Regulation of endothelin-1 gene expression by cell shape and
the microfilament network in vascular endothelium
Adel Moussa
Malek1,
Ike W.
Lee3,
Seth L.
Alper2, and
Seigo
Izumo3
1 Department of Neurosurgery,
Brigham and Women's Hospital, Children's Hospital, and Harvard
Medical School, Boston, 02115; and
3 Cardiovascular Division and
2 Molecular Medicine and Renal
Units, Beth Israel Deaconess Medical Center, and Departments of
Medicine and Cell Biology, Harvard Medical School, Boston,
Massachusetts 02215
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ABSTRACT |
Endothelial synthesis and release of
endothelin-1 (ET-1) are exquisitely regulated by external shear and
strain. We tested the hypothesis that manipulation of endothelial cell
shape can regulate ET-1 gene expression. Treatment of bovine aortic
endothelial cell (BAEC) monolayers with cytochalasin D disrupted
F-actin and induced cell retraction and rounding, in parallel
with time- and dose-dependent specific decreases in ET-1 mRNA levels.
Treatments with forskolin, phorbol 12-myristate 13-acetate,
staurosporine, and genistein also induced cell shape change and
decreased F-actin staining and ET-1 mRNA levels. BAEC plated onto
nonadhesive petri dishes coated with decreasing concentrations of
synthetic RGD polymer showed RGD dose-dependent decreases in
cell spreading and in F-actin microfilament elaboration. These changes
were specifically accompanied by decreases in ET-1 peptide secretion
(60%) and, via posttranscriptional mechanisms, ET-1 mRNA (94%)
and were not due to decreased cell-cell contact. We conclude that the
shape and microfilament network of endothelial cells are potent
posttranscriptional regulators of ET-1 gene expression.
morphometry; mechanotransduction; gene expression; cytoskeleton; F-actin
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INTRODUCTION |
THE ENDOTHELIAL CELL is continuously subjected to
hemodynamic forces that elicit, in turn, structural and functional
responses (6, 21). Numerous studies have documented that endothelial cell shape is determined by its external mechanical environment both in
vivo (17, 27) and in vitro (29). When
subjected to cyclic strain of the underlying extracellular matrix,
endothelial cell monolayers undergo a transition in cell shape from
polygonal to ellipsoidal and align along an axis perpendicular to the
strain vector (33, 34). In response to flow, endothelial cell
monolayers align in a parallel direction and assume a fusiform
morphology in areas of high shear and a polygonal one in areas of low
shear stress magnitude (11, 24). These differences in endothelial morphology in response to fluid shear stress are accompanied by changes
in F-actin and microfilament cytoskeleton structure (9, 10, 14, 22).
In addition to the externally applied mechanical stimuli of shear and
axial stress, endothelial cells have been shown to generate significant
tension against the substratum (18, 26); this phenomenon has been
observed by silicone-wrinkling techniques (13, 26) and quantified using
direct isometric measurement (18). The contractile force generated by
the endothelial cell is dependent on microfilament integrity and can be
abolished by treatment with cytochalasin D (CytD) (18). Fluid shear
stress applied in vitro also induces changes in the
functional state of the endothelial cell, including an altered pattern
of gene expression (6, 21) that is associated with chronic changes in
the cytoskeleton and cell morphology (22). Shear stress induces, in
addition, significant decreases in the rate of DNA synthesis (23) and
in intracellular pH (38). These effects resemble those previously
reported in endothelial cells grown in the round configuration (15,
16), with its associated decreased intracellular tension (1, 18).
Furthermore, fluid shear stress induces downregulation of the
vasoconstrictor endothelin-1 (ET-1) (20), a potent mitogen to vascular
smooth muscle cells and cardiac myocytes (35), which has been proposed
to play a central role in blood vessel homeostasis and structural
remodeling (32). Taken together, these results suggest a link between
external mechanical stimuli and endothelial gene expression that may be
important in the long-term adaptation to the hemodynamic stimulus in
vivo. Shear stress and mechanical strain also regulate
the microfilament network and cell shape. Direct roles of the
microfilament network and cell shape in control of gene expression
remain poorly defined.
In this report, we investigate the link between the cell shape and
microfilament network of endothelial cells and endothelial cell
expression of the ET-1 gene. Our findings, obtained in a setting free
of external mechanical stimulus, suggest that the status of
microfilaments and cell shape themselves play an important role in the
regulation of ET-1 production.
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MATERIALS AND METHODS |
Materials.
All reagents used were of the highest available grade.
CytD, phorbol 12-myristate 13-acetate (PMA), forskolin, genistein, and
staurosporine were obtained from Sigma Chemical (St. Louis, MO) and
ProNectin F from Protein Polymer Technologies (San Diego, CA).
Cell culture.
Bovine aortic endothelial cells (BAEC;
passages
6-15)
were harvested from descending thoracic aortas obtained from the local abattoir by collagenase digestion; >98% of the resulting cells displayed uptake of acetylated low-density lipoprotein
(Biomedical Technologies, Stoughton, MA). The cells were cultured in a
humidified incubator (37°C, 5%
CO2) in growth medium consisting
of Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Gaithersburg,
MD) supplemented with 10% calf serum (GIBCO BRL), 4 mML-glutamine, 25 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), 10 U/ml of penicillin, and 10 µg/ml of streptomycin. Changes in cell viability were assessed by exclusion of propidium iodide uptake using a fluorescence-activated cell sorter
(Becton Dickinson, Cockeysville, MD) and were found to be
<5% under all conditions at the stimulus durations used.
To control cell shape, 10-cm petri dishes (model 8-757-13,
Fisher Scientific, Pittsburgh, PA) with untreated nonadhesive surfaces were incubated at room temperature with solutions of RGD polymer (ProNectin F) at different concentrations (0, 0.01, 0.1, 0.5, and 2 µg/ml) for 30 min and then rinsed with phosphate-buffered saline
(PBS) and allowed to dry overnight under ultraviolet
irradiation for sterilization. BAEC suspensions (1 × 106 cells total, corresponding to
a final density of 17 × 103
cells/cm2) were then plated on
each RGD-coated dish in DMEM containing 0.5 or 1% calf serum and
allowed to spread for 18 h before analysis of cell shape and mRNA
content.
In experiments addressing the role of cell seeding density, confluent
monolayers of BAEC (3.25 × 106 cells) were trypsinized and
triturated to maximize cell dissociation. The cell suspension was
pelleted at low speed (200 g) and
then resuspended in growth medium (containing 10% calf serum) and
plated at serial dilutions from 1:16 (6.25% of initial density or 3.5 × 103
cells/cm2) to 1:1 (100% of
initial density or 55.5 × 103
cells/cm2) on 10-cm tissue
culture plates (model 3003, Falcon, Oxnard, CA) for 18 h prior to
analysis.
To study the effect of serum content in the medium, confluent BAEC
monolayers grown in 10-cm tissue culture plates were transferred to
DMEM supplemented with increasing concentrations of serum (0, 1, 2.5, 5, and 10%) for 18 h before analysis.
RNA isolation and hybridization.
The acid guanidium thiocyanate-phenol-chloroform method
(22) was used to isolate total cellular RNA. Northern blot
hybridization was performed with a random-primer
32P-labeled 1.9-kilobase (kb)
fragment of the bovine preproendothelin-1 cDNA (a kind gift of Dr.
Thomas Quertermous), a 3.7-kb EcoR I fragment of the bovine constitutive endothelial nitric oxide synthase (eNOS; a kind gift of Dr. Thomas Michel), and a 1.3-kb
PstI fragment of the rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. After
incubation, the blots were serially washed in 2× saline sodium
citrate (SSC)-1% sodium dodecyl sulfate (SDS) and 0.2× SSC-1%
SDS to a final temperature of 55°C for ET-1 and at 63°C for
GAPDH and then exposed to X-ray film (X-Omat-AR film, Kodak, Rochester,
NY) at
80°C. Autoradiograms exposed in the linear range were
subjected to two-dimensional densitometric scanning, and the signal
strength of the band of interest was normalized for each sample with
respect to the corresponding GAPDH mRNA signal.
Nuclear transcription runoff.
Nuclei were isolated from BAEC that had been seeded for 18 h on either
tissue culture plastic or petri dish surfaces coated with 0.01 µg/ml
of ProNectin F and assayed using a previously described protocol (20,
22).
Peptide measurement.
Medium supernatants were removed from BAEC plated on either tissue
culture plastic or increasing concentrations of ProNectin F for 18 h
and purified using Amprep Ethyl C2 columns from Amersham Life Science
(Buckinghamshire, UK). ET-1 content was then measured in triplicate
with a Biotrak enzyme-linked immunosorbent assay (ELISA) kit (Amersham
Life Science), using the manufacturer's protocol.
Immunostaining.
BAEC monolayers were washed three times with PBS, fixed
with 3.7% paraformaldehyde in PBS for 30 min, permeabilized in PBS containing 0.1% Triton X-100 for 15 min and then washed in PBS. The
fixed and permeabilized cells were then incubated with either monoclonal anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology, Lake Placid, NY), or monoclonal anti-paxillin antibody (clone 349, Transduction Laboratories, Lexington, KY) for 30 min in PBS
containing 1% bovine serum albumin (BSA), washed with PBS for 30 min
and then incubated with anti-mouse Cy3-conjugated anti-mouse immunoglobulin G secondary antibody (Jackson Laboratories, West Grove,
PA) for 30 min in PBS containing 1% BSA, washed with PBS for 30 min
and fixed. For F-actin visualization, fixed and permeabilized cells
were incubated with tetramethylrhodamine isothiocyanate (TRITC)-labeled
phalloidin (Sigma) for 10 min, washed three times in PBS and then fixed
with Mowiol (Calbiochem, San Diego, CA). Stained cells were visualized
in an epifluorescence microscope (model BH-2, Olympus, Tokyo, Japan)
and photographed with T-Max 400 film (Kodak).
Cell morphology analysis.
Cells were visualized on an inverted microscope
(Olympus model IMT-2) using phase-contrast or Hoffman phase-modulation
optics and were photographed using T-Max 400 film (Kodak). Images were then scanned two-dimensionally to serve as input into the image and
shape analysis program. Cell contour was traced manually, and
connectivity analysis was performed on the resulting set of blobs using
the Image Analyst software package (Automatix, Billerica, MA) on a
Macintosh II/fx computer system (Apple, Cupertino, CA). The algorithm
determined the best-fit ellipse through each cellular contour and
provided the corresponding cell surface area value.
Statistics.
Data are expressed as means ± SE. Statistical
analysis was performed by analysis of variance and the unpaired
Student's t-test.
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RESULTS |
CytD causes disruption of the F-actin cytoskeleton and induces cell
rounding.
Treatment of confluent endothelial monolayers with CytD
resulted in retraction of cell edges followed by cell rounding (Fig. 1A,
left). This phenomenon
was a time-dependent process that was accelerated at higher CytD
concentrations (data not shown). Staining with TRITC-phalloidin
revealed complete dissolution of the fine F-actin microfilament network
into a diffuse homogeneous staining pattern within the cell (Fig.
1A,
right). Morphometric image analysis of the CytD-treated BAEC monolayers and computation of the projected cell area revealed a time-dependent decrease, from 570 ± 25 to 270 ± 24 µm2/cell, which became
statistically significant as early as 15 min after treatment (Fig.
1B).

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Fig. 1.
Treatment of bovine aortic endothelial cells (BAEC) with cytochalasin D
(CytD) results in cell process retraction and rounding. A: phase-contrast micrographs
(left) of BAEC treated with CytD (0.2 µg/ml) for indicated times (0, 60, and 180 min) reveal
retraction of cell processes and cell rounding.
F-actin staining (right) with
tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin confirms rapid dissolution of F-actin fibers and cables with
concomitant edge retraction and eventual rounding.
B: quantitative morphometric analysis
of projected cell surface area illustrates a 2- to 2.5-fold decrease in
projected cell area in BAEC monolayers, occurring as soon as 15 min
after onset of CytD addition.
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CytD treatment induces decreased ET-1 mRNA expression in a time- and
dose-dependent manner.
Northern analysis performed on CytD-treated BAEC (0.2 µg/ml, 2 h)
revealed a time-dependent decrease in ET-1 mRNA content that contrasted
with unchanged GAPDH mRNA levels (Fig.
2A). The decrease in ET-1 mRNA was both time- and dose-dependent, with higher
doses of CytD resulting in earlier and more pronounced downregulation
of ET-1 mRNA levels (Fig. 2B).

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Fig. 2.
CytD treatment induces a time- and dose-dependent downregulation of
endothelin-1 (ET-1) mRNA expression.
A: Northern blot analysis of BAEC
monolayers treated with CytD (0.2 µg/ml) for 2 h shows a specific
decrease in ET-1 mRNA content without significant change in
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
B: confluent monolayers of BAEC were
treated with increasing concentrations of CytD (2 × 10 3 to 2 µg/ml) and
harvested at 0, 0.5, 2, and 6 h for mRNA analysis. Densitometric
analysis of ET-1 mRNA normalized with respect to GAPDH mRNA revealed a
time- and dose-dependent decrease in ET-1 mRNA detectable within 0.5 h
of treatment.
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Forskolin, PMA, genistein and staurosporine induce changes in cell
shape and F-actin network.
To further test the link between endothelial shape,
extent of cell spreading, and ET-1 mRNA content, we evaluated the
effects of several pharmacological agents on the endothelial
cytoskeleton. Forskolin and PMA both have been reported to induce BAEC
retraction and cytoskeletal remodeling (Fig.
3B),
with overall elongation, spindle formation, and significant decreases
in projected cell surface area compared with control (Fig.
3A). PMA also induced dissolution of
the coarse cable-like F-actin network (Fig.
3F) into a fine reticular staining
pattern (Fig. 3G). Forskolin (10 µM) induced mild changes in cellular outline (Fig.
3C), subtle changes in actin
staining pattern, and redistribution of F-actin stress fibers from the
center to the periphery (Fig. 3H).
Treatment with genistein (30 µM), a tyrosine kinase inhibitor, also
caused cell process retraction, partial rounding (Fig.
3D), and moderate alteration of
F-actin staining pattern with partial dissolution (Fig.
3I). Last, the protein kinase C
(PKC) inhibitor staurosporine (10 nM) was also noted to induce BAEC
rounding (Fig. 3E), which was nearly
as dramatic as that induced by CytD (Fig.
3I), as well as time-dependent
dissolution of actin filaments into a diffuse staining pattern (Fig.
3J). Thus four pharmacological
agents acting through different second messenger systems, and (in the
case of PMA and staurosporine) even having opposing stimulatory and
inhibitory actions, each induced in parallel BAEC rounding and F-actin
depolymerization.

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Fig. 3.
Phorbol 12-myristate 13-acetate (PMA), forskolin, genistein, and
staurosporine act on BAEC to induce cell shape change and F-actin
dissolution. Phase-contrast micrographs
(A-E)
and F-actin staining
(F-J)
of confluent BAEC monolayers treated for 6 h with the protein kinase C
(PKC) activator PMA (100 nM; B and
G), the adenylate cyclase activator
forskolin (10 µM; C and
H), the tyrosine kinase inhibitor
genistein (30 µM; D and
I), and the PKC inhibitor staurosporine (10 nM; E and
J), resulting in cell shape change including retraction and rounding compared with untreated controls (A and
F). Actin staining with
TRITC-phalloidin reveals significant remodeling and depolymerization of
F-actin network to an extent that varies with agent used.
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Cell shape and F-actin changes induced by forskolin, PMA, genistein
and staurosporine are accompanied by decreased expression of ET-1
mRNA.
BAEC treated with forskolin (10 µM), PMA (100 nM), genistein
(30 µM) and staurosporine (10 nM) were subjected to
Northern analysis with ET-1 and GAPDH cDNA probes. Figure
4 shows that all four agents induced
significant decreases in ET-1 mRNA within 6 h, without change in GAPDH
mRNA level. This decrease in ET-1 mRNA level accompanied the
endothelial cell shape retraction and F-actin staining loss in a manner
similar to that observed with CytD treatment. This correlation among
cell shape, the F-actin network, and ET-1 mRNA expression transcended
any specific second messenger system, since the above agents
individually acted via protein kinase A, PKC, tyrosine kinases, or
directly via the microfilament network.

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Fig. 4.
Endothelial cell shape change and rounding observed in response to
treatment with forskolin, PMA, genistein, and staurosporine are
accompanied by downregulation of ET-1 mRNA. Confluent
monolayers of BAEC were treated with forskolin (10 µM), PMA (100 nM),
genistein (30 µM), or staurosporine (10 nM) and then harvested after
6 h for Northern analysis. Results show that all 4 treatments resulted in decreased ET-1 mRNA content without significant decrease in GAPDH
mRNA, an effect that parallels cell rounding and cytoskeletal remodeling shown in Fig. 3.
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Steady-state endothelial cell shape and F-actin network can be
controlled by varying RGD density on RGD polymer-coated nonadhesive
petri dish surfaces.
We next evaluated the relationship between cell shape
and ET-1 mRNA level with an independent method for controlling
steady-state cell shape. RGD polymer-coated petri dishes were seeded
with BAEC, and cells were allowed to spread for 18 h. Figure
5 reveals that cells plated at a low RGD
polymer concentration ([RGD]) assumed a round morphology
(Fig. 5, top
left) at steady state, whereas those
plated at higher [RGD] appeared well spread and were
indistinguishable from cells plated on standard tissue culture plastic
(Fig. 5, bottom
right).

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Fig. 5.
Plating BAEC on various densities of synthetic RGD polymer on petri
dishes controls cell shape and extent of cell spreading. Uncharged nonadhesive petri dish surfaces were
preincubated with increasing concentrations of synthetic RGD polymer
(ProNectin F; 0, 0.01, 0.05, 0.1, 0.5, and 2 µg/ml) and then rinsed
with phosphate-buffered saline and allowed to air dry. Dispersed BAEC were then plated at a density of 17 × 103
cells/cm2 on RGD-coated surfaces
and allowed to spread for 18 h. Micrographs obtained using Hoffmann
phase-modulation optics illustrate a dose-dependent increase of cell
spreading with increasing RGD polymer density. Cells plated on surfaces
treated with 2 µg/ml of RGD polymer
(bottom right) were indistinguishable
morphologically from those plated on standard tissue culture plastic.
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The alteration of shape was also accompanied by changes in the
endothelial microfilament network. The diffuse homogeneous staining in
round cells plated at low [RGD] contrasted with the well-developed fibrillar F-actin network in spread cells plated at high
[RGD] (Fig. 6), which was
indistinguishable from that seen in cells plated on tissue culture
plastic.

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Fig. 6.
Control of steady-state BAEC shape by RGD polymer correlates with state
of F-actin network. Cells plated on various densities of synthetic RGD polymer on uncharged petri dishes were fixed after 18 h and stained with TRITC-phalloidin. Round cells plated on low
RGD peptide density (0.01 and 0.1 µg/ml) revealed diffuse staining, whereas spread endothelial cells plated on higher densities (0.5 and 2 µg/ml) showed filamentous F-actin network.
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Variation of RGD polymer concentration reveals correlation between
cell shape, F-actin network, and levels of ET-1 mRNA expression and
secreted peptide.
To test further the hypothesized link between cell shape,
F-actin network, and ET-1 expression, we performed Northern
analysis on BAEC cultured on RGD-coated nonadhesive petri dishes with
variable RGD polymer plating density. Northern blot analysis (Fig.
7) and densitometry (Fig.
8) demonstrated low levels of ET-1 mRNA in round cells (low [RGD]) and high levels of ET-1 mRNA in
spread cells (high [RGD]) that were indistinguishable from
tissue culture plastic. The level of ET-1 mRNA normalized with respect
to GAPDH decreased from 0.94 (arbitrary units) to 0.06, a 16-fold
variation. In contrast, the level of constitutive eNOS mRNA was not
significantly altered in the same cells, with normalization to GAPDH
mRNA levels showing a maximal variation of 15% across the entire range
of [RGD] used. Quantitative morphometric analysis revealed
an increase in projected cell surface area with increasing RGD polymer
plating density (Fig. 8) from 91 to 412 µm2. The clear correlation
between BAEC shape, extent of spreading, and ET-1 mRNA
content can be appreciated in Fig. 8. This correlation resembles that
observed in the pharmacological manipulation experiments presented
above (Fig. 4). The concentration of ET-1 peptide released into the
supernatant was measured in BAEC spread on ProNectin F-coated petri
dishes and quantitated using an ELISA kit. Although not as
dramatic as the decrease in ET-1 mRNA, decreasing cell spreading
resulted in an ~60% decrease in the amount of secreted ET-1 peptide
when plating on 2 and 0.01 µg/ml ProNectin F were compared,
indicating that the regulation is not limited to the RNA level but also
extends to the final rate of peptide biosynthesis in and release from
the endothelial cell (Fig. 9).

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Fig. 7.
Expression of ET-1 mRNA at steady state correlates closely with extent
of cell spreading and indirectly with state of F-actin network, whereas
endothelial nitric oxide synthase (eNOS) mRNA is independent.
BAEC plated at 1:5 were allowed to spread on RGD-coated petri dishes or on tissue culture plastic for 18 h.
Left: Northern blot analysis revealed
low levels of ET-1 mRNA in round cells ([RGD] = 0, 0.05, and 0.1 µg/ml) and high levels in flattened cells ([RGD] = 0.5 and 2 µg/ml and tissue culture plastic).
Right: in contrast, mRNA levels of
eNOS were not significantly different between cells that were round,
spread, or plated onto tissue culture plastic. GAPDH mRNA content was
not altered as a function of cell shape change.
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Fig. 8.
Steady-state correlation of endothelial cell shape with expression of
ET-1. Quantitative morphometric analysis ( ) and ET-1 mRNA content normalized with respect to GAPDH ( ) reveal concomitant increases in extent of spreading, as measured by projected cell area,
and in normalized ET-1 mRNA content, as a function of
[RGD]. In contrast, content of eNOS mRNA ( ) did not
change.
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Fig. 9.
Endothelial cell shape control affects release of ET-1 peptide.
BAEC plated at 1:5 were allowed to spread on RGD-coated
petri dishes or on tissue culture plastic for 18 h. Supernatant was then collected, purified, and quantitated, using an enzyme-linked immunosorbent assay kit for ET-1, and normalized with respect to value
at [RGD] = 2 µg/ml. Amount of ET-1 peptide released into medium is significantly lower (by 60%) in round cells
([RGD] = 0.01 µg/ml) compared with flattened cells
([RGD] = 2 µg/ml).
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The decrease in ET-1 mRNA is the result of posttranscriptional
control.
To determine the mechanism of regulation of the ET-1
mRNA by cell shape control, nuclear runoff transcription assays were performed and revealed that cells in the round morphology demonstrated a higher (up to 80%) relative rate of ET-1 transcription when normalized with respect to GAPDH compared with cells in the spread morphology (Fig. 10). This
finding points to a posttranscriptional mechanism, possibly that of
mRNA destabilization, to explain the decreased level of ET-1 mRNA in
round cells.

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Fig. 10.
Regulation of ET-1 mRNA level by cell shape control is not
transcriptionally mediated. BAEC plated at 1:5 were
allowed to spread in either well-spread morphology on tissue culture
plastic or to remain in round morphology by plating on RGD-coated
([RGD] = 0.01 µg/ml) petri dishes for 18 h. Nuclei were
then isolated and analyzed using a nuclear runoff transcription assay.
Unlike mRNA level and peptide release, nuclear transcriptional rate is actually higher in round cells compared with spread cells with respect
to not only GAPDH but also tubulin. pUC18, DNA vector hybridization
specificity control.
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Extent of endothelial cell spreading is reflected in the physical
distribution of paxillin and phosphotyrosine residues.
To determine the potential role of focal adhesion
contact-associated proteins in determining the relationship between
BAEC shape and ET-1 gene expression, we analyzed the distribution of paxillin (Fig. 11), a focal adhesion
contact-associated 70-kDa protein (3, 5) previously implicated in
fibroblast adhesion and spreading on fibronectin (3). Increasing
[RGD] resulted in increased cell spreading at steady state.
This was accompanied by a change in the distribution of both paxillin
and phosphotyrosine from a diffuse pattern to a punctate pattern with
enhancement at the cell periphery.

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Fig. 11.
Immunocytochemistry of paxillin and phosphotyrosine residues in BAEC
plated at increasing [RGD]. BAEC plated at
1:5 were allowed to spread on RGD-coated petri dishes at
[RGD] of 0.01 (top), 0.1 (middle) and 2 µg/ml
(bottom) for 18 h and then stained
with anti-paxillin antibody (left)
or anti-phosphotyrosine antibody (anti-p-tyr; right). Note diffuse staining in
round cells for both paxillin and tyrosine phosphorylation, which
becomes progressively more punctate and localized to the cell periphery
with increasing [RGD].
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Neither cell seeding density nor medium serum concentration can
account for shape-dependent regulation of ET-1 gene
expression.
To evaluate the contribution of reduced cell density to
the decreased ET-1 mRNA produced by cell rounding, secondary to CytD or
to low [RGD], confluent monolayers of BAEC were
trypsinized, triturated, replated in growth medium at cell seeding
densities ranging from 6.25% (1:16 split corresponding to
3.5 × 103
cells/cm2) to 100% (1:1 split
or 55.5 × 103
cells/cm2), and then harvested
after 18 h. Quantitative Northern blot analysis revealed a decrease in
ET-1 mRNA level at higher seeding density (Fig.
12A).
Because cells plated at low [RGD] have fewer cell-cell contacts, increased ET-1 mRNA expression at lower seeding density suggests that the decreased ET-1 expression at lower [RGD]
cannot be accounted for on the basis of cell density or cell-cell
contact.

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Fig. 12.
Effect of endothelial cell density and serum concentration on
expression of ET-1 gene. A: confluent
monolayers of BAEC were trypsinized and replated at densities ranging
from 3.5 × 103
cells/cm2 (6.25% of confluent
plate) to 55.5 × 103
cells/cm2 (100%). After 18 h,
total RNA was isolated and Northern blot analysis of ET-1 and GAPDH
mRNA was performed (inset).
Quantitative densitometric analysis of 3 independent experiments is
shown. Note that cells plated at lowest density exhibited 1.7-fold
higher levels of specific ET-1 mRNA content than did confluent
monolayers. B: confluent BAEC were
incubated for 18 h in presence of DMEM supplemented with 0, 1, 2.5, 5, or 10% serum. Total RNA was isolated and Northern blot analysis was
performed (inset). Quantitative densitometry of 3 independent experiments revealed 1.4-fold higher ET-1
mRNA in presence of 0 or 1% serum than at higher
concentrations.
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Similarly, because cells plated on RGD polymer-coated plates were grown
at low serum concentration (0.5-1%) to minimize the contribution
of fibronectin in serum, we evaluated the effect of low serum
concentration in the medium on specific ET-1 mRNA levels. Decreasing
serum concentration increased ET-1 mRNA content by 1.4-fold (Fig.
12B). Thus decreased ET-1 expression
at low [RGD] cannot be explained by decreased serum
concentrations.
 |
DISCUSSION |
This paper establishes a correlation between the abundance of ET-1 mRNA
in BAEC, the state of the actin cytoskeleton, cell spreading, and cell
shape. In particular, agents or maneuvers that depolymerize actin
stress fibers and decrease cell spreading also decrease ET-1 mRNA
levels. These effects occur in parallel not only in response to CytD,
acting directly on the cytoskeleton, but also in response to
perturbation of several distinct signaling pathways. These
perturbations include activation of certain isoforms of PKC by PMA,
activation of certain isoforms of adenylyl cyclase by forskolin,
blockade of unidentified serine/threonine kinases by staurosporine, and
blockade of unidentified tyrosine kinases by genistein. In addition,
graded increases in cell rounding and decreases of cell spreading
produced by plating cells onto various concentrations of RGD polymer
were also accompanied by graded decreases in cell spreading, by
depolymerization of the actin cytoskeleton, and by graded decreases in
ET-1 mRNA content.
Thus the correlation between ET-1 mRNA abundance, cell spreading, cell
shape, and the state of the actin cytoskeleton was maintained in a
variety of growth conditions, both during acute treatments with soluble
ligands and at nominally steady-state conditions 18 h after plating on
substrata of RGD polymer. Several observations supported the
specificity of this correlation with ET-1 mRNA. First, the level of
mRNA of eNOS, an important enzyme involved in vasomotor control and
previously shown to be upregulated by flow, was not affected by
RGD-mediated cell shape control. Second, the correlation was not a
primary consequence of changes in cell-cell contact, since decreased
plating density increased, rather than decreased, ET-1 mRNA level.
Third, the decreased serum concentrations in the RGD polymer
experiments did not explain the decrease in ET-1 mRNA, since, in cells
grown on tissue culture plastic, lowering serum concentration led to
increased, rather than decreased, ET-1 mRNA. In addition, the
regulation of ET-1 by cell shape was also found to extend to the
peptide level, pointing to this control mechanism as being of
functional significance in vasomotor control.
The mechanism by which the several examined stimuli downregulate ET-1
mRNA levels may be a reduction in the rate of ET-1 mRNA transcription
or an increase in the rate of ET-1 mRNA degradation. Downregulation of
ET-1 mRNA in BAEC secondary to application of laminar shear stress
results in a reduction in the rate of transcription and has been mapped
in the promoter of the ET-1 gene to a 5' flanking region between
~2.5 and 2.9 kb upstream of the transcriptional initiation site (20).
The nuclear run-on experiments performed in this study showed that cell
rounding increased the rate of ET-1 transcription relative to both
GAPDH and tubulin, suggesting posttranscriptional stability as the
major mode of control in response to cell shape changes. ET-1 mRNA
contains multiple AUUUA sequences and has previously been shown to
increase in response to protein synthesis inhibitors such as
cycloheximide (22). Whether this mechanism is shared with the one that
regulates ET-1 mRNA transcription in response to the varied agents that
depolymerize the actin cytoskeleton and cell shape modifiers remains to
be determined.
Cell shape has previously been shown to affect phenotype and gene
expression in other cell types. For instance, hepatocytes (2, 25) and
mammary gland cells (19) plated on substrata that allow attachment and
round cell morphology maintain tissue-specific gene expression
and an anabolic phenotype in the absence of cell proliferation. When
these cells are grown on a surface that allows spreading, they
dedifferentiate and enter the cell cycle. Recently, Hansen et al. (12)
have shown that cell spreading promotes and accelerates passage through
the G1-S transition of the cell
cycle and shuts off the intrinsic hepatocyte differentiation
program.
Though numerous signal transduction pathways appear to link the state
of the actin cytoskeleton to the abundance of ET-1 mRNA in BAEC, the
identity of the downstream steps remains unknown, as does the possible
existence of a downstream signal integrator. However, the results of
Kolodney and Wysolmerski linking endothelium-generated isometric force
to the state of the actin network (18) suggest intracellular tension,
transmitted throughout the microfilament network and acting at focal
adhesion contacts, as a possible candidate (5, 15). Recent work has
revealed clustering at focal adhesion complexes of growth factor
receptors, signaling molecules such as
pp60c-src,
phosphatidylinositol-3-kinase, phospholipase C, and the
Na+/H+
antiporter, and actin cytoskeleton binding proteins such as talin, vinculin, paxillin, and
-actinin. These findings, in conjunction with data of Wang et al. (37) showing that externally applied stresses
can be transmitted via focal adhesion contacts through the
cytoskeleton, have led to the proposal of a tensegrity-based model (7,
15). In this model, externally applied stresses or cell shape change
are translated into altered intracellular tension, which is borne
by the microfilament network and is transmitted to focal adhesion
contacts. There, intracellular tension may alter the conformation
or activity of one or more focal adhesion contact-associated signaling
molecules with subsequent downstream effects on gene expression and
cellular phenotype via a yet undefined signaling system.
Consistent with the intracellular tension hypothesis, we have observed
that lysophosphatidic acid, which activates Rho, a Ras-related
GTP-binding protein known to stimulate stress fiber induction, focal
adhesion formation, increased tyrosine phosphorylation (30), and
intracellular tension (4), induced actin stress fibers in confluent
BAEC and increased specific ET-1 mRNA content by twofold. In contrast,
compound H-7, a broad-range protein kinase inhibitor that inhibits
actomyosin contractility independently from its action on PKC (36) and
that has been shown to decrease stress fibers, focal adhesion
formation, and tyrosine phosphorylation (4), specifically decreased
ET-1 mRNA levels. Also consistent with this hypothesis, the recently
discovered natural cyclic peptide jasplakinolide, which binds to
F-actin at the phalloidin site (31), induced cell rounding, stress
fiber dissolution, and ET-1 mRNA decrease (unpublished data). The
altered distribution of paxillin and phosphotyrosine residues with cell
shape spreading at steady state (Fig. 11) provides a potential link
between cell shape and the signaling systems that may lead to
downstream altered ET-1 gene expression.
In conclusion, we have shown that cell shape and the state of the
microfilament network are potent regulators of ET-1 expression and have
presented data suggesting that this regulation may be mediated by
intracellular tension through posttranscriptional processing. Further
work will be required to identify the molecular links and mechanisms by
which intracellular tension may control gene regulation.
 |
ACKNOWLEDGEMENTS |
This work was supported by a Whitaker Foundation Grant, a Johnson & Johnson Foundation Research Grant (through the Harvard-Massachusetts Institute of Technology Health Sciences and Technology Division), National Institutes of Health Grants P30-HL-15157 (through the Boston Sickle Cell Center) and P60-DK-34854 (through the Harvard Digestive Diseases Center), and a National Institutes of Health Medical Scientist Training Program stipend to A. Malek.
 |
FOOTNOTES |
S. Izumo and S. L. Alper are Established Investigators of the American
Heart Association.
Current address of A. M. Malek: Neurointerventional Radiology,
University of California, San Francisco, Box 0628, L352, 505 Parnassus
Ave., San Francisco, CA 94143.
Address for reprint requests: S. Izumo, Rm. SL-201, or S. L. Alper, Rm.
RW-763, Beth Israel Deaconess Medical Ctr., East Campus, 330 Brookline
Ave., Boston, MA 02215.
Received 3 January 1997; accepted in final form 16 July 1997.
 |
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