Fluid shear-induced mechanical signaling in MC3T3-E1
osteoblasts requires cytoskeleton-integrin interactions
Fredrick M.
Pavalko1,
Neal X.
Chen2,
Charles H.
Turner2,3,
David B.
Burr2,
Simon
Atkinson4,
Yeou-Fang
Hsieh2,
Jinya
Qiu2, and
Randall L.
Duncan1,2
Departments of 1 Physiology and
Biophysics, 2 Anatomy, and
3 Orthopaedic Surgery and
4 Division of Nephrology,
Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
Mechanical stimulation of bone induces new bone formation in
vivo and increases the metabolic activity and gene expression of
osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of
mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused
reorganization of actin filaments into contractile stress fibers and
involved recruitment of
1-integrins and
-actinin to
focal adhesions. Fluid shear also increased expression of two proteins
linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the
early response gene product c-fos. Inhibition of actin stress fiber
development by treatment of cells with cytochalasin D, by expression of
a dominant negative form of the small GTPase Rho, or by microinjection
into cells of a proteolytic fragment of
-actinin that inhibits
-actinin-mediated anchoring of actin filaments to integrins at the
plasma membrane each blocked fluid-shear-induced gene expression in
osteoblasts. We conclude that fluid shear-induced mechanical signaling
in osteoblasts leads to increased expression of COX-2 and c-Fos through
a mechanism that involves reorganization of the actin cytoskeleton.
Thus Rho-mediated stress fiber formation and the
-actinin-dependent
anchorage of stress fibers to integrins in focal adhesions may promote
fluid shear-induced metabolic changes in bone cells.
mechanotransduction;
-actinin; gene expression
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INTRODUCTION |
THE ACTIN CYTOSKELETON and the integrin family of cell
adhesion molecules have been shown to play important roles in
mechanotransduction (23, 39). Integrins are heterodimeric adhesion
molecules composed of an
-subunit and a
-subunit that physically
link the extracellular matrix (ECM) and the actin cytoskeleton by
interacting with ECM proteins outside the cell and with bundles of
actin filaments (stress fibers) inside the cell (5, 19). The
organization of actin and myosin filaments into contractile stress
fibers is thought to increase internal tension in cells and is
regulated, in part, by the GTPase Rho (5, 32). Growing evidence
suggests that the development of internal tension by actin and myosin
plays a central role in signal transduction from the ECM to the nucleus to regulate gene expression (21, 34, 41). Ligand binding stimulates the
clustering of integrins in the membrane and causes the attachment of
stress fibers to integrins at specialized sites of cell-ECM attachment
called focal adhesions (5). Several linker proteins anchor actin
filaments to integrins at focal adhesions, including
-actinin,
vinculin, and talin (16, 24, 28, 33). In this study, we investigated
the roles Rho-mediated stress fiber development and the anchoring of
stress fibers to integrins had in the transmission of fluid shear
forces into intracellular signals that upregulate gene expression and
stimulate new bone formation.
Mechanically induced bone formation is preceded by expression of the
transcription factor c-Fos and prostaglandin production (8, 38). The
inducible isoform of the enzyme cyclooxygenase (COX-2) is important in
mechanotransduction, since selective inhibition of COX-2 eliminates
mechanically induced bone formation in the rat tibia (12). Mechanical
forces appear to be transduced to bone cells by fluid flow-induced
shear stress in the canaliculi and canals within the bone tissue (25,
35). The organization of the actin cytoskeleton and its attachments to
integrins may be part of a sensing apparatus used by cells to detect
and respond to mechanical signals. The term autobaric has been proposed
to describe a process in which cells use the cytoskeleton to apply an
internal load to themselves that is part of an intracellular signaling
mechanism (4). In this study, we found that application of fluid shear
to cultured osteoblasts induced cytoskeletal changes that were
consistent with an increase in internal load. These changes in response
to fluid shear included development of stress fibers and focal
adhesions and recruitment of
1-integrins and
-actinin
into focal adhesions. Fluid shear also increased expression of both
c-Fos and COX-2 in osteoblasts. Three lines of evidence support a
critical role for actin cytoskeleton reorganization, attachment of
actin stress fibers to integrins in focal adhesions, and the
development of internal tension in mediating fluid shear-induced mechanochemical signal transduction in osteoblasts. First, inhibiting fluid shear-induced cytoskeletal reorganization by treatment with cytochalasin D blocked the expression of COX-2. Second, microinjection of single cells with a fragment of
-actinin that prevents fluid shear-induced stress fiber development by competing with endogenous
-actinin for binding to integrin cytoplasmic tails (26) blocked COX-2 and c-Fos expression. Third, expression of COX-2 and c-Fos was
inhibited in cells transfected with a dominant negative mutant of the
GTPase Rho, which blocks fluid shear-induced stress fiber and focal
adhesion formation. Together, these results suggest that the
development of stress fibers and their anchorage to the membrane at
focal adhesions in response to fluid shear play a critical role in
transducing mechanical signals applied at the cell surface into
intracellular signals that are necessary for increased gene expression
and new bone formation.
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MATERIALS AND METHODS |
Cell culture and fluid flow.
The mouse osteoblast cell line MC3T3-E1 was cultured in
-MEM
containing 10% FCS and maintained in 5%
CO2 at 37°C. The MC3T3-E1 cell
line has a reproducible phenotype with a distinct proliferative stage
and, on reaching confluence, a differentiated stage in which osteoblastic markers such as alkaline phosphatase are expressed. For
microscopy, cells were grown on glass slides coated with 10 µg/ml
fibronectin (Sigma Chemical). Fluid flow was performed in chambers
using the flow loop designed by Frangos et al. (14) and marketed by
Cytodyne (San Diego, CA). This system produces laminar flow over a cell
monolayer. The system was maintained at 37°C, and the medium was
bubbled with 5% CO2-95% air
during flow experiments. Cells were exposed to fluid flow in
-MEM
containing 1% serum. A flow sensor (SWF-5 flowmeter, Zepeda
Instruments, Seattle, WA) incorporated into the flow loop was used to
monitor flow rate. The flow rate was 18 ml/min, which yielded a shear stress of 12 dyn/cm2. Control
cells were placed in
-MEM containing 1% serum but not subjected to
flow. For experiments using cytochalasin D, cells were pretreated for 1 h with 10 µM cytochalasin D in
-MEM with 10% serum and then
subjected to flow in
-MEM containing 10 µM cytochalasin D with 1% serum.
Antibodies.
Antibodies were obtained as follows: one integrin antibody (no. 763)
was generated by immunizing rabbits with a peptide corresponding to
residues 758-774 of the human
1-integrin cytoplasmic tail,
-actinin monoclonal antibody BM75 was purchased from Sigma, COX-2 and c-Fos antibodies were purchased from Santa Cruz, and all
fluorescently labeled secondary antibodies were purchased from Jackson Immunoresearch.
Western blot analysis.
For Western blotting, cells on glass slides were lysed in 1 ml of
Tris-buffered saline containing 1% Triton X-100, 1% deoxycholate, and
0.5% SDS, along with the protease inhibitors phenylmethylsulfonyl fluoride, aprotinin, and leupeptin. Cells were scraped from the substrate after 5 min in lysis buffer, and the insoluble material was
removed by centrifugation at 12,000 g
for 15 min. The supernatant was transferred to a fresh tube, and the
protein concentration was determined using bicinchoninic acid reagent
(Pierce Chemical); 10-µg samples were loaded onto 10% SDS-PAGE gels
for separation and transferred to nitrocellulose for immunoblot
analysis. Equal protein loadings were confirmed by Coomassie blue
staining of gels run in parallel. Each experiment was carried out a
minimum of four times, scanning densitometry of bands was performed
with the Bio-Rad Molecular Analyst program, and statistical analysis was made by Kruskal-Wallis one-way ANOVA.
Northern Blot analysis.
Total RNA was extracted from the cells using TRIzol reagent (GIBCO).
Ten micrograms of total RNA were run on a 1% agarose-0.44 M
formaldehyde gel, transferred to a Hybond nylon membrane (Amersham) by
capillary blotting, and fixed to the membrane by ultraviolet irradiation. Filters were hybridized for 2 h at 65°C in a rapid hybridization buffer (Amersham) containing radiolabeled cDNA probes. Rat COX-2, c-Fos, and glyceraldehyde-3-phosphate dehydrogenase cDNA
probes were labeled with
[32P]dCTP (specific
activity > 3,000 Ci/mmol; NEN), using a random prime labeling kit
(Boehringer Mannheim). Filters were washed and exposed to Fuji Rx film
at 80°C. Northern blot analysis was carried out in four separate
experiments, bands were analyzed by scanning densitometry, and the
statistical significance of differences in expression was confirmed by ANOVA.
Microinjection and fluorescence microscopy.
For microinjection, cells grown on glass slides were placed in a 100-mm
dish with DMEM containing 15 mM HEPES (pH 7.3). Microinjection was
performed at room temperature using a Leitz micromanipulator with
needles prepared from glass capillaries pulled on a two-stage needle
puller, as previously described (26). After injections, cells were
returned to the incubator at 37°C for 1 h before being subjected to
fluid shear. At the end of the flow period, cells were fixed in 4%
paraformaldehyde and processed for immunofluorescence microscopy as
previously described (29), using appropriate primary and fluorescently
labeled secondary antibodies, rhodamine-phalloidin or FITC-phalloidin
(Molecular Probes). Images were recorded on Tmax 400 film (Kodak) using
a Nikon Optiphot II microscope through either ×60 or ×100
planapo objectives (1.4 numerical aperture). The effect of
-actinin
fragment microinjection on cytoskeletal organization and gene
expression following fluid shear was confirmed by analysis of ~35
injected cells stained with phalloidin and 60 injected cells stained
with anti-c-Fos or anti-COX-2.
Transfection of MC3T3-E1 cells.
A pEXV expression vector containing N19 Rho was purified by CsCl
density gradient centrifugation. MC3T3-E1 cells were washed in
serum-free DMEM and transiently transfected using Lipofectin (GIBCO).
Cells were returned to
-MEM containing 10% FCS and cultured for 48 h before use in experiments. Transfected cells were identified using
the myc epitope tag antibody 9E10. Control cells were transfected with
a vector expressing the myc epitope tag only.
Purification and labeling of the 53-kDa
-actinin
fragment and intact
-actinin.
-Actinin was purified from chicken gizzard smooth muscle as
previously described (11). Purified
-actinin was digested with
thermolysin, and the 53-kDa integrin-binding fragment was purified on a
fast protein liquid chromatography MonoQ anion exchange column (26).
Intact
-actinin was conjugated to iodoacetaminotetramethyl rhodamine
(Molecular Probes), and the purified 53-kDa fragment was conjugated to
rhodamine isothiocyanate. Both proteins were dialyzed
extensively against microinjection buffer (75 mM KCL, 10 mM
KHPO4, and 0.1%
-mercaptoethanol) and concentrated to 5 mg/ml by ultrafiltration
before use for microinjection experiments.
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RESULTS |
Application of fluid shear to MC3T3-E1 osteoblasts induces
development of actin stress fibers and formation of focal adhesions
containing
1-integrins and
-actinin.
Figure 1 illustrates the
dramatic changes in actin filament and focal adhesion organization in
MC3T3-E1 osteoblasts that occur following fluid shear. Before fluid
shear, actin filaments, visualized by staining with FITC-phalloidin,
were poorly organized in MC3T3-E1 cells, with only a few thin stress
fibers being detectable (Fig. 1, A and
E). After the application of fluid
shear for 60 min at 12 dyn/cm2,
actin filaments became organized into stress fibers that were thicker
and more abundant than in nonflowed cells (Fig 1,
B and F). Immunostaining with antisera
directed against the integrin
1-subunit demonstrated that,
before fluid shear,
1-integrins were diffusely distributed over the surface of cells (Fig.
1C). However, after fluid shear,
1-integrins became concentrated
in the focal adhesions that formed at the termini of the stress fibers (Fig. 1D). Similarly,
-actinin
(Fig. 1, G and
H) was localized with a periodic
distribution along the stress fibers and was also present in the focal
adhesions at stress fiber termini (Fig.
1H) after fluid shear.

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Fig. 1.
MC3T3-E1 osteoblasts respond to fluid shear by developing prominent
stress fibers and recruiting
1-integrins to focal adhesions.
Cells that were not subjected to fluid shear
(A and
E) or were subjected to fluid shear
at 12 dyn/cm2 for 60 min
(B and
F) were stained with
rhodamine-phalloidin to visualize F-actin. Fluid shear induced
development of prominent F-actin stress fibers that were oriented
roughly parallel to long axis of cell, whereas stress fibers in cells
not subjected to flow were smaller and randomly oriented. Cells were
also labeled with antibodies against
1-integrin
(C and
D) or -actinin
(G and
H). Before fluid flow, each of these
proteins were diffusely localized in cells. However, after fluid flow,
1-integrins
(D) were recruited to focal
adhesions and -actinin (H) became
localized periodically along stress fibers and in focal adhesions
located at termini of prominent stress fibers. Scale bar, 20 µm.
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Osteoblasts upregulate COX-2 and c-Fos expression in response to
fluid shear.
Application of fluid shear (60 min at 12 dyn/cm2) to MC3T3-E1 osteoblasts
also induced a dramatic increase in the intensity of immunostaining for
the enzyme COX-2 (Fig. 2,
A and
B) and the nuclear protein c-Fos
(Fig. 2, C and
D). Western blotting of protein from
Triton X-100 extracts (Fig.
3A) and
Northern blot analysis of mRNA (Fig.
3B) from nonflowed and flowed cells
confirmed that fluid flow (1 h of flow at 12 dyn/cm2) significantly increased
expression of COX-2 and c-Fos protein and mRNA levels
(P < 0.005). In contrast to COX-2
and c-Fos, levels of the cytoskeletal proteins
-actinin, vinculin,
and talin were not significantly different after fluid flow compared
with nonflowed controls (Fig. 3). Figure 4
shows fields of cells at lower magnification than in Figs. 1 and 2 and
demonstrates 1) that fluid shear
induced changes in actin organization (increased stress fiber and focal adhesion development) in essentially all the cells in the field, 2) that fluid shear induced an
elongated cell shape compared with nonflowed controls, and
3) that essentially all cells
respond to fluid flow by increasing c-Fos expression.

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Fig. 2.
Fluid shear induces expression of COX-2 and c-Fos in osteoblasts.
Immunofluorescence microscopy of MC3T3-E1 osteoblasts using antibodies
against COX-2 (A and
B) or c-Fos
(C and
D) demonstrates increased expression
of both COX-2 and c-Fos proteins following fluid shear for 1 h at 12 dyn/cm2
(B and
D) compared with cells not subjected
to flow (A and
C). Scale bar, 20 µm.
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Fig. 3.
Fluid shear induces expression of COX-2 and c-Fos protein and mRNA in
osteoblasts. A: immunoblot analysis of
protein extracts from cells not subjected to fluid shear (No flow) or
cells subjected to fluid shear for 1 h at 12 dyn/cm2 (1 h flow) confirms
increased expression of both c-Fos and COX-2 protein following fluid
shear. Expression of cytoskeletal proteins -actinin, vinculin, and
talin, in contrast, were not altered by fluid shear. Ten micrograms of
protein were loaded onto each lane of gel.
B: Northern blot analysis of mRNA from
control cells not subjected to fluid shear or from cells subjected to
fluid shear at 12 dyn/cm2 for 1 or
3 h demonstrated increased COX-2 and c-Fos mRNA expression following
fluid shear. A probe to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) confirmed equivalent mRNA loadings on gel.
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Fig. 4.
Increased expression of c-Fos correlates with stress fiber development
following fluid shear. A and
B: viewed at low magnification,
F-actin staining with rhodamine-phalloidin demonstrates high proportion
of cells that develop stress fiber bundles following fluid flow
(B; flow was from
right to
left), whereas nonflowed cells have
randomly oriented F-actin structures that are not organized in bundles
(A).
C and
D: overlaid F-actin and c-Fos
immunofluorescence images demonstrate that a high proportion of cells
that develop stress fibers in response to fluid shear
(D) also express higher levels of
c-Fos than nonflowed control cells
(C). Scale bar, 40 µm.
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Disruption of microfilaments with cytochalasin D inhibits fluid
shear-induced increases in COX-2 protein expression.
To evaluate the role of the actin cytoskeleton in fluid shear-induced
upregulation of gene expression, cells were pretreated with 10 µM
cytochalasin D for 1 h and then subjected to fluid shear for 30 min or
1 h in the presence of 10 µM cytochalasin D. Western blot analysis of
protein extracts from untreated control cells and cytochalasin
D-treated cells demonstrated that microfilament disassembly by
cytochalasin D resulted in decreased COX-2 expression compared with
untreated controls (Fig. 5).
Analysis by scanning densitometry of four separate experiments
demonstrated that COX-2 expression increased an average of 2.7-fold
following 1 h of fluid shear compared with nonflowed controls and was
significant as determined by ANOVA (P < 0.05). Pretreatment
of cells with cytochalasin D caused COX-2 expression following 1 h of
fluid shear to not be significantly different from nonflowed controls.
Immunofluorescence microscopy confirmed the inability of fluid shear to
induce reorganization of the actin cytoskeleton (Fig.
6, A and
B) or to increase the intensity of
immunostaining for COX-2 (Fig. 6, C
and D) when cells were treated with
cytochalasin D. Unlike COX-2, c-Fos protein expression was relatively
unaffected by treatment with cytochalasin D (not shown).

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Fig. 5.
Inhibition of actin cytoskeleton with cytochalasin D inhibits
fluid shear-induced upregulation of COX-2 protein. Protein extracts
were prepared from cells not subjected to fluid shear (No flow) or from
cells subjected to fluid shear at 12 dyn/cm2 for 30 min
(30-min flow) or 60 min (60-min flow) in either absence ( ) or
presence (+) of 10 µM cytochalasin D, as described in MATERIALS
AND METHODS. Equal amounts of protein were loaded onto SDS-PAGE
gels, transferred to nitrocellulose, and blotted for COX-2.
Top: Coomassie blue staining of a gel to confirm equivalent
protein loadings. MM, molecular mass. Bottom: an immunoblot
for COX-2 that demonstrates decreased levels of expression in cells
subjected to either 30 or 60 min of fluid flow in presence of
cytochalasin D compared with controls not treated with drug.
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Fig. 6.
Immunofluorescence microscopy of cells subjected to fluid shear in
presence of cytochalasin D fail to develop stress fibers or upregulate
COX-2 protein expression. Staining with rhodamine-phalloidin confirms
that, in absence (A and
C) or presence
(B and
D) of fluid flow over cells treated
with 10 µM cytochalasin D, stress fibers fail to develop
(A and
B) and cells also fail to upregulate
COX-2 protein expression (C and
D). Scale bar, 20 µm.
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Microinjection of the integrin-binding domain of
-actinin inhibits linkage of actin filaments to
integrins and blocks fluid shear-induced cytoskeletal reorganization
and gene expression.
We next used an alternative approach to inhibit reorganization of the
actin cytoskeleton that avoids the potential pharmacologic complications frequently associated with use of cytochalasin D. For
these experiments, we microinjected a proteolytic fragment of the
microfilament-integrin linker protein
-actinin into cells to inhibit
the function of endogenous
-actinin.
-Actinin can be cleaved by
proteolysis into major fragments of 27 and 53 kDa using the enzyme
thermolysin. The 27-kDa fragment contains an actin-binding site, and
the 53-kDa fragment binds to the cytoplasmic domain of
1-,
2-, and
3-integrins. When microinjected
into cells such as fibroblasts that normally contain well-developed stress fibers, the 53-kDa integrin-binding fragment of
-actinin causes disassembly of existing stress fibers by competitively displacing the endogenous
-actinin from focal adhesion and causing detachment of stress fibers from the focal adhesions (26). Figure 7 illustrates that
microinjection of the 53-kDa integrin-binding fragment of
-actinin
completely blocked the fluid shear-induced development of stress fibers
in osteoblasts. Observation of ~45 microinjected cells stained with
phalloidin showed that stress fibers never formed, strongly suggesting
that
-actinin plays a critical role in mediating the anchorage of
actin filaments to integrins in focal adhesions that is required for
stress fiber formation to occur.

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Fig. 7.
Microinjection of 53-kDa -actinin fragment into osteoblasts blocks
fluid shear-induced development of stress fibers. Cells microinjected
with rhodamine-labeled 53-kDa fragment
(A and
C) were returned to incubator for 1 h before being subjected to fluid shear at 12 dyn/cm2 for 1 h and then fixed and
stained with FITC-phalloidin to visualize F-actin
(B and
D). Cells injected with 53-kDa
fragment failed to develop stress fibers in response to fluid shear.
Scale bar, 20 µm.
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We predicted that development of stress fibers and the consequent
increase in intracellular tension may play a role in signaling to the
nucleus that leads to increased COX-2 expression. To test this
hypothesis, cells were microinjected with the 53-kDa
-actinin fragment before the application of fluid shear for 1 h at 12 dyn/cm2 to block shear-induced
stress fiber formation. As illustrated in Fig.
8, analysis of ~60 cells microinjected
with the 53-kDa
-actinin fragment confirmed that injected cells did
express lower levels of COX-2 after 1 h of fluid shear than uninjected
neighboring cells.

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Fig. 8.
Microinjection of 53-kDa -actinin fragment blocks fluid
shear-induced COX-2 protein expression in osteoblasts. Cells
microinjected with rhodamine-labeled 53-kDa fragment
(A and
C) were returned to incubator for 1 h before being subjected to fluid shear at 12 dyn/cm2 for 1 h and then fixed and
stained with an antibody against COX-2, followed by a FITC-labeled
secondary antibody (B and
D). Microinjected cells, which are
outlined, failed to upregulate COX-2 protein in response to fluid
shear. Scale bar, 20 µm.
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Similarly, analysis of ~60 cells microinjected with the 53-kDa
-actinin fragment and stained for c-Fos protein expression following
fluid shear for 1 h confirmed that c-Fos expression was completely
blocked (Fig. 9). The effect of the 53-kDa
fragment on stress fiber formation and on COX-2 and c-Fos expression
was not a general effect of the microinjection procedure, since
microinjection of intact
-actinin had no effect on stress fiber
formation (Fig. 10,
A and
B), COX-2 expression (Fig. 10,
C and
D), or c-Fos expression (Fig. 10,
E and
F).

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Fig. 9.
Microinjection of 53-kDa -actinin fragment blocks fluid
shear-induced c-Fos protein expression. Cells microinjected with
rhodamine-labeled 53-kDa fragment (A
and C) were returned to incubator
for 1 h before being subjected to fluid shear at 12 dyn/cm2 for 1 h and then fixed and
stained with an antibody against c-Fos, followed by a FITC-labeled
secondary antibody (B and
D). Microinjected cells failed to
upregulate c-Fos protein in response to fluid shear. Scale bar, 20 µm.
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Fig. 10.
Microinjection of intact -actinin does not inhibit fluid
shear-induced stress fiber formation or COX-2 or c-Fos protein
expression. Cells microinjected with rhodamine isothiocyanate-labeled
intact -actinin (A,
C, and
E) were returned to incubator for 1 h before being subjected to fluid shear for 1 h at 12 dyn/cm2 and then fixed. Cells were
stained with FITC-phalloidin (B),
COX-2 antibody (D), or c-Fos
antibody (F), followed by an
FITC-labeled secondary antibody. Scale bar, 20 µm.
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Expression of a dominant negative Rho mutant blocks stress fiber
formation and decreases expression of COX-2 and c-Fos protein in
osteoblasts.
To further investigate the relationship between fluid shear-induced
stress fiber/focal adhesion formation and gene expression in
osteoblasts, cells were transfected with a dominant negative Rho mutant
(N19 Rho) as an alternate method of inhibiting stress fiber formation.
Cells expressing N19 Rho were detected by staining with an antibody
directed against a myc epitope tag engineered into the expressed
protein. After fluid shear for 60 min at 12 dyn/cm2, cells transfected with
N19 Rho failed to develop stress fibers (Fig.
11, A
and B) and did not increase
expression of COX-2 (Fig. 11, C and
D) or c-Fos (Fig. 11,
E and
F). Thus Rho activity
is required for flow-induced stress fiber development and increased
expression of both COX-2 and c-Fos proteins. Control cells transfected
with the myc-tagged vector alone did not affect stress fiber formation or gene expression.

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Fig. 11.
Expression of a dominant negative Rho mutant (N19 Rho) inhibits fluid
shear-induced stress fiber formation and blocks fluid shear-induced
COX-2 and c-Fos protein expression. Cells were transfected with N19
Rho, cultured for 48 h, subjected to fluid shear at 12 dyn/cm2 for 1 h, and then fixed.
Transfected cells were detected using an antibody against a myc epitope
tag (A,
C, and
E). Cells were stained with
FITC-phalloidin (B), an antibody
against COX-2 (D), or an antibody
against c-Fos (F). Scale bars (in
A for
A-D,
in E for E
and F), 20 µm.
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DISCUSSION |
In this study we showed that MC3T3-E1 osteoblasts respond to fluid
shear by increasing expression of the enzyme COX-2 and the early
response gene product c-Fos. Increased expression of these proteins
correlated with changes in the organization of the actin cytoskeleton,
including the development of contractile actin stress fibers and
increased formation of focal adhesions that contain
1-integrins. Three distinct
approaches to inhibiting cytoskeletal function, namely, treatment with
cytochalasin D, microinjection of cells with the 53-kDa fragment of
-actinin, and transfections of cells with a dominant negative Rho,
each blocked the increased expression of COX-2 following fluid shear, whereas c-Fos expression was inhibited by the 53-kDa
-actinin fragment and dominant negative Rho. These results suggest that the
actin cytoskeleton plays a critical role in the transmission of fluid
shear-mediated mechanical signals that lead to increased COX-2 and
c-Fos expression.
Because
-actinin is an actin-binding protein that can cross-link
actin filaments and interact directly with the cytoplasmic domain of
integrin subunits,
-actinin appears to be capable of dual functions:
bundling actin filaments into stress fibers and linking stress fibers
to integrins at the cytoplasmic face of the plasma membrane in focal
adhesions. The intracellular distribution of
-actinin is consistent
with these dual functions. When microinjected into cells, fluorescently
labeled intact
-actinin is incorporated both into the focal
adhesions and along stress fibers with a periodic distribution (26,
29). After microinjection into fibroblasts, the 53-kDa integrin-binding
fragment of
-actinin, however, initially concentrates in focal
adhesions and then rapidly causes the disassembly of preexisting stress
fibers and focal adhesions (26).
The inability of osteoblasts that were microinjected with the
integrin-binding 53-kDa fragment of
-actinin to develop stress fibers or focal adhesions or to upregulate COX-2 or c-Fos protein suggests that
-actinin plays a critical role in transmitting mechanical signals across the membrane to the cytoskeleton. Experiments with the dominant negative Rho mutant (N19 Rho) further suggest that
stress fiber formation mediated by Rho and linkage of stress fibers to
integrins at the cytoplasmic face of focal adhesions are necessary for
the transmission of mechanical signals received at the cell surface
into intracellular signals necessary for increased gene expression and
new bone formation. This suggests that osteoblasts may experience an
increase in intracellular tension resulting from the formation of
actin/myosin-containing stress fibers. The linkage of stress fiber to
integrins, in response to fluid shear, may play a critical role in
transducing mechanical signals into intracellular signals that precede
increased gene expression and new bone formation. Additionally, it is
possible that actin filaments anchored to the cell membrane may
function to facilitate the trafficking of signaling molecules from the
membrane to the nucleus.
Mechanotransduction in bone tissue involves several steps:
1) mechanochemical transduction of
the signal (the subject of this study),
2) cell-to-cell signaling, and
3) increased number and activity of
osteoblasts (10). Cell-to-cell signaling after a mechanical stimulus
involves prostaglandins, especially those produced by the inducible
isoform of cyclooxygenase (COX-2) (8, 12), and nitric oxide (13, 38).
Prostaglandins induce new bone formation by promoting both
proliferation and differentiation of osteoprogenitor cells. The
selective inhibition of COX-2 in the rat tibial four-point bending
model eliminates mechanically induced bone formation (12). A role for
the actin cytoskeleton and
1-integrins in the metabolic
response of osteoblasts and osteosarcoma cells to mechanical strain has
also been observed (6). Thus the direct linkage between contractile
actin stress fibers and mechanically induced COX-2 and c-Fos expression
observed in the present study is relevant to mechanically induced bone formation in vivo. It should be noted that these experiments were carried out under conditions of steady fluid flow. Bearing in mind that
cells in vivo may experience fluid movements that are pulsatile
and/or oscillatory in nature, the response of cells in our in
vitro system will also need to be examined under conditions of
nonsteady fluid flow.
There is now evidence of at least two mechanochemical transduction
pathways within osteoblasts. One clearly involves the actin cytoskeleton. For example, expression of osteopontin in embryonic chick
osteoblasts, which is upregulated in response to mechanical stimuli, is
dependent on an intact actin cytoskeleton, since treatment with the
drug cytochalasin D blocks osteopontin expression (34). Furthermore,
focal adhesion kinase (p125FAK)
is tyrosine phosphorylated and becomes associated with the actin cytoskeleton in response to mechanical stimuli. This strongly suggests
that recruitment of focal adhesion proteins to sites of integrin
aggregation may play a role in promoting mechanically induced
cytoskeletal reorganization. These results are consistent with the
existence of an internal loading mechanism that detects and responds to
external mechanical signals as previously proposed (4, 39). Electron
microscopy of alveolar bone has revealed actin filaments in situ that
are organized into bundles that appear to be similar to the stress
fibers of cultured cells, suggesting that stress fibers are relevant in
vivo (40).
A second pathway involving G protein-linked mechanosensors in the cell
membrane, calcium, and the cytoskeleton may also be involved in
mechanical signaling (1, 9, 18, 19). In bone cell culture, fluid shear
increases production of prostaglandins and nitric oxide within minutes
(20, 30). This stimulation of prostaglandins can be 70-80%
blocked by the G protein inhibitors guanosine
5'-O-(2-thiodiphosphate) and
pertussis toxin, indicating that a G protein-associated
mechanotransducer attached to the cell membrane may be responsible for
prostaglandin production (31). Constitutive isoforms of cyclooxygenase
(COX or prostaglandin synthase) and nitric oxide synthase are typically
bound to the cell membrane and thus would be available for
mechanochemical transduction involving G proteins (36, 37). It is
possible that the cytoskeletal and G protein-linked mechanosensor
pathways are tightly coupled; our experiments do not yet address the
interrelationships between these pathways.
This study provides direct evidence that linkage of actin stress fibers
to integrins in focal adhesions via
-actinin is a critical link in
the mechanical signal transduction pathway in osteoblasts, since
blocking this linkage prevents fluid shear-induced COX-2 and c-Fos
expression. The selective inhibition of
-actinin-integrin interaction by the 53-kDa
-actinin fragment is a useful approach for
assaying the role of stress fiber formation and increased intracellular
tension in mechanically induced gene induction. Interpreting the
results of experiments using pharmacologic inhibition of actin filament
integrity or kinase activity in mechanically induced signal
transduction is complicated by possible unintended side effects of the
drugs on signaling pathways not involving the cytoskeleton. Competitive
inhibition of
-actinin-mediated stress fiber linkage to integrins,
however, is less likely to have unanticipated effects on
noncytoskeletal intracellular signaling pathways. Interpreting the
results of the present studies is therefore simplified, since
-actinin fragment microinjection is unlikely to directly affect this
or other non-cytoskeleton-mediated signaling pathways.
The Ras-like GTPase, Rho, has been shown to control stress fiber and
focal adhesion formation in fibroblasts (32) in response to growth
factor and Ras signaling. We have shown that N19 Rho, a dominant
negative mutant of Rho, blocks stress fiber and focal adhesion
formation in response to shear stress. The Rho effector Rho-kinase has
been shown to mediate Rho-dependent stress fiber formation, probably by
increasing phosphorylation of the myosin II regulatory light chain (2,
3, 22). Moreover, Rho-kinase induces transcriptional activation of the
c-Fos serum response element (7). Rho-kinase is therefore a likely
candidate for mediation of shear stress-induced stress fiber and focal
adhesion formation in osteoblasts.
How then does Rho activity affect COX-2 and c-Fos expression? It is
possible that Rho regulates stress fiber formation, on the one hand,
and expression of COX-2 and c-Fos, on the other, via distinct pathways,
perhaps both involving Rho-kinase. In fibroblasts, disruption of the
actin cytoskeleton by treatment with cytochalasin D (15) did not
inhibit the ability of Rho-mediated signaling, stimulated by
lysophosphatidic acid (LPA), to induce c-Fos transcription, suggesting
that LPA-stimulated Rho signaling to the nucleus is not mediated by
intact cytoskeletal structures. The rapid response of osteoblasts to
fluid shear also argues that c-Fos and COX-2 expression in response to
mechanical stimuli may be mediated by a regulatory pathway that is
distinct from LPA-mediated signaling. In our studies, fluid
shear-induced expression of COX-2 and c-Fos was inhibited by both N19
Rho and by injection of the 53-kDa
-actinin fragment, suggesting
that Rho could influence expression of the genes for COX-2 and c-Fos
via stress fiber formation in response to a mechanical stimulus.
However, although cytochalasin D inhibited fluid shear-induced COX-2
expression, surprisingly, this drug did not inhibit c-Fos expression.
Although we cannot yet explain the different response of these two
genes to cytochalasin D, it is possible that the mechanical signaling
pathways leading to increased expression of COX-2 and c-Fos are
distinct. Indeed, other pharmacologic effects of cytochalasin D,
unrelated to the actin cytoskeleton, may stimulate c-Fos expression
through a mechanism that is independent of a mechanical stimulus.
Further experiments will be required to clarify these points.
Recent evidence of linkages between the ECM and nucleoplasm involving
the cytoskeleton (23) predict that mechanotransduction through
integrin-cytoskeleton complexes may regulate gene expression. The
studies described here provide direct evidence to support this
prediction. Specifically, our results indicate that Rho-dependent stress fiber formation in response to fluid shear is involved in the
mechanically induced upregulation of COX-2 and c-Fos in MC3T3-E1
osteoblasts. Furthermore, the increased intracellular tension that
results from stress fiber formation is likely to be an important part
of this signaling pathway, since the selective inhibition of
-actinin-mediated stress fiber-integrin linkage blocked fluid
shear-induced COX-2 and c-Fos protein expression. It is also possible
that changes in intracellular contractility alter the conformation of
the nuclear matrix and activate matrix-associated transcription
factors. Future studies should aim to determine the mechanisms of
transcriptional control that are affected by the contractile actin
cytoskeleton in mechanically responsive cells such as osteoblasts.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H. Kamioka for technical assistance in the initial
stages of this study and Drs. P. Gallagher, B. P. Herring, and S. Sawyer for helpful discussions and critical reading of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grant GM-4733 and a Grant-in-Aid from the American Heart
Association (F. M. Pavalko), by National Institute of Arthritis and
Musculoskeletal and Skin Diseases (NIAMS) Grant AR-43222 and National
Aeronautics and Space Administration Grant NAG5-4917 (R. L. Duncan), by
NIAMS Grant T32 AR-07581 (D. B. Burr), and by NIAMS Grant AR-43730 (C. H. Turner).
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: F. M. Pavalko, Dept. of Physiology and
Biophysics, Indiana University School of Medicine, 635 Barnhill Dr.,
Indianapolis, IN 46202-5120.
Received 3 June 1998; accepted in final form 24 August 1998.
 |
REFERENCES |
1.
Aderem, A.
Signal transduction and the actin cytoskeleton: the roles of MARCKS and profilin.
Trends Biochem. Sci.
17:
438-443,
1992[Medline].
2.
Amano, M.,
K. Chihara,
K. Kimura,
Y. Fukata,
N. Nakamura,
Y. Matsuura,
and
K. Kaibuchi.
Formation of actin stress fibers and focal adhesions enhanced by rho-kinase.
Science
275:
1308-1311,
1997[Abstract/Free Full Text].
3.
Amano, M.,
M. Ito,
K. Kimura,
Y. Fukata,
K. Chihara,
T. Nakano,
Y. Matsuura,
and
K. Kaibuchi.
Phosphorylation and activation of myosin by rho-associated kinase (rho-kinase).
J. Biol. Chem.
271:
20246-20249,
1996[Abstract/Free Full Text].
4.
Banes, A. J.,
M. Tsuazaki,
J. Yamamoto,
T. Fischer,
B. Brigman,
T. Brown,
and
L. Miller.
Mechanoreception at the cellular level: the detection, interpretation, and diversity of responses to mechanical signals.
Biochem. Cell Biol.
73:
349-365,
1995[Medline].
5.
Burridge, K.,
and
M. Chrzanowska-Wodnicka.
Focal adhesions, contractility and signaling.
Ann. Rev. Cell Dev. Biol.
12:
463-519,
1996[Medline].
6.
Carvalho, R. S.,
J. E. Scott,
and
E. H. Yen.
The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells.
Arch. Oral Biol.
40:
257-264,
1995[Medline].
7.
Chihara, K.,
M. Amano,
N. Nakamura,
T. Yano,
M. Shibata,
T. Tokui,
H. Ichikawa,
R. Ikebe,
M. Ikebe,
and
K. Kaibuchi.
Cytoskeletal rearrangements and transcriptional activation of c-fos serum response element by Rho-kinase.
J. Biol. Chem.
272:
25121-25127,
1997[Abstract/Free Full Text].
8.
Chow, J. W.,
and
T. J. Chambers.
Indomethacin has distinct early and late actions on bone formation induced by mechanical stimulation.
Am. J. Physiol.
267 (Endocrinol. Metab. 30):
E287-E292,
1994[Abstract/Free Full Text].
9.
Duncan, R. L.,
N. Kizer,
E. L. Barry,
P. A. Friedman,
and
K. A. Hruska.
Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.
Proc. Natl. Acad. Sci. USA
93:
1864-1869,
1996[Abstract/Free Full Text].
10.
Duncan, R. L.,
and
C. H. Turner.
Mechanotransduction and the functional response of bone to mechanical strain.
Calcif. Tissue Int.
57:
344-358,
1995[Medline].
11.
Feramisco, J. R.,
and
K. Burridge.
A rapid purification of
-actinin, filamin and a 130,000-dalton protein from smooth muscle.
J. Biol. Chem.
255:
1194-1199,
1980[Abstract/Free Full Text].
12.
Forwood, M. R.
Inducible cyclo-oxygense (Cox-2) mediates the induction of bone formation by mechanical loading in vivo.
J. Bone Miner. Res.
11:
1688-1693,
1996[Medline].
13.
Fox, S. W.,
T. J. Chambers,
and
J. W. Chow.
Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E955-E960,
1996[Abstract/Free Full Text].
14.
Frangos, J. A.,
L. V. McIntire,
and
S. G. Eskin.
Shear stress induced stimulation of mammalian cell metabolism.
Biotechnol. Bioeng.
32:
1053-1060,
1988.
15.
Hill, C. S.,
J. Wynne,
and
R. Treisman.
The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF.
Cell
81:
1159-1170,
1995[Medline].
16.
Hitt, A. L.,
and
E. J. Luna.
Membrane interactions with the actin cytoskeleton.
Curr. Opin. Cell Biol.
6:
120-130,
1994[Medline].
17.
Hung, C. T.,
F. D. Allen,
S. R. Pollack,
and
C. T. Brighton.
What is the role of the convective current density in the real-time calcium response of cultured bone cells to fluid flow?
J. Biomech.
29:
1403-1409,
1996[Medline].
18.
Hung, C. T.,
F. D. Allen,
S. R. Pollack,
and
C. T. Brighton.
Intracellular Ca2+ stores and extracellular Ca2+ are required in the real-time Ca2+ response of bone cells experiencing fluid flow.
J. Biomech.
29:
1411-1417,
1996[Medline].
19.
Hynes, R. O.
Integrins: versatility, modulation and signaling in cell adhesion.
Cell
69:
11-25,
1992[Medline].
20.
Johnston, D. L.,
T. N. McAllister,
and
J. A. Frangos.
Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts.
Am. J. Physiol.
271 (Endocrinol. Metab. 34):
E205-E208,
1996[Abstract/Free Full Text].
21.
Juliano, R. L.,
and
S. Haskill.
Signal transduction from the extracellular matrix.
J. Cell Biol.
120:
577-585,
1993[Medline].
22.
Kimura, K.,
M. Ito,
M. Amano,
K. Chihara,
Y. Fukata,
M. Nakafuku,
B. Yamamori,
J. Feng,
T. Nakano,
K. Okawa,
A. Iwamatsu,
and
K. Kaibuchi.
Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase).
Science
273:
245-248,
1996[Abstract].
23.
Maniotis, A. J.,
C. S. Chen,
and
D. E. Ingber.
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure.
Proc. Natl. Acad. Sci. USA
94:
849-854,
1997[Abstract/Free Full Text].
24.
Otey, C. A.,
F. M. Pavalko,
and
K. Burridge.
An interaction between
-actinin and the
1 integrin subunits in vitro.
J. Cell Biol.
111:
721-730,
1990[Abstract].
25.
Owan, I,
D. B. Burr,
C. H. Turner,
J. Qiu,
Y. Tu,
J. E. Onyia,
and
R. L. Duncan.
Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain.
Am. J. Physiol.
273 (Cell Physiol. 42):
C810-C815,
1997[Abstract/Free Full Text].
26.
Pavalko, F. M.,
and
K. Burridge.
Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of
-actinin.
J. Cell Biol.
114:
481-491,
1991[Abstract].
27.
Pavalko, F. M.,
and
S. M. LaRoche.
Activation of human neutrophils induces an interaction between the integrin
2 subunit (CD18) and the actin-binding protein
-actinin.
J. Immunol.
151:
3795-3807,
1993[Abstract/Free Full Text].
28.
Pavalko, F. M.,
and
C. A. Otey.
Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton.
Proc. Soc. Exp. Biol. Med.
205:
282-293,
1994[Abstract].
29.
Pavalko, F. M.,
G. Schneider,
K. Burridge,
and
S. S. Lim.
Immunodetection of
-actinin in focal adhesions is limited by antibody inaccessibility.
Exp. Cell Res.
217:
534-540,
1995[Medline].
30.
Reich, K. M.,
and
J. A. Frangos.
Effect of flow on prostaglandin E2 and inositol triphosphate levels in osteoblasts.
Am. J. Physiol.
261 (Cell Physiol. 30):
C428-C432,
1991[Abstract/Free Full Text].
31.
Reich, K. M.,
T. N. McAllister,
S. Gudi,
and
J. A. Frangos.
Activation of G proteins mediates flow-induced prostaglandin E2 production in osteoblasts.
Endocrinology
138:
1014-1018,
1997[Abstract/Free Full Text].
32.
Ridley, A. J.,
and
A. Hall.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70:
389-399,
1992[Medline].
33.
Sastry, S. K.,
and
A. F. Horwitz.
Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling.
Curr. Opin. Cell Biol.
5:
819-831,
1993[Medline].
34.
Toma, C. D.,
S. Ashkar,
M. L. Gray,
J. L. Schaffer,
and
L. C. Gerstenfeld.
Signal transduction of mechanical stimuli is dependent on microfilament integrity: identification of osteopontin as a mechanically induced gene in osteoblasts.
J. Bone Miner. Res.
12:
1626-1636,
1997[Medline].
35.
Turner, C. H.,
M. R. Forwood,
and
M. W. Otter.
Mechanotransduction in bone: do bone cells act as sensors of fluid flow?
FASEB J.
8:
875-878,
1994[Abstract/Free Full Text].
36.
Turner, C. H.,
M. R. Forwood,
J. Rho,
and
T. Yoshikawa.
Mechanical loading thresholds for lamellar and woven bone formation.
J. Bone Miner. Res.
9:
87-97,
1994[Medline].
37.
Turner, C. H.,
Y. Takano,
I. Owan,
and
G. A. C. Murrell.
Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats.
Am. J. Physiol.
270 (Endocrinol. Metab. 33):
E634-E639,
1996[Abstract/Free Full Text].
38.
Turner, C. H., Y. Tu, and J. E. Onyia.
Mechanical loading of bone in vivo causes bone formation through
early induction of c-fos but not c-jun of c-myc.
Annals of Biomed. Eng. 24, Suppl 1: S-74, 1996.
39.
Wang, N.,
J. P. Butler,
and
D. E. Ingber.
Mechanotransduction across the cell surface and through the cytoskeleton.
Science
260:
1124-1127,
1993[Medline].
40.
Watanabe, H.,
H. Yamamoto,
H. Agematsu,
K. Miake,
and
J. Sasaki.
Electron microscopic study of the cytoskeleton of osteoblasts in rat alveolar bone: microfilaments and intermediate filaments as demonstrated by detergent perfusion.
Bull. Tokyo Dent. Coll.
34:
89-94,
1993[Medline].
41.
Werb, Z.,
P. M. Tremble,
O. Behrendtsen,
E. Crowley,
and
C. H. Damsky.
Signal transduction through the fibronectin receptor induces collagenase and stromelysin gene expression.
J. Cell Biol.
109:
877-889,
1989[Abstract].
Am J Physiol Cell Physiol 275(6):C1591-C1601
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