AP-1 mediates stretch-induced expression of HB-EGF in bladder
smooth muscle cells
John M.
Park1,
Rosalyn M.
Adam1,
Craig A.
Peters1,
Paul D.
Guthrie1,
Zijie
Sun2,
Michael
Klagsbrun3, and
Michael R.
Freeman3
1 Urologic Laboratory,
Department of Urology,
3 Laboratory for Surgical
Research, Children's Hospital, and Department of Surgery, Harvard
Medical School, Boston, Massachusetts 02115; and
2 Departments of Surgery and
Genetics, Stanford University School of Medicine, Stanford,
California 94305
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ABSTRACT |
Mechanical induction of growth factor synthesis
may mediate adaptive responses of smooth muscle cells (SMC) to
increases in physical load. We previously demonstrated that cyclic
mechanical stretch induces expression of the SMC, fibroblast, and
epithelial cell mitogen heparin-binding epidermal growth factor-like
growth factor (HB-EGF) in bladder SMC, an observation that suggests
that this growth factor may be involved in compensatory bladder
hypertrophy. In the present study we provide evidence that the
activator protein-1 (AP-1) transcription factor plays a critical role
in this mechanoinduction process. Rat bladder SMC were transiently
transfected with a series of 5' deletion mutants of a promoter-reporter
construct containing 1.7 kb of the mouse HB-EGF promoter that was
previously shown to be stretch responsive. The stretch-mediated
increase in promoter activity was completely ablated with deletion of
nucleotide positions
1301 to
881. Binding of AP-1, as
evaluated by electrophoretic mobility shift assay, to a synthetic
oligonucleotide containing an AP-1 binding site increased in response
to stretch, and binding was inhibited by excess unlabeled DNA
corresponding to nucleotides
993 to
973 from the HB-EGF
promoter, a region that contains a previously recognized composite
AP-1/Ets site. Stretch-induced promoter activity was significantly
inhibited by site-directed mutagenesis of the AP-1 or Ets components of
this site. Consistent with the promoter and gel-shift studies,
curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA
induction after stretch. Stretch also specifically increased mRNA
levels for matrix metalloproteinase (MMP)-1, the promoter of which
contains a functional AP-1 element, but not for MMP-2, the promoter of
which does not contain an AP-1 element. The stretch response of the
MMP-1 gene was also completely inhibited by curcumin. Collectively,
these findings indicate that AP-1-mediated transcription plays an
important role in the regulation of gene expression in bladder muscle
in response to mechanical forces.
heparin-binding epidermal growth factor; mechanical signaling; gene
expression; activator protein
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INTRODUCTION |
A VARIETY OF CELL TYPES have been shown to respond to
mechanical forces by activating multiple signal transduction cascades, altering their program of gene expression and increasing their rates of
protein and/or DNA synthesis (9, 10, 15, 16, 18, 26, 29, 30). These
physiological responses have been observed in culture systems in which
cells are subjected to quantitative application of forces of various
kinds, such as radial stretch and shear stress. In vitro systems have
provided evidence that pathophysiological tissue remodeling seen in
vivo, such as cardiac hypertrophy in response to hemodynamic overload,
is the result of regulated mechanochemical signaling within specific
cellular compartments (13, 31).
Bladder hypertrophy occurs as an adaptive response to physical or
neuromuscular obstruction of the lower urinary tract arising in adult
men primarily from age-related growth of the prostate gland and in
children in association with several congenital uropathic syndromes.
The bladder detrusor muscle increases in size as a compensatory
response to urine outflow obstruction. Experiments in animal models
have indicated that compensatory bladder growth follows a highly
regulated, idiosyncratic course, which includes alterations in the mRNA
levels of some growth-related genes, an early proliferative response by
the uroepithelium and fibromuscular cells of the lamina propria, and
hypertrophic as well as hyperplastic expansion of the bladder muscle
(reviewed in Refs. 5 and 14). The molecular mechanisms that underlie
these physiological changes observable in vivo and that ultimately
result in bladder decompensation and functional failure, are largely unknown.
Repetitive stretch and relaxation applied to bladder smooth muscle
cells (SMC) in vitro has been used to model increases in urodynamic
load experienced by the bladder detrusor muscle under conditions of
bladder outlet obstruction. In a recent report from our laboratory,
Park et al. (22) used such an in vitro system to identify the mitogen
heparin-binding epidermal growth factor-like growth factor (HB-EGF) as
a stretch-responsive protein in bladder SMC. HB-EGF is an activating
ligand for the ErbB1 receptor tyrosine kinase (EGF receptor) and a
growth factor with a mitogenic potency for SMC that is greater than
that seen with other cognate ligands for the same receptor, such as EGF
(reviewed in Ref. 25). HB-EGF is synthesized by the detrusor muscle in
vivo, and HB-EGF mRNA and protein expression increase predominantly in
the muscle compartment during acute urinary obstruction in mice
(unpublished observations; 8). Consequently, HB-EGF is a
potential physiological mediator of SMC growth in the urinary tract.
A repetitive stretch-relaxation stimulus applied to bladder SMC adhered
to a deformable membrane resulted in increased expression of HB-EGF
mRNA and protein in bladder SMC by a mechanism that involved secretion
of the peptide hormone ANG II and activation of the angiotensin
receptor type 1 (AT1) (22). In
these experiments, stretch-induced increases in HB-EGF mRNA levels
could be completely accounted for by activation of the HB-EGF promoter,
suggesting that the primary mechanism of HB-EGF induction under these
conditions is transcriptional activation. In the present experiments we
have analyzed the HB-EGF promoter in bladder SMC in a search for
cis-acting DNA elements that mediate
the stretch response.
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MATERIALS AND METHODS |
Cell culture.
Rat bladder SMC were harvested by an enzymatic dispersion method, as
described previously (22). Cells were cultured in medium 199 (GIBCO)
supplemented with 20% fetal bovine serum (FBS; Hyclone Laboratory),
penicillin (100 U/ml), and streptomycin (100 µg/ml) and were
maintained in a humidified 5%
CO2-95% air atmosphere at
37°C. All experiments were performed on cells between
passages 2 and
4.
Application of cyclical stretch-relaxation.
Bladder SMC were grown on six-well silicone Elastomer-bottomed culture
plates that had been coated with collagen type I (Bioflex, Flexcell,
McKeesport, PA). Cells were rendered quiescent by incubation in medium
199 supplemented with 0.5% FBS for 48 h. The cells were then subjected
to continuous cycles of stretch and relaxation by use of a
computer-driven, vacuum-operated stretch-inducing device (Flexercell
Strain Unit FX-3000) for variable duration as indicated. Each cycle
consisted of 5 s of stretch and 5 s of relaxation (0.1 Hz). The vacuum
induced an ~20% maximum radial stretch at the periphery of the membrane.
RT-PCR.
Semiquantitative RT-PCR assays were performed to assess relative mRNA
levels. Total RNA extraction was performed using Tri-Reagent (Molecular
Research, Cincinnati, OH). RT was performed using Maloney's murine
leukemia virus reverse transcriptase with oligo(dT) as the first-strand
primer. Primers were selected from the previously published rat HB-EGF,
matrix metalloproteinase (MMP)-1 and -2, and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) sequences (19, 24). A 413-bp HB-EGF product was
amplified using the sense primer 5'-TCC CAC TGG AAC CAC AAA CCA
G-3' (nt 157-178) and the antisense primer 5'-CCC ACG
ATG ACA AGA AGA CAG AC-3' (nt 570-548). A 494-bp MMP-1
product was amplified using the sense primer 5'-AGG TGA AAA GGC
TCA GTG CTG C-3' (nt 1119-1140) and the antisense primer
5'-GAT CCT TGG GGC TCT CAA TTT C-3' (nt 1591-1612). A
591-bp MMP-2 product was amplified using the sense primer 5'-TTC AGA AGG TGC CCC ATG TG-3' (nt 1502-1521) and the antisense
primer 5'-TTC CCT GCG AAG AAC ACA GC-3' (nt
2073-2092). A 571-bp GAPDH product was amplified using the sense
primer 5'-TCA CCA TCT TCC AGG AGC G-3' (nt 245-263)
and the antisense primer 5'-CTG CTT CAC CAC CTT CTT GA-3'
(nt 816-797). PCR reactions were performed in a total volume of 25 µl, containing 22 µl of PCR SuperMix (GIBCO), 0.5 µl each of
sense and antisense primers (20 pmol/µl), 0.1 µl of
[32P]dCTP (3,000 Ci/mmol, Amersham), and 2 µl of cDNA. PCR amplification was performed
for 26-28 cycles. PCR products were subjected to size separation
by PAGE. All samples were normalized to GAPDH mRNA levels, and a
limiting dilution method was used to make semiquantitative comparisons.
HB-EGF promoter deletion and mutation.
The full-length HB-EGF promoter-luciferase construct (pHB-EGF-luc) has
been described previously (6). It contains a ~1.7-kb Mbo
I-Not I fragment derived from the
5'-untranslated region of a murine HB-EGF genomic clone
(corresponding to sequences
1837 to
155 relative to the
translation initiation site) ligated upstream of a luciferase reporter
construct pGL2Basic (Promega, Madison, WI). A series of deletion mutant
constructs from
1837 to
477, generated by PCR, were
previously used to demonstrate the requirement for sequence upstream of
position
912 to elicit HB-EGF promoter activation in response to
Raf-1 activation (20). To examine the possible role of a composite
AP-1/Ets binding site at nucleotide position
988 to
974
in stretch-induced activation of the HB-EGF promoter, we performed
site-directed mutagenesis of the
1301pHB-EGF-luc deletion mutant
from this series. The numbering convention we have adopted is based on
that of Chen et al. (6); however, sequencing of the deletion mutant
1301pHB-EGF-luc revealed several discrepancies with the reported
sequence, and we have amended the sequence and numbering accordingly.
With use of a PCR-based approach, two fragments were generated for each
transcription factor binding site mutant to be created. The upstream
fragment was created using a wild-type sense primer and an appropriate antisense mutant primer. The downstream fragment was created using an
antisense wild-type primer and an appropriate sense mutant primer. This
resulted in the generation of two fragments that overlapped in the
region of the desired mutation. The appropriate fragments were then
mixed in a 1:1 molar ratio and used as a template for primer extension
with use of wild-type primers wt-S and wt-AS and subsequent
amplification to regenerate the full-length constructs incorporating
mutations in the AP-1 or Ets component of the site or in both
components. All PCR reactions were performed using the Expand
High-Fidelity PCR System (Boehringer Mannheim), and products were
purified using the High Pure PCR Product Purification Kit (Boehringer
Mannheim). Incorporation of the desired mutations was confirmed
by DNA sequencing, and products were digested with Nhe
I and Bgl
II restriction enzymes before ligation into
Nhe I/Bgl
II-digested
1301 pHB-EGF-luc deletion construct. Putative recombinants were confirmed by analytic digests, and large-scale preparation of transfection-quality plasmid was performed using the
Qiagen Maxi-Prep Kit.
Transfection and luciferase assay.
Cells were grown to 60-70% confluence (~1 × 105/well) on Bioflex plates.
DNA-Superfect (Qiagen) mixtures (total plasmid 2 µg) were added to
cells and incubated for 1 h at 37°C. Cells were washed and
incubated for 24 h in the standard culture medium containing 20% FBS.
Cells were then rendered quiescent by incubation in 0.5% FBS
containing medium 199 for 48 h and subjected to stretch stimulation for
12 h. Cell lysates were harvested, and luciferase activity was measured
immediately after the addition of luciferase substrate. A sample of
cells was similarly transfected with the promoterless pGL2-basic and
served as control.
Preparation of nuclear extracts.
Quiescent bladder SMC were subjected to cyclical stretch and relaxation
for 2 h. Cells were collected in ice-cold PBS and centrifuged. The cell
pellet was resuspended and incubated for 15 min on ice in a buffer
containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM dithiothreitol (DTT), and
1 mM phenylmethylsulfonyl fluoride (PMSF). NP-40 (3% vol/vol) was
added and mixed vigorously for 10 s to lyse cell membranes. After
centrifugation at 25,000 g for 30 s,
the resulting nuclear pellet was resuspended and incubated for 30 min
on ice in the extraction buffer containing 20 mM HEPES (pH 7.9), 0.42 M
NaCl, 1 mM EDTA, 1 mM EGTA, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 1 mM DTT, and 1 mM PMSF. After centrifugation at 25,000 g for 5 min, supernatant was collected
as nuclear extracts and stored at
80°C.
Electrophoretic mobility shift assay.
Nuclear extract (2 µg) was incubated with 0.5 µg of poly(dI-dC)
(Pharmacia) and 104 cpm
32P-labeled DNA fragment in 20 µl of buffer containing 20 mM HEPES (pH 7.9), 50 mM KCl, 5 mM EDTA, 1 mM DTT, 3 mM MgCl2, and 5%
glycerol. After incubation on ice for 20 min, the binding reaction was
analyzed by electrophoresis on 4% polyacrylamide gel (19:1
bisacrylamide) in 0.25× Tris-boric acid-EDTA buffer. A
double-stranded oligonucleotide, 5'-CGCTTGATGACTCAGCCGGAA-3', containing a putative AP-1
binding element was labeled with
32P by use of T4 polynucleotide
kinase and used as a probe. Competition experiments were performed by
preincubating nuclear extracts with a 50-fold molar excess of unlabeled
competitor DNA fragment at 4°C for 10 min. An unlabeled
oligonucleotide,
5'-TCT
TCTTCCTGT-3', derived from the HB-EGF promoter region (nt
993 to
973)
containing the implicated AP-1 binding site (underscored), was used as
a competitor. A c-Fos polyclonal rabbit antibody (Santa Cruz
Biotechnology) was used in the supershift experiments to identify AP-1.
Rabbit polyclonal IgG served as a control. Nuclear extracts were
incubated with antibody for 10 min before the
32P-labeled DNA fragments were added.
 |
RESULTS |
Stretch stimulates HB-EGF expression in bladder SMC.
Quiescent bladder SMC were grown to near confluence on collagen-coated
silicone membranes and subjected to continuous cycles of stretch and
relaxation for 0, 1, 2, 4, and 8 h. Relative HB-EGF mRNA
levels were assessed using semiquantitative RT-PCR with
normalization to GAPDH mRNA levels. With stretch, HB-EGF transcript
levels increased rapidly, reaching the peak levels after 4 h (Fig.
1). This pattern was reproducible in three
independent experiments, consistent with our previously published
results (22).

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Fig. 1.
Effect of mechanical stretch on heparin-binding epidermal growth
factor-like growth factor (HB-EGF) mRNA expression in bladder smooth
muscle cells (SMC). Quiescent cells were grown to near confluence on
collagen-coated silicone membranes and subjected to cyclical
stretch-relaxation for 0, 1, 2, 4, and 8 h. Relative mRNA levels were
assessed by semiquantitative RT-PCR with normalization to
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels.
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HB-EGF promoter activation by stretch requires a proximal AP-1
element.
We demonstrated previously that mechanical stretch stimulates HB-EGF
promoter activity (22). Bladder SMC that had been transiently transfected with a DNA reporter construct containing >1.7 kb
5'-flanking region of the mouse HB-EGF gene cloned upstream to a
luciferase reporter gene demonstrated a significant increase in
promoter activity after stretch compared with nonstretch controls. This DNA fragment encompasses positions
1885 to
155 upstream
of the translation initiation codon in exon 1 and contains possible
binding sites for nuclear factor-
B (NF-
B), AP-1, Ets, E-box,
Pit-1, and SP-1 transcription factors on the basis of consensus
sequence analysis (6). To identify
cis-acting elements within this
promoter region that confer stretch responsiveness to the HB-EGF gene, we transiently transfected bladder SMC with a series of deletion mutant
constructs cloned upstream of luciferase and subjected the transfected
cells to cyclical stretch and relaxation for 12 h (Fig.
2). Successive deletion of the promoter
from nucleotides
1885 to
1301 had no discernible effect
on promoter activation by mechanical stretch compared with the
full-length construct. However, deletion of the sequences between
nucleotides
1301 and
881 resulted in an almost complete
loss of promoter activity after mechanical stretch. Consistent with
this finding, further deletions through nucleotide
477 were
similarly unresponsive to stretch. These results suggested that DNA
sequences between positions
1302 and
881 were required
for full activation of the HB-EGF promoter by mechanical stretch.

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Fig. 2.
Deletion analysis of HB-EGF promoter after mechanical stretch.
Top: structure of murine HB-EGF
promoter region as defined previously (6), with numbering amended as
described in MATERIALS AND METHODS.
Bottom: base pair position of deletion
mutants used in this study and relative levels of promoter activation
after stretch. Positions of potential transcription factor binding
sites are indicated. Constructs were transiently transfected into
bladder SMC, and cells were subjected to continuous cycles of stretch
and relaxation for 12 h. Cell lysates were subsequently analyzed for
luciferase activity. NF- B, nuclear factor- B; AP-1, activator
protein-1.
|
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Next we investigated whether specific transcription factor binding
sites within this region were required for activation of the HB-EGF
promoter after stretch. A composite AP-1/Ets transcription factor
binding site located at positions
988 and
974 was
previously identified by DNA sequence analysis (6). The Ets component of this site has been demonstrated to be functional in NIH/3T3 cells in
response to ligand-dependent activation of a conditionally activatable
Raf-1 kinase fusion protein (20); however, AP-1 binding to this site
has not been demonstrated. We were not able to identify another
consensus transcription factor binding site in the
1301 to
881 region of the published sequence by computer analysis (Fig.
3). Therefore, we introduced specific
mutations into the
1301 deletion construct at the AP-1/Ets site
in an attempt to alter the stretch response. We used mutations shown
previously to ablate Raf-1-induced transcription at this site (20).
During DNA sequencing to confirm incorporation of the desired mutations into the promoter, we identified several discrepancies with the published sequence. These discrepancies were previously noted by
McCarthy et al. (20); however, these investigators did not amend the
published sequence. The new sequence data are presented in Fig. 3, with
the numbering amended accordingly.

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Fig. 3.
Nucleotide sequence of mouse HB-EGF promoter region between 1301
and 881. Location of putative binding site for AP-1
transcription factor is indicated.
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Bladder SMC were transiently transfected with wild-type and mutant
variants of the
1301 construct in which the AP-1 and Ets components of the targeted AP-1/Ets site were modified (Fig.
4A). Transfected cells were subjected to cyclical stretch and relaxation for
12 h (Fig. 4B). The cells
that had been transfected with the partially deleted wild-type
construct demonstrated full promoter activation in response to
mechanical stretch, whereas the constructs transfected with the mutant
AP-1 and mutant Ets constructs, and a double mutant in which both sites
were modified, showed a significant attenuation (
70%) of stretch
responsiveness (Fig. 4B). Mutation of the AP-1 component resulted in a quantitatively significant decrease
in unstimulated transcription, suggesting that AP-1 is required for
significant levels of basal expression of the HB-EGF gene. These
results indicate that full activation of the HB-EGF promoter by the
stretch signal requires the presence of the AP-1/Ets site located at
nucleotides
988 and
974.

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Fig. 4.
Site-specific mutation of the AP-1/Ets binding site at 988
through 974 of the HB-EGF promoter abrogates stretch
reponsiveness. A: indicated mutations
were introduced into AP-1 site (mAP-1) in context of wild-type
1301 deletion construct by substituting nucleotides as indicated
using a PCR-based approach. B:
constructs were transiently transfected into bladder SMC. Cells were
then subjected to continuous cycles of stretch and relaxation for 12 h.
Nonstretch control cells were prepared and treated in parallel but
without stretch stimulation. Cell lysates were subsequently analyzed
for luciferase activity.
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We then determined whether an increase in binding activity of the AP-1
transcription factor could be detected at this site after mechanical
stretch by performing electrophoretic mobility shift assay (EMSA).
Quiescent bladder SMC were subjected to cyclical stretch and relaxation
for 2 h. Compared with nonstretch controls, cells that had been
stretched demonstrated a marked increase in AP-1 binding activity (Fig.
5). This binding was completely inhibited when nuclear extracts were coincubated with unlabeled competitor oligonucleotides corresponding to positions
993 and
973
of the HB-EGF promoter, which include the AP-1 site at position
988 to
982 and flanking sequences. Incubation of nuclear
proteins before EMSA with anti-c-Fos antibody resulted in a
supershifted complex as well as diminution of AP-1 binding intensity.
Preincubation of nuclear extracts with a control antibody (rabbit IgG)
did not result in supershift of the complex.

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Fig. 5.
Electrophoretic mobility shift assay of HB-EGF promoter. A 21-bp
double-stranded oligonucleotide containing an AP-1 binding site was
labeled with 32P by use of T4
polynucleotide kinase. Nuclear extract (2 µg) from nonstretched and
2-h stretched bladder SMC were mixed with 0.5 µg of poly(dI-dC) and 1 × 104 cpm labeled probe.
They were then analyzed by electrophoresis on a 4% polyacrylamide gel.
SS, supershifted complex. A 50-fold molar excess of an unlabeled,
double-stranded oligonucleotide corresponding to 993 and
973 positions of HB-EGF promoter was used as competitor (HB). A
polyclonal antibody for c-Fos family members supershifted DNA protein
complex; a negative control antibody (rabbit IgG) had no effect.
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Curcumin, an AP-1 inhibitor, suppresses stretch-induced HB-EGF
expression.
Previous studies have demonstrated that curcumin (diferuloylmethane), a
potent inhibitor of tumor promotion, inhibits AP-1-mediated transcription (11). As an additional test of the involvement of AP-1 in
the stretch response of the endogenous HB-EGF promoter, we examined the
effect of curcumin on HB-EGF mRNA expression in bladder SMC after
mechanical stretch. Quiescent bladder SMC were subjected to cyclical
stretch and relaxation for 4 h, in the presence or absence of 20 µM
curcumin, and the relative levels of HB-EGF mRNA were assessed by
semiquantitative RT-PCR. Stretch-induced expression of HB-EGF was
almost completely attenuated in the presence of curcumin (Fig.
6). This finding is consistent with the
conclusion that AP-1 activation is required for transcriptional
induction of the HB-EGF gene by stretch.

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Fig. 6.
Effect of curcumin, an AP-1 inhibitor, on expression of HB-EGF, matrix
metalloproteinase (MMP)-1, and MMP-2 after mechanical stretch.
Quiescent bladder SMC were subjected to continuous cycles of
stretch-relaxation for 4 h, and relative levels of HB-EGF, MMP-1, and
MMP-2 mRNA were assessed by semiquantitative RT-PCR with normalization
to GAPDH mRNA levels. Representative results are shown from 2 of a
total of 4 independently performed experiments.
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The above findings led us to test the hypothesis that the presence of
functional AP-1 cis-elements in the
promoter region could predict stretch responsiveness of other genes in
this system. We therefore examined the effect of mechanical stretch on
the expression of two MMP genes, one with a functional AP-1 binding site and one without an AP-1 binding site in its promoter. MMPs are
frequently upregulated in vivo at sites of tissue remodeling (reviewed
in Ref. 21) and metalloproteinase activation has been previously
associated with acceleration of rates of soluble HB-EGF secretion (7,
28). The rat interstitial collagenase (MMP-1) gene has been shown to
contain a functional AP-1 element in its promoter in response to basic
fibroblast growth factor stimulation (1). In contrast, the rat 72-kDa
type IV collagenase (MMP-2) gene does not contain any known AP-1
binding site in its promoter (3). Quiescent bladder SMC were subjected
to cyclical stretch and relaxation for 4 h. By semiquantitative RT-PCR,
steady-state MMP-1 mRNA levels increased significantly (~6-fold)
after stretch, and the level of MMP-2 mRNA was not affected by stretch.
Furthermore, the AP-1 inhibitor curcumin completely attenuated the
stretch-induced increase in MMP-1 levels, but it had no discernible
effects on MMP-2 mRNA expression (Fig. 6).
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DISCUSSION |
In this study we have identified the AP-1 transcription factor as an
essential element in the mechanism of transcriptional activation of the
HB-EGF gene in response to a repetitive mechanical stimulus. These
studies and other recent reports from our group (unpublished
observations; 8, 22) have identified HB-EGF as a mechanically regulated
factor of potential physiological relevance to bladder physiology.
HB-EGF was first identified as a stretch-responsive gene in bladder SMC
(22). In this first report, induction of HB-EGF gene expression by
stretch was found to be under partial control of signaling through the
angiotensin receptor AT1. HB-EGF is synthesized by the bladder detrusor muscle on the basis of immunohistochemical analysis of human and mouse bladder tissue (unpublished observations; 8). In these studies the membrane-anchored precursor of mature HB-EGF (proHB-EGF) was immunolocalized to the
detrusor muscle with use of two different antibodies directed against
the proHB-EGF cytoplasmic tail domain, as well as antibodies to the
mature growth factor. HB-EGF synthesis was also shown in the
uroepithelium, and HB-EGF was identified as an autocrine uroepithelial cell growth factor (8). HB-EGF mRNA and protein expression were also
observed to increase, primarily in the muscle compartment, after
bladder outlet obstruction in mice (unpublished observations). The
predominant cognate receptor for HB-EGF, the ErbB1 receptor tyrosine
kinase, is present in the detrusor muscle and in the epithelial layer
of the bladder mucosa (2; unpublished observations). These findings,
taken together, suggest a role for HB-EGF in the bladder's response to
injury and/or as a potential mediator of bladder wall thickening in
compensatory hypertrophy.
The system we used for the present and an earlier study (22) employs a
vacuum device that deforms, in a highly controlled fashion, the
substrate on which adherent cells are plated. A similar or identical
system has been used by other groups to examine various responses of
other cell types, including cardiac myocytes and fibroblasts, vascular
SMC and endothelial cells, and glomerular mesangial cells, to
mechanical stimuli. Many fewer studies have been attempted using
bladder SMC, and few details of the molecular mechanism of compensatory
bladder hypertrophy are known. Cardiac hypertrophy is a condition where
tissue remodeling is believed to proceed as a compensatory response of
the heart muscle to increases in hemodynamic load. This tissue response
bears at least a superficial resemblance to the response of the bladder
wall to outlet obstruction. A series of studies using cardiac myocytes
have identified a variety of molecular mediators of stretch-induced
intracellular signaling. Stretch of cardiac myocytes or fibroblasts by
use of a system similar to that used here induces the activation of
mitogen-activated protein kinase cascades and the expression of
immediate-early genes as well as genes the expression of which is
characteristic of fetal development of the heart (13, 18, 31). These
results have been interpreted to reflect the response of the cardiac
muscle during cardiac hypertrophy. Importantly, the vasoregulatory
peptide ANG II, signaling through the
AT1 angiotensin receptor, has been identified as a mechanically induced mediator of cardiac hypertrophy (31). These findings are consistent with our previously reported results in bladder SMC and suggest that mechanical forces regulate similar sets of genes in both cell types. We previously identified ANG
II as a peptide secreted by bladder SMC in response to the repetitive
stretch stimulus used in the present study (22). Blockade of
angiotensin receptors inhibited induction of HB-EGF mRNA promoter
activity and DNA synthesis by bladder SMC in response to stretch,
suggesting that the angiotensin system is a mediator of the stretch
response. This signaling system may be relevant to bladder hypertrophy
in vivo, because angiotensinogen, the ANG II precursor, and
AT1 are present in the bladder
wall (27).
In the present study we identified AP-1 as an essential mediator of the
stretch-induced stimulation of HB-EGF expression by 1) deleting the region of the HB-EGF
promoter containing a composite AP-1/Ets binding site, showing loss of
the stretch response; 2) mutational
analysis of the putative binding site, demonstrating that inactivating
mutations abolished the stretch response;
3) EMSA, demonstrating an increase
in AP-1 binding activity with stretch, and specific ablation of this
response by competition with an oligonucleotide corresponding to the
putative stretch-responsive region of the HB-EGF promoter; and
4) pharmacological inhibition of
HB-EGF induction by stretch with curcumin, an AP-1 inhibitor. Additional evidence implicating AP-1 as a mediator of the stretch signal was provided by our demonstration that the rat MMP-1 gene, the
promoter of which contains an AP-1 site demonstrated in previous studies to be activated in response to growth factor stimulation, is
also responsive to stretch in this system, whereas the MMP-2 gene,
which is not regulated by AP-1, does not respond to stretch or to
curcumin. Like the HB-EGF gene, stretch-induced activation of MMP-1
mRNA is inhibitable by curcumin. Consistent with our present results,
AP-1 has been identified as a mediator of mechanical stretch in
vascular SMC, endothelial cells, and cardiac myocytes (10, 13, 30).
Consequently, AP-1 appears to be an important mediator of mechanical
regulation of gene expression in several cell types.
In our study, introduction of an inactivating mutation into the Ets
component of the AP-1/Ets site also abolished the stretch signal,
suggesting that an Ets family transcription factor cooperates with AP-1
to mediate the stretch response. Preliminary results indicate that
stretch activates Ets binding activity in the bladder SMC system
(unpublished observations); however, we have not been able to confirm
that the responsible transcription factor at the AP-1/Ets site in the
HB-EGF promoter is Ets-2, which has been shown to bind this site in
NIH/3T3 cells. The promoter for tissue inhibitor of MMP-1 (TIMP-1), an
endogenous inhibitor of MMP-1 and other metalloproteinases that is
often coordinately regulated with MMPs during tissue remodeling
conditions, contains a composite AP-1/Ets site in which AP-1 and Ets-1
act synergistically to activate TIMP-1 transcription (17). Preliminary
data from our laboratory indicate that TIMP-1 is also a
stretch-responsive gene in bladder SMC (unpublished observations). This
finding may have physiological relevance to bladder disease, because
our laboratory has previously demonstrated that upregulation of TIMP-1
was associated with bladder hypertrophy in an in vivo model of bladder
outlet obstruction (23). An attractive hypothesis is that the AP-1/Ets
site in the TIMP-1 promoter is a critical mediator of the stretch
response and participates in the dysregulation of the proteolytic
balance associated with obstruction-induced bladder fibrosis.
It remains to be determined how the stretch signal, which may be
converted to a biochemical pathway by membrane-bound receptors, possibly integrins (18), is transmitted to the nucleus in bladder SMC.
One possibility is the JNK pathway, a known regulator of AP-1 via
phosphorylation of the Jun component of the heterodimeric AP-1
transcription factor by the SAPK/JNK mitogen-activated protein kinase.
Activation of the JNK pathway by stretch has been demonstrated in
cardiac myocytes (13, 16) and vascular SMC (9) and by shear stress in
endothelial cells (15).
It will also be of interest to determine whether stretch-responsive
genes identified in other cell systems, particularly in SMC, are
similarly stretch responsive in bladder SMC. Such a result would
suggest that mechanoinduction of biochemical pathways related to tissue
growth are conserved across several cell types and organ systems.
Consistent with this possibility, Igura et al. (12) demonstrated that
HB-EGF is rapidly induced in neointimal cells in rat carotid arteries
in response to balloon injury, possibly in response to a stretch
signal. An alternative possibility is that mechanical signals activate
cell-specific pathways and responses. This question remains to be
resolved. Studies in the literature provide evidence that induction of
gene expression in response to mechanical signals can vary with cell
type, the strength of the mechanical stimulus, and the extracellular
matrix composition of the substratum (18, 26, 29). For example, in
contrast to our findings, Yang et al. (32) recently reported that
platelet-derived growth factor- or tumor necrosis factor-
-induced
synthesis of MMP-1 was suppressed by small mechanical strains in
vascular SMC. These studies suggest that mechanoregulation of gene
expression may be cell and context dependent.
In conclusion, these studies have identified HB-EGF as a mechanically
regulated gene in bladder SMC and have determined that this
mechanochemical regulatory mechanism requires stretch-induced activation of the transcription factor AP-1. We also provide
preliminary evidence that this transcriptional mechanism may apply to a
network of similarly regulated genes. One important goal will be to
identify other genes in this network so as to better understand
mechanical signaling of growth-regulatory pathways in the bladder wall
and possibly in other hollow organs such as the heart. We propose that
our studies suggest that HB-EGF may play a role in obstruction-induced increases in DNA and protein synthesis in the bladder wall in animal
models of bladder outlet obstruction and in the human disease.
 |
ACKNOWLEDGEMENTS |
This study was funded by National Institutes of Health Grants RO1
DK-47556, RO1 DK-47582, and RO1 CA-77386.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. R. Freeman,
Enders Research Laboratories, Rm. 1151, Children's Hospital, 300 Longwood Ave., Boston, MA 02115 (E-mail:
freeman_m{at}a1.tch.harvard.edu).
Received 23 February 1999; accepted in final form 26 April 1999.
 |
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