Mechanosensitive tyrosine phosphorylation of paxillin and
focal adhesion kinase in tracheal smooth muscle
Dachun
Tang,
Dolly
Mehta, and
Susan J.
Gunst
Department of Physiology and Biophysics, Indiana University School
of Medicine, Indianapolis, Indiana 46202-5126
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ABSTRACT |
We investigated
the role of the integrin-associated proteins focal adhesion kinase
(FAK) and paxillin as mediators of mechanosensitive signal transduction
in tracheal smooth muscle. In muscle strips contracted isometrically
with ACh, we observed higher levels of tyrosine phosphorylation of FAK
and paxillin at the optimal muscle length
(Lo) than at
shorter muscle lengths of 0.5 or 0.75 Lo. Paxillin
phosphorylation was also length sensitive in muscles activated by
K+ depolarization and adjusted
rapidly to changes in muscle length imposed after contractile
activation by either ACh or K+
depolarization. Ca2+ depletion did
not affect the length sensitivity of paxillin and FAK phosphorylation
in muscles activated with ACh, indicating that the mechanotransduction
process can be mediated by a
Ca2+-independent pathway. Since
Ca2+-depleted muscles do not
generate significant active tension, this suggests that the
mechanotransduction mechanism is sensitive to muscle length rather than
tension. We conclude that FAK and paxillin participate in an
integrin-mediated mechanotransduction process in tracheal smooth
muscle. We propose that this pathway may initiate alterations in smooth
muscle cell structure and contractility via the remodeling of actin
filaments and/or via the mechanosensitive regulation of
signaling molecules involved in contractile protein activation.
cytoskeleton; focal adhesion proteins; mechanotransduction; length-tension curve; contractility; signal transduction
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INTRODUCTION |
THE PHYSIOLOGICAL BASIS FOR the length-tension behavior
of smooth muscle tissues is not understood (12). Although the
length-tension behavior of striated muscles has been attributed to
differences in the overlap of contractile filaments (10), it is not
clear that analogous mechanisms can explain the length-tension behavior of smooth muscle. Smooth muscle exhibits a plasticity of its mechanical response that is not well accounted for on the basis of the traditional sliding filament paradigm. The physical length of smooth muscle at the
time that contractile activation is initiated has long-lasting and
persistent effects on its mechanical properties for the duration of the
period of contractile activation, even when those properties are
subsequently measured under identical mechanical conditions (11, 14,
15). We have hypothesized that the plasticity of the mechanical
response of smooth muscle may result from an ability of the muscle cell
to remodel the organization of its contractile apparatus in response to
changes in external stress or strain (13, 14). Actin filament
remodeling at the time of contractile activation might be a mechanism
by which smooth muscle cells adjust the organization of their
contractile apparatus to accommodate to changes in their mechanical
environment (13, 14, 22).
The actin filaments of smooth muscle cells link to the membrane at
dense plaque sites that are structurally similar to the focal adhesion
plaques of cultured cells (4). Smooth muscle dense plaques and focal
adhesion sites contain cytoskeletal proteins, including talin,
vinculin, and
-actinin, that connect actin filaments to
transmembrane integrins (4, 8) and thereby enable the transmission of
tension between the actin cytoskeleton and the extracellular matrix
(35, 36). The adhesion plaques of cultured cells also form a locus for
the interaction of signaling molecules that regulate processes involved
in adhesion-induced changes in cell physiology, such as cytoskeletal
assembly and actin remodeling (3). In cultured cells, focal adhesion
kinase (FAK) and paxillin localize to focal adhesion sites and are
thought to play a critical role in mediating these signaling processes
(3, 21). Both paxillin and FAK undergo phosphorylation during
integrin-mediated cell adhesion and during stimulation by a variety of
mitogens and growth factors. The phosphorylation of these proteins has been correlated with the assembly of actin stress fibers and with focal
adhesion formation (1, 5, 6, 24, 28, 31).
There is growing evidence that transmembrane integrins can function as
mechanotransducers and that the regulation of cellular responses to
mechanical stimuli is coordinated by the complex of cytoskeletal
proteins that associate with the cytoplasmic domains of integrin
molecules (30). In cultured endothelial cells, FAK and paxillin undergo
tyrosine phosphorylation in response to periods of repetitive
mechanical strain (38). In a number of cultured cell types, including
endothelial cells and airway smooth muscle cells, cyclic mechanical
strain has been shown to induce the alignment of actin filaments along
the axis perpendicular to the force vector (16, 29, 32). Paxillin and
FAK have been proposed to play an integral role in these strain-induced
morphological changes (38).
The contractile activation of tracheal smooth muscle elicits the
tyrosine phosphorylation of paxillin (22, 37), indicating that some of
the signaling processes that occur in the dense plaques of smooth
muscle cells in response to contractile stimuli are similar to those
that occur in the adhesion plaques of cultured cells. We therefore
hypothesized that paxillin and FAK may be components of an
integrin-mediated mechanotransduction pathway in smooth muscle. Such a
pathway might initiate signaling events that lead to the modulation of
smooth muscle cell shape and contractility. The mechanosensitive
modulation of smooth muscle cell contractility might result from the
remodeling of actin filaments or from the modulation of signaling
pathways that regulate contractile protein activation.
The objectives of the present study were to determine whether a
mechanosensitive signal transduction pathway mediated by
integrin-associated dense plaque proteins is present in smooth muscle
tissue and to evaluate the sensitivity of this pathway to acute changes
in muscle length and tension. Changes in the tyrosine phosphorylation
of FAK and paxillin were used as indexes of activation of an
integrin-mediated mechanotransduction signaling pathway.
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METHODS |
Preparation of tissue.
Mongrel dogs (20-25 kg) were anesthetized with pentobarbital
sodium and quickly exsanguinated. A 12- to 15-cm segment of
extrathoracic trachea was immediately removed and immersed in
physiological saline solution (PSS) at 22°C (in mM: 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4,
and 5.6 glucose). The solution was aerated with 95%
O2-5%
CO2 to maintain a pH of 7.4. Rectangular strips of tracheal muscle 2-3 mm in diameter and
12-15 mm in length were dissected from the trachea after removal
of the epithelium and connective tissue layer. Each muscle strip was
placed in PSS at 37°C in a 25-ml organ bath and attached to a Grass
force transducer. At the beginning of each experiment, the optimal
length for maximal active force
(Lo) was
determined by increasing muscle length progressively until the active
force in response to 10
5 M
ACh (Sigma) reached a maximum for that stimulus
(Fmax). All subsequent changes
in muscle length were calibrated as fractions of
Lo.
General procedures.
Up to 14 muscle strips from a single trachea were contracted
isometrically with ACh or KCl at muscle lengths of
Lo, 0.75 Lo, or 0.5 Lo. Tissues were
quickly frozen at desired time points after contractile stimulation,
using a liquid nitrogen-cooled clamp, for the determination of the
tyrosine phosphorylation of paxillin or FAK. Strips stimulated with KCl
and uncontracted strips were studied in the presence of
10
7 M atropine to block the
potential effects of neurotransmitters released from intramural nerves
in the tissue.
Ca2+
depletion of muscle strips.
In some protocols, smooth muscle strips were depleted of intracellular
Ca2+ before stimulation with
contractile agonists. After
Lo was
determined, strips were incubated in
Ca2+-free PSS containing 0.1 mM
EGTA for 10 min for the removal of extracellular
Ca2+. No change in resting tension
occurred when the bath was changed from PSS to
Ca2+-free PSS containing 0.1 mM
EGTA. Muscle strips were then stimulated for 5 min by adding
10
5 M ACh to the
Ca2+-free PSS. This step was
repeated three to four times with
10
5 M ACh. Between each
stimulation, the strips were incubated in Ca2+-free PSS containing 0.1 mM
EGTA for 10 min. Stimulation with 10
5 M ACh initially
produced a force of 70% Fmax, but
subsequent stimulations resulted in progressively smaller contractions.
At the end of the depletion protocol, force in response to
10
5 M ACh was <10% of
Fmax.
Extraction of muscle proteins.
Frozen muscle strips were pulverized under liquid nitrogen, and the
powder was transferred to dry ice-cooled centrifuge tubes. While the
tubes were on dry ice, 180 µl of extraction buffer were added to the
tubes; then the tubes were quickly vortexed. The extraction buffer
contained 20 mM Tris (pH 7.4), 2% Triton X-100, 0.2% SDS, 2 mM EDTA,
phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and
2 mM sodium pyrophosphate), and protease inhibitors (2 mM benzamidine,
0.5 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Each sample
of extract was boiled for 5 min to inactivate phosphatases and
proteases, and then it was kept at 4°C for 1 h. The supernatant was
collected after centrifugation at 14,000 rpm for 20 min at 4°C. For
the extraction of FAK, the concentration of SDS in the extraction
buffer was increased to 2%. After extraction, the SDS content was
readjusted to 0.2% before the determination of protein concentration.
The concentration of protein in each sample of supernatant was
determined using a standard bicinchoninic acid protein assay kit (Pierce).
Immunoprecipitation of paxillin and FAK.
Muscle extracts containing equal amounts of protein were precleared for
30 min with 50 µl of 10% protein A-Sepharose. The precleared
extracts were collected after centrifugation at 14,000 rpm for 2 min,
and monoclonal antibodies against paxillin (clone 349, Transduction
Labs) or FAK (clone 77, Transduction Labs) were added to them. The
extracts were incubated with antibodies overnight and then incubated
with 125 µl of a 10% suspension of protein A-Sepharose beads
conjugated to rabbit anti-mouse IgG for 2 h. Immunocomplexes were
washed four times in Tris-buffered saline containing 0.1% Triton
X-100. All procedures of immunoprecipitation were performed at 4°C.
Analysis of protein phosphorylation.
Whole muscle extracts and immunoprecipitates of paxillin or FAK were
boiled in sample buffer (1.54% dithiothreitol, 2% SDS, 80 mM Tris, pH
6.8, 10% glycerol, and 0.01% bromphenol blue) for 5 min and separated
by 7.5% (for FAK) or 10% (for paxillin) SDS-PAGE. Proteins were
transferred to nitrocellulose, blocked with 2% gelatin, and probed
with antibody to phosphotyrosine (PY20, ICN Pharmaceuticals), followed
by horseradish peroxidase conjugated to anti-mouse IgG (Amersham Life
Science) for visualization by enhanced chemiluminescence (ECL).
Nitrocellulose membranes were then stripped of bound antibodies and
reprobed with monoclonal antibodies against paxillin or FAK to confirm
the location of each protein and normalize for minor differences in
protein loading. Scanning densitometry of phosphotyrosine blots and
paxillin or FAK blots was used to quantitate proteins after the
visualization by ECL. The tyrosine phosphorylation of paxillin was
analyzed from immunoblots of whole muscle extracts. In each protocol,
the results were confirmed for selected points from immunoblots of
paxillin immunoprecipitates. No differences were observed between
results obtained by analysis of immunoblots of whole muscle extracts
and by analysis of immunoblots of paxillin immunoprecipitates (Fig.
1). Phosphorylation of FAK was quantitated from immunoblots of immunoprecipitated FAK. Changes in the tyrosine phosphorylation of paxillin or FAK were expressed as
multiples of the phosphorylation of resting tissues at
Lo. Each
measurement of paxillin phosphorylation represents the average from
duplicate muscle strips in a single experiment.

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Fig. 1.
Immunoblots illustrating effect of muscle length on paxillin (Pax)
tyrosine phosphorylation in tracheal smooth muscle strips. Results
obtained from immunoblots of whole muscle extracts
(A) and paxillin immunoprecipitates
(B) are compared. Canine tracheal
smooth muscle strips were frozen at optimal muscle length
(Lo) or 0.5 Lo after 5 min of
stimulation with 10 4 ACh or
without stimulation (Resting) to determine paxillin tyrosine
phosphorylation. Immunoblots from muscle extracts
(A) or paxillin immunoprecipitates
(B) were probed with
antiphosphotyrosine antibody (Ab), stripped, and reprobed with
anti-paxillin Ab. Numbers at left are
molecular mass (in kDa) of standard molecular mass markers. Paxillin
tyrosine phosphorylation is higher in strips stimulated with ACh at
Lo than in strips
stimulated at 0.5 Lo. Muscle length
had no effect on paxillin tyrosine phosphorylation in unstimulated
muscle strips. No significant differences were detected in measurements
of paxillin phosphorylation determined from immunoblots of paxillin
immunoprecipitates and muscle extracts. Mean increases in paxillin
phosphorylation induced by ACh (as multiples of control) were 3.18 ± 0.08 at Lo
and 2.17 ± 0.09 at 0.5 Lo when measured
from immunoblots of whole muscle extracts
(n = 5). Mean increases in paxillin
phosphorylation measured from immunoblots of paxillin
immunoprecipitates (n = 3; as
multiples of control) were 3.21 ± 0.06 at
Lo and 2.00 ± 0.17 at 0.5 Lo.
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Statistical analysis.
All statistical analysis was performed using SigmaSTAT software.
Comparison among multiple groups was performed by one-way ANOVA or
Kruskal-Wallis one-way ANOVA. Differences between pairs of groups were
analyzed by Student-Newman-Keuls test or Dunn's method. Values of
n refer to the number of experiments
used to obtain each value. P < 0.05 was considered to be significant.
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RESULTS |
Mechanosensitivity of the tyrosine phosphorylation of paxillin in
ACh-stimulated muscle strips.
Canine tracheal smooth muscle strips were isometrically contracted with
10
4 M ACh at muscle lengths
of Lo or 0.5 Lo and then
frozen for the analysis of paxillin tyrosine phosphorylation 1, 5, or
10 min after contractile stimulation. Uncontracted strips at
Lo were also
frozen in the presence of
10
7 M atropine to determine
the effect of muscle length on the resting levels of paxillin tyrosine phosphorylation.
The tyrosine phosphorylation of paxillin was higher at
Lo than at 0.5 Lo during
isometric contraction with ACh, but muscle length had no effect on
paxillin tyrosine phosphorylation in resting muscles (Fig. 1). The
length sensitivity of paxillin tyrosine phosphorylation was evident as
early as 1 min after stimulation with ACh and persisted for the 10-min
duration of the contraction (Fig. 2).
Differences in paxillin tyrosine phosphorylation between Lo and 0.5 Lo were
statistically significant at all time points during ACh stimulation
(n = 4).

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Fig. 2.
Time course of paxillin tyrosine phosphorylation in muscle strips
stimulated with ACh at
Lo or 0.5 Lo. Active force
(A) and paxillin tyrosine
phosphorylation (B) were
significantly lower when strips were stimulated with ACh at a muscle
length of 0.5 Lo
than at a length of
Lo. All values
are means ± SE (n = 4). Active
force is quantitated as percent of maximal response to
10 4 M ACh at
Lo. Paxillin
phosphorylation is quantitated as multiples of phosphorylation level
obtained in unstimulated tissues at
Lo (open circle
with dot). Paxillin tyrosine phosphorylation remained higher in muscle
strips contracted at
Lo for entire
10-min duration of contraction. * Statistically significant
differences in force or paxillin phosphorylation between
Lo and 0.5 Lo
(P < 0.05).
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Mechanosensitivity of tyrosine phosphorylation of paxillin in
unstimulated muscle strips.
The length sensitivity of paxillin tyrosine phosphorylation was
compared in unstimulated muscle strips and in muscle strips activated
with 10
4 M ACh for 5 min
(Fig. 3). The tyrosine phosphorylation of
paxillin was significantly lower in muscles contracted at lengths of
0.75 Lo or 0.5 Lo than in
muscles contracted at
Lo
(n = 5). However, paxillin tyrosine
phosphorylation was not significantly different in the unstimulated
strips at muscle lengths of
Lo, 0.75 Lo, and 0.5 Lo
(n = 5).

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Fig. 3.
Mean total force (A) and paxillin
tyrosine phosphorylation (B) in
unstimulated muscle strips and in muscle strips contracted
isometrically with 10 4 M
ACh for 5 min at lengths of 0.5 Lo, 0.75 Lo, and
Lo
(n = 5). Tyrosine phosphorylation of
paxillin increased at longer muscle lengths between 0.5 Lo and
Lo in
ACh-stimulated muscle strips but not in uncontracted muscle strips.
Force is quantitated as percent of total force (active and passive) at
Lo 5 min after
contraction with 10 4 M ACh.
Paxillin phosphorylation is quantitated as multiples of level in
unstimulated tissues at
Lo (open circle
with dot). Values are means ± SE. * Values at each length are
significantly different from each other
(P < 0.05).
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To determine whether paxillin tyrosine phosphorylation can be
stimulated by tension alone, uncontracted muscle strips were stretched
to 1.3 Lo, at
which length the passive force was 81 ± 5% of the active force
elicited in response to 10
4
M ACh at Lo.
Stretching the unstimulated strips to 1.3 Lo caused a
slight increase in paxillin phosphorylation over that in unstimulated strips at Lo;
however, paxillin phosphorylation in the passively stretched strips
remained significantly lower than that in tissues contracted actively
to comparable levels of tension (Fig. 4).

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Fig. 4.
Effect of tension on paxillin tyrosine phosphorylation. Paxillin
tyrosine phosphorylation was compared in unstimulated smooth muscle
strips that were stretched to 1.3 Lo in presence of
10 7 M atropine and in
muscle strips stimulated with
10 4 M ACh at
Lo. Although
tension in passively stretched strips was similar to that in strips
activated at Lo
with ACh, paxillin tyrosine phosphorylation in unstimulated strips at
1.3 Lo was
significantly lower than that in ACh-stimulated strips at
Lo
(n = 3, P < 0.05). * Significantly
different in unstimulated stretched strips compared with actively
contracted strips (P < 0.05).
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Effects of muscle length on paxillin tyrosine phosphorylation during
isometric contraction with KCl.
To evaluate whether the mechanosensitive modulation of paxillin
phosphorylation requires receptor activation, canine tracheal smooth
muscle strips were contracted isometrically for 5 or 15 min with 60 mM
KCl at muscle lengths of
Lo or 0.5 Lo for the
determination of paxillin phosphorylation (Figs.
5 and 6).
Uncontracted strips were also quickly frozen at
Lo to determine
the resting level of paxillin tyrosine phosphorylation. Additional
strips were contracted at
Lo for 5 min and
quickly shortened to 0.5 Lo and allowed to recontract isometrically for 1 or 10 min and then frozen for the measurement of paxillin tyrosine phosphorylation.

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Fig. 5.
Immunoblot illustrating effect of muscle length on paxillin tyrosine
phosphorylation in tracheal smooth muscle strips stimulated with KCl.
Canine tracheal smooth muscle strips were frozen at
Lo or 0.5 Lo after 5 min of
stimulation with 60 mM KCl or without stimulation (Resting) to
determine paxillin tyrosine phosphorylation. An additional strip was
contracted at Lo
for 5 min and then quickly shortened to 0.5 Lo and allowed to
recontract isometrically for 1 min. Immunoblots of paxillin
immunoprecipitates were probed with antiphosphotyrosine Ab, stripped,
and reprobed with anti-paxillin Ab. Numbers at
left are molecular mass (in kDa) of
standard molecular mass markers. Paxillin tyrosine phosphorylation is
higher in strips stimulated with KCl at
Lo than in strips
at 0.5 Lo,
whether strips are shortened to 0.5 Lo before or
after stimulation with KCl.
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Fig. 6.
Effect of muscle length and length step on paxillin tyrosine
phosphorylation in strips contracted isometrically with 60 mM KCl at
lengths of Lo or
0.5 Lo
(n = 4). Additional strips were
contracted at Lo
for 5 min and then quickly shortened to 0.5 Lo and allowed to
recontract isometrically for an additional 1 or 10 min
(n = 4). Active force
(A) and paxillin tyrosine
phosphorylation (B) were
significantly higher in muscle strips contracted at
Lo than in strips
contracted at 0.5 Lo
(P < 0.05). When muscles were
shortened from Lo
to 0.5 Lo,
paxillin tyrosine phosphorylation in contracted muscle strips decreased
to level obtained at 0.5 Lo within 1 min
of length change. Force is quantitated as percent of maximal response
to 60 mM KCl at
Lo. Paxillin
phosphorylation is quantitated as multiples of level obtained from
unstimulated strips at
Lo (open circle
with dot). * Values at 0.5 Lo that are
significantly different from corresponding values at
Lo
(P < 0.05).
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During stimulation with KCl, paxillin tyrosine phosphorylation was
significantly higher in muscle strips contracted at
Lo than in strips
contracted at 0.5 Lo, indicating
that the length sensitivity of paxillin phosphorylation does not
require receptor activation (n = 4).
When the activated muscle was quickly shortened from
Lo to 0.5 Lo, paxillin
tyrosine phosphorylation decreased to the level obtained at 0.5 Lo within 1 min
of the length change, demonstrating that paxillin phosphorylation can
adjust rapidly in response to changes in muscle length
(n = 4).
Length sensitivity of paxillin phosphorylation in
Ca2+-depleted
muscle strips stimulated with ACh.
The mechanosensitivity of paxillin phosphorylation was studied in
Ca2+-depleted muscle strips
stimulated with ACh to evaluate its dependence on active tension and
intracellular Ca2+.
Ca2+-depleted muscle strips were
stimulated with 10
4 M ACh
for 5 or 10 min at muscle lengths of
Lo or 0.5 Lo. Additional Ca2+-depleted muscle strips were
activated with 10
4 M ACh at
0.5 Lo and then
stretched to Lo,
at which length paxillin phosphorylation was determined 1 or 5 min
after the stretch.
After Ca2+ depletion, stimulation
with 10
4 M ACh increased
active force by <10% of maximal force under all conditions; however, the length sensitivity of paxillin tyrosine phosphorylation was unaffected (Figs. 7 and
8). The tyrosine phosphorylation of
paxillin was significantly lower in the strips stimulated with ACh at
0.5 Lo than in
the strips stimulated at
Lo
(n = 5). When strips were stimulated
with ACh at 0.5 Lo and then
stretched to Lo,
paxillin tyrosine phosphorylation increased to the level obtained at
Lo within 1 min
of the length increase (n = 5; Fig.
8).

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Fig. 7.
Effect of Ca2+ depletion on
paxillin tyrosine phosphorylation in paxillin immunoprecipitates from
extracts of muscle strips stimulated with
10 4 M ACh for 5 min at
muscle lengths of
Lo or 0.5 Lo. Immunoblots
of paxillin immunoprecipitates were probed with anti-phosphotyrosine Ab
and then stripped and reprobed with anti-paxillin Ab. Paxillin tyrosine
phosphorylation was significantly lower during stimulation of
Ca2+-depleted strips with ACh at
0.5 Lo than at
Lo. In
unstimulated muscle strips, muscle length had no significant effect on
paxillin tyrosine phosphorylation.
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Fig. 8.
Effect of Ca2+ depletion on
sensitivity of paxillin tyrosine phosphorylation to muscle length.
Ca2+-depleted muscle strips were
stimulated for 5 or 10 min with
10 4 M ACh at muscle lengths
of Lo or 0.5 Lo. Additional
strips were contracted at 0.5 Lo for 5 min and
then quickly lengthened to
Lo and allowed to
contract isometrically for an additional 1 or 5 min. Paxillin tyrosine
phosphorylation was lower in strips stimulated with ACh for 5 or 10 min
at 0.5 Lo than in
strips stimulated at
Lo
(n = 5). In strips stimulated at 0.5 Lo and stretched
to Lo, paxillin
tyrosine phosphorylation increased to level obtained at
Lo within 1 min
of length increase. ACh caused little or no active force development in
Ca2+-depleted muscle strips
(n = 5). Active force
(A) is quantitated as percent of
maximal response to 10 4 M
ACh at Lo.
Paxillin phosphorylation is quantitated as multiples of level obtained
in resting undepleted tissues at
Lo (open circle
with dot). * Values at
Lo that are
significantly different from corresponding values at 0.5 Lo
(P < 0.05).
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Figure 9 compares the effect of muscle
length on paxillin tyrosine phosphorylation and active tension in
Ca2+-depleted tissues and in
undepleted tissues after 5 min of stimulation. The tyrosine
phosphorylation of paxillin in strips stimulated at
Lo was not
significantly different in undepleted strips and in
Ca2+-depleted strips. In both
groups of strips, paxillin tyrosine phosphorylation was reduced at 0.5 Lo compared with
Lo, but the decrease was more pronounced in
Ca2+-depleted than in undepleted
muscle strips. Although there was a large difference in active tension
at Lo and at 0.5 Lo in undepleted muscles, there was little effect of muscle length on tension in the
Ca2+-depleted muscle strips. Thus
the differences in paxillin phosphorylation at
Lo and 0.5 Lo were not
correlated with differences in active tension.

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Fig. 9.
Effect of muscle length on force and paxillin phosphorylation in
Ca2+-depleted and undepleted
muscle strips stimulated with ACh for 5 min. Although there was minimal
active tension in response to ACh in
Ca2+-depleted strips stimulated at
Lo (solid bar) or
0.5 Lo (hatched
bar), tyrosine phosphorylation of paxillin was similar at
Lo under both
conditions. In both undepleted and
Ca2+-depleted muscle strips,
tyrosine phosphorylation of paxillin was lower in strips stimulated
with ACh at 0.5 Lo than in strips
stimulated at Lo.
* Values of paxillin phosphorylation or force that are
significantly lower at 0.5 Lo than at
Lo.
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Length sensitivity of the tyrosine phosphorylation of FAK during
stimulation with ACh.
The length sensitivity of the tyrosine phosphorylation of FAK was
measured in smooth muscle strips stimulated with
10
4 M ACh for 5 min. The
tyrosine phosphorylation of FAK was higher in muscles stimulated at
Lo than at 0.5 Lo (Figs.
10 and
11). The tyrosine phosphorylation of
FAK did not change significantly over a 5-min period of contraction
with ACh. FAK phosphorylation at Lo increased by
5.9 ± 0.3-fold over resting levels by 1 min of stimulation with ACh
and remained elevated by 5.1 ± 0.2-fold after 5 min of stimulation
(n = 3; data not shown).

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Fig. 10.
Tyrosine phosphorylation of focal adhesion kinase (FAK)
immunoprecipitates in extracts from undepleted
(A) and
Ca2+-depleted
(B) muscle strips stimulated with
10 4 M ACh for 5 min at
muscle lengths of
Lo or 0.5 Lo. Tyrosine
phosphorylation of FAK in both undepleted and
Ca2+-depleted muscle strips
stimulated with ACh was lower at 0.5 Lo than at
Lo.
Ca2+ depletion did not affect
tyrosine phosphorylation of FAK in unstimulated muscle strips.
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Fig. 11.
Mean tyrosine phosphorylation of FAK immunoprecipitates from undepleted
and Ca2+-depleted muscle strips
stimulated with 10 4 M ACh
for 5 min at Lo
(solid bar) or 0.5 Lo (hatched bar).
Tyrosine phosphorylation of FAK was lower at 0.5 Lo than at
Lo in both
Ca2+-depleted and undepleted
muscle strips. * Values of FAK phosphorylation that are
significantly lower at 0.5 Lo than at
Lo
(n = 3, P < 0.05).
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A similar protocol was performed to evaluate the effect of muscle
length on FAK tyrosine phosphorylation in
Ca2+-depleted muscle strips. The
tyrosine phosphorylation of FAK was also lower in
Ca2+-depleted muscle strips
stimulated with ACh at 0.5 Lo than in strips
stimulated at Lo
(Figs. 10 and 11).
The length sensitivity of FAK tyrosine phosphorylation was compared in
Ca2+-depleted muscle strips and in
undepleted tissues stimulated with ACh (Fig. 11). The tyrosine
phosphorylation of FAK was not significantly different in undepleted
strips and Ca2+-depleted tissues
at a muscle length of
Lo. However, at a
muscle length of 0.5 Lo, FAK tyrosine
phosphorylation was significantly higher in undepleted strips than in
Ca2+-depleted tissues
(n = 3).
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DISCUSSION |
Summary.
In this study, we demonstrate that tyrosine phosphorylation of the
integrin-associated proteins paxillin and FAK is sensitive to muscle
length during the contractile stimulation of tracheal smooth muscle. In
addition, this is the first demonstration that the contractile
stimulation of a smooth muscle tissue elicits the tyrosine
phosphorylation of FAK. The phosphorylation of both paxillin and FAK is
higher when tracheal muscles are stimulated isometrically at a long
muscle length than at a short length. Differences in the tyrosine
phosphorylation of paxillin can be distinguished in muscles contracted
at lengths of 0.5 Lo, 0.75 Lo, and
Lo (Fig. 3). The
length sensitivity of paxillin tyrosine phosphorylation is observed for
the entire duration of a 10-min period of contractile stimulation (Fig.
2). A length step imposed on the muscle after contractile activation
results in a rapid change in paxillin tyrosine phosphorylation,
indicating that the mechanosensitive response mediated by paxillin can
occur rapidly in response to an acute change in the mechanical
environment of the muscle (Figs. 5, 6, and 8). These results suggest
that integrin-mediated mechanotransduction may be an important
mechanism by which smooth muscle cells can modulate signaling pathways
in response to changes in their external mechanical environment.
Ca2+
dependence of mechanosensitive protein phosphorylation.
We have previously demonstrated that the phosphorylation of paxillin is
Ca2+ sensitive (19, 37). We and
others have also shown that intracellular Ca2+ and myosin light chain (MLC)
phosphorylation are modulated in response to changes in muscle length
in tracheal smooth muscle (20, 39). We therefore evaluated whether the
mechanosensitivity of paxillin phosphorylation can be mediated by
changes in intracellular Ca2+ by
assessing paxillin phosphorylation in muscles activated by K+ depolarization at different
muscle lengths. Our results show that paxillin phosphorylation is
mechanosensitive when muscles are activated by
K+ depolarization, indicating that
the length sensitivity of paxillin phosphorylation can be mediated by a
Ca2+-sensitive pathway (Figs. 5
and 6).
We evaluated whether intracellular
Ca2+ is the primary regulator of
the mechanosensitive modulation of FAK and paxillin phosphorylation by
analyzing the length sensitivity of their phosphorylation in Ca2+-depleted tracheal tissues. We
have previously shown that ACh elicits high levels of paxillin
phosphorylation in Ca2+-depleted
tracheal tissues without stimulating MLC phosphorylation or active
tension development (19). In the present study, we found that
Ca2+ depletion did not alter the
length sensitivity of paxillin or FAK phosphorylation, indicating that
the mechanical modulation of the phosphorylation of these proteins does
not depend on a Ca2+-sensitive
pathway (Figs. 8, 9, and 11). Thus the integrin-linked regulation of
FAK and paxillin phosphorylation may be components of a primary
mechanotransduction process for the mechanosensitive regulation of
signaling pathways in smooth muscle.
Tension vs. length as the stimulus for mechanosensitive signal
transduction.
Although paxillin and FAK phosphorylation is mechanosensitive in
actively contracted muscle strips, their phosphorylation is unaffected
by muscle length in unstimulated smooth muscle strips (Fig. 3). This
observation suggested that tension per se might be the stimulus for the
mechanosensitive regulation of FAK and paxillin phosphorylation in this
tissue. However, we observed that the length sensitivity of paxillin
and FAK phosphorylation is similar in
Ca2+-depleted and in undepleted
muscles, despite the absence of active tension in the
Ca2+-depleted tissues (Figs. 9 and
11). As the differences in tension at muscle lengths of
Lo and 0.5 Lo are much
smaller in Ca2+-depleted tissues
than in undepleted tissues, this indicates that mechanosensitive signal
transduction does not result from a tension-sensitive mechanism. The
contractile stimulation of
Ca2+-depleted tissues does not
increase MLC phosphorylation significantly (19); thus these results
also demonstrate that the activation of contractile proteins is not
required for mechanosensitive signal transduction. Our observation that
paxillin phosphorylation increased only slightly when high levels of
passive tension were generated by stretching uncontracted muscles
strips (Fig. 4) provides further support for our conclusion that muscle
length rather than tension is the primary stimulus for mechanosensitive
signal transduction in tracheal smooth muscle.
Molecular mechanism for mechanosensitive signal transduction.
The mechanically induced changes in the phosphorylation of paxillin and
FAK observed in this study may be mediated by transmembrane integrins.
In cultured cells, paxillin and FAK colocalize with integrin molecules
in focal adhesion complexes at the membrane termini of actin stress
fiber bundles (3, 33). Extracellular matrix proteins bind to the
extracellular domain of integrins, whereas the cytosolic domain of
integrin molecules binds to cytosolic proteins, including talin,
-actinin, and vinculin, that link the integrin molecules to actin
filaments (3, 4). These complexes serve as loci for the transmission of
tension between the actin cytoskeleton and the extracellular matrix
(27, 35, 36). Mechanical strain or tension applied directly to the
extracellular domain of integrins results in increased protein tyrosine
phosphorylation, cytoskeletal stiffening, and the activation of
downstream signaling pathways, suggesting that integrins can function
as mechanotransducers (27, 30, 35, 36).
In cultured fibroblasts, integrin activation caused by adhesion to
extracellular matrix proteins or by antibody-mediated integrin cross-linking leads to the increased tyrosine phosphorylation of both
FAK and paxillin (5, 17). The integrin-mediated induction of the
tyrosine phosphorylation of FAK requires the clustering of integrin
receptors and cannot occur without the cytoplasmic domain of the
-integrin subunit (2, 18). The tyrosine phosphorylation of FAK is
associated with the recruitment of Src and/or Fyn protein tyrosine kinases to the integrin-associated complex; the FAK-Src-Fyn kinase complex then catalyzes the tyrosine phosphorylation of paxillin
(21). The phosphorylation of FAK and paxillin is correlated with the
formation of actin stress fibers and focal adhesions under many
conditions of activation (3, 5, 6, 24, 28). FAK and paxillin also
participate in signaling processes leading to the activation of the
Ras-mitogen-activated protein kinase (MAP kinase) pathways (7, 26).
Paxillin itself appears to function as a molecular adaptor, directing
structural and regulatory proteins into a complex that can coordinate
multiple signaling pathways and nucleate cytoskeletal organization (3,
25, 34).
In cultured endothelial cells, the imposition of cyclical cycles of
mechanical strain induces the tyrosine phosphorylation of paxillin and
FAK and the reorientation of actin stress fibers (38). Similar events
have been demonstrated in cultured airway smooth muscle cells, in which
mechanical strain induces the reorientation of the actin stress fibers
in the direction of the strain (32). Thus, in cultured cells, the
evidence suggests that mechanical strain sensed by integrin molecules
induces the tyrosine phosphorylation paxillin and FAK, which act in
conjunction with other focal adhesion proteins to coordinate downstream
events leading to cytoskeletal reorganization and the realignment of
actin filaments in response to the strain.
Role of FAK and paxillin in the regulation of smooth muscle
contraction.
We have hypothesized that the stimulation of smooth muscle cells with
contractile agonists initiates active processes that regulate the
organization of the actin cytoskeleton and the attachment of actin
filaments to the membrane at membrane-associated dense plaque sites
(13, 14, 22). According to our hypothesis, these processes occur in
parallel to the activation of contractile proteins and enable force
development to be optimized to the mechanical environment of the smooth
muscle cell at the time of contractile activation.
Our present results provide support for a mechanotransduction process
in fully differentiated smooth muscle tissues that is analogous to that
in other cell types. We propose that mechanical strain sensed by
integrin receptors modulates the receptor-mediated activation of FAK
and paxillin and perhaps also other proteins in the smooth muscle dense
plaque. This complex of dense plaque proteins might then regulate the
activation of downstream molecules involved in actin filament
remodeling and thereby modulate contractility by adjusting the
orientation of actin filaments in response to changes in external strain.
Strain-sensitive signaling cascades mediated by integrins might also
play a role in modulating contractile protein activation. In smooth
muscle tissues, intracellular Ca2+
and MLC phosphorylation are sensitive to muscle length (20, 23, 39).
The activation of MAP kinase has also been shown to be length sensitive
in vascular smooth muscle tissue (9).
In conclusion, our results demonstrate the presence of a
mechanosensitive Ca2+-independent
signaling pathway in airway smooth muscle that is mediated by the dense
plaque-associated proteins FAK and paxillin. This pathway is sensitive
to changes in muscle length in the presence of a contractile stimulus.
The mechanotransduction mechanism does not depend on the generation of
tension or on the activation of contractile proteins. Paxillin and FAK
may participate in an integrin-mediated mechanotransduction process
that initiates alterations in cell structure and contractility via the
remodeling of actin filaments. It is also possible that FAK and
paxillin participate in the mechanosensitive regulation of signaling
molecules involved in contractile protein activation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grant HL-29289.
 |
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: S. J. Gunst, Dept. of Physiology and
Biophysics, Indiana University School of Medicine, 635 Barnhill Dr.,
Indianapolis, IN 46202-5126.
Received 1 July 1998; accepted in final form 29 September 1998.
 |
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