This document summarizes a study that investigated how mechanical forces applied to integrin receptors control intracellular signaling in osteoblasts. The researchers found that cyclic forces applied to the beta-1 integrin subunit at 1 Hz were more effective at stimulating calcium responses in osteoblasts than continuous forces. Cyclic forces also induced increased tyrosine phosphorylation of cytoskeleton-anchored proteins and greater activation of focal adhesion kinase and mitogen-activated protein kinase compared to continuous forces. These responses depended on an intact cytoskeleton and the presence of intracellular calcium. Analysis of spatial calcium signals revealed they originated near the stressed receptors, indicating cells can sense local stress via integrins.

1. The Mode of Mechanical Integrin Stressing Controls
Intracellular Signaling in Osteoblasts
HAGEN POMMERENKE,1,2
CHRISTIAN SCHMIDT,1,2
FRIEDA DU¨ RR,1
BARBARA NEBE,1
FRANK LU¨ THEN,1
PETRA MU¨ LLER,1
and JOACHIM RYCHLY1
ABSTRACT
Following the idea that integrin receptors function as mechanotransducers, we applied defined physical forces
to integrins in osteoblastic cells using a magnetic drag force device to show how cells sense different modes of
physical forces. Application of mechanical stress to the ␤1-integrin subunit revealed that cyclic forces of 1 Hz
were more effective to stimulate the cellular calcium response than continuous load. Cyclic forces also induced
an enhanced cytoskeletal anchorage of tyrosine-phosphorylated proteins and increased activation of the focal
adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. These events were dependent on an intact
cytoskeleton and the presence of intracellular calcium. Analyses of the intracellular spatial organization of the
calcium responses revealed that calcium signals originate in a restricted region in the vicinity of the stressed
receptors, which indicates that cells are able to sense locally applied stress on the cell surface via integrins. The
calcium signals can spread throughout the cell including the nucleus, which shows that calcium also is a
candidate to transmit mechanically induced information into different cellular compartments. (J Bone Miner
Res 2002;17:603–611)
Key words: mechanical forces, integrin, calcium, signal transduction, osteoblast
INTRODUCTION
PHYSICAL FORCES are a fundamental physiological factor
in bone and may be the principal functional determinant
of adult bone mass.(1,2)
Experiments have revealed that
physical forces act directly on the cellular level and bone
cells are able to respond physiologically to mechanical
stress.(3–5)
It was shown further that cyclic mechanical load-
ing appeared to be more effective to induce proliferation
and gene expression than static loading.(6)
In all these ex-
periments, different types of cell deformation were induced
by stretching or compressing the whole cell, which did not
reveal any information on how the cell is able to perceive
and transduce mechanical stimuli. Therefore, despite accu-
mulating data of cellular responses due to mechanical stress,
which support the physiological relevance of physical
forces, the molecular basis explaining how cells sense me-
chanical stimuli and realize signal transduction remains
obscure. In the tissue, the extracellular matrix may represent
the site where external forces are transmitted to the cell.
Therefore, integrin receptors that mediate the interaction
between cells and the extracellular matrix(7)
are supposed to
act as mechanotransducers.(8–10)
This view is supported by
the findings that integrins can be induced to form a physical
link to the cytoskeleton and therefore are able to transmit
forces to intracellular structures.(11,12)
Recently, we have
developed a method that enables the application of drag
forces on specific cell surface receptors.(13)
We have found
that mechanical stressing of the integrin subunits ␤1 or ␣2,
with forces in physiological ranges, induced a significantThe authors have no conflict of interest.
1
Department of Internal Medicine, University of Rostock, Rostock, Germany.
2
These authors contributed equally to this work.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 17, Number 4, 2002
© 2002 American Society for Bone and Mineral Research
603

2. intracellular calcium response in osteoblasts, which was not
observed when a nonadhesion receptor was stressed and
indicates that calcium plays a role in the regulation of
mechanotransduction.(13)
Intracellular calcium is a key sig-
naling molecule in pathways induced by a variety of exter-
nal factors. It acts as a regulator of different cellular pro-
cesses including gene expression and cell proliferation.(14)
Therefore, we were interested in whether intracellular cal-
cium and the subsequent activation of further signaling
events represent a molecular basis to sense different modes
of integrin-mediated mechanical loading.
MATERIALS AND METHODS
Cell preparation
In general, cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Life Technologies, Eggenstein,
Germany) containing 10% fetal calf serum at 37°C and in a
5% CO2 atmosphere.
For analyses of intracellular calcium, primary osteoblas-
tic cells were used. Trabecular bone from the femoral head
of orthopedic patients was obtained from the Orthopedic
Clinic of the University of Rostock, Rostock, Germany (Dr.
T. Schuhr). The bone pieces were minced and collected in
DMEM. To 1 vol of this suspension, 1 vol of collagenase (2
mg/ml) and two parts of dispase (2.4 U/ml) were added and
cultured for 2 h under continuous shaking. The supernatant
containing osteoblastic cells was centrifugated and cells
were plated into plastic culture vessels and then passaged
twice before being seeded into wells of a 96-well Fluoro
Nunc module or a chamber coverglass (Nunc A/S, Roskilde,
Denmark).
For biochemical analyses, the osteosarcoma cell line U-2
OS was used. One hundred microliters of cells in the me-
dium containing 1 ϫ 105
cells was seeded into wells of a
96-well culture module and grown to near confluence. Two
hours before mechanical strain was applied, the cells were
depleted of serum.
Preparation of beads
Preparation of beads and incubation are described else-
where.(13)
In brief, paramagnetic microbeads (2.8 ␮m in
size), then streptavidin or sheep anti-mouse antibody coated
(Dynal, Hamburg, Germany) were used. These beads were
coated with biotin-labeled anti-integrin ␤1 antibodies
(Southern Biotechnologies Associates, Inc., Birmingham,
AL, USA) or with unlabeled anti-integrin ␤1 antibodies
(both are nonblocking; Immunotech, Hamburg, Germa-
ny),(15)
respectively, or for controls with anti-CD71 (trans-
ferrin receptor) antibody (Immunotech). Fifty microliters of
phosphate-buffered saline (PBS) containing beads (25 ␮g)
were added to the cell monolayer in each well and incubated
for 20 minutes at room temperature. On average, 2–7 mi-
crobeads were attached to the ␤1-integrin subunit on the
dorsal surface of one cell.
Application of physical stress
The magnetic device has been described in detail.(13)
Briefly, the device consists of a coil system containing a
ferrite core of which the two poles are modeled differently
to generate an inhomogeneous magnetic field. A culture
well containing the prepared cells was located between the
two poles of the device. Drag forces in the horizontal
direction act on the magnetic beads that are attached to the
receptors on the cell surface. The forces subjected to one
bead were adjusted to 2 ϫ 10Ϫ10
N. For calcium measure-
ments, the device was mounted on a stage of an inverted
confocal laser scanning microscope (LSM-410; Carl Zeiss
Jena, Germany). A continuous or cyclic stress was applied
for 10 minutes. The frequency of the cyclic stress was 1 Hz
(0.5 s on and 0.5 s off) or 0.1 Hz (5 s on and 5 s off). For
biochemical analyses, the procedure was performed as de-
scribed earlier.(16)
Drag forces were applied for 30 minutes
in continuous or cyclic mode (1 Hz). Application of a cyclic
magnetic field did not increase the temperature of the me-
dium, as measured with a laboratory thermometer. For
comparison, cells were incubated with anti-integrin
antibody–coated beads for 50 minutes for clustering.
Measurement of intracellular calcium
Before the cell monolayer was incubated with magnetic
microbeads, cells were loaded with the Ca2ϩ
indicator
fluo-3 according to a modified method of Vandenberghe and
Ceuppens.(17)
Briefly, 50 ␮l of 0.5 ␮M of fluo-3/
acetoxymethylester and Pluronic F-127 (Molecular Probes,
Inc., Eugene, OR, USA) in PBS were added to the mono-
layer, incubated for 20 minutes at 37°C, and diluted with
HEPES buffer (1:5). During mechanical receptor stressing,
the global calcium responses were detected with the confo-
cal microscope using a 10ϫ Plan-Neofluar objective. For
excitation, a 488-nm argon-ion laser was used and the
emission was detected at 515 nm. By analyzing a full frame
(512 ϫ 512), images of 20 individual cells were taken every
8 s using the “time series” software. The fluorescence in-
tensities were related to the basic level detected with un-
stimulated fluo-3 loaded cells. For imaging of spatial intra-
cellular calcium signals, a 40ϫ oil immersion objective
Plan-Neofluar was used. In intervals of 2 s, images were
taken and the recorded fluorescence intensities were ex-
pressed in false colors representing 256 gray values.
Preparation of Triton X-100–soluble and –insoluble
fractions
The cell monolayers were washed in PBS and incubated
with cell extraction buffer containing 1% Triton X-100, 20
mM of imidazole, 2 mM of MgCl2, 80 mM of KCl, and 2
mM of EGTA for 5 minutes at 4°C (pH 7.8). For the
analysis of the soluble fraction, the supernatants were col-
lected and precipitated with 1% trichloracetic acid for 15
minutes on ice. The pellets were washed in ice-cold acetone,
dried, and boiled in sodium dodecyl sulfate (SDS) sample
buffer. The Triton-nonsoluble fractions were collected in
SDS sample buffer by Laemmli containing 50 mM of Tris/
HCl, pH 8.0, 6% (vol/vol) ␤-mercaptoethanol, and 5%
(wt/vol) SDS. Lysates were subjected to gel electrophoresis
as described below.
604 POMMERENKE ET AL.

3. Immunoprecipitation
To analyze activation of the focal adhesion kinase (FAK)
and mitogen-activated protein (MAP) kinases such as ERK-1
and ERK-2, cells were lysed in precipitation buffer containing
50 mM of Tris/HCl, pH 7.4, 100 mM of NaCl, 50 mM of NaF,
40 mM of glycerophosphate, 5 mM of EDTA, 1 mM of
sodium orthovanadate, 100 ␮M of phenylmethylsulfonyl flu-
oride (PMSF), 1 ␮M of leupeptin, 1 ␮M of pepstatin A, and
0.1% (vol/vol) Triton X-100 and clarified by centrifugation at
13,000g for 5 minutes at 4°C. The supernatant was incubated
with 100 ␮l of anti–ERK-1 (p44) antibody (clone C-16), which
also detects ERK-2, or 100 ␮l of anti-FAK antibody (both
from Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA)
for 1 h at room temperature followed by adding 50 ␮l of
protein A agarose. After centrifugation, the pellet was washed
in precipitation buffer and analyzed for phosphotyrosine, FAK,
and MAP kinases by immunoblotting as described in the
following section.
Gel electrophoresis and immunoblotting
The samples were subjected to a 7.5% SDS-
polyacrylamide gel electrophoresis (PAGE). The proteins
were transferred to polyvinylidene difluoride (PVDF)
membranes. To block nonspecific binding, membranes
were incubated with buffer containing 5% milk powder.
Immunoblotting for phosphotyrosine and different pro-
teins was performed with alkaline phosphatase–labeled
anti-phosphotyrosine antibody (dilution, 1:20,000), anti-
FAK (clone A-17), anti–ERK-1, and anti-actin (clone
C-11; all diluted 1:1,000; all from Santa Cruz Biotech-
nology), respectively. To analyze activation of MAP
kinases by the mobility shift assay, the precipitates were
electrophoresed using a 14% SDS-polyacrylamide gel
and subjected to immunoblotting with anti–ERK-1 (p44).
All immunoblots were visualized with chemilumines-
cence (CDP star; Roche Molecular Biochemicals, Mann-
heim, Germany).
To compare total protein levels associated with the cy-
toskeleton after different treatments of the cells, aliquots of
Triton X-100–nonsoluble fractions were subjected to a 14%
electrophoresis gel. Then, the gel was fixed with 70% acetic
acid for 2 h followed by staining with 1% Coomassie
brilliant blue G 250 in 70% acetic acid for 2 h. To remove
the background staining, the gel was washed in 50% acetic
acid overnight.
Treatment with cytochalasin and calcium chelator
To disrupt the actin filaments of the cytoskeleton, the cell
monolayer was treated with 25 nM of cytochalasin D (Sig-
ma, Deisenhofen, Germany) for 20 minutes at 37°C. Cells
were washed and incubated with microbeads to perform the
procedure for mechanical loading as described.
For chelating intracellular calcium, the cells were prein-
cubated with 5 ␮M of 1,2-bis-(o-aminophenoxy)-ethane-
N,N,NЈ,NЈ-tetracetic acid, acetoxymethyl ester (BAPTA-
AM) for 15 minutes. Mechanical strain was then applied in
the presence of 5 ␮M of BAPTA.
Statistics
Data for global intracellular calcium responses of in-
dividual cells were presented as mean Ϯ SD and sig-
nificance of different treatments were determined by
analysis of variance (ANOVA) followed by unpaired
Student’s t-test.
RESULTS
Differential calcium responses by continuous and
cyclic integrin loading
Cells were stressed at the ␤1-integrin subunit and the
global calcium signals of individual cells were recorded
FIG. 1. Characteristic intracellular global calcium reactions of a
representative osteoblastic cell during application of physical forces to
the ␤1-integrin subunit with a frequency of 1 Hz for 10 minutes (ࡗ).
The onset of the application of forces was at time zero and the changes
in relative fluorescence intensities were recorded over the indicated
time. Controls: anti–␤1-integrin coated beads but was not exposed to
drag forces (f), cells without beads exposed to the magnetic field
(similar line but not shown).
FIG. 2. Global intracellular calcium responses are dependent on the
strength of the mechanical load applied to the ␤1-integrin. Analyses of
the maximal calcium amplitudes of individual osteoblasts during a
cyclic stress for 10 minutes (n ϭ 13–20 cells). Note that the lowest
force we were able to apply induced a calcium signal. Significantly
higher signals were obtained with forces of 2 ϫ 10Ϫ10
N/bead. Con-
trols: Ϫ, untreated cells; Ϫ/1 Hz, cells without beads but exposed to
magnetic field.
605MECHANICAL INTEGRIN STRESSING

4. microscopically over a period of 10 minutes. The majority
of cells responded with oscillating calcium signals, which
had a maximum of eight spikes during the observation time
regardless of whether a continuous or cyclic stress was
applied. In Fig. 1, a characteristic calcium response of a cell
is shown showing spikes with a duration of ϳ1 minute. In
controls, when anti-integrin antibody–coated beads were
incubated without subsequent mechanical stress or when
pure cells without beads were subjected to the magnetic
field, no significant calcium signals were detected. To test
which pulling strength is required to induce a calcium
signal, we reduced the forces applied to integrins (Fig. 2).
The results revealed that the minimal force we were able to
apply with our device (i.e., 2.5 ϫ 10Ϫ11
N/bead) was suffi-
cient to induce a calcium response.
Application of a cyclic stress with 1 Hz compared with a
continuous stress revealed two differences (Fig. 3). First,
mechanical stress evoked a higher maximal calcium reac-
tion because of a 1-Hz stress (Fig. 3A), and, second, the
onset of the calcium reaction was earlier (Fig. 3B). An
influence on the frequency of the calcium spikes was not
observed in all the experiments (Fig. 3C). The results indi-
cate that the calcium response is controlled by the mode of
application, that is, whether a continuous or a cyclic me-
chanical stress is applied. In an additional experiment, we
compared the effect of 0.1 Hz with 1 Hz and a continuous
stress. This experiment showed that compared with a 1-Hz
stress, application of a stress with 0.1 Hz provoked a max-
imal calcium response of 58%, which was similar to a
continuous stress (52% of the reaction with 1 Hz; data not
shown).
Enhanced cytoskeletal anchorage of tyrosine-
phosphorylated proteins by cyclic mechanical stress
The results revealed that cyclic mechanical forces with a
frequency of 1 Hz at the ␤1-integrin subunit induced a
higher amount of tyrosine-phosphorylated proteins in the
cytoskeletal fraction than static forces (Fig. 4A). Coomassie
staining of total proteins after Triton X-100 extraction (Fig.
4B) indicated that the increased tyrosine phosphorylation
was caused by an immobilization of these activated pro-
teins, predominantly in the higher molecular weight range.
A Western blot experiment (Fig. 4C) identified FAK, a
representative protein in integrin-mediated signaling, in the
insoluble fraction because of integrin stress and most effec-
tively after cyclic stress. The immobilization of FAK to the
cytoskeleton was confirmed by the shift of FAK from the
soluble to the nonsoluble fraction (Fig. 4D). Disruption of
the cytoskeleton by cytochalasin D supported the finding
that the actin cytoskeleton serves as a structure for the
immobilization of activated proteins because of integrin
stress (Figs. 4A–4C). This process requires intracellular
calcium as shown by treatment with the calcium chelator
BAPTA-AM (Figs. 4A–4C). Together, the results suggest
that the mode of physical integrin stress regulates the cy-
toskeletal immobilization of activated proteins.
FIG. 3. Analyses of the intracellular global calcium responses during
continuous and cyclic mechanical stress (1 Hz) applied to the ␤1-
integrin subunit. The time courses of calcium responses in individual
cells (as shown in an example in Fig. 1) were analyzed with respect to
maximal calcium amplitude, the onset of the first calcium signal,
and the number of calcium spikes. Data of three independent experi-
ments (1–3; n ϭ 20 cells) were evaluated. (A) Maximal calcium
amplitudes (in general the first spike). Significantly higher calcium
amplitudes during cyclic integrin stress were obtained in experiments 1,
2, and 3 (p Յ 0.05, p Յ 0.001, and p Յ 0.001, respectively). (B) Time
of the initial calcium response after the onset of the mechanical loading.
Significantly earlier initial calcium signals were obtained during cyclic
stress in all three experiments (p Յ 0.001) (C) Number of the calcium
spikes during the experimental time. Significant differences only in
experiment 3 (p Յ 0.01).
606 POMMERENKE ET AL.

5. Enhanced activation of FAK and MAP kinases by
cyclic integrin loading
Activation of FAK and MAP kinases are significant
events in integrin signaling. Our results revealed that cyclic
forces were more effective to activate both FAK and MAP
kinase ERK-2 than a continuous load detected by tyrosine
phosphorylation of these proteins (Figs. 5A and 5B). En-
hanced activation of MAP kinases caused by cyclic forces
was confirmed also by the mobility shift in SDS-PAGE
(Fig. 5C). Clustering of the receptors by incubation with
beads had a lower effect and stressing the transferrin recep-
FIG. 4. Cytoskeletal anchorage of proteins caused by differential
mechanical loading of ␤1-integrin. (A) Tyrosine-phosphorylated pro-
teins in the Triton X-100–insoluble fraction: cyclic stress with 1 Hz (s
or 1Hz), permanent stress (p), clustering with anti-integrin antibody
coated beads (c), or not treated (Ϫ). For comparison, the CD71 receptor
was mechanically stressed or cells were pretreated with BAPTA-AM
(BAPTA) and cytochalasin D (CCD), respectively. Controls: cells
without beads were exposed in the magnetic field to a frequency of 1
Hz and to a constant field (p). Note a higher level of phosphotyrosine
because of cyclic stress. Stressing of the CD71 receptor and exposure
of untreated cells to the magnetic field had no effect. BAPTA-AM and
cytochalasin D blocked the enhanced tyrosine phosphorylation. Rep-
resentative blot of three independent experiments. (B) Total protein
content in the Triton X-100–insoluble fraction after different treat-
ments of the cells as described in panel A. The highest protein content
in the cytoskeletal fractions was obtained because of cyclic mechanical
integrin loading (1 Hz). Note that the immobilization was selective and
proteins of the higher molecular weight range became anchored to the
cytoskeleton (M, molecular weight marker). (C) FAK and actin content
in the Triton X-100–insoluble fraction after different treatments of the
cells as described in panel A. The highest level of FAK was detected
because of cyclic stress to ␤1-integrins (1 Hz). Stress to the CD71
receptor had no effect, and BAPTA-AM and cytochalasin D blocked
the immobilization of FAK. The level of actin remained unaffected
because of the different treatments. (D) Movement of FAK from the
Triton X-100–soluble fraction to the Triton X-100–nonsoluble frac-
tion. Cells were treated as described in panel A. In controls (Ϫ), after
clustering (c) and permanent stressing (p) of the integrins, FAK was
detected predominantly in the soluble fraction. After application of
cyclic stress (1 Hz), FAK was detected in the insoluble fraction, which
was paralleled with a loss of FAK in the soluble fraction.
FIG. 5. Activation of FAK and MAP kinases because of differential
mechanical loading of the ␤1-integrin. Treatments are described in Fig.
4. (A) Tyrosine phosphorylation of FAK; (B) tyrosine phosphorylation
of MAP kinases. Note that the most profound tyrosine phosphorylation
of FAK and MAP kinases (ERK-2) was obtained due to a cyclic
integrin stress. No effect due to mechanical stressing of the CD71
receptor and exposure of untreated cells to the magnetic field.
BAPTA-AM and cytochalasin D blocked the enhanced activation. (C)
Mobility shift assay for MAP kinases. Note that cyclic forces induced
the slow migrating fraction of activated ERK-2 protein (ERK-P*).
Representative blots of three experiments.
607MECHANICAL INTEGRIN STRESSING

6. tor had no effect. Tyrosine phosphorylation of both FAK
and MAP kinases was blocked completely in the presence
of the Ca2ϩ
chelator BAPTA-AM, which shows the role of
intracellular calcium. Similarly, using cytochalasin D, the
results indicate the requirement of an intact cytoskeleton for
activation of FAK and MAP kinases.
Application of local mechanical stress determines the
spatial origin and induces a transcellular spreading of
calcium
To further evaluate the role of intracellular calcium, we
investigated the spatiotemporal calcium responses in in-
dividual cells by image analysis. Figure 6 illustrates a
representative calcium reaction in an osteoblast. The
beads that stress the receptors indicate the localization of
the mechanical load. The calcium signals were initiated
only near beads that stressed integrins. This indicates that
for the local origin of the calcium signal, the presence of
a bead was required. The calcium reaction of the cell
shows that a primary calcium impulse may originate
independently in different restricted regions of the cell.
First, the calcium signal arose at the upper margin of the
cell (Fig. 6, arrow 1), visible after 14 s of stress appli-
cation. Although this calcium wave spreads two other
waves originated in different regions of the cell after 18 s
(Fig. 6, arrows 2 and 3). As shown in this cell, in the
majority of the cells, the individual waves spread over an
extended area and combined to fill the entire cell interior.
The climax of the calcium reaction may be accompanied
by an accumulation of the highest concentration of cal-
cium in the nucleus. The following decline of the calcium
level in the nucleus paralleled that of the cytoplasm.
To analyze the temporal course of the calcium response in
different regions of the cell, we set four gates and deter-
mined the time-dependent alterations in calcium concentra-
tion (Fig. 7). These data show that the individual local
calcium reactions do not have a simultaneous timing. In
addition, as shown in two regions (blue and red areas), a
lower calcium response may be initiated and then declined
again before the onset of the main signal. Analyses of the
spatiotemporal calcium reactions further revealed that in
ϳ10% of the cells, the calcium signals remained restricted
to the region where the calcium waves were initiated. An
example is shown in Fig. 8, where a calcium signal origi-
nated due to an integrin stress in a very restricted region
directly at the location of the bead after 12 s with a maxi-
mum after 20 s. The signal then declined again without
spreading over the cell.
FIG. 6. Imaging of the intracel-
lular calcium organization during
a cyclic mechanical stress of 1 Hz
to the ␤1-integrin subunit. Typi-
cal time course of the cytosolic
calcium propagation in a repre-
sentative cell. Beads that are at-
tached to the receptors are visible
as yellow dots (autofluores-
cence). The onset of the applica-
tion of mechanical stress is at
time zero. After 16 s, a calcium
signal initiates near a bead (arrow
1). After 18 s, two other beads
induce a local calcium response
(arrows 2 and 3). The calcium
waves spread over the cell with a
climax at 24 s and then decline.
The images are presented in
pseudocolor. The blue color rep-
resents the lowest and white rep-
resents the highest concentration
of calcium. The time in seconds
is indicated in the images (bar ϭ
10 ␮m).
608 POMMERENKE ET AL.

7. DISCUSSION
Our experiments revealed that a physical stress to inte-
grins already with a low intensity evoked significant cal-
cium signals. This confirms previous findings that show
intracellular calcium is an early response in integrin signal-
ing.(18)
Most of these studies examined integrin-mediated
calcium during cell spreading or adhesion to other cells that
probably is a more complex mechanism that involves cell
shape changes.(19,20)
In our experiments we could not ob-
serve, at least by light microscopy that the cell membrane
was distorted, although the forces applied at the receptor-
bound beads might induce a small highly localized distor-
tion of the membrane. Previous studies have shown that the
integrin- and stress-mediated rise of intracellular calcium is
caused by both release from intracellular stores and an
extracellular entry.(21,22)
Calreticulin, which is able to inter-
act with cytoplasmic domains of ␣-integrin subunits, is
essential for integrin-mediated calcium entry and mediates
adhesion.(23,24)
When we compared the calcium reactions in individual
cells during continuous and cyclic integrin stress with a
frequency of 1 Hz, we found that cyclic stress was a
stronger stimulus. Controls without beads but exposed to a
cyclic magnetic field revealed no effect on the calcium
reaction, which indicates that possible random electrical
currents are not the cause for an increased calcium reaction
because of cyclic forces. A lower frequency of 0.1 Hz had
a similar effect as a continuous stress, which is consistent
FIG. 7. Time courses of calcium responses in different regions of the
cell during mechanical integrin stress. Four gates were set into the cell
shown in Fig. 6. (A) Time courses of the calcium responses in these
marked regions (gates). (B) The onset of the mechanical stress is at
time zero. The curves show different intensities of the calcium re-
sponses in the four regions. It is further notable that the spikes of the
signals are not synchronous (e.g., the cyan curve precedes the red
curve). In the red and blue areas, transient calcium signals occurred
before the onset of the main signals (bar ϭ 10 ␮m).
FIG. 8. Spatially restricted calcium response in a representative cell
because of mechanical integrin stress. The yellow dot represents the
bead attached to the ␤1-integrin subunit. The onset of the mechanical
load is at time zero. After 12 s, an increase in the intracellular calcium
response is visible with a maximum after 20 s (black area; arrow). This
response declines and remains locally restricted. The images represent
the substraction of the image before the onset of the mechanical load
from the images during the mechanical load (bar ϭ 30 ␮m).
609MECHANICAL INTEGRIN STRESSING

8. with studies of mechanical loading of rat tibias showing no
effect on bone formation at frequencies below 0.5 Hz but
increasing induction of bone mass up to 2 Hz.(25)
The actin cytoskeleton appears to be of major impor-
tance for integrin-mediated mechanotransduction.(10,26)
A mechanical coupling between integrins and the cy-
toskeleton has been established(11,27)
and, furthermore,
the cytoskeleton serves as a structural scaffold to assem-
ble signaling molecules to interact in biochemical
reactions.(28,29)
Here, we could show that a cyclic
integrin stress is more effective to anchor tyrosine-
phosphorylated proteins at the cytoskeleton than a con-
tinuous stress. These proteins also included the FAK,
which is essential for the formation of the cytoskeletal
signaling structures and their functional activities be-
cause FAK recruits SH2 and SH3 domain–containing
signaling proteins and is required for further signal-
ing.(30,31)
Earlier immunofluorescence analyses revealed
that the accumulation of cytoskeletally associated pro-
teins was strongest at the site of the stressing beads, but
also in the ventral regions of the cell, a visible assembly
of proteins was detected.(32)
The cytoskeletal association
of activated proteins depended on intracellular calcium,
which stresses the relevance of calcium for mechano-
transduction and forming a cytoskeletal signaling com-
plex. In addition, this is supported by the finding that
cytoskeletal linkage of ␤1-integrin was blocked by che-
lating of intracellular calcium.(12)
Previous studies have shown that application of physical
stress to cells induced an activation of FAK and MAP
kinases.(9,33)
We showed that mechanical stress applied to a
defined integrin receptor activated FAK and MAP kinases
and the magnitude was dependent on the mode of stress.
MAP kinase activation could be dependent on cytoskel-
etally associated FAK because cytochalasin treatment in-
hibited both activation of FAK and MAP kinases, which
supports earlier findings.(34)
However, experiments also
have indicated that activation of MAP kinases can be inde-
pendent of FAK activation.(35)
Analyses of the spatiotemporal distribution of calcium
inside the cell because of a mechanical integrin stress re-
vealed three main results: (1) the site of application of
physical forces determined the spatial origin of the evoked
calcium response; (2) calcium may spread over the cell
including the nucleus, whereas spatially restricted spikes
could precede the spreading of calcium over the entire cell;
and (3) the calcium signal could remain confined to a
narrow region in the cytoplasm. One conclusion is that cells
are able to sense the localization of the applied stress on the
cell surface. This is supported by findings that have shown
a spatially restricted transmission of mechanical forces to
the cytoskeleton using an optical trap(36)
and highly local-
ized responses of the actin and microtubule cytoskeleton to
applied stress.(27)
This local mechanotransduction appears
also to have local physiological consequences because me-
chanical stress to the cell surface induced a relocation of the
apparatus for protein synthesis to the site of signal recep-
tion.(37)
Spreading of calcium caused by mechanical integrin load-
ing in our experiments showed that calcium is an excellent
candidate to transmit information into different compart-
ments of the cell interior. Calcium can act directly at target
sites in different cellular compartments.(38,39)
The physio-
logical relevance of calcium in the nucleus was shown in an
elegant study showing that gene expression is controlled
differently by nuclear and cytoplasmic calcium.(40)
On the
other hand, mechanical stress applied at the cell surface also
can be transmitted by a direct mechanical coupling to the
nucleus, which was mediated by a linkage between cy-
toskeleton and nucleus.(41)
Although our results suggest a key role of intracellular
calcium in the signaling pathway of mechanical integrin
stress, it is reasonable that multiple routes exist that may not
depend on calcium. For example, intracellular calcium in-
duction by ligation of ␣v-integrin did not contribute to cell
adhesion, whereas ␣5␤1 mediated cell adhesion without a
calcium reaction.(20)
Taken together, our results indicate that mechanical load-
ing of integrins, notably, the mode of the stress controlled
integrin signaling at different levels of the cascade by quan-
titative modulation of signaling events. We suggest that
such quantitative modulations imply functional conse-
quences that were supported by experiments in myoblasts,
which established that quantitative changes in integrin-
mediated activation of paxillin, FAK, and MAP kinases
decided whether the cells proliferated or withdrew from the
cell cycle.(42)
ACKNOWLEDGMENT
This work was supported by a grant from Bundesminister
fu¨r Bildung, Forschung und Technologie (BMBF;
01ZZ9601), and by a grant from the Deutsche Forschungs-
gemeinschaft (GK-Br 1255/4–1).
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Address reprint requests to:
Dr. Joachim Rychly
Department of Internal Medicine
Ernst-Heydemann-Str. 6
University of Rostock
18055 Rostock, Germany
Received in original form November 16, 2000; in revised form
September 3, 2001; accepted November 12, 2001.
611MECHANICAL INTEGRIN STRESSING