Rapid Induction and Translocation of Egr-1 in Response to
Mechanical Strain in Vascular Smooth Muscle Cells
Henning Morawietz, Yunn-Hwa Ma, Franklin Vives, Emily Wilson, Vikas P. Sukhatme,
Jürgen Holtz, Harlan E. Ives
Abstract—The effect of mechanical strain on transcription and expression of the immediate-early genes, early growth
response gene-1 (Egr-1), c-jun, and c-fos, was investigated in neonatal rat aortic vascular smooth muscle (VSM) cells.
Cells grown on silicone elastomer plates were subjected to cyclic mechanical strain (1 Hz) at various durations and
magnitudes. Egr-1 mRNA increased rapidly in response to cyclic strain, reached a maximum of 10-fold after 30 minutes,
and returned to baseline after 4 hours. c-jun exhibited a similar pattern, whereas c-fos mRNA expression was unaffected
by strain. Cycloheximide prolonged the increase in Egr-1 and c-jun mRNA and caused superinduction of both. The
threshold level of continuous cyclic strain needed to induce expression was 5% for Egr-1 and c-jun. Even a single cycle
of mechanical strain that lasted 1 second was sufficient to induce Egr-1 and c-jun mRNA. Strain also increased
expression of a transiently transfected Egr-1 promoter-reporter construct. The effect of varying extracellular matrices on
strain-induced Egr-1 and c-jun mRNA was examined. In contrast to collagen type 1- and pronectin-coated plates, strain
did not significantly alter expression of Egr-1 and c-jun was less induced on laminin-coated plates. On collagen type
1, strain increased Egr-1 protein levels by 2.1-fold at 60 minutes. Immunofluorescence microscopy revealed
translocation of Egr-1 to the nucleus in response to strain. These observations indicate that Egr-1 expression and
translocation are sensitive to mechanical perturbation of the cell. c-jun is also induced by strain, but c-fos is not. The
signal for this induction may involve specific cell-matrix interactions. (Circ Res. 1999;84:678-687.)
Key Words: mechanical stimulation n cyclic strain n genes n muscle, smooth, vascular n transcription
modified nuclear factor (NF)-kB site found in the promoter
region of several genes induced by shear stress in endothelial
cells.9,10 Recently, the TPAresponsive element and the binding
site of early growth response gene-1 (Egr-1) were found to be
involved in shear stress–induced gene expression in endothelial
cells.11,12 In cardiac myoctes, Egr-1, c-jun, and c-fos expression
are increased by mechanical forces.13–15
On the basis of VSM cells transfected with various
PDGF-A– chain promoter truncations and subjected to strain,
data from this laboratory identified a 92-bp region of the
promoter that is responsive to strain6 This region contains a
binding site for the Egr-1 protein.
Egr-1 is thought to be involved in the regulation of mitosis
and differentiation.16,17 Egr-1, like c-jun and c-fos, belong to
the class of transcription factors called immediate-early
genes. The expression of these transcription factors is induced
by a variety of stimuli, including growth factors, serum, or
vasoconstrictors.17,18 After activation, Egr-1 protein is translocated from the perinuclear space to the nucleus, at which it
can bind to specific promoters and regulate gene
expression.19,20
V
ascular smooth muscle (VSM) cells in large arteries are
exposed to substantial mechanical forces during the
cardiac cycle. These forces may participate in developmental
processes and in the vascular hypertrophy associated with
disease states.1,2 Recently, the application of mechanical
strain to VSM cells in vitro has yielded a variety of descriptive data on alterations in the growth and phenotype of VSM
cells subjected to strain.3
Previous work from this laboratory has shown that cyclic
mechanical strain, applied by a vacuum to cells cultured on
silicone elastomer membranes, induces DNA synthesis and
expression of gene transcripts, such as PDGF-A, PDGF-B,
and the PDGF-b receptor.4 –7 Under certain conditions,
smooth muscle myosin SM-1 and SM-2 are induced.8 These
responses vary depending on the extracellular matrix. On
laminin (LN), DNA synthesis and expression of PDGF are
minimal5 but induction of smooth muscle myosins SM-1 and
SM-2 become more apparent.8
To date, only minimal information exists on the transcription
factors that transduce mechanical signals into gene activity. One
example is the shear stress–responsive element, which is a
Received November 20, 1998; accepted January 7, 1999.
From the Cardiovascular Research Institute and Division of Nephrology (H.W., Y-H.M., F.V., E.W., H.E.I.), University of California, San Francisco,
Calif; Institute of Pathophysiology (H.M., J.H.), Martin Luther University Halle-Wittenberg, Halle, Federal Republic of Germany; and the Beth Israel
Hospital and Harvard Medical School (V.P.S.), Boston, Mass.
Correspondence to Emily Wilson, PhD, Assistant Professor, Department of Medical Physiology, Texas A&M Health Science Center Medical School,
College Station, TX 77843. E-mail emilyw@tamu.edu
© 1999 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
678
Morawietz et al
In VSM cells, the effect of mechanical strain on Egr-1 and
c-jun is unknown. The cellular induction of c-fos by mechanical strain is controversial.21,22 We therefore examined the
induction of Egr-1, c-jun, and c-fos expression in VSM cells
subjected to varying mechanical forces. We find that Egr-1
and c-jun but not c-fos mRNA expression is dramatically
upregulated by exposure to even brief mechanical stimuli.
Extracellular matrix composition may determine both the
magnitude and direction of the effect of strain on Egr-1 and
c-jun transcription.
Materials and Methods
Materials
All materials were purchased from Sigma Chemical Co unless
otherwise specified. BSA was fraction V, fatty acid poor (Miles Inc).
Flex plates were from FlexCell Inc. Transfection reagent (DOTAP)
was purchased from Boehringer Mannheim, and Bicinchoninic Acid
solution protein assay reagent was purchased from Pierce Corp. The
Egr-1 antibody used is a rabbit affinity-purified polyclonal antibody
raised against a peptide of 14 amino acids mapping at the carboxy
terminus of mouse Egr-1 p82 (Santa Cruz Biotechnology, Inc). The
Egr-1 antibody was shown to react with the rat Egr-1 p82 by Western
blot analysis. b -Galactosidase assay kit was purchased
from Promega.
Cell Culture
Primary cultures of VSM cells from newborn rat aorta were
established by Dr Peter Jones (University of Southern California,
Los Angeles, Calif). From these primary cultures, the cell line R22
D was established23 and at passage 15 generously supplied by Dr
Jones. The cells were maintained in medium with 10% serum (MEM
with 10% FBS, 2% tryptose phosphate broth, 50 U/mL penicillin,
and 50 U/mL streptomycin) in a humidified atmosphere of 5% CO2
at 37°C. Culture medium was changed every other day until cells
were confluent. Confluent cells were subcultured with trypsinversenes. Cells were used from passages 17 to 25 for these studies.
In most experiments, cells were plated on 6 well (5 cm2 per well)
collagen-type I (Col I) silicone elastomer plates (Flex I, FlexCell Inc)
in medium with 10% serum and cultured until confluent. Three days
before experimentation, the medium was replaced with serum-free
medium (MEM with 0.5 g/L BSA, 0.5 mg/L apo-transferrin, 2%
tryptose phosphate broth, 50 U/mL penicillin, and 50 U/mL streptomycin). Medium was subsequently replaced with fresh serum-free
medium every 24 hours and again 3 hours before the strain
experiment. In experiments on different extracellular matrices, confluent cells in conventional plastic flasks were detached with
trypsin-versenes and trypsin was then inactivated with serumcontaining medium. The cells were then centrifuged for 5 minutes at
1000 rpm, washed once with serum-free medium, and the cells of
one 9-cm plastic dish were plated in serum-free medium on one
6-well silicone elastomer plate coated with Col I, pronectin (FN, a
fibronectin-like poly RGD matrix), or LN. Serum-free medium was
replaced 24 hours later (3 hours before application of mechanical
strain).
Application of Cyclic Strain to Cultured Cells
Confluent VSM cells on the silicone elastomer culture plates were
subjected to mechanical deformation with the Flexercell Stress Unit
(FlexCell Inc). The stress unit is a modification of the unit initially
described by Banes and coworkers24,25 and consists of a computercontrolled vacuum unit and a base plate to hold the culture dishes.
Vacuum is repetitively applied to the rubber-bottomed dishes
through the base plate, which is placed in a humidified incubator
with 5% CO2 at 37°C. The computer system controls the frequency
of deformation and the negative pressure applied to the culture
plates. A negative pressure of 20 kPa results in a maximal 25%
elongation of cells at the periphery of the dishes. After 24 hours of
April 2, 1999
679
continuous cyclic mechanical strain at the maximum attainable level,
no increase in lactate dehydrogenase activity (In vitro toxicology
assay kit, Sigma) was measurable in the supernatant (H.M., H.E.I.,
unpublished data, 1998).
Immunofluorescence Microscopy
VSM cells were grown on Col I– coated silicone elastomer plates and
subjected to 30 minutes of cyclic mechanical strain (1 Hz, 25%
strain) followed by 30 minutes in the resting state. In other
experiments, cells were subjected to 60 minutes of continuous cyclic
strain. After being rinsed briefly with PBS, cells were incubated with
4% paraformaldehyde/PBS at room temperature for 10 minutes
followed by incubation with methanol at 220°C for 6 minutes. All
subsequent steps were performed in a humidified chamber at room
temperature. The fixed cells were incubated with 10% serum/PBS
for 20 minutes, washed with 1% BSA/PBS, and then incubated with
rabbit polyclonal anti–Egr-1 antibody (2 mg/mL, Santa Cruz Biotechnology, Inc) for 1 hour. After the primary antibody was
removed, the cells were gently washed with 1% BSA/PBS, incubated
with a fluorescein isothiocyanate– conjugated goat anti-rabbit IgG
(1:200 dilution, Sigma) in the dark for 45 minutes and washed with
PBS. The silicone bottoms of the culture plates were removed and
trimmed to allow the material to be flattened for microscopic
observation. The silicone disks were then mounted in gel/mount
(Biomeda) on a glass slide. The stained cells were visualized with a
320 Fluor objective (Nikon) with epifluorescence illumination.
Photographs were taken with the use of Kodak Tmax 100 ASA film.
cDNA Clones
The Egr-1 cDNA clone contains a 1.6 kb BglII mouse Egr-1 cDNA
fragment cloned into the BamHI sites of pUC19 (Stratagene).16 The
Egr-1 plasmid DNA was isolated with the QIAGEN Plasmid Maxi
Kit (QIAGEN Inc), digested with HindIII and EcoRI, and the 1.6-kb
Egr-1 fragment was eluted after agarose gel electrophoresis with the
QIAquick Gel Extraction Kit (QIAGEN Inc). The c-jun cDNA clone
is the mouse 2.6-kb JAC.1 in the pGEM-2 vector (ATCC). The c-fos
cDNA clone includes the pTRI– c-fos/exon 2 from mouse (Ambion
Inc). In addition, a human c-fos cDNA fragment was cloned from
human umbilical vein endothelial cells by reverse-transcriptase PCR
and its identity confirmed by DNA sequencing (H.M., H.E.I.,
unpublished data, 1998). The rat GAPDH clone is a 250-bp EcoRV
GAPDH fragment cloned into the pT7Blue vector (Novagen Inc).26
The Egr-1 promoter deletion chloramphenicol acetyltransferase
(CAT) construct pEgr-1 P1.2 contains the “full-length” Egr-1 promoter (from position 2957 to 1248 bp, relative to the Egr-1
promoter transcriptional start site) fused with the reporter gene
CAT.27 The Egr-1 promoter-deletion CAT construct pE50 contains
the minimal Egr-1 promoter (from position 250 to 165 bp, relative
to the Egr-1 promoter transcriptional start site) fused with the
reporter gene CAT.28 The pRSV.CAT plasmid29 was used as a
positive control for CAT protein after transfection, and the pRSVb-Gal plasmid30 was used as a control for transfection efficiency.
RNA Isolation and Northern Blot Analysis
Total RNA was isolated from VSM cells with the RNA STAT-60
reagent (Tel-Test “B,” Inc). RNA samples (10 mg per lane) were
separated by electrophoresis through 1.2% agarose gels after denaturation of the RNA with glyoxal and dimethylsulfoxide.31 RNA was
transferred with 203 SSC buffer to Hybond-N nylon membranes
(Amersham Life Science Inc). The isolated cDNA fragments were
labeled with an oligolabeling kit (Pharmacia Biotech Inc) with
[a-32P]dCTP (Amersham Life Science Inc), purified with MicroSpin
Columns (Pharmacia Biotech Inc), and hybridized with the RNA
membranes in hybridization solution (1 mol/L NaCl, 1% SDS, 10%
dextran sulfate, and 100 mg/mL denatured salmon sperm DNA) at
65°C for 16 hours. The membranes were washed twice for 15
minutes with 23SSC, 0.1% SDS at 60°C and once for 15 minutes
with 0.23SSC, 0.1% SDS at 55°C and exposed to Hyperfilm MP
(Amersham Life Science Inc) at 280°C. Blots were quantified by
680
Immediate-Early Gene Induction by Mechanical Strain
scanning of autoradiographs with a laser densitometer (Molecular
Dynamics).
Protein Isolation and Western Blot Analysis
After mechanical strain, cells were washed and harvested in PBS and
centrifuged for 5 minutes at 3000 rpm at 4°C. The cell pellet was
lysed in lysis buffer (0.5% SDS in PBS) with a syringe, boiled for 10
minutes, and centrifuged for 10 minutes at 13 000 rpm and 4°C. The
protein concentration was determined with the BCA protein assay
reagent. Proteins (10 mg per lane) were separated in SDS-PAGE
(7.5%) and transferred to Hybond ECL nitrocellulose membranes
(Amersham Life Science Inc). The membranes were incubated with
a primary Egr-1 antibody (Santa Cruz Biotechnology, Inc), secondary horseradish peroxidase–linked rabbit Ig, and detected with ECL
Western blotting detection reagent (Amersham Life Science Inc).
Transfections and CAT Assays
Transfections were performed with the transfection reagent DOTAP.
VSM cells were grown on Col I-coated silicone elastomer plates
until 60% to 80% confluent. They were then transfected for 10 hours
with DOTAP; 5 mg RSV—b-galactosidase–DNA; and either (1) 10
mg Egr-1 promoter CAT-DNA, (2) 10 mg pRSV-CAT, or (3) without
CAT-DNA. Cells were then incubated in Opti-Mem I culture
medium (Gibco-BRL Life Sciences) for 48 hours and subjected to
cyclic mechanical strain (1 Hz, 25% strain) for 8 hours or 30 minutes
strain after incubation in the relaxed position for 7.5 hours. The cells
were lysed, and the protein concentration was determined with the
Coomassie plus protein assay reagent (Pierce Corp). The
b-galactosidase activity of the equal protein amounts of the cell
lysate was measured with the b-galactosidase enzyme assay system
(Promega), and the CAT protein was determined with CAT ELISA
(Boehringer Mannheim).
Statistics
Dimensionless quantities (ie, band densities) from multiple similar
experiments were combined by calculation of the fold increase (or
decrease) versus control under each experimental condition. Data are
given as mean6SEM (n$3 in all cases). Statistical analysis was
performed with Student’s t test. A value of P,0.05 was considered
statistically significant.
Results
Egr-1 and c-jun mRNA Induction by Continuous
Cyclic Mechanical Strain
Exposure of cultured neonatal aortic VSM cells to varying
time periods of cyclic mechanical strain (1 Hz, 25%) resulted
in a dramatic upregulation of Egr-1 and c-jun mRNA expression (Figure 1). Increased Egr-1 and c-jun mRNA were first
detectable at 15 minutes (Egr-1: 6.161.3-fold increase in
band density versus control, P,0.001, n56; c-jun: 1.460.09fold increase versus control, P,0.001, n54), reached its
maximum for both genes after 30 minutes (Egr-1: 9.761.1fold increase versus control, P,0.001, n56; c-jun: 5.160.6fold increase versus control, P,0.001, n54), and returned to
baseline after 4 hours (Egr-1) or 2 hours (c-jun) continuous
exposure to cyclic strain. In contrast, c-fos mRNA was not
significantly affected by mechanical strain in VSM cells. This
result was confirmed with a independently cloned human
c-fos cDNA probe (data not shown).
To assess the role of de novo protein synthesis in the
downregulation of Egr-1 and c-jun mRNA during continuous
exposure to strain, the strain protocol was repeated in the
presence of cycloheximide (Figure 2). Cycloheximide
(35 mmol/L) prevented the downregulation of Egr-1 and c-jun
mRNA expression and caused superinduction of both genes
Figure 1. Time course of immediate-early mRNA induction during exposure to continuous cyclic mechanical strain. VSM cells
on Col I– coated silicone elastomer plates were exposed to
cyclic (1 Hz) mechanical strain (25%) for the indicated times. A,
Total RNA was harvested and 10-mg samples were subjected to
electrophoresis and hybridized to cDNA probes for Egr-1, c-jun,
c-fos, and GAPDH. B, Densitometric analysis of experiments as
described in A. The Egr-1, c-jun, and c-fos mRNA expression (in
relative units) was normalized versus GAPDH mRNA. Bars represent the mean6SEM for 5 similar experiments. *P,0.05,
***P,0.001 vs basal.
Morawietz et al
April 2, 1999
681
Figure 2. Cycloheximide prolongs and enhances
induction of Egr-1 and c-jun by strain. VSM cells
on Col I– coated silicone elastomer plates were
exposed to cyclic (1 Hz) mechanical strain (25%)
for the indicated times, and RNA was analyzed as
in Figure 1A. Cells were exposed to 35 mmol/L
cycloheximide (CHX1) or no inhibitor (CHX2) during the strain period only. In the control (0) with
cycloheximide, cells were incubated with the inhibitor for 240 minutes.
at times .1 hour and for at least 4 hours. In the absence of
strain, cycloheximide caused only a minimal induction of
Egr-1 and c-jun mRNA at 4 hours (Figure 2). In contrast,
even in the presence of cycloheximide, the expression of c-fos
in response to strain was not affected (data not shown). Thus,
downregulation of Egr-1 and c-jun mRNA expression during
continuous exposure to strain requires de novo protein
synthesis.
The threshold magnitude of strain necessary to induce
Egr-1 and c-jun mRNA was determined by exposing cells to
5% to 25% cyclic strain for 30 minutes (Figure 3). Induction
of Egr-1 mRNA was first detectable with 5% cyclic strain
(2.460.3-fold increase in band density versus unstrained
control, P,0.05, n53), increased rapidly up to 10% strain
(6.960.4-fold increase versus control, P,0.001, n53), and
increased slightly further (9.460.6-fold versus control,
P,0.001, n53) with strain #25%. The threshold level of
c-jun induction by cyclic mechanical strain was similar (5%
strain: 1.460.02-fold induction versus control, P,0.001,
n54), which at 10% strain reached a level of induction
(4.260.3-fold induction versus control, P,0.001, n54) that
did not increase significantly #25% strain (4.560.6-fold
induction). Therefore, Egr-1 mRNA is '2-fold higher induced than c-jun (each versus control) in response to mechanical strain in VSM cells.
Induction of Egr-1 and c-jun mRNA by a Single
Cycle of Strain/Relaxation
We next determined the minimum number of strain/relaxation cycles necessary to induce Egr-1 and c-jun mRNA
expression. Because the maximal expression of Egr-1 mRNA
was previously found to occur after 30 minutes of continuous
cyclic strain (Figure 1), we exposed cells to strain (25%) for
a varied number of strain/relaxation cycles and then incubated cells in the relaxed position to achieve a total incubation time of 30 minutes (Figure 4). Surprisingly, even a single
cycle of strain/relaxation that lasted only 1 second was
sufficient to induce Egr-1 (4.661.2-fold versus control,
P,0.05, n54) and c-jun (2.460.3-fold versus control,
P,0.001, n56) mRNA. Egr-1 (5.760.4-fold) and c-jun
(3.760.4-fold) mRNA was further increased after application
of 5 minutes of cyclic strain and reached its maximum after
30 minutes of continuous cyclic strain (Egr-1; 9.660.1-fold
versus control, P,0.001, n54; c-jun; 5.660.6-fold induction, compared with control without strain, P,0.001, n56).
The threshold percent strain needed from single mechanical stimulus to induce Egr-1 mRNA was 10% (1.560.07-fold
induction versus control, P,0.05, n53) and to induce c-jun
mRNA was 20% (1.760.2-fold induction versus control,
P,0.01, n53; Figure 5). Expression was dose-dependently
increased and reached its maximum after a single deformation of 25% (Egr-1; 4.960.3-fold induction versus control,
P,0.01, n53; c-jun; 3.460.8-fold induction versus control,
P,0.05, n53). Higher degrees of mechanical deformation
were not tested because they were not relevant to the in vivo
situation.3 The c-fos mRNA was not affected by a single
mechanical stimulus of increasing magnitude (data not
shown).
We next examined the induction of Egr-1 by a single cycle
of strain/relaxation in more detail. The time course of Egr-1
mRNA induction after a single cycle of mechanical strain
(1-second duration, 25% strain; Figure 6) was similar to the
time course during continuous cyclic strain (Figure 1).
Increased Egr-1 mRNA was first detectable after 15 minutes
(2.660.5-fold induction versus control, P,0.05, n56),
reached its maximum level at 30 minutes (4.061.0-fold
induction versus control, P,0.05, n56), and returned to
baseline 2 hours after the single mechanical stimulus.
Induction of Egr-1 Promoter CAT Construct by
Mechanical Strain
In later studies, we examined the effect of mechanical strain
on Egr-1 in more detail. To determine whether increased
Egr-1 mRNA expression in response to strain is due to
increased transcription, we examined the effect of strain on
expression of transiently transfected Egr-1 promoter CAT
constructs (Figure 7). The full-length Egr-1 promoter construct (pEgr-1P1.2) was induced by mechanical strain
(5.960.7-old versus control without strain, P,0.05, n54). A
similar induction of pEgr-1P1.2 was detectable after 30
minutes of cyclic strain after incubation in the relaxed
position to reach a total time of 8 hours (4.560.7-fold versus
control without strain; P,0.05, n54, data not shown).
Neither a truncated Egr-1 promoter construct (pE50) nor
pRSV-CAT was induced by strain. Thus, Egr-1 transcription
is induced by mechanical strain at least in part through an
element located between 2957 to 250 bp of the promoter.
Effect of Extracellular Matrix Protein on
Induction of Egr-1 and c-jun mRNA by Strain
Previous studies from our laboratory showed that mechanical
strain of VSM cells is sensed by interactions with specific
682
Immediate-Early Gene Induction by Mechanical Strain
Figure 3. Threshold of Egr-1 and c-jun induction by mechanical
strain. VSM cells were grown on Col I– coated silicone elastomer
plates and subjected to continuous cyclic strain of the indicated
magnitude for 30 minutes. A, Total RNA was isolated and analyzed as in Figure 1A. B, Densitometric analysis of experiments
as described in panel A. Bars represent the mean6SEM for 3
similar experiments. *P,0.05, ***P,0.001 vs basal.
extracellular matrix proteins.5 Therefore, we examined the
effect of extracellular matrix protein composition on the
induction of Egr-1 and c-jun mRNA by mechanical strain
(Figure 8). To minimize the potential impact of extracellular
matrix protein synthesis by the cells after plating, experiments were performed 24 hours after plating, compared with
72 hours after reaching confluence for the experiment re-
Figure 4. Induction of Egr-1 and c-jun mRNA by a single cycle
of strain/relaxation. VSM cells on Col I– coated silicone elastomer plates were exposed to the indicated number of strain
cycles (1 Hz strain/relaxation) and then incubated in the relaxed
position to achieve a total incubation time of 30 minutes. A,
Total RNA was isolated and hybridized as in Figure 1A. B, Densitometric analysis of 3 similar experiments as described in
panel A. Data are given as mean6SEM. *P,0.05, ***P,0.001 vs
basal.
ported above. This led to lower levels of Egr-1 and c-jun
induction after strain than was observed above. Egr-1 and
c-jun mRNA were induced on Col I– (Egr-1; 2.860.2-fold
induction, P,0.01, n53; c-jun; 2.460.1-fold induction versus control on the same matrix without strain, P,0.001, n54)
Morawietz et al
April 2, 1999
683
Figure 6. Time course of Egr-1 mRNA induction after exposure
to a single cycle of mechanical strain. VSM cells on collagencoated silicone elastomer plates were exposed to a single cycle
(1 second) of mechanical strain (25%) and then incubated in the
relaxed position for the indicated times. A, Total RNA was harvested and hybridized with Egr-1 and GAPDH as in Figure 1A.
B, Densitometric analysis of experiments as described in panel
A. Bars represent the mean6SEM for 3 similar experiments.
*P,0.05 vs basal.
Figure 5. Threshold of Egr-1 and c-jun induction by a single
cycle of mechanical strain. VSM cells were grown on collagencoated silicone elastomer plates and subjected to a single cycle
(duration, 1 second) of mechanical strain of the indicated magnitude followed by 30 minutes incubation in the relaxed position.
A, Total RNA was isolated and analyzed as in Figure 1A. B,
Densitometrical analysis of 3 similar experiments as described
in panel A. Data are given as mean6SEM. *P,0.05, **P,0.01,
***P,0.001 vs basal.
and FN- (Egr-1; 2.660.4-fold induction: c-jun; 2.160.2-fold
induction versus control; P,0.001, n54) coated silicone
elastomer plates after 30 minutes of cyclic mechanical strain
(1 Hz, 25% strain). The time course of Egr-1 mRNA
induction by cyclic strain was also similar on Col I- and
FN-coated plates (unpublished data, 1998). In contrast, Egr-1
mRNA was not significantly induced by strain in cells
cultured on LN (0.960.1-fold expression versus control on
the same matrix without strain, n53). c-jun mRNA was
induced by cyclic strain on LN-coated plates (1.660.1-fold
induction versus control on LN without strain, P,0.001,
n54) to a significantly reduced extent compared with Col
I– coated plates (2.460.1-fold induction; P,0.01, n53) and
FN-coated plates (2.160.2-fold induction, P,0.05, n54).
This was due in part to increased basal expression of Egr-1
and c-jun mRNA on LN-coated plates (Egr-1; 1.760.3-fold;
c-jun; 1.460.3-fold expression versus expression without
strain on Col I– or FN-coated plates, P,0.05, n53). Despite
this fact, expression of Egr-1 after strain on LN-coated plates
was lower than on Col I– or FN-coated plates (LN: 1.660.4fold; Col I; 2.860.1-fold; and FN; 2.660.2-fold induction
versus expression without strain on Col I; LN versus collagen
684
Immediate-Early Gene Induction by Mechanical Strain
Figure 7. Induction of an Egr-1 promoter CAT construct by
mechanical strain. VSM cells on Col I– coated silicone elastomer
plates were transiently transfected with RSVb-Gal and either
RSV.CAT (RSV) or 1 of the Egr-1 promoter constructs pE50 or
p1.2, whose structures are indicated. 48 hours after transfection, cells were subjected to cyclic mechanical strain for 8
hours. CAT expression was measured as described. At this time
point, Egr-1 promoter-induced CAT protein expression was
maximal28 without reaching saturation (H.M., H.E.I., unpublished
data, 1998). A similar induction of Egr-1 promoter-induced CAT
protein expression was found after 30 minutes of mechanical
strain following incubation in the relaxed position to reach a
total incubation time of 8 hours. Bars represent the mean6SEM
for 4 similar experiments. *P,0.05 vs basal.
or FN, P,0.05, n53). In contradiction, the expression level
of c-jun after 30 minutes of mechanical strain was similar on
all 3 different extracellular matrices (Col I; 2.560.2-fold; FN;
2.360.2-fold; and LN; 2.160.2-fold induction versus expression without strain on Col I, n54, respectively).
Figure 8. Effect of extracellular matrix protein on induction of
Egr-1 and c-jun mRNA by strain. VSM cells were plated in serumfree medium on Col I–, FN-, or LN-coated silicone elastomer plates
and were subjected to 30 minutes of continuous cyclic mechanical
strain (1) or no strain (2). Total RNA was isolated and analyzed as
in Figure 1A. Densitometrical analysis of experiments was performed on Northern blots. Bars represent the mean6SEM for 3
similar experiments. *P,0.05, **P,0.01 vs basal.
space. After 30 minutes of mechanical strain, there was no
change in Egr-1 distribution at the center of the strain dishes,
at which strain on the cells is nearly zero.32 However, at the
periphery of the dishes, at which strain is maximal, Egr-1
protein became uniformly distributed in the nucleus. Similar
translocation was observed after 1 hour of continuous mechanical strain (data not shown). Thus, both Egr-1 expression
and translocation are highly sensitive to mechanical forces in
VSM cells.
Increase of Egr-1 Protein Expression After
Mechanical Strain
We next examined Egr-1 protein expression after exposure of
cells to various periods of continuous cyclic mechanical
strain on Col I– coated plates. Western blots (Figure 9)
demonstrated first increases in Egr-1 protein after 30 minutes
of cyclic strain (1.360.01-fold versus control without strain;
P,0.01, n55) and a maximal 2.160.2-fold increase at 60
minutes (P,0.001, n55). Protein expression subsequently
declined toward basal levels. This pattern of expression was
temporally similar to that observed for the mRNA in Figure 1.
Translocation of Egr-1 Protein to the Nucleus in
Response to Mechanical Strain
Immunofluorescence imaging of Egr-1 protein was performed after 30 or 60 minutes of cyclic mechanical strain
(Figure 10). In resting controls, immunofluorescence demonstrated a granular pattern of Egr-1 protein in the perinuclear
Figure 9. Expression of Egr-1 protein in response to mechanical
strain. VSM cells on collagen-coated silicone elastomer plates
were subjected to cyclic strain for the indicated times. A, Protein
was harvested from total cell lysates and Western blots were performed with an Ab to Egr-1. B, Bars represent the mean6SEM for
3 similar experiments. **P,0.01, ***P,0.001 vs basal.
Morawietz et al
Figure 10. Intracellular translocation of Egr-1 protein in
response to mechanical strain. VSM cells were subjected for 30
minutes to mechanical strain in the center (B) or at the periphery
of the plate followed by maintenance in the relaxed state at
37°C for 30 minutes (A, control without strain). Immunofluorescence imaging of the Egr-1 protein illustrates that in the resting
state, the protein clusters around the perinuclear space (A).
After mechanical strain of 25% (periphery of the stretch plate),
the Egr-1 protein is diminished in the perinuclear space and
became uniformly distributed in the nucleus (C). However, in the
center of the well (,5% strain), no obvious difference of Egr-1
location was observed after 30 minutes of mechanical strain (B).
Discussion
This study shows that mechanical strain of cultured neonatal
rat VSM cells induces immediate-early gene transcription,
protein synthesis, and protein translocation from the perinuclear space to the nucleus, respectively.
The time and the level of maximum induction of the
immediate-early gene Egr-1 mRNA in VSM cells exposed to
cyclic strain is similar to the induction found in stretched
cardiac myocytes33 and in cyclically stretched mesangial
cells.34 While performing a more detailed time course in
VSM cells, we found that Egr-1 and c-jun transcription after
mechanical strain starts between 5 and 15 minutes, reaches its
maximum at 30 minutes, and returns to baseline after 4 hours
or after 2 hours of continuous cyclic mechanical strain. The
rapid decline in Egr-1 and c-jun mRNA during continuous
cyclic strain might be due to the subsequent binding of
transcription factors that downregulate the transcription of
both genes and raises the possibility of an enzyme that
degrades these mRNA molecules. This hypothesis is supported by the prolongation and superinduction of Egr-1 and
c-jun mRNA after exposure to strain in the presence of the
inhibitor of de novo protein biosynthesis, cycloheximide.
This effect might be due to the inhibition of de novo synthesis
of proteins that downregulate the transcription of both
immediate-early genes. In addition, cycloheximide prevents
synthesis of a protein, which degrades the poly (A) tail of
certain mRNAs35 and thus superinduces many immediateearly response genes.36,37
Evidence that the induction of Egr-1 mRNA by strain is
due to increased transcription was obtained with a 957-bp
full-length promoter CAT construct. Known responsive elements in this promoter region include 2 AP-1 sites, 4 SP-1
sites, 2 cAMP response elements, 6 CArG-boxes or serum
response elements, and 4 Egr-1 binding sites.38 Any of these,
or as yet undefined sequences, could be “strain responsive
elements.” Because the AP-1 transcription factor is composed
of c-jun homodimers or c-jun/c-fos heterodimers, the induc-
April 2, 1999
685
tion of c-jun but not c-fos by mechanical strain suggests the
activation of c-jun homodimers in response to strain in VSM
cells. The involvement of c-jun in the cellular response to
mechanical strain is supported by recent data from this
laboratory, which show the activation of c-jun amino terminal
kinase by cyclic strain in VSM cells.39 In additional preliminary studies, we could show that the SP-1 protein is induced
by strain, possibly also implicating SP-1 sites in the strain
response.6 Recently, a functional interplay between Egr-1 and
SP-1 in the PDGF-A– chain promoter has been elegantly
demonstrated in bovine aortic endothelial cells in response to
shear stress.12 In these studies, the binding of Egr-1 to the
PDGF-A– chain promoter was induced by shear stress, which
displaced SP-1 from their overlapping recognition elements.
Ongoing work from our laboratory suggests a similar binding
of the Egr-1 protein to its binding site in response to
mechanical strain in VSM cells (E.W., H.E.I., unpublished
data, 1998).
In this work, we attempted to determine the minimal
mechanical stimulus needed to activate Egr-1 gene activity. A
single, transient, mechanical stimulus that lasted 5 minutes
was shown to be sufficient to induce DNA synthesis in adult
human VSM cells.40 In cardiac myocytes, c-fos mRNA was
induced by as little as 1 minute of continuous mechanical
strain.15 Surprisingly, in our system, even a single cycle of
strain/relaxation that lasted only 1 second was sufficient to
induce Egr-1 and c-jun mRNA. To our knowledge, this is the
shortest duration of any mechanical perturbation reported to
induce gene activity. The minimal magnitude of strain necessary to induce Egr-1 or c-jun mRNA by single cycle of
strain/relaxation was between 5% and 10% (or 15% and 20%
at the dish periphery). Because the strain profile on the dishes
we used is not homogeneous and many of the cells are
actually exposed to lesser degrees of strain, this value of 10%
sets the upper limit of strain necessary to elicit a cellular
response. After application of a single stretch that lasted 1
second to VSM cells grown on uniformly distensable silicone
membranes and after incubation of these cells for 30 minutes
in the relaxed position, we found a similar threshold level of
Egr-1 mRNA induction in response to strain (H.M., D.D.,
H.E.I., unpublished data, 1998). The threshold strain level
needed to induce Egr-1 mRNA by continuous cyclic strain
was 5% lower than for a single perturbation. Although the
system we studied cannot be directly compared with VSM
cells in vivo, the threshold strain levels we determined are
comparable to strains of 6% to 22% observed in certain intact
blood vessels.3 It is also possible that the in vivo threshold for
Egr-1 induction is reached only during injury to the vessel
wall or in severe hypertension, when strain of the vessel wall
is abnormally increased.41
Additional studies were aimed at determining the mechanism by which strain is sensed in VSM cells. Previous work
from this laboratory indicates that strain is sensed by specific
cell– extracellular matrix interactions.5 Induction of DNA
synthesis and activation of MAP kinase was found in neonatal VSM cells stretched on collagen or FN but not in cells on
LN5,39 This is not due to a failure of the cells to detect strain
on LN, because strain increased expression of smooth muscle
myosin isoforms8 and activated c-jun amino terminal kinase39
686
Immediate-Early Gene Induction by Mechanical Strain
in cells on LN. Thus, cells respond differently to strain when
they are plated on different matrix proteins.
In the current work, we demonstrate that the induction of
Egr-1 follows the pattern previously observed for the proliferative response to strain. Egr-1 mRNA was significantly
increased in cells on Col I and the fibronectin-like protein FN
but was not increased on LN. The maximal magnitude of
Egr-1 induction on collagen I and FN was somewhat lower in
this group of experiments than in the earlier experiments on
Col I. This was probably due to the shorter period (24 hours
versus 72 hours) allowed for the cells to achieve quiescence.
The shorter time period was chosen to minimize de novo
production of extracellular matrix proteins, which might have
interfered with the impact of the specific matrix proteins used
to coat the silicone dishes. We do not yet know the significance of the higher basal (without strain) expression of Egr-1
in cells plated on LN, but it is consistent with higher basal
rates of thymidine incorporation in cells plated on LN than on
Col I or fibronectin.5 The basal expression of c-jun was
increased on LN in a similar way. In contrast, c-jun mRNA
expression after application of mechanical strain is similar on
all 3 extracellular matrices tested. Thus, the effect of strain on
Egr-1 expression is closely correlated with its effect on DNA
synthesis in cells on various extracellular matrix proteins.
This observation supports the hypothesis that Egr-1 may
contribute to the induction of PDGF-A transcription and
ultimately secretion of PDGF in response to strain.4,6 On the
other hand, c-jun mRNA transcription and c-jun amino
terminal kinase are activated on collagen, FN, and LN. This
pattern of activation might reflect a matrix-independent, more
general response to extracellular stress in VSM cells. VSM
cells in the media of large arteries in vivo (eg, in the aorta, in
which mechanical stimuli of 6% to 22% strain are relevant)
are mainly surrounded by collagen (mainly type I).42 In
pathophysiological stages, mainly in late-stage atherosclerosis, in which increased mechanical forces act on the cells of
the vessel wall, smooth muscle cells dedifferentiate more,
start to grow, and increase DNA synthesis. During this
process, they synthesize and secrete connective tissue matrix
molecules such as collagen to potentiate this change of
smooth muscle cell phenotype. Typical smooth muscles of
this active and proliferating type can be found in the fibrous
cap of the fibrous plaque. Therefore, the induction of mitogenic gene products like Egr-1 and c-jun by increased
mechanical strain found on Col I– coated plates in vitro might
be involved in these pathophysiological changes of the vessel
wall in vivo.
Finally, we examined the expression and translocation of
Egr-1 protein in response to strain. The induction of the Egr-1
protein after strain reaches its maximum after 1 hour is
delayed only slightly compared with the maximum Egr-1
mRNA transcription. The 2.1-fold increase in Egr-1 protein
by mechanical strain (Figure 9) is less than the '10-fold
induction of Egr-1 transcription (Figure 1). This difference
may have several explanations, including regulation at the
posttranscriptional and posttranslational levels, and the presence of a pool of Egr-1 protein present in the basal state, as
observed in immunofluorescence images of unstrained cells
(Figure 10).
The physiological relevance of the induction of Egr-1 by
strain was further demonstrated by the translocation of Egr-1
protein from the perinuclear space to the nucleus after
exposure to 30 to 60 minutes of continuous cyclic strain.
Interestingly, this Egr-1 translocation was only detected at the
periphery of the Flexcell plates at which strain was maximal
(25%) and not at the center of the plates, at which strain was
less than 3%. This finding suggests an initial direct effect of
strain on Egr-1 translocation rather than an indirect effect of
secreted factors, such as PDGF. PDGF has previously been
shown to be secreted into the medium after several hours
exposure of VSM cells to strain.4 In addition, these data
suggest that translocation of Egr-1 requires strain, which, as
noted above for mRNA induction, is in the range observed in
intact blood vessels.
In summary, our results indicate that Egr-1 and c-jun
expression and translocation are exquisitely sensitive to
mechanical strain in VSM cells. The signal transduction
pathway for this induction involves specific cell/matrix interactions. In response to strain, Egr-1 and c-jun may then
participate in the regulation of other genes as part of developmental processes or in the adaptive response to injury or
hypertension. The inducible expression of Egr-1– dependent
genes has been proposed as a paradigm of transcriptional
activation in vascular endothelium and VSM cells.43 In
particular, we propose that early induction of Egr-1 in
response to strain participates in the induction of PDGF-A
expression, which subsequently leads to secretion of PDGF
and proliferation of cells exposed to cyclic mechanical strain.
Acknowledgments
This work was supported by National Institutes of Health grants
HL48474 (H.E.I.) and DK07219 (Y.-H.M.) and the German Academic
Exchange Service and the Deutsche Forschungsgemeinschaft (H.M.).
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