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J. Biol. Chem., Vol. 277, Issue 48, 46493-46503, November 29, 2002
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From the Department of Medicine, Baylor College of Medicine,
Houston, Texas 77030
Received for publication, April 16, 2002, and in revised form, September 4, 2002
In the diaphragm muscle we tested the hypothesis
that MAP kinase signaling pathways are activated by mechanical stress
and such signaling pathways are dependent on the direction in which mechanical stress is applied. Although equal magnitudes of
mechanical stress were applied axially and transversely a greater level
of activation of ERK1/2, p38, Raf-1, p90 RSK, Elk-1, and the DNA binding activity of AP-1 transcription factor was produced when the
muscle was stretched transversely than when stretched axially. A
significant up-regulation in protein tyrosine phosphorylation was
observed in axially or transversely loaded diaphragm muscles and the
activation of ERK1/2 was completely inhibited by genistein (protein-tyrosine kinase inhibitor). Pretreatment of muscles with wortmannin (phosphoinositide 3-kinase inhibitor), TMB-8 (antagonist of
intracellular calcium release), GF109203X (PKC inhibitor), or PD98059
(MEK1/2 inhibitor) blocked the activation of ERK1/2 kinases in
response to axial but not to transverse loading. On the other hand,
pretreatment of muscles with protein kinase A inhibitors H-7 and KT5720
completely suppressed the activation of ERK1/2 in response to
transverse loading only. Taken together with the alterations of MAP
kinases and the findings of elevations of downstream transcription
targets, our data are consistent with two distinct MAP kinase signal
transduction pathways in response to mechanical stress.
In vivo, skeletal muscles function in a
three-dimensional mechanical environment in which forces are generated
along the muscle fibers and transmitted transversely across the lateral
surfaces of the muscle cell, as well as at the ends of the muscle cell (1). Any elastic sheet of muscle that is mechanically loaded in the
direction of the muscle fibers may have a length-tension relationship
that is altered from when the sheet is also mechanically loaded in the
direction orthogonal to the muscle fibers (2). Force transmission in
skeletal muscles varies greatly with muscle fiber architecture (3, 4).
Muscle fibers may be organized in parallel, in series, or in a complex
multipinnate arrangement. Muscle fibers in the mouse diaphragm span the
entire length of the muscle and they are arranged in parallel. However,
in vivo, the diaphragm unlike most other skeletal muscles is
mechanically loaded biaxially. That is, in vivo, the
diaphragm is subjected to stresses in the direction of the muscle
fibers and in the direction perpendicular to the fibers in the plane of
the muscle sheet. Therefore, length tension in the direction of the
muscle fibers and transverse to the fibers are important in mediating
signal transduction.
The network of mitogen-activated protein
(MAP)1 kinases is a very
complex signaling system and it acts in many different physiological processes, such as cellular proliferation, differentiation,
inflammatory responses, apoptosis, and development. Recent evidence has
shown that mechanical stresses or forces alter the growth and
differentiation of a number of tissues and cells (5-7). The mechanism
that relates the mechanical forces with the response of skeletal muscle
cells has been investigated recently and several signaling molecules have been demonstrated to play significant roles in mechanical force-initiated signal transduction (8). Among them are the MAP
kinases, which are activated by mechanical stresses. Activation of MAP
kinases may link the effects of mechanical stress to the biochemical
responses and gene expression (9-11). The MAP kinases act via the
regulation of several transcription factors initiating the expression
of a variety of immediate and delayed response genes (12, 13). In
mammalian cells, three parallel pathways of MAP kinases have been
described, i.e. extracellular signal related kinase
(ERK1/2), protein kinase 38 (p38), and c-Jun-N-terminal kinases (JNKs).
These kinases require dual phosphorylation on threonine and tyrosine
residues by specific upstream protein kinase. JNK and p38 are often
referred to as stress-activated protein kinases (SAPK1/JNK and
SAPK2/p38), and ERK1/2 are often described as the kinases involved in
growth factor stimulation (14, 15).
The physiologic significance of activation of MAP kinases by mechanical
forces is not yet understood. In particular, it is not known whether in
skeletal muscles mechanical forces activate specific transcription
factors, e.g. activator protein-1 (AP-1). AP-1 is a
redox-sensitive transcription factor that plays an important role in
cell proliferation, differentiation, and response to various environmental stimuli (16, 17). AP-1 consists of a homodimer and
heterodimers of members of the Jun family (c-Jun, JunB, and JunD) and
heterodimers of the Jun and Fos (c-Fos, FosB, Fra1, and Fra2) families
(18). Activation of AP-1 leads to its binding to the DNA at consensus
sites that in turn regulate the transcription of
AP-1-dependent genes (18, 19). Accumulating evidence
suggests that the activity of AP-1 is regulated at least in part by the MAP kinase signaling pathways (20, 21). The transcriptional activity of
several components of AP-1 transcription factor such as c-Jun,
activating transcription factor 2, c-Fos, and Elk-1 is increased
upon their phosphorylation by ERK1/2 and JNKs (20).
Although, recently it has been established that mechanical stress
activates the ERK1/2, JNKs, and p38 MAP kinases in skeletal muscle
cells (22-30), the pathways that are involved in the activation of
these kinases in response to mechanical stress remain unknown. Furthermore, most of the studies on the activation of MAP kinases were
carried out in vitro using hind limb muscle cells, which are
loaded in vivo along the direction of the long axis of the muscle fibers. In contrast, the diaphragm muscle is mechanically loaded
in vivo with transdiaphragmatic pressure, and therefore, its
muscles are subjected to mechanical forces not only in the direction of
the muscle fibers but also in the direction transverse to the fibers
(1). It is therefore important to investigate the effects of mechanical
forces applied to the diaphragm muscle axially and transversely. In the
present study we investigated the mechanism of activation of ERK1/2 in
response to constant static stretch applied either in the axial
direction of the muscle fibers or in the transverse direction of the
fibers. Additionally, we also report the effects of mechanical stretch
on the activity of AP-1 and Elk-1 transcription factors in muscle
fibers of the diaphragm.
Materials--
PD98059, wortmannin, H-7, and histone H1 were
obtained from Sigma. Genistein, KT5720, KT-93, TMB-8, and GF109203X
were from Calbiochem (San Diego, CA). Anti-JNK1 antibody was obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). GST-Jun-(1-79)
was a kind gift from Dr. B. Su of the M. D. Anderson Cancer Center (Houston, TX). Normal as well as phospho-specific rabbit polyclonal anti-Elk-1 (Ser383), anti-p44/42
(Thr202/Tyr204), anti-p38
(Thr180/Tyr182), anti-Raf-1
(Ser259), and monoclonal phosphotyrosine (Tyr(P)-100)
antibodies were obtained from Cell Signaling Technology (Beverly, MA).
AP-1 consensus oligonucleotide was obtained from Promega (Madison, WI).
Poly(dI·dC) was from Amersham Biosciences.
[ Mice and Tissue Preparation--
Mice (SV129 strain) were housed
and fed in stainless steel cages on a 12 h on and 12 h off
lighting schedule. The animal facility is a virus-free facility.
Experimental protocols have been approved by the Animal Protocol Review
Committee of the Baylor College of Medicine Animal Program (animal
welfare assurance number A-3823-01) and are assigned protocol number
AN-1727. All procedures were conducted in strict accordance with the
United States Public Health Service animal welfare policy. Mice
weighing 21-24 g were anesthetized with an intravenous injection of
pentobarbital (0.5-0.7 ml/kg). The diaphragmatic muscle was excised
from each animal and immediately immersed into a muscle bath containing
a modified Krebs-Ringers solution (in mM: 137 NaCl, 5 KCl,
1 NaH2PO4, 24 NaHCO3, 2 CaCl2, 1 MgSO4, pH 7.4) bubbled with 95%
O2, 5% CO2. The solution was maintained
at a temperature of 25 °C throughout the muscle preparation and
experimental phase of this study. We excised hemidiaphragms that
included a portion of lower ribs and the central tendon. To obtain the
length-tension relationship, four silk suture position markers (7-0 or
8-0 Surgilene) were sutured on the abdominal surface of the left costal
hemidiaphragm. To minimize the boundary effects, all markers were
placed in the central region of the muscle. The markers were placed in
an approximate 1-mm2 square configuration, two pairs of
marker were aligned in the direction along the fibers and two pairs
were aligned in the direction transverse to the muscle fibers.
Ex Vivo Mechanical Loading of the Diaphragm--
We used a
biaxial tissue testing apparatus to apply mechanical stress to the
tissue along the muscle fiber direction as well as transverse to the
fiber direction. The detailed description of this apparatus was
described previously (1). Using the entire costal muscle of the left
hemidiaphragm mechanical loading was applied by passively stretching
the diaphragm in the fibers direction by applying passive tension of
about 0.4 N/cm. This is equivalent to a passive mechanical stretch of
about 50% from the unstressed state. Unstressed length or length of
excised muscle is the shortest length of the muscle and it is
equivalent to the in vivo length of the diaphragm at total
lung capacity. Optimal length is about 125% of this unstressed length.
Therefore, our stretch of 50% places the diaphragm at about 120% of
optimal muscle length. In a different mechanical loading protocol we
stretched the diaphragm muscle in the direction transverse
to the muscle fibers by applying tension of 0.4 N/cm. This is
equivalent to a mechanical stretch of about 20% from the unstressed
state. In each of these two protocols the muscle was held in the
stretched state for 15 min. The total time postmortem did not exceed
1 h.
Thickness Measurements and Data Acquisition--
Unstressed
muscle thickness measurements were obtained from the excised diaphragm
and a digital image of the muscle surface was created as described
previously (31). Briefly, the surface area of the excised tissue was
determined by using Image Tool (version 2.0, ddsdx.uthscsa.edu). Excess
water was gently removed from the surface of the tissue with a
cotton-tipped swab, and the tissue sample was immediately weighed.
Thickness was then computed as t = m/Ad, where
t is muscle thickness in cm, m is muscle mass in
grams (g), A is the surface area of the muscle sample in
cm2, and d is muscle density Strain Calculations--
Mechanical strains were calculated
based on methods established in our previous work (32). Briefly,
coordinates for any three markers are denoted xi and
yi (i = 1, 2, or 3), where three
markers define a triangle in a plane. Subsequently, the four-sutured
markers would define four triangles. For example, triangle A would be
defined by markers 1, 2, and 3 whereas triangle B would be defined by
markers 1, 2, and 4. The displacements of the markers from an
unstressed state to higher stressed states of loading are denoted
ui for along the fibers and vi for transverse to the fibers, for example, as follows.
Assay of ERK1/2, p90 RSK, Elk-1, p38, PKC, and
Raf-1--
After muscles were stretched for 15 min, samples were
washed with phosphate-buffered saline and homogenized in lysis buffer A
(20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.3% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
benzamidine, 1 mM dithiothreitol, and 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM c-Jun Kinase Assay--
The c-Jun kinase assay was performed by
a method described earlier with minor modifications (33). After the
application of mechanical stress to the muscles for 15 min at 25 °C
the muscles were washed with phosphate-buffered saline and cell
extracts were prepared by grinding the muscle in lysis buffer
containing 20 mM HEPES, pH 7.4, 2 mM EDTA, 250 mM NaCl, 0.3% Nonidet P-40, 2 µg/ml leupeptin, 2 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml
benzamidine, and 1 mM dithiothreitol. Cell extracts
(600-700 µg of protein/sample) were immunoprecipitated with 0.5 µg
of anti-JNK1 antibody for 4-6 h at 4 °C. Immune complexes were
collected by incubation with protein A-Sepharose beads for 1 h at
4 °C. The beads were washed three times with lysis buffer followed
by two additional washes with kinase buffer (20 mM HEPES, pH 7.4, 1 mM dithiothreitol, 50 mM NaCl).
Kinase assays were performed for 15 min at 30 °C with
GST-Jun-(1-79) as a substrate (2 µg/sample) in 20 mM
HEPES, pH 7.4, 10 mM MgCl2, 1 µM
dithiothreitol, and 10 µCi of [ Electrophoretic Mobility Shift Assays--
To determine AP-1
activation, electrophoretic mobility shift assays were carried as
described previously (34) with some modification. The muscles, after
being mechanically stretched, were weighed frozen using dry ice and
ground using a manual grinder in low salt lysis buffer containing 10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin,
2.0 µg/ml aprotinin, and 0.5 mg/ml benzamidine (18 µl
buffer/milligram of muscle tissue). The tissue extract in low salt
lysis buffer was allowed to swell on ice for 10 min, vortexed
vigorously for 10 s, followed by two freeze-thaw cycles. The
homogenate was centrifuged for 10 s at 4 °C, the supernatant was removed, and the nuclear pellet was resuspended (5 µl/mg of original tissue weight) in ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 420 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10 mM MgCl2, 20%
glycerol, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 2.0 µg/ml leupeptin, 2.0 µg/ml
aprotinin, 0.5 mg/ml benzamidine) and incubated on ice for 30 min with
intermittent vortexing. Samples were centrifuged for 5 min at 4 °C,
and the supernatant (nuclear extract) was either used immediately or
stored at Statistical Analysis--
All experiments were repeated at least
three times unless otherwise indicated. Results are expressed as
mean ± S.D. Statistical analysis used Student's t
test or analysis of variance to compare quantitative data populations
with normal distribution and equal variance. A value of
p < 0.05 was considered statistically significant unless specified.
Stress-Strain Relationship Along and Transverse to the Muscle
Fibers--
A representative set of uniaxial length tension
relationships of the left costal muscle sheet of the diaphragm in
either the muscle fiber direction or transverse to the muscle fibers is
shown in Fig. 1. These data
demonstrate that tension along the muscle fibers and transverse to the
fibers increase continuously with increasing mechanical stretch.
However, the length tension of the diaphragm in the axial direction is
shifted to the right compared with the length-tension curve in the
transverse plane, that is, the normal diaphragm muscle has a greater
extensibility in the direction of the muscle fibers than transverse to
the fibers. The nonlinear behaviors in the two directions are somewhat
different. The stress-strain relation in the muscle direction is only
mildly nonlinear over the sizable range of strains that our data cover. The nonlinear stiffening in the transverse direction appears to be more
pronounced. Using all normal diaphragms, we computed the average
extension ratio, Activation of ERK1/2 and p90 RSK by Mechanical
Stretch--
Because the diaphragm muscle in the transverse direction
of the muscle fibers is less compliant as compared when loaded axially in the fiber direction (Fig. 1), we investigated the effect of mechanical stress applied axially as well as transversely on the activation of MAP kinases. Our preliminary experiments showed that
mechanical stress of the diaphragm either axially or transversely activates the MAP kinases as early as 5 min at the onset of mechanical stress with maximum activation occurring at 15 min of mechanical loading (data not shown). Therefore, in all MAP kinase assays we
stretched the muscles for 15 min and then the tissue was immediately lysed in lysis buffer. As assayed by phosphospecific immunoblot analysis, stretching of the muscles either axially or transversely resulted in the induction of ERK1/2 phosphorylation (Fig.
2A). However, the level of
phosphorylation of ERK1/2 proteins in response to the applied load
appears to be dependent on the direction along which the mechanical
load was applied. Phosphorylation level of ERK1/2 was significantly
greater in response to mechanical stretch applied in the transverse
direction of the muscle fibers than that applied in the direction of
the muscle fibers.
Because ERK1/2 phosphorylate cytosolic protein p90 RSK (35, 36) we also
studied the effect of mechanical stress on the phosphorylation level of
p90 RSK protein. As shown in Fig. 2B (upper
panel), mechanical stress of muscles led to an elevated level of
phosphorylation of p90 RSK with a significantly higher level of
phosphorylation occurring in those muscles stretched in the direction
transverse to the muscle fibers. The increased phosphorylation level of
ERK1/2 and p90 RSK in response to mechanical stress was not attributed
to an alteration in the level of these proteins in muscle tissues,
because the total amount of these proteins before and after the
application of mechanical stress remained essentially the same (Fig. 2,
A and B, lower panels). Because
mechanical stress was applied equally in the direction of the muscle
fibers and transverse to the fibers (refer to "Experimental Procedures") the difference in the level of activation of ERK1/2 and
p90 RSK was not because of the difference in the magnitude of
mechanical stress between stress applied in the fiber direction and
stress applied in the transverse direction to the muscle fibers. These
data thus clearly show that mechanical stresses activate the ERK1/2 and
p90 RSK to different levels depending on the direction in which
mechanical stresses are applied.
Effect of Mechanical Stretch on the Activity of JNK1 and p38 MAP
Kinase--
Because the level of phosphorylation of ERK1/2 was
dependent on whether mechanical stretch was applied to the diaphragm
muscle axially or transversely, we also studied the effect of
mechanical stretch applied in either direction on the activity of JNK1
and p38 MAP kinase. The JNK1 activity was assayed by phosphorylation of
the GST-c-Jun protein. Interestingly, the mechanical
loading of the muscles led to a drastic increase in the JNK1 activity, however, in contrast to ERK1/2, there was no difference in the level of
activation of JNK1 between the axially stretched and those transversely
stretched diaphragm muscles (Fig.
3A). The activity of p38
kinase was also increased in response to both axial and transverse
stress with significantly higher levels of activation in those
transversely loaded diaphragms (Fig. 3B). The effects of
mechanical loading on MAP kinase activation were specific, as the
activity of a cell cycle kinase cyclin-dependent kinase-2
(cdk2) was not altered (Fig. 3C). Mechanically stretching the diaphragm muscle either axially or transversely did not affect the
total cellular level of JNK1, p38, or cdk2 (data not shown).
PD98059 Inhibits the Phosphorylation of ERK1/2 and
p90 RSK in Response to Axial Loading but Not to Transverse
Loading--
P98059 is a specific inhibitor of the MAP kinase kinase
(MEK1/2), a kinase that phosphorylates ERK1/2 in the classical ERK1/2 signaling pathway (37). We investigated the effect of PD98059 on the
phosphorylation of ERK1/2. Published reports have shown that 25-50
µM PD98059 can completely inhibit the activity of MEK1/2 kinases in all cells including skeletal muscles (37, 38). We found that
pretreatment of the diaphragm for 30 min with 50 µM
PD98059 completely inhibited the phosphorylation of ERK1/2 in axially
loaded muscles (Fig. 4A). On
the other hand, there was no effect of PD98059 on the level of
phosphorylation of ERK1/2 kinases in the transversely loaded muscles.
These results show that MEK1/2 are involved in the activation of ERK1/2
in response to axial stress, whereas different mechanisms of signaling
are involved in the activation of ERK1/2 in response to transverse stress. PD98059 did not have any significant effect on the activation of JNK and p38 MAP kinases (data not shown). The effect of PD98059 on
the phosphorylation level of p90 RSK in the diaphragm was also investigated. Interestingly, pretreatment of the diaphragm with PD98059
completely inhibited the phosphorylation of p90 RSK in those muscles
that were axially loaded, whereas the p90 RSK phosphorylation in
response to transverse loading remained unchanged (Fig. 4B). These results suggest that p90 RSK is only phosphorylated by an ERK1/2-dependent mechanism in response to axial mechanical
stress. It further confirms that MEK1/2 are not involved in the
activation of ERK1/2 in response to transverse loading of the
diaphragm.
Activation of Raf-1 in Response to Axial or Transverse
Stress--
Raf-1, a serine/threonine protein kinase is an important
component of the mitogen- and mechanical-induced signal transduction response (4, 14). To further delineate the pathways involved in the
activation of MAP kinases in response to mechanical loading, the effect
of mechanical stress on the activation of Raf-1 kinase was also
studied. Although not statistically significant an equal amount of
stress applied axially and transversely seems to activate the Raf-1
kinase with somewhat higher level of activation (not statistically
significant) in transversely loaded muscle (Fig. 5).
Protein Tyrosine Phosphorylation Is Required for the
Activation of ERK1/2 in Response to Mechanical
Loading--
Protein tyrosine phosphorylation by both receptor and
nonreceptor protein-tyrosine kinases is known to trigger downstream signaling events in response to various stimuli (39). We next investigated whether mechanical loading of diaphragm muscle can alter
the protein tyrosine phosphorylation in diaphragm muscle. Immunoblotting with phosphotyrosine monoclonal antibody showed that a
significant increase in phosphorylation of approximately 67-kDa protein
was observed in response to both axial and transverse loading of
diaphragm muscles (Fig 6A).
The phosphorylation level was much higher in transversely loaded
diaphragm muscle as compared with those loaded axially. Furthermore, an
additional protein of approximately 85 kDa seems to be phosphorylated
in response to transverse loading only (Fig. 6A).
To investigate whether the protein tyrosine phosphorylation
is required for the activation of ERK1/2 in response to
mechanical loading, the diaphragm muscles were preincubated for 45 min
with 50 µM genistein (a protein-tyrosine kinase
inhibitor) followed by mechanical stretching for 15 min. As shown in
Fig. 6B, pretreatment of diaphragm muscles with genistein
appears to completely inhibit the activation of ERK1/2 in response to
both axial and transverse loading. These data strongly suggest that
protein-tyrosine kinases are involved in the activation of ERK1/2 in
response to both axial and transverse mechanical stress.
Phosphoinositide 3-Kinase (PI3K) Is Involved in Activation of
ERK1/2 in Response to Axial Loading--
Using a specific
and potent inhibitor of PI3K, wortmannin, we investigated the role of
PI3K in the activation of ERK1/2 in response to mechanical loading of
the diaphragm. Pretreatment of muscles with 400 nM
wortmannin resulted in a significant inhibition in the phosphorylation
of ERK1/2, whereas wortmannin did not have any effect on the
phosphorylation level of ERK1/2 in response to transverse loading (Fig.
7).
Role of Intracellular Ca2+,
Calmodulin-dependent Protein Kinase II (CaMKII), and
Stretch-activated (SA) Channels in Activation of ERK1/2
in Response to Mechanical Loading--
Ca2+ ions mediate a
large number of cellular responses by binding to specific intracellular
proteins, which may be considered Ca2+ receptors (40, 41).
The Ca2+ concentration in the cells in response to
mechanical stress is increased by either release of Ca2+
ions from intracellular stores and/or by Ca2+ influx from
the extracellular environment mainly through SA channels (7, 42).
Pretreatment of diaphragm muscle with TMB-8 (an intracellular
Ca2+ antagonist) suppressed the activation of ERK1/2 in
response to axial loading but not to transverse loading (Fig.
8A). The CaMKII appears not to
be involved in the activation of ERK1/2 in response to either axial or
transverse stress, as the inhibition of CaMKII activity by KN-93
(a specific inhibitor of CaMKII) did not have any effect on the
activation of ERK1/2 (Fig. 8, right panel). Furthermore,
pretreatment of diaphragm muscle with 20 µM gadolinium III chloride (an SA channel blocker) or removing the extracellular Ca2+ from the physiologic Krebs-Ringer solution did not
affect the activation of ERK1/2 in response to either axial or
transverse mechanical stress (data not shown). This indicates that
either SA channels or extracellular Ca2+ influx is not
required for the stress-induced activation of ERK1/2 in
diaphragm muscles.
Protein Kinase C Is Required for the Activation of
ERK1/2 in Response to Axial Whereas Protein Kinase A
(PKA) Is Needed for Activation of ERK1/2 in Response to
Transverse Loading--
One of the early steps in cell signaling is
the activation of various isoforms of PKC and PKA (43-45). Because
intracellular Ca2+ mobilization was required for the
activation of ERK1/2 in response to axial loading, we investigated
whether the protein kinase C or protein kinase A have any role in
mechanotransduction in skeletal muscle. Interestingly, H-7, an
inhibitor of protein kinase A completely blocked the activation of
ERK1/2 in response to transverse loading while there was only a
marginal inhibition in response to axial loading (Fig.
9A). Because H-7 is also known
to inhibit the activity of PKC at higher concentrations, we used
KT5720, a very specific inhibitor of PKA having no effects on PKC to
study the activation of ERK1/2, and found that KT5720 only inhibited
the activation of ERK1/2 in response to transverse loading but not to
axial loading, suggesting that PKA is required for the activation of
ERK1/2 in response to transverse stress (Fig. 9B).
GF109203X, a specific inhibitor of PKC, on the other hand inhibited the
activation of ERK1/2 only in response to axial loading, and there was
no effect on the activation of ERK1/2 in response to transverse loading (Fig. 9C). These data thus suggest that protein kinase C
activation is required for the activation of ERK1/2 in response to
axial stress, whereas PKA is needed in transverse stress.
Activation of AP-1 and Elk-1 Transcription Factors by Mechanical
Stretch--
Our data demonstrate a greater level of activation of MAP
kinases in response to transverse stretch than axial stretch (Figs. 2
and 3). We wondered if such activation would lead to the activation of
transcription factors that are the phosphorylation targets of MAP
kinases. Using the electrophoretic mobility shift assay, we
investigated the effect of mechanical stretch of diaphragm muscle
sheets in either the fiber or transverse fiber direction on the
activation of the AP-1 transcription factor. As shown in Fig.
10 an increased DNA binding activity of
AP-1 was observed as a result of mechanical stress. Furthermore, the
level of activation of AP-1 was greater in the transversely loaded
muscles as compared with the axially loaded muscles.
We also investigated the effect of mechanical stress on the activation
of Elk-1. As demonstrated in Fig. 11,
Elk-1 is strongly activated when the diaphragm muscle sheet was
stretched in the transverse fiber direction than when stretched in the
axial direction. These results thus show that a greater level of
activation of MAP kinases in the transversely loaded diaphragms is
linked to activation of additional downstream targets of this signal
transduction pathway.
We show that the diaphragm muscle responses to mechanical
stress by activating the ERK1/2, JNKs, and p38 MAP kinases.
Furthermore, the mechanical stress activates the Raf and the p90
ribosomal S6 kinase (p90 RSK) as well as the transcription factors AP-1 and Elk-1. We also show that there are two distinct signaling pathways
activated in response to mechanical stress applied axially and
transversely to muscle fibers of the diaphragm. In particular, our data
show that PI3K, PKC, and MEK1/2 are activated when the mechanical
stress of the diaphragm is applied axially but not activated when
stress is applied transversely. On the other hand, cyclic
AMP-dependent PKA is activated only in response to
transverse mechanical stress. This is despite the fact that we applied
the same magnitude of mechanical stress axially and transversely to the
muscle fibers.
Mechanism of Activation of ERK1/2 Signaling Pathways by
Axial and Transverse Loading of Diaphragm Muscle--
The diaphragm is
mechanically loaded in vivo with transdiaphragmatic
pressure, and therefore, its muscles are subjected to mechanical forces
not only in the direction of the muscle fibers but also in the
direction transverse to the fibers. Therefore, it is important to study
mechanical signal transduction not only when mechanical stress is
applied along the muscle fibers but also when mechanical stress is
applied in the transverse direction to the fibers. Our experiments were
conducted such that mechanical stress was applied in either the
direction of the muscle fibers or transverse to the fibers. In our
experimental protocols axial stress applied to the muscle was
essentially the same as that applied transversely, and data in Fig. 1
show that this resulted in mechanical stretch that is about twice as
much in the axial direction than in the transverse fiber direction. The
stress-strain relationships shown in Fig. 1 clearly demonstrate that
muscle compliance is small and extensibility is less in the transverse direction to the muscle fibers compared with those in the direction of
the muscle fibers. That is when mechanical stress was applied equally
in either direction the resistance to mechanical stretch is greater in
the transverse direction of the muscle fibers than in the direction of
the fibers. The greater activation of ERK1/2, p38, and Raf-1 kinases,
as well as the transcription factors, AP-1 and Elk-1, in response to
transverse mechanical stress are consistent with greater transverse
muscle stiffness compared with axial stiffness.
Earlier reports have shown that mechanical tension alone can regulate
cell growth and metabolism and the intracellular signaling proteins may
link changes in the tension to this cellular adaptation (46). Recently
it has been shown that mechanical stress stimulates the signaling
pathways of ERKs, JNKs, and p38 MAP kinases in a variety of cell lines
including skeletal muscle cells (8, 9, 22, 23, 29). In all these
studies, however, the investigators applied axial stress to myocytes
isolated from hind limb muscles. In agreement with these studies we
found that axial mechanical stress activates ERK1/2, JNK1, and p38 MAP
kinase. Additionally, we have shown that mechanical stress activates
p90 RSK and Elk-1, the downstream targets of ERK1/2.
In recent years, the effect of mechanical axial stress on cell growth
and regulation of expression of several genes has been investigated.
However, there is little published work directed toward understanding
the biochemical signaling pathways initiated by different mechanical
stresses. The ERK pathways involve the sequential phosphorylation and
activation of serine kinase Raf, MAP kinase (MEK1/2), and two ERK
isoforms (ERK1/ERK2). The ERK1/2 phosphorylate and activate cytosolic
substrates such as p90 RSK and nuclear transcription factors such as
c-Myc, Elk-1, and c-Fos (14, 18). The use of specific inhibitors
has been a powerful tool in delineation of the downstream MAP signaling
in response to numerous stimuli. It has been reported that PD98059 is a
highly specific inhibitor of MEK1/2 with very little inhibition
reported on other members of MAP kinase family (37, 38). Our results using PD98059 suggest the involvement of MEK1/2 in the activation of
ERK1/2 only in response to axial stress. Interestingly, PD98059 did not
have any effect on the phosphorylation level of ERK1/2 in response to
transverse stress indicating that different kinases might be involved
in the phosphorylation of ERK1/2 during transverse loading of the
diaphragm muscle. This is further supported by a higher level of
activation of ERK1/2 in response to transverse stress as compared with
axial stress. Although, ERK1/2 have been proposed to be the direct
phosphorylation target of the MEK1/2 in the classical signal
transduction, this pathway is not a universal phenomenon. Signal
transduction in cells involves cross-talk, interconnection between
signaling pathways, and shared substrates among multiple enzymes (47,
48). The increased phosphorylation of ERK1/2 in response to either form
of mechanical stress also led to an increase in the activation of p90
RSK. Similar to ERK1/2 treatment with PD98059 completely inhibited the
activation of p90 RSK in response to axial stress, whereas no effect
was observed in the case of transverse loading. These results suggest
that application of mechanical stress triggers the activation of p90 RSK via the ERK1/2 mechanism. PD98059 did not affect the activity of
JNK1 and p38 MAP kinase (data not shown), suggesting that ERK1/2 are
the only MAP kinases that are phosphorylated by MEK1/2 in response to
axial mechanical stress and the activity of JNKs and p38 MAP kinase is
regulated by different pathways. Indeed, the cell signaling pathways
that activate JNK1 and p38 MAP kinases in response to various stimuli
have been well established (14), however, the pathways that lead to the
activation of JNKs and p38 MAP kinase in response to mechanical loading
remain unknown.
Despite a great deal of research in the last decade, the
mechanotransduction remains poorly understood. In the absence of specific cell surface receptors, it remained unclear how mechanical forces are converted into intracellular signals coupled with nuclear gene expression. Some investigators have proposed that
transduction of mechanical signals to biological signals occur via the
extracellular matrix/integrin/cytoskeletal axis (7, 49). Mechanical
stress causes deformation of the sarcolemma, which may directly or
indirectly cause conformational changes in protein (and subsequent
activation of them) that are anchored to the inner surface of cell
membranes, or in transmembrane proteins. Several effectors enzyme such
as protein-tyrosine kinases, phospholipases, and protein kinase C isoenzymes, ion channels, such as the SA channels, are examples of
sarcolemmal proteins that might be affected by mechanical stresses (7).
To understand the upstream events that lead to the activation of MAP
kinases in response to axial and transverse mechanical loading, we
investigated the role of several effector molecules and second
messengers. Interestingly, there was a significant increase in the
protein tyrosine phosphorylation of an approximately 67-kDa protein
upon the application of mechanical stress to the diaphragm muscle. The
phosphorylation level, however, was much higher in transversely loaded
diaphragm muscle as compared with axially loaded muscle. Furthermore,
there was an additional tyrosine phosphorylation of an approximately
85-kDa protein in transversely loaded diaphragm muscle (Fig.
6A). The protein tyrosine phosphorylation seems to be
required for the activation of ERK1/2 in response to both axial and
transverse loading as the inhibition of protein-tyrosine kinases
activity by genistein abrogated the mechanical stress-induced phosphorylation of ERK1/2 in diaphragm muscle (Fig. 6B).
What role protein-tyrosine kinase plays in the activation of ERK1/2 in
response to mechanical stress remains a subject of much speculation. Some recent reports, however, suggested that focal adhesion kinase, a
nonreceptor tyrosine kinase, localized at focal adhesion is involved in
mechanosensing in fibroblast and endothelial cells (50, 51). Several
other reports also suggested that members of Src family of
protein-tyrosine kinases such as c-Src constitute a part of the
mechanotransduction in endothelial cells in response to shear stress
(Ref. 52, and references therein). Although, the nature of
protein-tyrosine kinase is not known at present, our data suggest that
protein-tyrosine kinase are involved in stress-induced activation of
ERK1/2 in the diaphragm muscle.
Another striking observation of the present investigation is the
involvement of PKC in response to axial stress whereas PKA was involved
in response to transverse stress. The activation of ERK1/2 of the
diaphragm muscle in response to applied axial but not transverse
mechanical stress requires the intracellular mobilization of
Ca2+ ions as antagonist of intracellular Ca2+
TMB-8 blocked the activation of ERK1/2 (Fig. 8). ERK1/2 activation in
response to either axial or transverse stress does not involve SA
channels or the extracellular Ca2+ influx (data not shown).
Furthermore, the inhibition of PKC activity also led to the inhibition
of ERK1/2 activation in response to axial loading only (Fig.
9C), suggesting that Ca2+ ions plays an
important role in mediating the cellular response in axial loading of
skeletal muscle fibers. In addition to increasing the activity of PKC,
elevation of free Ca2+ level in the cells also results in
its binding and activation of calmodulin (CaM), which consequently
modulates the activity of protein kinases and phosphatases (40, 41).
One such enzyme Ca2+/calmodulin-dependent
protein kinase II (CaMKII) that has broad substrate specificity and
mediates various biological responses (53) is, however, not involved
because the inhibition of CaMKII with KT-93 did not alter the
activation of ERK1/2 in response to mechanical stress (Fig. 8,
right panel).
In contrast to axial loading, the activation of Ca2+ ions
or PKC is not required for the activation of ERK1/2 in response
to transverse loading. Instead, cyclic AMP-dependent PKA is
needed for the activation of ERK1/2 in response to transverse loading. PKA mediates several of the effects of the elevated levels of cAMP in
the cells and has been reported to both activate and inhibit the ERK1/2
signaling pathway either directly or through the activation of Rap1
(45). Although both PKA and PKC can modulate the activity of ERK1/2
pathways by phosphorylation of Raf kinases, PKA preferentially targets
B-Raf, whereas PKC alters the activity of the Raf-1 isoform (7, 45,
54, 57).
PI3K, a lipid kinase complex, is another important regulatory protein
that is involved in the initiation of different signaling pathways and
regulating the major functions of the cell (55, 56). The precise role
of PI3K in MAP kinase signaling pathways is not very clear. Both
PI3K-dependent and PI3K-independent activation of MAP
kinases has been reported (57-61). Ikeda et al. (62) have shown that the activation of ERK1/2 in endothelial cells in response to
cyclic strain is dependent on the PI3K and p21ras. Similar to
these findings our results with wortmannin suggest that the
activation of ERK1/2 pathways in response to axial stress of the
diaphragm involves PI3K (Fig. 7). However, the same is not true
in the case of transverse loading. We did not get any inhibition in the
activation of ERK1/2 in response to transverse loading by wortmannin
even when it was used at higher concentrations (up to 1 µM, not shown). How PI3K regulates the activation of ERK/12 in response to axial mechanical stress remains speculative. The
main effectors of PI3K include the mitogen transducing signaling proteins (protein kinase C, phosphoinoside-dependent
kinases, and small G-proteins) that are activated either via their
interaction with the lipid products of PI3K such as
phosphatidylinositol 3,4,5-P3 or PI3K-dependent
phosphorylation of proteins. Activation of PI3K activates
phosphoinositide-dependent kinase-1, which can directly or
along with phosphatidylinositol 3,4,5-P3 modulate the
activity of various isoform of PKC (7, 44, 57). There are also reports that suggest that lipid products of PI3K can cause the release of
Ca2+ stores and hence activate the
Ca2+-dependent signaling pathways (42, 44).
Activation of AP-1 Transcription Factor by Mechanical
Stress--
The present study for the first time demonstrates that
mechanical stress can activate the DNA binding activity of the AP-1 transcription factor in skeletal muscles. Although the role of AP-1 in
mechanical stress response of skeletal muscles is still recondite, the
activity of AP-1 has been shown to be very important for various
cellular responses (18) and may have a role in mechanotransduction through modulation of the late load-response genes through an as yet
undefined transcription cascade mechanism. Indeed, increased mRNA
levels of several genes including insulin-like growth factor I (63),
fibroblast growth factor (64), myogenic regulatory growth factor (65),
c-Jun (66), and smpx (67) have been reported in response to mechanical
stretch in skeletal muscle cells. Vandenburgh et al. (68)
have shown that the activity of cyclooxygenase increases within 4 h of initiating mechanical stretch in skeletal muscle cells. Similarly,
the expression of the nitric-oxide synthase (NOS) gene is augmented
following mechanical stimulation of rat skeletal muscle cells (69). The
promoter region in either genes of COX and NOS has been reported to
contain the consensus binding sites for the AP-1 transcription factor (70-72). The activation of AP-1 by mechanical stress thus suggests that one mechanism by which mechanical loading could lead to the increased expression of either COX or NOS is through the activation of
AP-1 transcription factor.
The exact mechanism of activation of AP-1 in response to mechanical
stress is not understood, however, we observed a significantly higher
level of AP-1 activity in transversely loaded muscles as compared with
those loaded axially. AP-1 is a collective term referring to dimeric
transcription factors composed of Jun, Fos, Elk-1, or activating
transcription factor subunits that bind to a common DNA site, the
AP-1-binding site (18). The transcriptional activity of several AP-1
subunits is increased upon their phosphorylation by MAP kinases,
especially ERK1/2 and JNK1 (20, 73). We also found that the
phosphorylation level of Elk-1, a physiological substrate of ERK1/2, is
increased by application of axial or transverse loading (Fig. 11). It
is therefore possible that the increased activation of AP-1 in response
to mechanical stress is a direct result of the activation of MAP
kinases. Based on our findings in this report we provide here a
schematic model of the activation of MAP kinases and AP-1 transcription
factor (Fig. 12). The schematic show
clearly that there are two distinct signaling pathways in the diaphragm
as function of the direction of applied mechanical stress. It is very
possible that those distinct pathways exist in all skeletal muscles
that are mechanically loaded in vivo with axial and
transverse stress and those include the diaphragm, abdominal wall, and
intercostal muscles.
In summary, our study provides the first evidence that mechanical
signaling is directional in the skeletal muscle cells of the
diaphragm and also demonstrates an up-regulation in the activity of
specific transcription factors in response to mechanical stress. Because most of the intracellular signaling pathways lead to an altered
gene expression, our results support the hypothesis that gene
expressions can be differentially regulated depending on the direction
in which mechanical forces are applied.
We thank Dr. Tony Eissa for discussion about
the data and review of the manuscript. We also thank Dr. Deshen Zhu for
invaluable technical assistance.
*
This work was supported by National Institutes of Health
Grants RO1-63134 and RO1-46230 (to A. B.) and HL45721 (to M. B. R.).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. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, September 6, 2002, DOI 10.1074/jbc.M203654200
The abbreviations used are:
MAP, mitogen-activated protein;
ERK1/2, extracellular-regulated kinase-1 and
2;
JNK1, c-Jun N-terminal kinase;
p90 RSK, p90 ribosomal S6 kinase;
MAPK, mitogen-activated protein kinase;
AP-1, activator protein-1;
PI3K, phosphoinositide 3-kinase;
PKC, protein kinase C;
PKA, protein
kinase A;
CaMKII, calmodulin-dependent protein kinase II;
SA, stretch-activated;
FAK, focal adhesion kinase;
N, newton;
GST, glutathione S-transferase;
MEK1/2, mitogen-activated protein
kinase kinase kinase;
PI3K, phosphoinositide 3-kinase.
Distinct Signaling Pathways Are Activated in Response to
Mechanical Stress Applied Axially and Transversely to Skeletal Muscle
Fibers*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (specific activity, 3000 (111 TBq) Ci/mmol)
was obtained from PerkinElmer Life Sciences (Boston, MA).
1.06
g/cm3 (31). Each loading cycle began at the unstressed
length (defined as the muscle length that produced no passive tension).
Stress was computed as applied tension divided by muscle thickness of the unstressed tissue (stress = tension/thickness), where stress is in N/cm2. Tension is computed by the measured force in
grams and divided by the width of the tissue clamp and is measured in
N/cm. In the direction of the muscle fibers clamp width was 10 mm,
whereas muscle width in the transverse fiber direction was 4 mm. The
force data for either axis was collected at a sample rate of 10 Hz
using a data acquisition board (model Lab-PC-1200/AI, National
Instruments) and LabVIEW software (version 4.0). The force data were
stored in an ASCII file for post-analysis of the length-tension relationships.
This equation, with known values of the displacements and the
coordinates of three markers substituted for ui,
xi, and yi, provides a set of
three equations for the three coefficients a1,
a2, and a3. Similarly,
the coordinates of the markers provide information for the following
equation.
(Eq. 1)
The values of the coefficients a2,
a3 etc. were used to find the partial
derivatives that were substituted into the following equations defined
to calculate strains.
(Eq. 2)
(Eq. 3)
(Eq. 4)
(Eq. 5)
x denotes strain developed along the fibers,
y denotes strain developed transverse to the fibers, and xy denotes shear strain. Strains were computed relative to
unstressed length. Unstressed length was defined as the length of the
markers in the absence of applied forces. The final strains computed as
the average of the strains from the four triangles. For small strains,
x and y are equivalent to the fractional change
of muscle fiber length relative to the unstressed length in their
respective direction. Our mechanical data are plotted using extension
ratios (1.0 + strain values).
-glycerophosphate). The protein concentration of each
sample was measured using the Bio-Rad protein assay reagent. An 80-µg
aliquot of protein was resolved on each lane on 10% SDS-PAGE,
electrotransferred onto nitrocellulose membrane, and probed with the
phosphospecific anti-p44/42 MAPK
(Thr202/Tyr204) or anti-p38
(Thr180/Tyr182) MAP kinase or anti-Raf-1
(Ser259), anti-Elk-1 (Ser383), or p90 RSK
(Ser380) antibodies (Cell Signaling Technology, Inc.)
raised in rabbits and detected by chemiluminescence (ECL, Amersham
Biosciences). The bands obtained were quantitatively assessed with
Personal Densitometer Scan version 1.30 using ImageQuant software
version 3.3 (Amersham Biosciences). Total amount of these
kinases in the cell extracts was determined by using antibodies, which
bind to both phosphorylated and unphosphorylated proteins.
-32P]ATP. Reactions
were stopped with the addition of 20 µl of 2× SDS sample buffer,
boiled for 5 min, and subjected to 10% SDS-PAGE. GST-Jun-(1-79) was
visualized by staining with Coomassie Brilliant Blue, and the dried gel
was exposed to x-ray film to see the radioactive bands of
GST-c-Jun-(1-79).
70 °C. The protein content was measured using the
Bio-Rad protein assay reagent. Electrophoretic mobility shift assays
were performed by incubating 20 µg of nuclear extract with 16 fmol of
the 32P-end-labeled AP-1 consensus oligonucleotide
5'-CGCTTGATGAGTCAGCCGGAA-3' (underline indicates AP-1
binding sites) for 15 min at 37 °C. The incubation mixture included
2.5 µg of poly(dI·dC) in a binding buffer (25 mM HEPES,
pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 1%
Nonidet P-40, 5% glycerol, 50 mM NaCl). The DNA-protein
complex thus formed was separated from free oligonucleotide on 7.5%
native polyacrylamide gels using buffer containing 50 mM
Tris, 200 mM glycine, pH 8.5, and 1 mM EDTA.
The gel was then dried and the radioactive bands were visualized by
exposing the gel to the x-ray film for a suitable amount of time. The
bands obtained were quantitatively assessed with Personal Densitometer
Scan version 1.30 using ImageQuant software version 3.3 (Amersham
Biosciences). The specificity of the AP-1 band was confirmed by cold
competition assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, at tension of ~0.4 N/cm along the fiber
direction and found to be greater in the direction of the muscle
fibers than transverse to the fibers (along fibers, = 1.5;
transverse to fibers, = 1.2).
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Fig. 1.
In vitro length-tension
relationships of a normal mouse diaphragm. Muscle length is
expressed as a fraction of muscle unstressed length (length at which no
passive force is applied). The uniaxial data along the direction of the
muscle fibers is shown with 
and 
. The open circles
indicate the stretching of the muscle tissue along the fibers, and the
closed circles indicate the unloading in the direction along
muscle fibers. Uniaxial data for loading transverse to the direction of
the muscle fibers are shown with 
and +. The diamonds
indicate the stretching curve of the muscle transverse to fibers, and
crosses indicate the unloading curve. The data demonstrate
that extensibility and compliance is much smaller when stresses are
applied in the transverse direction of the muscle fibers than when
applied in the axial direction of the fibers.
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Fig. 2.
Activation of ERK/12 (A) and
p90 RSK (B) by loading of muscles in the direction
along and transverse to the fiber orientation. The diaphragm
muscles were exposed to different passive mechanical loadings for 15 min followed by their lysis in lysis buffer. Equal amounts of proteins
were subjected to immunoblot analysis with phosphorylated ERK1/2 and
p90 RSK antibodies. The total amount of the proteins was determined by
using normal antibodies against ERK1/2 and p90 RSK proteins. A
representative blot and the quantitation of multiple experiments shown
here shows that loading of muscles along or transverse to the direction
of fiber orientation activates ERK1/2 and p90 RSK (top
panels) and an equal amount of ERK1/2 and p90 RSK protein was
present in each lane (bottom panels).
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Fig. 3.
Effect of mechanical loading of diaphragm
muscles on the activity of JNK1 (A) and p38 MAP kinase
(B), and cyclin-dependent kinase 2 (cdk2)
(C) activity. Diaphragm muscles were stretched
for 15 min in the direction along or transverse to the fiber
orientation. Muscles were then immediately lysed in the lysis buffer.
The JNK1 and cdk2 activity were determined by immunoprecipitating equal
amounts of proteins with anti-JNK1 and anti-cdk2 antibodies,
respectively, followed by in vitro kinase assays using
GST-c-Jun-(1-79) for JNK1 or histone H1 for cdk2 as substrates. The
activity of p38 MAP kinase was determined by immunoblot analysis with
phosphospecific p38 MAP kinase antibodies. The result presented here
shows that both longitudal and transverse loading of the diaphragm
muscle activate JNK1 and p38 MAP kinase, whereas no effect was observed
on the activity of cdk2.
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Fig. 4.
Effect of PD98509 on the activation of ERK1/2
(A) and p90 RSK (B). The
diaphragm muscles were treated for 30 min in 50 µM
PD98509 followed by stretching of the muscles either axially or
transversely for 15 min. The muscles were lysed in lysis buffer and
equal amounts of proteins were subjected to immunoblot analysis using
phospho-ERK1/2 and phospho-p90 RSK antibodies. The representative
blot and the quantitation of multiple experiments using the
densitometer presented here show that PD98059 inhibited the
activation of ERK1/2 and p90 RSK in response to axial stress but not in
response to transverse stress.
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Fig. 5.
Effect of mechanical loading of diaphragm
muscles on the activity of Raf-1. Muscles were mechanically loaded
in either the axial or transverse direction of the diaphragm muscle
fibers and the muscles were immediately lysed in a lysis buffer at the
end of a 15-min constant mechanical stretch. The cell extracts were
analyzed by immunoblotting with phospho-Raf-1 antibodies. The
representative blot and the quantitation from three experiments show
that the Raf-1 activity is increased by either axial or transverse
loading with somewhat slightly higher levels of Raf-1 activation in
transversely loaded muscles as compared with axially loaded
muscles.
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Fig. 6.
Involvement of protein-tyrosine kinases in
mechanical stretch-induced activation of ERK1/2. A,
diaphragm muscles were mechanically loaded for 15 min in either the
axial or transverse direction of the muscle fibers followed by its
lysis in lysis buffer. The cell extracts were analyzed by
immunoblotting with phosphotyrosine (Tyr(P)-100) monoclonal antibodies.
The representative blot shows that the protein tyrosine phosphorylation
is increased upon mechanical loading. B, diaphragm muscles
were treated with 50 µM genistein for 45 min followed by
loading of muscles along or transverse to the direction of the muscle
fiber for 15 min. A representative blot and quantitation from two
independent experiments show that genistein inhibits the
phosphorylation of ERK1/2 in response to either axial or transverse
mechanical loading.
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Fig. 7.
Effect of wortmannin on the activation of
ERK1/2 in response to mechanical loading. Diaphragm muscles were
treated with 400 nM wortmannin for 30 min followed by
loading of muscles along or transverse to the direction of muscle fiber
for 15 min. The tissue extracts were made and equal amounts of protein
were subjected to immunoblot analysis using phospho-ERK1/2 antibodies.
A representative blot and the quantitative data from three independent
experiments presented here clearly show that wortmannin inhibits the
ERK1/2 phosphorylation when muscles are loaded in the direction of the
muscle fibers but not in the transverse direction of the fibers.
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Fig. 8.
Involvement of Ca2+, and CaMKII
in the activation of ERK1/2 in response to mechanical loading.
Diaphragm muscles were pretreated with 20 µM TMB-8 or 10 µM KN-93 for 45 min followed by the application of
mechanical loading of muscles in the direction of the muscle fibers or
transverse to the fiber for 15 min. Data presented here show that TMB-8
(an intracellular Ca2+ antagonist) inhibits the activation
of ERK1/2 only in response to axial loading. KN-93 (an inhibitor of
CaMKII) did not affect the activation of ERK1/2 in response to either
axial or transverse mechanical loading.
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Fig. 9.
Role of PKC and PKA in the activation of
ERK1/2 in response to mechanical loading. Diaphragm muscles were
pretreated with 20 µM H-7 (A), 1 µM KT5720 (B), or 5 µM GF109203X
(C) for 45 min followed by mechanical loading of muscles in
either the fiber or transverse to the fiber direction for 15 min.
Consequently we conducted immunoblotting with phospho-ERK1/2
antibodies. Representative data from two independent experiments
suggest that PKA inhibitors H-7 and KT5720 inhibit the activation of
ERK1/2 in response to transverse stress, whereas PKC inhibitor
GF109203X inhibits the activation of ERK1/2 in response to axial
stress.
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Fig. 10.
Effect of mechanical loading of diaphragm
muscle on the DNA binding activity of AP-1. Diaphragm muscles
stretched along the muscle fibers or transverse to the fibers were
subjected to extraction of nuclear proteins. Equal amounts of nuclear
proteins were allowed to bind with 32P-labeled
oligonucleotide containing AP-1 consensus sites. The DNA-protein
complex was analyzed using the electrophoretic mobility shift assay as
described under "Experimental Procedures." The loading of muscles
along the muscle fibers (lane 3) as well as transverse to
the fibers (lane 5) increased the DNA binding activity of
AP-1. Furthermore, there is a higher level of activity when muscle was
loaded transversely than when muscle was loaded axially. The
specificity of the bands was confirmed by using an excess of unlabeled
oligonucleotide in the DNA-protein complex of transversely loaded
muscles.
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Fig. 11.
Effect of mechanical loading of diaphragm
muscles on the activity of Elk-1. Diaphragm muscles were subjected
to mechanical stress applied either axially in the muscle fiber
direction or transverse to the fibers. The muscles were immediately
lysed in a lysis buffer. The activity of Elk-1 was determined by
immunoblot analysis with phospho-specific Elk-1 antibodies. The data
shown here is representation of three experiments carried out
independently.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 12.
Possible mechanism of activation of
ERK1/2 and AP-1 in skeletal muscles. Schematic representation of
the potential mechanism of activation of ERK1/2 and AP-1
transcription factor in response to mechanically loading of skeletal
muscles either axially or transversely as supported by the data.
Despite activation of ERK1/2 by either form of mechanical stress, the
MEK1/2, PI3K, and PKC are involved in the activation of ERK1/2 in
response to axial mechanical stress whereas PKA is involved only in
response to applied transverse stress.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Pulmonary and Critical
Care, Suite 520B, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030. Fax: 713-798-3619; E-mail:
boriek@bcm.tmc.edu.
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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