From the Department of Internal Medicine, University of Rostock,
18055 Rostock, Germany
Physical forces play a fundamental role in the
regulation of cell function in many tissues, but little is known about
how cells are able to sense mechanical loads and realize signal
transduction. Adhesion receptors like integrins are candidates for
mechanotransducers. We used a magnetic drag force device to apply
forces on integrin receptors in an osteoblastic cell line and studied
the effect on tyrosine phosphorylation as a biochemical event in signal
transduction. Mechanical stressing of both the
1 and the
2
integrin subunit induced an enhanced tyrosine phosphorylation of
proteins compared with integrin clustering. Application of cyclic
forces with a frequency of 1 Hz was more effective than a continuous
stress. Using Triton X-100 for cell extraction, we found that
tyrosine-phosphorylated proteins became physically anchored to the
cytoskeleton due to mechanical integrin loading. This cytoskeletal
linkage was dependent on intracellular calcium. To see if mechanical
integrin stressing induced further downstream signaling, we analyzed
the activation of mitogen-activated protein (MAP) kinases and found an
increased phosphorylation of MAP kinases due to mechanical stress. We
conclude that integrins sense physical forces that control gene
expression by activation of the MAP kinase pathway. The cytoskeleton
may play a key role in the physical anchorage of activated signaling molecules, which enables the switch of physical forces to biochemical signaling events.
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INTRODUCTION |
Application of physical forces to cells induces gene expression
and proliferation in a variety of cell types (1-5, 9). Therefore,
mechanical forces are a fundamental physiological factor in regulating
structure and function in many tissues. In bone, mechanical loading
stimulates the increase of bone mass (6-8) and plays an important role
in the therapy of osteoporosis. The cellular mechanisms of mechanically
induced signal transduction are largely unknown. Above all, it has
remained elusive how cells are able to sense physical forces.
Indications exist that integrin receptors may serve as
mechanotransducers (10-14). Integrins are heterodimeric transmembrane
molecules by which cells adhere to the extracellular matrix (15). The
subunit is combined with one of the different
subunits, and
both extracellular domains are involved in ligand binding. Engagement
and clustering of these receptors induce signal transduction, which
involves integrin linkage to the cytoskeleton and the generation of
second messengers and biochemical events (13, 16, 17). Activation of
protein tyrosine kinases appears to play a central role in
integrin-mediated signaling (18-20).
Because mechanical strain may act in different frequencies and
strength, which appears to have relevance in regulating cell physiology
(21-23), an important question is whether perception of physical
forces by integrin receptors induces a differential intracellular
signal transduction. Recently, we developed a method to mechanically
stress cell surface receptors (14). Using magnetic beads, drag forces
in defined strength and frequency can be applied to receptors of cells
in a monolayer. The method enables the application of physical forces
to defined integrins, which allows the evaluation of the relevance of
specific integrin subunits in mechanical signal transduction. Herein,
we report that in the osteosarcoma cell line U-2 OS, mechanical
stressing of integrins induces an increased tyrosine phosphorylation of
proteins, including the MAP1
kinase, compared with integrin clustering and depending on whether a
permanent or intermittent stress is applied. We also observed an
increased physical anchorage of tyrosine-phosphorylated proteins at the
cytoskeleton, which suggests that the cytoskeleton may serve as a
structure where mechanical signals can switch into a chemical-signaling
pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
The osteosarcoma U-2 OS cell line was obtained
from American Type Culture Collection (Rockville, MD). 96-well Fluoro
Nunc modules from Nunc A/S (Roskilde/Denmark) were used for plating the
cells. Paramagnetic microbeads (size 2.8 µm, coated with
streptavidin) were purchased from Dynal (Hamburg, Germany). For coating
of the microbeads, biotinylated anti-
1 (clone 2A4) and anti-
2
(clone AK7) integrin antibodies were from Southern Biotechnology
Associates, Inc. (Birmingham, AL); biotinylated anti-CD71 (transferrin
receptor) antibody was from Oncogene Science, Inc. (Uniondale, NY). For immunoprecipitation of MAP kinases, anti-ERK-1 (p44) antibody (clone
C-16), which also reacts with ERK-2 (p42), was used from Santa Cruz
Biotechnology. Protein A-agarose was also purchased from Santa Cruz
Biotechnology. Recombinant anti-phosphotyrosine antibody (clone RC-20)
conjugated with alkaline phosphatase was from Transduction
Laboratories. CDP-star for chemiluminescence was obtained from
Boehringer Mannheim.
Cell Culture--
U-2 OS cells were cultured in Dulbecco's
modified Eagle's medium and supplemented with 10% fetal calf serum at
37 °C and in 5% CO2 atmosphere. For the experiments,
100 µl of cells in complete medium containing 105 cells
were seeded into wells of a 96-well culture module and grown to near
confluence. 2 h before mechanical strain was applied, the cells
were depleted of serum.
Mechanical Receptor Stressing--
The procedure to strain
integrin receptors was described in detail elsewhere (14). In brief,
the cell monolayer was incubated with paramagnetic microbeads coated
with anti-
1 or anti-
2 antibodies. This is termed here as
clustering. In average, five beads bound at the surface of one cell. To
apply mechanical drag forces, each of the wells was then placed between
the poles of the magnetic device, which induces an inhomogeneous
magnetic field (14). The forces subjected to one bead were adjusted to
10 dyne/cm2. Analyses revealed that this strength was
exerted to most of the beads, whereas a smaller part of the beads in
the vicinity of the flat pole was subjected to lower forces. The forces
were applied either with a frequency of 1 Hz if not otherwise mentioned or permanently during the indicated time. In general, the following experimental designs were compared: 1) control cells without any treatment (
); 2) clustered samples (c), i.e. the cell
monolayer was incubated with anti-integrin antibody-coated beads for 50 min; 3) mechanically stressed samples, i.e. the cell
monolayer was incubated (in a control experiment with anti-CD71) with
anti-integrin antibody-coated beads for 20 min followed by application
of forces for 30 min.
Cell Lysis and Immunoblotting--
After integrin stimulation as
described above, adherent cells in each well were washed in
phosphate-buffered saline and lysed in 10 µl of SDS sample buffer.
Lysates of several wells were pooled and then boiled. The samples were
subjected to a 7.5% SDS-polyacrylamide gel electrophoresis. For
immunoblotting, the proteins were transferred to polyvinylidene
difluoride membranes. To block nonspecific binding, membranes were
incubated with 5% milk powder in buffer containing 0.01% Tween 20, 100 mM Tris/HCl, pH 9.0, 155 mM NaCl for 30 min at 37 °C. Immunoblotting was performed with alkaline
phosphatase-labeled anti-phosphotyrosine antibody at a dilution of
1:20,000 and then visualized with chemiluminescence (CDP
star).
Immunoprecipitation--
To analyze activation of MAP kinases,
cells were lysed in precipitation buffer containing protease inhibitor,
SDS, and Triton X-100 (0.1%). After centrifugation, the supernatant
was incubated with 100 µl of anti-ERK antibody for 1 h at room
temperature followed by adding 50 µl of protein A-agarose. After
centrifugation, the pellet was washed in precipitation buffer and
analyzed for phosphotyrosine as described above.
Preparation of Cytoskeletal Fractions--
The cell monolayer
was incubated with cell extraction buffer containing 1% Triton X-100,
20 mM imidazole, 2 mM MgCl2, 80 mM KCl, 2 mM EGTA for 5 min at 4 °C. The
Triton nonsoluble fractions were then collected in SDS sample buffer
and subjected to gel electrophoresis as described above.
Treatment with Cytochalasin and Calcium Chelator--
To disrupt
the actin filaments of the cytoskeleton, the cell monolayer was treated
with 25 nM cytochalasin D for 20 min at 37 °C. The cells
were then washed and incubated with the microbeads to perform the
procedure for mechanical loading.
For chelating intracellular calcium, the cells were preincubated with 5 µM of
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, acetoxymethyl ester (BAPTA-AM) for 15 min. Mechanical strain was
then applied in the presence of 5 µM of BAPTA.
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RESULTS |
The osteosarcoma cell line expressed the
1 as well as the
2
integrin subunits on the cell surface (14). Therefore, we examined the
effect of mechanical stress applied to both integrin subunits on
tyrosine phosphorylation of proteins as a mechanism in
integrin-mediated signal transduction. First, we were interested in the
time course of tyrosine phosphorylation due to clustering of the
1
integrin subunit by incubation of the cells with anti-
1-coated microbeads. We observed an increase of phosphorylation during the time
of incubation, which reached the maximum after 60 min (Fig.
1). Based on this finding, mechanical
stress was applied to integrins for 30 min after an incubation time of
20 min to bind the beads to the receptors. Application of forces to the
1 as well as to the
2 subunit induced an increased tyrosine phosphorylation of proteins compared with integrin clustering alone
(Fig. 2). Stressing the
1 chain, the
effect was more pronounced than with
2. To prove whether the
mechanically induced cellular reactions are specific for integrins, we
stressed the transferrin receptor (CD71) for comparison. Although a
slightly increased tyrosine phosphorylation was observed compared with
untreated cells, the effect was distinctly lower than after stressing
an integrin receptor (Fig. 3). Next we
compared the effect of permanent mechanical loading with an
intermittent stress of 1 Hz on tyrosine phosphorylation. Application of
a stress with a frequency of 1 Hz induced a more profound
phosphorylation than permanent drag forces (Fig. 2). To exclude the
possibility that the influence of different modes of magnetic field
application alone and not the mechanical receptor stress provoked the
differences in tyrosine phosphorylation, we compared controls in which
pure cells were subjected to a permanent and a cyclic magnetic field.
This experiment clearly demonstrated that the magnetic field alone did
not influence tyrosine phosphorylation, independent of the mode of the
magnetic field (Fig. 3).

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Fig. 1.
Time dependence of tyrosine phosphorylation
after clustering of the 1-integrin subunit. Cells were
incubated with anti- 1-coated microbeads for the indicated periods in
minutes. A total cell lysate was then electrophoresed and blotted
against an anti-phosphotyrosine antibody. Tyrosine phosphorylation
increased up to 60 min and then decreased.
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Fig. 2.
Tyrosine phosphorylation induced by
mechanical stressing of integrins. Cells in a monolayer were
incubated with microbeads to bind at the 1 or 2 integrin subunit
followed by application of drag forces (s). These samples
are compared with clustering of the integrins by incubation with the
microbeads without subsequent mechanical loading (c), and
untreated control cells ( ). Total cell lysates were electrophoresed
and blotted for anti-phosphotyrosine. Most pronounced differences are
observed in the 40-kDa region. In all cases, physical forces induced a
significantly enhanced tyrosine phosphorylation compared with
clustering and controls. Mechanical stressing of 1 induced a higher
response than loading the 2 subunit (compare 1/1 Hz/s
versus 2/1 Hz/s; or 1/P/s versus 2/P/s).
A cyclic stress with a frequency of 1 Hz was more effective than a
permanent stress in both integrin subunits (compare 1/1 Hz/s
versus 1/P/s; and 2/1 Hz/s versus
2/P/s). The results are representative of four independent
experiments.
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Fig. 3.
Comparison of tyrosine phosphorylation due to
mechanical stress to integrins, mechanical load to the transferrin
receptor, and application of the magnetic field alone to the
cells. Cells in a monolayer were mechanically stressed at the 1
integrin with 1 Hz as described above (lane 2) or stressed
in the same manner at the transferrin receptor (CD71) (lane
3). Cells in a monolayer without magnetic beads were subjected to
a permanent magnetic field (lane 4) or a cyclic magnetic
field with 1 Hz (lane 5) for 30 min. Untreated cells were
also examined (lane 1). Total cell lysates were
electrophoresed and blotted for anti-phosphotyrosine. Compared with
application of stress to the 1 integrin, mechanical stress to the
transferrin receptor induced a detectable but significantly lower level
of tyrosine phosphorylation. Different modes of the magnetic field
applied to untreated cells had no effect on tyrosine phosphorylation.
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To evaluate the role of the cytoskeleton in mechanically induced
tyrosine phosphorylation, we examined whether tyrosine-phosphorylated proteins are anchored to the cytoskeleton. After clustering and mechanically loading of the integrins, cells were extracted with Triton
X-100, and the detergent insoluble fraction analyzed for tyrosine-phosphorylated proteins (Fig.
4). Both clustering and additional stress
induced a linkage of tyrosine-phosphorylated proteins. Again,
mechanical load was more effective than clustering. This observation
concerned the
1 subunit, whereas the linkage of
2 to the
cytoskeleton was similar comparing the effect of clustering and
additional mechanical load. Furthermore, cytoskeletally anchored
phosphorylated proteins were preferably detected in the higher
molecular weight range. The anchorage of phosphorylated proteins to the
cytoskeleton was further studied by disruption of the actin filaments
by cytochalasin D (Fig. 5). Treatment
with cytochalasin D abolished the cytoskeletal linkage of
tyrosine-phosphorylated proteins indicating the requirement of actin
polymerization.

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Fig. 4.
Cytoskeletal anchorage of
tyrosine-phosphorylated proteins due to mechanical stress. After
treatment of the cells by integrin stressing (1 Hz) (s),
clustering (c), or without treatment ( ), the cells were
extracted with Triton X-100 to obtain the detergent-insoluble fraction.
This cytoskeletal fraction was then processed for anti-phosphotyrosine
immunoblotting. For 2, similar quantities of phosphorylated proteins
were found after mechanical stress and clustering. For 1, mechanical
stress induced a significant enhancement of cytoskeletally linked
tyrosine-phosphorylated proteins compared with clustered integrins. The
anchorage of tyrosine-phosphorylated proteins was observed in the
region of 130 kDa but not in the lower molecular weight range.
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Fig. 5.
Inhibition of the cytoskeletal anchorage of
tyrosine-phosphorylated proteins by cytochalasin D. Cells were
treated by mechanical stressing of the 1 integrin subunit (1 Hz)
(s), clustering of 1 (c), or without treatment
in the presence of cytochalasin D (+) to disrupt the actin filaments of
the cytoskeleton. For comparison, the cells were treated in the same
way in the absence of cytochalasin D ( ). The cells were then
extracted with Triton X-100 to obtain the insoluble cytoskeletal
fraction. This fraction was then processed for anti-phosphotyrosine
immunoblotting. Cytochalasin D dramatically blocked the cytoskeletal
linkage of tyrosine-phosphorylated proteins. In the absence of
cytochalasin D, mechanical stressing of integrins induced a distinctly
enhanced anchorage of phosphorylated proteins. This represents the
typical results of four independent experiments. (To obtain a
background in the cytochalasin-treated samples, the blots in these
experiments were exposed longer to the film.)
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Because intracellular calcium is induced by integrin stimulation, we
determined whether intracellular calcium plays a role in the
association of tyrosine-phosphorylated proteins to the cytoskeleton.
During mechanical loading of integrins, cells were treated with the
intracellular calcium chelator BAPTA-AM. The following analysis of
cytoskeletally anchored proteins, which are tyrosine-phosphorylated,
revealed that BAPTA significantly reduced the physical anchorage of
these proteins to the cytoskeleton (Fig.
6). This indicates that calcium regulates
the linkage of activated proteins to the cytoskeleton during
integrin-mediated mechanically induced signal transduction.

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Fig. 6.
Influence of the calcium chelator BAPTA-AM on
the anchorage of tyrosine-phosphorylated proteins. Cells were
treated by mechanical stressing of the 1 integrin subunit (1 Hz)
(s), clustering of 1 (c), or without treatment
( ) in the presence of BAPTA-AM (+BAPTA) to chelate
intracellular calcium. The cells were then extracted with Triton X-100,
and the insoluble fraction was processed for anti-phosphotyrosine
immunoblotting. BAPTA-AM significantly inhibited the anchorage of
tyrosine-phosphorylated proteins to the cytoskeleton, which is most
obvious in the higher molecular weight range. For comparison, in the
absence of BAPTA-AM ( BAPTA), the most profound tyrosine
phosphorylation of cytoskeletally linked proteins was found due to
mechanical stressing of the 1 integrin subunit. The results are
representative of four independent experiments.
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Last, we were interested in whether the increased tyrosine
phosphorylation of proteins due to mechanical integrin stressing may
lead to downstream signaling, which could be relevant for gene
expression. Therefore, we examined whether among the
tyrosine-phosphorylated proteins the MAP kinases are also activated.
Immunoprecipitation of MAP kinases and analysis of tyrosine
phosphorylation demonstrated that mechanical loading of both the
1
and the
2 integrin subunits induced a distinctly higher degree of
activation of the MAP kinases compared with integrin clustering (Fig.
7). This suggests that regulation of gene
expression by physical forces is controlled by differential activation
of MAP kinases and mediated by integrins.

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Fig. 7.
Induction of the activation of MAP kinases by
mechanical stressing of integrin receptors. Cells were treated by
mechanical stressing (1 Hz) of the 1 or the 2 integrin subunits
(s), clustering of the subunits (c), or without
treatment ( ). After cell lysis, MAP kinases were immunoprecipitated
with anti-ERK antibody. After separation in the electrophoresis, the
samples were probed with anti-phosphotyrosine antibody. Mechanical
stressing of both the 1 and the 2 integrin subunit induced an
enhanced tyrosine phosphorylation of MAP kinases p42 (ERK-2) and p44
(ERK-1) compared with controls and clustering of the integrins. The
results are representative of three independent experiments.
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DISCUSSION |
Tyrosine phosphorylation of several cellular proteins appears to
play an essential role in integrin-mediated signal transduction because
its inhibition blocks gene expression (24). The mechanisms by which
extracellular interactions of integrins regulate tyrosine phosphorylation remains elusive. We demonstrate that application of
physical forces to integrin receptors enhanced the tyrosine phosphorylation of proteins compared with integrin clustering alone.
These results support the idea that integrin receptors function as
mechanosensors (11, 12, 25). It indicates that physical forces applied
to integrin receptors determine the degree of tyrosine phosphorylation
that may have consequences in gene expression. Furthermore, tyrosine
phosphorylation was also quantitatively influenced by the mode of
receptor stressing. Intermittent forces are more effective than a
permanent stress. Several reports have shown that application of an
intermittent mechanical load on bones is more effective to stimulate
growth than a permanent strain (26, 27). Therefore, increased tyrosine
phosphorylation due to cyclic integrin stressing possibly provides a
cellular-signaling mechanism for the differential effect of mechanical
loads on bones. One of the initial effects of a mechanical stress to
integrins is the induction of their cytoskeletal anchorage. This has
been demonstrated in migrating cells by integrin cross-linking or
mechanical twisting of the integrins (11, 28, 29). It is an intriguing aspect that the strength of this linkage is variable and depends on
external forces. Using an optical trap to restrain integrin bound beads
resulted in a proportional strengthening of the integrin cytoskeletal
linkage (30). In the line of these findings, we suggest that in our
experiments mechanical forces induced an increased strength of the
integrin cytoskeletal linkage. Other experiments have revealed that the
characteristics of the ligand and receptor-ligand interaction control
components of the integrin-mediated signal transduction resulting in
functional consequences (31-33). For example, integrin clustering
induced a restricted accumulation of signaling molecules into the
cytoskeletal complex compared with additional occupancy of the
functional receptor epitope, which led to a model of a hierarchy in
signal transduction (34). Furthermore, the physical characteristics of
the collagen matrix was affecting integrin activation and signal
transduction, which inhibited proliferation (32). This clearly suggests
that how integrins interact with a ligand is of importance for the
induced signal transduction. Although the
and
subunits of
integrins form a dimer, we have found a difference in mechanically
induced tyrosine phosphorylation between the two integrin subunits.
Mechanical load on the
subunit provoked a more profound tyrosine
phosphorylation than stressing the
subunit. We cannot exclude that
this finding was due to a higher expression of
1 compared with
2
that we have found in these cells (14), but it could reflect a
different mechanism of the intracellular interaction of the cytoplasmic domains of both integrin subunits. The
chain can directly interact with the cytoskeletal proteins
actinin and talin (35, 36). In
addition, the integrin-linked kinase can associate with the
1
cytoplasmic domain and mediate further downstream signaling (37).
Mechanical stress to the
subunit provoked a significant anchorage
of tyrosine-phosphorylated proteins to the cytoskeleton, which was
increased compared with integrin clustering. Tyrosine phosphorylation
of cytoskeletally anchored proteins could be a prerequisite to form the
cytoskeletal complex (38), and a higher degree of phosphorylation may
be a prerequisite for the higher strengthening between receptors and
cytoskeleton. Regarding the factors that determine the association of
activated signaling molecules to the cytoskeleton, we have found that
intracellular calcium is obviously an important regulator of the
immobilization of proteins to the cytoskeleton. This concerns not only
intracellular-signaling proteins but also the cytoskeletal anchorage of
integrins to the cytoskeleton (28). The role of calcium for a
mechanically induced signal transduction is also stressed by data that
have shown that intracellular calcium concentrations correlated with
increasing force levels applied to integrins (39). However, our
previous experiments suggest that the differential cytoskeletal
anchorage of tyrosine-phosphorylated proteins and integrin subunits due to stimulation of
1 compared with the
subunit is not controlled by differences in the magnitude of the calcium response. Incubation of
cells with anti-integrin antibodies prior to mechanical stimulation of
the cells (13), as well as preliminary results concerning the
comparison of the calcium responses due to mechanical stress applied
with magnetic beads to
1 and
2, revealed no quantitative differences in calcium signaling.
Concerning downstream signaling, we argue that the cytoskeleton could
represent a structure where physical forces are transformed into a
biochemical signal pathway. The differential anchorage of
tyrosine-phosphorylated proteins due to physically stimulated integrins
may regulate downstream intracellular-signaling events.
One of these events is the activation of MAP kinases as a key mechanism
to control the activation of transcription factors, which therefore
mediates gene expression. The involvement of this pathway in integrin
signaling has been established (40, 41). We found that activation of
the MAP kinases was significantly increased due to physical forces
compared with integrin clustering. Due to the key role of the MAP
kinases, our finding emphasizes that physical forces transduced by
integrins differentially regulate cell proliferation and the expression
of genes through the MAP kinase cascade. The fact that activation of
MAP kinase by integrins depends on an intact cytoskeleton (41, 42) and
the involvement of cytoskeletally associated signaling molecules like
focal adhesion kinase (43) highlights the significance of a controlled
cytoskeletal anchorage of tyrosine-phosphorylated proteins for
consequences in cell behavior. Because the integrin-mediated MAP kinase
pathway converges with growth factor-induced pathways (44), our result suggests a synergistic effect of mechanical forces and cytokines in the
regulation of cell function.
In conclusion, integrins mediate physical forces and may regulate
physiological consequences in the cell by a well tuned induction of the
degree of tyrosine phosphorylation of proteins. A significant aspect is
the cytoskeletal anchorage of activated signaling proteins, which
depends on the mobilization of intracellular calcium. The functional
relevance of these mechanisms is supported by the result of an enhanced
activation of MAP kinases due to mechanical integrin stimulation.