrosine phosphorylation is involved in the pathway of Ang II (Fig.
3A). Similar results were
obtained when genistein, another tyrosine kinase inhibitor was used
(54 ± 1.1 to 82 ± 5.3% inhibition of the Ang II response,
depending on the promoter construct used; mean ± S.E. of three
experiments).
We have previously shown that Ang II signals through a
lipoxygenase-dependent pathway to increase
macrophage-mediated oxidative modification of low density lipoprotein
(61). Therefore, we were interested to see if Ang II would also signal
through that pathway to increase IGF-1R transcription. Indeed, ETYA
almost completely inhibited the stimulatory effect of Ang II on IGF-1R transcription (representative experiments as follows: for
It is documented that Ang II activates the MAPK pathway in vascular
smooth muscle cells (62, 63) and that this activation is partially
dependent on protein kinase C (62) and apparently requires prior
activation of a Ca2+-dependent tyrosine kinase
(64). Co-transfection experiments with dominant negative Ras, Raf, and
MEK1 suggested that the Ras-Raf-MAPKK pathway is involved in the
transcriptional activation of the IGF-1R by Ang II because all
inhibited the luciferase response induced by Ang II (Fig.
4), whereas the empty vectors had no
effect (data not shown). Similarly, the specific MAPKK inhibitor PD
98059 (100 µM) blocked the Ang II response to control
levels in all promoter constructs without having any effect on basal
luciferase activity (
We have seen that the Ang II-induced IGF-1R gene expression is
calcium-dependent and is mediated via a redox-sensitive
pathway.2 Indeed,
intracellular Ca2+ chelation using BAPTA/AM (10 µM) decreased the stimulatory effect of Ang II to control
levels without having any effect on basal luciferase activity,
suggesting that intracellular Ca2+ is required (
While both Ang II and bFGF stimulated IGF-1R gene transcription (Fig.
1), the signal pathway by which these two growth factors mediate the
increase in IGF-1R expression showed common but also distinct features.
Thus, tyrphostin A25 reduced the stimulatory effect of bFGF on IGF-1R
transcription by 44.4 ± 3.8% (
More recently, a novel nuclear signaling pathway has been described
that regulates a large family of transcription factors called STATs
(76). This pathway, initially described for the interferon receptors,
has subsequently been shown to be involved in hormone and growth factor
signaling, such as growth hormone (77), Ang II (36), or bFGF (45, 46).
We have previously shown that Ang II directly stimulates the Jak/STAT
pathway in rat aortic smooth muscle cells by phosphorylation of the
intracellular Jak2 kinase and its substrates STAT1 and STAT2 (36). We
therefore investigated whether kinase-deficient Jak2 or dominant
negative STAT1 Tyr701 and STAT3 Tyr705 mutants
could inhibit the increase in IGF-1R transcriptional activity induced
by Ang II or bFGF. While all mutants had no effect on the Ang II
response (data not shown), they greatly reduced the effect seen with
bFGF (Fig. 9), suggesting that the
Jak/STAT pathway is involved in the response to bFGF but not to Ang II in this cell model.
Similarly to the transcriptional assays, the MAPKK inhibitor PD 98059 and the protein tyrosine kinase inhibitor tyrphostin A25 decreased the
stimulatory effect of Ang II or bFGF on IGF-1R protein expression,
whereas the lipoxygenase inhibitor ETYA blocked only the Ang II
response. Furthermore, the p38 MAPK inhibitor SB 203580 did not inhibit
the Ang II or bFGF effect (Fig. 10), confirming the results observed in the reporter assays.
It has previously been demonstrated that growth factors such as
PDGF, thrombin, Ang II, and bFGF increase IGF-1R on vascular smooth
muscle cells and that this effect is transcriptionally regulated (23,
25, 28). Furthermore, the ability of Ang II to up-regulate IGF-1R is a
critical determinant of its mitogenic activity on vascular cells, since
the Ang II-induced increase in DNA synthesis was inhibited by
IGF-1R-specific antisense oligonucleotides (29). The present studies
show by which mechanisms and signaling pathways Ang II and bFGF
increase IGF-1R gene transcription. By deletional analysis of the
IGF-1R promoter region, we determined that the Ang II-responsive region
is located in the proximal promoter, between nucleotides There has been significant interest generated by the observation that
growth factors and cytokines, which possess structurally different
receptors, with or without intrinsic tyrosine kinase activity, may
signal through a common pathway to the nucleus. In order to define the
mechanisms and the signaling cascade involved in the Ang II or bFGF
regulation of IGF-1R gene expression, we transiently transfected
various IGF-1R promoter constructs into CHO-AT1 or CHO-K1
cells and used different approaches to block the signaling pathways at
different levels. Our findings clearly show that Ang II and bFGF share
common but also quite distinct pathways. Thus, both Ang II and bFGF
increase IGF-1R transcriptional activity via the Ras-Raf-MAPKK-MAPK
pathway, since transfection of dominant negative expression constructs
for Ras, Raf, or MEK1 dose-dependently reduced the
stimulatory effects of these growth factors on IGF-1R promoter
activity, whereas they had no effect on IGF-1R promoter activation in
the absence of Ang II or bFGF. Further evidence for the involvement of
this signaling pathway in the activation of the IGF-1R promoter by Ang
II or bFGF was provided by experiments using PD 98059. This compound,
which is a specific inhibitor of MAPKK phosphorylation and activation
(78, 79), completely reversed the stimulatory effect on luciferase activity induced by Ang II or bFGF. Furthermore, analysis at the protein level clearly demonstrated that both Ang II and bFGF induced a
rapid phosphorylation of MAPK, which was inhibited by upstream blockade
of MAPKK by PD 98059, and inhibition of the MAPKK reduced the
stimulatory effect of Ang II and bFGF on IGF-1R protein levels. Thus,
the Ras-Raf-MAPK pathway is clearly required for Ang II and bFGF
induction of IGF-1R gene and protein expression.
Although the Ang II AT1 receptor does not possess intrinsic
tyrosine kinase activity, its activation leads to intracellular second
messenger protein tyrosine phosphorylation by cytosolic tyrosine
kinases (80). Thus, our finding that the protein-tyrosine kinase
inhibitors genistein and tyrphostin A25 inhibited the Ang II-induced
stimulation of IGF-1R gene expression and phosphorylation of MAPK
demonstrates a requirement for protein-tyrosine kinase(s) in Ang
II-stimulated IGF-1R expression. We have previously shown that
lipoxygenases may be involved in the signaling pathway of Ang II (61).
Our present study demonstrates that Ang II-induced activation of the
IGF-1R promoter requires lipoxygenase activity, since this stimulation
was blocked by ETYA, a lipoxygenase inhibitor (81). ETYA and genistein
not only reduced the stimulatory effect of Ang II on IGF-1R
promoter transcriptional activity to basal levels but also inhibited
the Ang II-induced phosphorylation of MAPK, suggesting that protein
tyrosine phosphorylation and lipoxygenase activation is upstream of
MAPK activation. Consistent with the results observed in the
transcriptional assays, tyrosine kinase and lipoxygenase inhibition
abolished the increase in IGF-1R protein level induced by Ang II.
The regulation of IGF-1R transcription is not well understood. The
IGF-1R gene promoter lacks TATA or CAAT motifs; thus, transcription starts from a unique initiator sequence (82). The present experiments are therefore of interest in characterizing the signaling pathway of
Ang II or bFGF in stimulating the IGF-1R promoter and in determining the interaction between transcription factors and the IGF-1R promoter. The region of the IGF-1R promoter extending from nucleotide Another important mitogenic cascade that is activated by cytokines and
growth factors involves the Jak family of cytoplasmic tyrosine kinases
(76, 88). Jak-mediated tyrosine phosphorylation of STATs promotes the
translocation of these growth factors to the nucleus, where they bind
to specific DNA motifs and induce c-fos gene transcription
(76, 88-90). Marrero et al. (36) have previously
demonstrated that Ang II stimulates tyrosine phosphorylation of Jak
isoforms, tyrosine kinase activity of Jak2, and tyrosine phosphorylation of STATs in vascular smooth muscle cells. Using a
kinase-deficient Jak2 or dominant negative STAT1 or STAT3, we were
unable to inhibit the Ang II-induced stimulation of the IGF-1R promoter
in transient transfection assays using CHO-AT1 cells, indicating that the Jak/STAT pathway is not involved in Ang II-induced IGF-1R gene expression. Quite in contrast to Ang II, kinase-deficient Jak2, dominant negative STAT1, and STAT3 completely inhibited the
stimulatory effect of bFGF on IGF-1R transcription, whereas neither
empty vectors nor both dominant negative expression constructs had any
effect on the IGF-1R transcriptional activity in the absence of bFGF.
These findings demonstrate that the Jak/STAT pathway and more precisely
Jak2, STAT1, and STAT3 are required in the transcriptional activation
of the IGF-1R promoter by bFGF but are not involved in the stimulation
by Ang II.
The involvement of the transcription factor NF- In conclusion, we have characterized the regulation of IGF-1R gene
expression by Ang II and bFGF and the main signaling pathways by which
these growth factors increase IGF-1R transcriptional activity and
IGF-1R protein expression. Both growth factors increase IGF-1R promoter
activity by acting on the proximal promoter region upstream of the
transcription start site. Although Ang II and bFGF possess structurally
different receptors, they transduce signaling through common pathways,
notably the Ras-Raf-MAPKK-MAPK and c-Jun pathways. However, they also
use unique signaling pathways, such as lipoxygenase-mediated MAPK
activation or the involvement of the transcription factor NF-B is
redox-sensitive. While co-transfection of dominant negative I
B
mutant completely inhibited the Ang II-induced increase in
transcription, it had no effect on the bFGF signaling. In contrast,
co-transfection studies indicated that the transcription factors STAT1,
STAT3, and c-Jun and the Janus kinase 2 kinase are required in the
signaling pathway of bFGF, whereas only dominant c-Jun inhibited the
Ang II-induced effect. In summary, these data demonstrate that Ang II
and bFGF increase IGF-1R gene transcription via distinct as well as
shared pathways and have important implications for understanding growth-stimulatory effects of these growth factors on vascular cells.
INTRODUCTION
Top
Abstract
Introduction
References
1, platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and its receptor (5, 7, 12-14). Similarly,
basic fibroblast growth factor (bFGF) has been implicated in the
vascular injury response. In particular, bFGF increases endothelial
cell migration and proliferation and also stimulates angiogenesis
in vitro and in vivo (15, 16). The role of bFGF in vessel injury and repair is further supported by evidence that bFGF
is released from vessel wall cells after injury (17) and that bFGF
mRNA is up-regulated in atherosclerotic lesions (18).
and phospholipase A2 activation (40, 41), activation
of the MAPK pathway (42-44), and activation of the Jak/STAT cascade (45, 46). Similarly to Ang II, bFGF has been shown to induce the
expression of the early response gene c-fos (47). However, the signaling pathways by which Ang II or bFGF increase transcriptional activity of the IGF-1R gene are unknown, with the exception that the
bFGF, but not the Ang II effect, is protein kinase
C-dependent (48).
EXPERIMENTAL PROCEDURES
-subunit
of the IGF-1 receptor was purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA).
2350/+640-Luc) and a shorter promoter construct
(
476/+640-Luc) were a generous gift from Dr. H. Werner (National
Institutes of Health, Bethesda, MD). Deletion fragments were made from
the full-length promoter construct and subcloned upstream of the
firefly luciferase cDNA, resulting in fragments extending from
nucleotides
476 to +21,
416 to +21,
330 to +21,
270 to +21, and
135 to +21. The following constructs have previously been described:
dominant negative mutant p21ras (N17) (50), dominant negative
Raf (301) (51), dominant negative MAPKK 1 (MEK1) mutant A221 (52),
dominant negative Jun kinase (stress-activated extracellular
signal-regulated kinase (SEK1)) (53), dominant negative c-Jun (Tam67)
(54), dominant negative STAT1 Tyr701 (55), dominant
negative STAT3 Tyr705 (56), kinase-deficient Jak2 kinase
(57), and dominant negative I
B
K21/22R (58). To control for
transfection efficiency and interwell variation, cells were
co-transfected with the internal control vector pRL-TK according to the
manufacturer (Herpes simplex virus thymidine kinase promoter region
driving Renilla luciferase expression). Cells were plated in
24-well plates and transfected with 1 µg of reporter plasmid and 5 ng
of pRL-TK/well with LipofectAMINE reagent. In co-transfection
experiments with dominant negative Ras, Raf, MEK1, STAT1, STAT3, Jak2,
c-Jun, SEK1, and the I
B
mutant, cells were transfected as
described above with the addition of increasing amounts of the above
mentioned plasmids. The total amount of DNA transfected was kept
constant using salmon sperm DNA. Twenty hours after transfection, the
DNA-containing medium was changed to Ham's F-12, and the cells were
treated with or without Ang II (100 nM) or bFGF (10 ng/ml).
To determine the signaling pathways, transfected cells were incubated
with inhibitors for 1 h prior to the addition of Ang II or bFGF.
After 24 h, cells were washed and lysed according to the
manufacturer's instruction. Firefly and Renilla luciferase
activities were measured using an EG & G Berthold luminometer (Bad
Wildbad, Germany). Firefly luciferase activity was normalized to the
internal control Renilla luciferase (Luc/Ren).
-aminocaproic acid, 1 mM sodium orthovanadate, 0.1 M okadaic acid, 0.1 µM aprotinin, 10 µg/ml
leupeptin, and 10 mM NaF. Lysates were subjected to
SDS-PAGE on 7.5 or 12% gels, and separated proteins were transferred
to polivinylidene fluoride membranes. Blots were blocked with 5% dry
milk; incubated with polyclonal anti-MAPK, anti-phosphospecific MAPK
(p44, p42), or anti-IGF-1R
antibodies; and then incubated with
peroxidase-conjugated donkey anti-rabbit antibody. Immunopositive bands
were visualized by enhanced chemiluminescence. Purified MAPK protein
was included as positive control; nonimmune rabbit IgG was used as
negative control.
RESULTS
476/+640-Luc)), and a lesser
response occurred with the construct containing a shorter 5'-UTR. It
appeared that there were some negative regulatory elements between
nucleotides
2350 and
476, because the longest reporter construct
responded less to Ang II stimulation, compared with (
476/+640-Luc).
When the sequence between nucleotides
270 and
135 was deleted, the
stimulatory effect of Ang II was greatly diminished, although not
completely, suggesting that the major Ang II-responsive element may be
located between nucleotides
270 and
135 of the 5'-flanking
region.
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Fig. 1.
Regulation of IGF-1R promoter activity by Ang
II and bFGF. A, IGF-1R promoter constructs used.
CHO-AT1 cells were transiently transfected with 1 µg of
the various reporter plasmids and 5 ng of pRL-TK and 20 h later
incubated with or without Ang II (100 nM) (B) or
bFGF (10 ng/ml) (C) for 24 h. The luciferase values are
normalized to Renilla luciferase activity and presented as
mean ± S.E. from five separate experiments. Hatched
bars, control cells; filled bars,
cells stimulated with Ang II or bFGF, respectively.
476 and
188 of the 5'-flanking region (59). In our
CHO-AT1 cells, the
476/+640 construct responded well to
bFGF (~3-fold increase in luciferase activity). In contrast to Ang II, there was no evidence of a repressor sequence in the larger construct (
2350/+640). The removal of the majority of the 5'-UTR resulted similarly in a reduction in basal activity, but the bFGF response persisted. Progressive deletion of the cis-acting sequence up
to
135/+21 still yielded a ~2-fold increase in luciferase activity
after bFGF treatment (Fig. 1C).
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Fig. 2.
Western blot analysis of the
effect of Ang II and bFGF on IGF-1R levels in CHO-AT1
cells. After 24 h of serum starvation, cells were treated
with 100 nM Ang II or 10 ng/ml bFGF for the times
indicated. Total proteins from cell lysates were subjected to SDS-PAGE
under reducing conditions on 7.5% gels and transferred to
polyvinylidene fluoride membranes. Membranes were then probed with an
antibody recognizing the -subunit of the IGF-1R. Lanes 1 and
5, control cells; lanes 2-4 and 6-8, cells
were incubated with Ang II or with bFGF for 4, 8, and 24 h,
respectively. Fifty µg of protein were loaded.
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Fig. 3.
Effect of protein tyrosine kinase inhibition
and lipoxygenase inhibition on the Ang II-induced increase in IGF-1R
transcription and on AngII-induced MAPK phosphorylation.
A, CHO-AT1 cells were transfected with the
p( 476/+640-Luc) and the p(
270/+21-Luc) construct and the pRL-TK
plasmid to correct for interwell variation. Twenty hours after
transfection, cells were incubated with or without tyrphostin A25 (10 µM) for 1 h prior to the addition of Ang II (100 nM). Twenty four hours later, cells were lysed, and
luciferases were measured. Data are presented as Luc/Ren and are
mean ± S.E. of three separate experiments. Tyrphostin A25 alone
had no effect on basal luciferase activity. Tyrphostin A25 had the same
inhibitory effect on all other IGF-1R promoter constructs (data not
shown). B, cells were treated with or without genistein (60 nM) or ETYA (10 µM) prior to the addition of
Ang II for 24 h. Ten micrograms of total protein were subjected to
SDS-PAGE on a 12% reducing gel and probed with anti-phosphospecific
p44/p42 antibody.
476/+640, control, 9.5; Ang II, 35.6; and ETYA/Ang II, 17.9 Luc/Ren,
respectively; or for
416/+21, control, 5.4; Ang II, 16.5; and
ETYA/Ang II, 7.2 Luc/Ren), whereas it had no effect on the increase
induced by bFGF (
2350/+640: control, 10.1 ± 3.6; bFGF,
16.8 ± 1.4; ETYA, 14.7 ± 4.8; and ETYA/bFGF, 19.8 ± 5.9, Luc/Ren respectively (mean ± S.D. of two experiments). ETYA
alone had no effect on basal luciferase activity (data not shown). In
agreement with the above mentioned experiments using genistein or ETYA
to block the Ang II stimulation of IGF-1R gene transcription, both
blockers also inhibited Ang II-induced MAPK phosphorylation, suggesting
that a tyrosine kinase- and a lipoxygenase-dependent step
are upstream of MAPK activation (Fig. 3B).
2350/+640: control, 3.3 ± 0.8; Ang II,
13.9 ± 2.7; PD 98059/Ang II, 5.1 ± 1.2 Luc/Ren,
respectively;
135/+21: control, 6.8 ± 1.1; Ang II, 20.7 ± 2.2; and PD 98059/Ang II, 8.6 ± 1.9 Luc/Ren, respectively
(mean ± S.E. of five independent experiments)). In contrast,
while the response induced by Ang II required the p44/p42 MAPK
activation, the p38 MAPK inhibitor SB 203580 had no effect on the Ang
II response, suggesting that p38 MAPK was not involved (
476/+21:
control, 6.5 ± 0.6; SB 203580, 5.7 ± 0.6; Ang II, 28.8 ± 1.8; and SB 203580/Ang II, 24.2 ± 1.7 Luc/Ren, respectively (mean ± S.E. of four experiments)). To confirm the specificity of
these findings, cells were treated with or without Ang II for various
times, and total proteins were immunoblotted with phosphospecific antibodies against p44 and p42 (extracellular signal-regulated kinases
1 and 2). Ang II induced a rapid phosphorylation of p44/p42 already
after 2 min, with a maximum at 5 min. PD 98059 completely inhibited the
Ang II-induced phosphorylation of p44/p42 (data not shown). It is known
that MAPKs in turn phosphorylate numerous cellular proteins, including
c-Jun among many others (65). When dominant negative c-Jun (Tam67) was
co-transfected with the p(
476/+21-Luc), it completely reduced the
stimulatory effect of Ang II to control values, whereas the empty
vector had no effect (Fig. 5). This inhibitory effect of dominant negative c-Jun could also be observed with the smaller IGF-1R promoter constructs p(
270/+21-Luc) and p(
135/+21-Luc) (data not shown).
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Fig. 4.
The Raf-Ras-MAPK kinase pathway is required
in the transcriptional stimulation of the IGF-1R promoter and IGF-1R
protein by Ang II. CHO-AT1 cells were transfected with
the p( 476/+640-Luc) IGF-1R promoter construct and increasing doses of
dominant negative Raf, Ras, and MEK1. After transfection, cells were
then stimulated with or without Ang II (100 nM) for 24 h. The experiments were performed twice with essentially identical
results.
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Fig. 5.
Effect of dominant negative c-Jun (Tam67) on
the Ang II activation of IGF-1R promoter activity. Cells were
transfected with the p( 476/+21-Luc) construct and increasing doses of
Tam67. Twenty hours later, cells were stimulated with or without Ang
II. Data are expressed as mean ± S.E. of three experiments.
476/+640:
control, 10.8 ± 1.3; BAPTA/AM, 13.4 ± 1.9; Ang II,
31.5 ± 2.5; and BAPTA/AM/Ang II, 8.7 ± 7.0 Luc/Ren, respectively (mean ± S.D. from two experiments)). Also, reactive oxygen species have been shown to act as second messengers in Ang
II-induced signaling (66) and activation of the transcription factor
NF-
B is redox-sensitive (67). Co-transfection of the IGF-1R promoter
construct p(
476/+640-Luc) with K21/22R, the I
B
mutant that
shows a defect in degradation and in ubiquitin conjugation and
therefore inhibits translocation of NF-
B to the nucleus (58), completely inhibited the Ang II-induced increase in IGF-1R
transcription (Fig. 6A). This
was also true when the shorter construct p(
270/+21-Luc) was used
(data not shown). Interestingly, the I
B
mutant had no effect on
the Ang II response when the short IGF-1R promoter construct
p(
135/+21-Luc) was used, suggesting that a putative NF-
B site is
located 5' of the nucleotide
135 (Fig. 6B). Quite in
contrast to Ang II, however, co-transfection with I
B
lysine mutant did not decrease the bFGF-induced activation of IGF-1R expression (data not shown).
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Fig. 6.
Effect of I B mutant on the Ang II-induced
increase in IGF-1R transcriptional activity. CHO-AT1
cells were transfected with the p(
476/+640-Luc) (A) or the
short p(
135/+21-Luc) (B) construct and increasing doses of
dominant I
B (K21/22R) mutant. After transfection, cells were
stimulated with or without Ang II for 24 h. The I
B mutant
significantly and dose-dependently (p = 0.02, Student's t test) reduced the stimulatory effect of Ang II.
Note that the inhibitory effect of K21/22R is lost when the short
construct is used. Data are mean ± S.E. of three
experiments.
476/+21: control, 3.8 ± 0.10; tyrphostin A25, 3.1 ± 0.08; bFGF, 11.8 ± 0.46; and
tyrphostin A25/bFGF, 6.5 ± 0.29, respectively (mean ± S.E.
of four experiments)). The Ras-Raf-MAPK pathway seemed also to be
required in the transcriptional activation of the IGF-1R by bFGF, as we
found with Ang II. Dominant negative Ras, Raf, and MEK1 inhibited the
bFGF-induced increase in luciferase activity to a similar degree as
seen with Ang II (Fig. 7). Accordingly, the MAPKK inhibitor PD 98059 (100 µM) completely
abrogated the bFGF-induced stimulation of IGF-1R transcription and
inhibited the bFGF-induced phosphorylation of p44/p42 MAPK (data not
shown). Furthermore, BAPTA/AM inhibited the bFGF-induced stimulation of IGF-1R expression to control values (representative experiment
476/+640: control, 9.9; bFGF, 33.3; and BAPTA/bFGF, 13.7 Luc/Ren, respectively); however, the p38 MAPK inhibitor SB 203580 did not inhibit the bFGF response similarly to Ang II-stimulated IGF-1R expression (data not shown). The c-Jun N-terminal
kinase/stress-activated protein kinase (JNK/SAPK) has been shown to
phosphorylate and regulate the activity of several transcription
factors including c-Jun, ELK-1, and ATF-2 (68-72). The JNK/SAPK is
phosphorylated, resulting in its activation by JNK kinase (JNK
kinase/SEK1) (53, 73-75). Increasing doses of dominant negative SEK1
expression construct produced a dose-dependent decrease in
the IGF-1R transcriptional activity induced by bFGF when the
p(
476/+21-Luc) reporter construct was used, suggesting that the
SEK1/JNK/SAPK pathway was involved (Fig.
8A). It is of interest that
the same dominant negative SEK1 did not have any effect on IGF-1R
transcriptional stimulation by Ang II (data not shown), despite the
inhibitory effect of dominant negative c-Jun in the response to Ang II
(Fig. 5). It is of note that dominant negative c-Jun also
dose-dependently reduced the stimulatory effect seen with
bFGF (Fig. 8B).
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Fig. 7.
Involvement of the Ras-Raf-MAPK kinase
pathway in the stimulation of IGF-1R transcriptional activity by
bFGF. CHO-AT1 cells were transfected with the
p( 270/+21-Luc) reporter construct and increasing doses of dominant
negative Ras, Raf, and MEK1. Cells were then treated with or without
bFGF (10 ng/ml) for 24 h. Values are mean ± S.E. of three
experiments.
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Fig. 8.
Effect of dominant negative SEK1
and c-Jun on the bFGF-induced stimulation of the IGF-1R
promoter. A, increasing amounts of dominant negative
SEK1 were transfected with the p( 476/+21-Luc) IGF-1R promoter
construct, and 20 h later cells were stimulated with or without
bFGF (10 ng/ml). B, CHO-AT1 cells were
transfected with dominant negative c-Jun (Tam67) and with
p(
476/+21-Luc). Transfected cells were then incubated with or without
bFGF for 24 h. Data are mean ± S.E. of three
experiments.
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Fig. 9.
Inhibitory effect of dominant negative STAT1,
STAT3, and Jak2 on the bFGF-induced stimulation of the IGF-1R
promoter. Increasing amount of dominant negative STAT1 Y701F or
STAT3 Y705F and the p( 270/+21-Luc) as well as kinase-deficient Jak2
together with p(
476/
21-Luc) were transfected as described. Twenty
hours later, cells were treated with or without bFGF (10 ng/ml). Data
are mean ± S.E. of three experiments.
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Fig. 10.
Effect of various inhibitors on the Ang II-
or bFGF-induced increase in IGF-1R protein expression. Total
protein from cells treated with or without Ang II or bFGF were
subjected to SDS-PAGE under reducing conditions on 7.5% gels,
transferred to polyvinylidene fluoride membranes, and then blotted with
anti-IGF-1R . Lane 1, control cells
(Con); lane 2, Ang II or bFGF,
respectively; lane 3, PD 98059/Ang II and PD
98059/bFGF, respectively; lane 4, tyrphostin
A25/Ang II and tyrphostin A25/bFGF, respectively; lane
5, SB 203580/Ang II and SB 203580/bFGF, respectively;
lane 6, ETYA/Ang II and ETYA/bFGF, respectively.
In all lanes, 50 µg of protein were loaded.
DISCUSSION
270 and
135 upstream of the transcription start site, as is the
bFGF-responsive element. In addition, stimulation of IGF-1R gene
promoter activity by Ang II or bFGF in transient transfection
experiments correlates well with its effect on endogenous IGF-1R
protein levels. Both increased IGF-1R protein expression after 8-24 h.
This is in good agreement with the previous reports of Du et
al. (48) and Ververis et al. (25), which showed that Ang II and bFGF caused a significant increase in IGF-1R mRNA
peaking at 3 h and 6-9 h, respectively. Of note,
Hernandez-Sanchez et al. (59) reported that the
bFGF-responsive element was located between nucleotides
476 and
188. These findings are somewhat different from ours; however, our
studies were performed using different cells, and our deletion
constructs contained less 5'-UTR sequence. Our data indicate loss of
basal activity between nucleotides
476 and
135 but conservation of
a bFGF-responsive element.
2350 in
the 5'-flanking region to nucleotide +640 in the 5'-UTR, contains putative consensus sequences for a number of well defined regulatory elements, including Egr-1 (83) and Sp1 (84), as well as a PDGF-responsive element (85) and potential AP-2 (86), AP-1, and NF-
B
sites.3 To gain insights into
the promoter region of the IGF-1R gene responsive to Ang II or bFGF,
cells were transfected with expression vectors encoding dominant
negative forms of I
B (lysine mutant), JNK kinase, c-Jun, and the
transcription factors STAT1 and STAT3. The results of these experiments
suggest that one of the other common pathways by which Ang II or bFGF
increases IGF-1R gene transcription was the involvement of c-Jun. Thus,
our data indicated that dominant negative c-Jun
dose-dependently inhibited the Ang II- as well as the
bFGF-induced increase in IGF-1R promoter activity. c-Jun is one of the
components of the transcription factor AP-1, its best known partner
being c-Fos (87). However, the activity known as AP-1 can consist of
heterodimers between any of the Jun proteins and any of the Fos
proteins (87). The finding that dominant negative c-Jun inhibited the
increase in IGF-1R promoter activity by Ang II or bFGF, whereas
dominant negative JNK kinase (SEK1), which activates JNK/SAPK and
ultimately activates c-Jun, dose-dependently blocked the
effect induced by bFGF and not by Ang II can be explained by
differential pathways whereby these two growth factors signal. It is
possible that although Ang II and bFGF both activate MAP kinases, Ang
II may predominantly induce c-Fos through the MAPK pathway, whereas
bFGF induces c-Jun through activation of the JNK/SAPK pathway.
Obviously, more data are needed to fully comprehend and define this difference.
B seems to be
restricted to the stimulatory effect induced by Ang II and not by bFGF,
since the I
B
mutant only inhibited the activation of luciferase
activity by Ang II. Furthermore, it was found that by deleting
nucleotides between
270 and
135, the inhibitory effect of the
I
B
mutant on the Ang II-induced effect was lost, implying that a
putative NF-
b site is located between nucleotides
270 and
135.
These results suggest that Ang II and bFGF utilize distinct and
specific transcription factors to stimulate the IGF-1R gene promoter.
B in
the case of Ang II or the JNK/SAPK cascade and the Jun kinase and
Jak/STAT pathway in the bFGF-induced regulation of IGF-1R gene
expression. These studies have important implications for understanding
growth-stimulatory effects of these growth factors.
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ACKNOWLEDGEMENTS |
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We thank Dr. B. Cenni for helpful discussions, Dr. H. Werner for the generous supply of the full-length IGF-1R promoter constructs, Dr. M. Birrer (National Institutes of Health, Bethesda, MD) for the gift of dominant negative c-Jun (Tam67), and Dr. D. M. Wojchowski (Pennsylvania State University) for the supply of kinase-deficient Jak2.
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FOOTNOTES |
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* These studies were supported by NHLBI, National Institutes of Health, Grants HL 47035 and HL 45317, the Swiss Cardiology Foundation, Swiss National Science Foundation Grant FNSR3100-050799.97, and the Gerbex-Bourget Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Div. of Cardiology, University Hospital of Geneva, Rue Micheli-du-Crest 24, 1211 Geneva 14, Switzerland. Tel.: 41 22 372 7192; Fax: 41 22 372 7229; E-mail: Patrice.Delafontaine{at}hcuge.ch.
The abbreviations used are: Ang II, angiotensin II; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; bFGF, basic fibroblast growth factor; PDGF, platelet-derived growth factor; ETYA, eicosatetrayonic acid; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; STAT, signal transducer and activator of transcription; UTR, untranslated region; PAGE, polyacrylamide gel electrophoresis; SEK, stress-activated extracellular signal-regulated kinase; Jak, Janus kinase; BAPTA/AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester.
2 J. Du, T. Peng, K. Scheidegger, and P. Delafontaine, submitted for publication.
3 J. Du, unpublished results.
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REFERENCES |
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