1 Department of Integrative
Biology, Components of signaling pathways for mechanotransduction during
load-induced enlargement of skeletal muscle have not been completely
defined. We hypothesized that loading of skeletal muscle would result
in an adaptive increase in the expression of two focal adhesion complex
(FAC)-related proteins, focal adhesion kinase (FAK) and paxillin, as
well as increased FAK activity. FAK protein was immunolocalized to the
sarcolemmal region of rooster anterior latissimus dorsi (ALD) myofibers
in the middle of the ALD muscle. FAK (77 and 81%) and paxillin (206 and 202%) protein concentrations per unit of total protein in Western
blots increased significantly after 1.5 and 7 days, but not after 13 days, of stretch-induced hypertrophy-hyperplasia of the ALD muscle. FAK autokinase activity in immunoprecipitates was increased after 1.5, 7, and 13 days in stretched ALD muscles. To determine whether increased
FAK and paxillin protein concentrations are associated with hypertrophy
and/or new fiber formation, two additional experiments were performed.
First, during formation of primary chicken myotubes (a model of new
fiber formation), FAK protein concentration (63%), FAK activity
(157%), and paxillin protein concentration (97%) increased compared
with myoblasts. Second, FAK (112% and 611%) and paxillin (87% and
431%) protein concentrations per unit of total protein in the soleus
muscle increased at 1 and 8 days after surgical ablation of the
synergistic gastrocnemius muscle (a model of hypertrophy without
hyperplasia). Thus increases in components of the FAC occur in
hypertrophying muscle of animals and in newly formed muscle fibers in
culture. Furthermore, increased FAK activity suggests a possible
convergence of signaling at the FAC in load-induced growth of skeletal muscle.
hypertrophy; mechanotransduction; focal adhesion complex
THE PRIMARY TRANSMEMBRANE components of focal adhesion
complexes (FACs) are integrins, a large family of transmembrane
heterodimers (7). Members of this protein superfamily act as receptors
for extracellular matrix components on the outside of the cell and interact with the cytoskeletal components of focal adhesions on the
cell interior (7). Integrin aggregation results in accumulation of the
focal adhesion proteins tensin and focal adhesion kinase (FAK) at FACs
(17, 40). Occupancy of the integrin receptor by extracellular matrix
proteins results in the accumulation of vinculin, talin, and
FAK is a phosphoprotein, the phosphorylation state of which can be
altered by a variety of stimuli. FAK, in concert with integrins, has a
central role in focal adhesion assembly (7). Indeed, it is widely
accepted that FAK may be a point of convergence in the actions of
integrins and growth factors (11, 19, 20). Increased tyrosine
phosphorylation of FAK has been observed in cell culture after an
integrin-dependent transduction of force by cell attachment (8), by
mechanical strain/stress to cells (16, 38, 44, 47), or by growth factor
signals such as oncogenes, insulin, vascular endothelial growth factor,
and some neuropeptides (1, 5, 53). A mechanical effect has not yet been
reported in skeletal muscle. Tyrosine phosphorylation at distinct
residues of FAK can modify biological activity by stimulating its
tyrosine kinase activity (26) and modulating FAK interaction with
downstream signaling molecules (41). Increased FAK phosphorylation
promotes cytoskeleton reorganization, which influences transcriptional
activity (13) and translation (28). A more immediate target of
phosphorylated FAK is the phosphorylation of paxillin, a 68-kDa
cytoskeletal protein that is recruited to FACs when integrins cluster
because of occupancy of their receptors (18).
The fact that loading increases the mass of skeletal muscle has been
appreciated for >2,000 years (2). How mechanical overload is
converted to chemical signals that induce skeletal muscle enlargement (50) is not completely understood. Ingber's tensegrity model (19)
predicts that living cells and nuclei are "hard wired" and
respond immediately to mechanical stresses transmitted to FACs. FACs
function as molecular bridges between the extracellular matrix,
integrin receptors, and actin microfilaments, by stabilizing cell
adhesions and providing a continuous path for mechanical signal
transfer across the cell membrane (20, 37). This information caused us
to speculate that loading of skeletal muscle would increase the
transmission of mechanical forces per focal adhesion from outside and
inside the muscle cell. We further speculated that this process would
be associated with a compensatory increase in the quantity of FACs as
the muscle cell adapted to normalize the force per FAC. To test some of
this speculation, we hypothesized that the concentrations of two FAC
components, FAK and paxillin proteins, as well as FAK activity, would
increase when skeletal muscle of living animals enlarged in response to
mechanical loading. To optimize the possibility of a positive finding,
the first model tested was a hypertrophy-hyperplasia model, i.e.,
stretch-induced enlargement of the anterior latissimus dorsi (ALD)
muscle of young roosters. After an increase in FAK and paxillin protein
concentrations was observed in the stretched ALD muscle, two additional
models were employed to establish whether these increases in FAK and paxillin also occur in hypertrophy alone and/or in new muscle fiber formation.
Animal care.
The Institutional Animal Welfare Committee at the University of
Texas
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-actinin to link integrins to the cytoskeleton at FAC, resulting in
the phosphorylation of tensin and FAK (31, 37). To estimate changes in
FAC density during load-induced enlargement of skeletal muscle, we
measured the densities of the focal adhesion proteins FAK and paxillin
in loaded muscles of animals. During muscle contraction, skeletal
muscle fiber transmits loads from its cytoskeleton to the extracellular
matrix via focal adhesions (35). It is thus reasonable to speculate
that increased loading of muscle would be transmitted through focal adhesions.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Health Science Center at Houston (UTHSCH) had approved all
protocols used in these experiments. Young roosters (White Leghorn,
Texas A & M University, College Station, TX) were received at 5-7
wk of age. They were housed in a run and received chicken chow and
water ad libitum in the animal care facilities at the UTHSCH. The left
wing was loaded with a weight corresponding to 10% of their body
weight for 1.5, 7, or 13 days, as previously described by Carson et al.
(9, 10). The ALD muscle was harvested after anesthesia [ketamine
HCl, xylazine, and acepromazine (100, 4, and 6 mg/kg body wt sc,
respectively)], snap-frozen in liquid nitrogen, and stored in
sealed tubes at
80°C until use.
80°C until they were further processed. Animals were thus distributed into one of four groups (n = 7/group), with muscles being harvested at 1 or 8 days after
gastrocnemius ablation or a sham operation.
Reagents. Polyclonal FAK antiserum was raised in rabbits against an acrylamide gel-purified GST-FAK fusion protein [FAK amino acids 749-1052 (17)]. The serum recognized specifically an ~125,000-molecular weight protein in Western blots of mouse, human, and chicken cell extracts. Furthermore, in vitro kinase assays of immunoprecipitates with the serum revealed phosphorylation of the same-sized protein exclusively on the amino acid tyrosine (data not shown). Affinity-purified anti-FAK serum was prepared by absorption to bead-coupled GST-FAK fusion protein followed by washing and low-pH elution according to standard methods. The reactivity of affinity-purified serum was confirmed by Western blotting and in vitro kinase assay. Paxillin antibody P13520 (Transduction Laboratories) has been characterized previously (28). Monoclonal vinculin antiserum from mouse was a gift of Dr. M. A. Glukhova (Paris, France) and has also been characterized previously (14). Secondary horseradish peroxidase (HRP)-conjugated antibodies were obtained from Amersham.
Cell culture.
Primary embryonic myoblast cultures were established from 11-day
chicken embryos, as described previously (24). Cell plates were
periodically examined using a microscope and harvested at 24 h
(myoblast stage) or 96 h after plating (myotube stage) by washing twice
with PBS and scraped in cold Mueller buffer (50 mM HEPES, pH 7.4, 0.1%
Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM
Na4P2O7 · 10 H2O, 100 mM -glycerophosphate,
25 mM NaF, 1 mM Na3VO4,
0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 0.3 µg/ml aprotinin). For selective depletion of replicating cells including fibroblasts, 10 µM cytosine arabinoside was added for 24 h at 48 h
after plating (36).
Isolation of protein fractions. Total protein homogenates were prepared by homogenizing frozen rooster ALD and rat soleus muscles three times for 20 s each with a Polytron homogenizer (Kinematica) on ice at low setting. Muscles were homogenized in Mueller buffer (5 ml buffer/700 mg tissue). Protein concentration was estimated using a Lowry-based protein assay (DC protein assay, Bio-Rad), and aliquots corresponding to 50 µg of total protein each were run on an 8% SDS-PAGE with subsequent Coomassie blue staining to check estimated protein concentration and to verify the integrity of extracted proteins. For 1% Triton X-100 extraction of proteins, 50 µl of the total protein homogenate (1 mg of total protein) were diluted to 500 µl with cold immunoprecipitation buffer (10 mM Tris · HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 1 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin) and vortexed, and tubes were end-over rocked for 40 min at 8°C. After centrifugation (3 min at 10,000 g at 8°C), the 1% Triton X-100-soluble fraction was separated from the pelleted insoluble matter. Protein concentration was estimated and verified, as described above.
SDS-PAGE, Western blotting, and immunodetection.
Samples were solubilized as 1 µg/µl in 1× SDS loading buffer
(50 mM Tris · HCl, pH 6.8, 10% glycerol, 2% SDS,
2% -mercaptoethanol) by vortexing, passaging three times through 18 × 1 in. and 21 × 0.5 in. needles, respectively, and
boiling. Aliquots (50 µl for rooster and 40 µl for rat) were run on
an 8% SDS-PAGE, the wet gels were Western blotted (in 25 mM Tris base,
pH ~8.3, 192 mM glycine, 20% methanol) onto a nitrocellulose
membrane that was stained with Ponceau S to verify equal loading and
transfer. The membrane was blocked for 1 h at 25°C in blocking
solution (2.5% nonfat dry milk, 1% BSA) in TTBS (20 mM Tris base, pH
7.5, 150 mM NaCl, 0.05% Tween 20). FAK antibody (1:2,000) or preimmune control of the same rabbit or anti-paxillin antibody (1:5,000 for
rooster and 1:10,000 for rat) was added in blocking solution for a
total of 2 h at room temperature. After the membrane was washed with
TTBS, it was incubated with HRP-conjugated donkey anti-rabbit antibody
for FAK (1:7,500) or anti-mouse antibody for paxillin (1:10,000 for
rooster and 1:5,000 for rat; Amersham) in blocking solution for 1 h at
room temperature. The membrane was next washed with TTBS, and the
signal was detected by enhanced chemiluminescence by using Kodak XAR5
film. The intensity of FAK and paxillin signals (proportional to the
amount of protein) was quantified by densitometry scanning (Bio Image,
Millipore, Ann Arbor, MI) as integrated optical density (IOD).
Immunohistochemistry.
Excised muscles were embedded (Tissue Tek), frozen in
nitrogen-isopentane, and stored until use at 70°C.
Cryosections (10 µm) were cut from the center of the ALD muscle and
layered on glass plates (Superfrost Plus), air dried, and fixed in cold
acetone; after the section was wetted in PBS, tissue peroxidase
activity was quenched (10 min, 0.6%
H2O2
in methanol), and sections were washed in PBS. Sections were blocked
with 10% FCS in PBS and incubated with affinity-purified anti-FAK or
anti-vinculin antiserum (1:5 and 1:20 dilution in PBS, respectively).
After the sections were washed with PBS, they were incubated with
secondary antibody (1:400 dilution in PBS) of goat anti-rabbit
IgG-biotin or goat anti-mouse IgG (catalog nos. E0432 and E0433,
respectively, Dako) and then washed with PBS. Sections were incubated
with a PBS solution of diluted avidin-biotin complex coupled to HRP (30 min; catalog no. K0355, Dako) and washed in PBS, and immunoreactivity
was detected with diaminobenzidine substrate (6 mg diaminobenzidine in
10 ml PBS + 0.03%
H2O2).
After the color reaction was stopped with water, nuclei were
counterstained with hematoxylin (30 s), the slide was washed in PBS,
and sections were embedded (Aquatex, Merck). The stain was visualized
and documented on slide film (Kodak ES100) by use of a
microscope-photograph system (Leica Leitz DMRD/DMRB).
FAK immunoprecipitation and autokinase assay.
Proteins in 1% Triton X-100-soluble fraction (extraction of 1 mg
protein of total protein homogenate) were divided into two aliquots:
one was incubated with FAK antiserum and the other with preimmune serum
from the same animal for 2 h each with end-over rocking at 8°C.
Precipitation of antigen-antibody complexes was initiated with the
addition of 50 µl of a 10% slurry of Staphylococcus aureus cell suspensions (Sigma Chemical; equilibrated
with immunoprecipitation buffer) for 1 h at 8°C. After
centrifugation (1 min at 8,000 g at
8°C), the precipitate was washed with three cycles of repetitive pipetting of 1 ml of cold immunoprecipitation buffer and brief centrifugation (as described above). The immunoprecipitate was equilibrated in 400 µl of cold kinase A buffer (20 mM Tris base, pH
7.5, 10 mM MgCl2, 2 mM
MnCl2) and divided into two
aliquots, and both were centrifuged for 1 min at 8,000 g. One pellet was solubilized in 30 µl of 2× SDS loading buffer [100 mM
Tris · HCl, pH 6.8, 20% glycerol, 4% SDS, 4%
(vol/vol) 2-mercaptoethanol, 0.2% bromphenol blue] and analyzed
for FAK protein by Western blotting and immunodetection. The other
pellet was used for autokinase activity: Autophosphorylation of
immunoprecipitated proteins was initiated by the addition of 30 µl of
cold kinase A buffer supplemented to 8 µM ATP and 0.33 µCi/µl
[-32P]ATP for 15 min at 30°C. The reaction was stopped with the addition of 10 µl
of 4× SDS-loading buffer (see above), and the sample was run on
5% SDS-PAGE. The gels were exposed wet at
80°C to Kodak
XAR5 film, and intensity of the FAK autophosphorylation signal
(proportional to amount of incorporated phosphate) was quantified by
densitometry scanning (Bio Image) as IOD.
Statistical analysis. An ANOVA test with Fisher's post hoc test was employed using repeated measures where appropriate. For the rooster ALD data, two independent experiments that had been performed on different dates were normalized to the mean of their respective controls and pooled. P < 0.05 was selected as the significance level. Values are means ± SE.
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RESULTS |
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Antibody specificity.
The specificity of our FAK antibody had not been previously
characterized in rooster or rat skeletal muscle. The antiserum, but not
the preimmune serum, detected FAK protein in total protein homogenate
of rooster ALD muscle as a ~125-kDa polypeptide (Fig. 1A).
No other protein band was detected using the conditions employed. A
recombinant FAK protein was also recognized by the FAK antiserum (data
not shown). FAK has previously been identified in chicken embryo cells
as a 125-kDa polypeptide (40). In rat soleus muscle homogenate, our FAK
antiserum detected two bands of ~120 and 125 kDa (see Fig.
5A) that were not detectable with
the preimmune serum. Similar to the rooster ALD homogenate, no other
protein was detected by the FAK antiserum. A doublet-banding pattern
during Western blot analysis has been similarly seen for FAK protein in
human platelets (42) and human kidney cells (45). Paxillin antibody
(28) detects a diffuse protein band migrating at the previously
described molecular weight of paxillin protein
[65,000-70,000, relative to marker BSA (48)] in
rooster ALD muscle (Fig. 1B) and rat
soleus muscle (see Fig. 5B).
|
FAK and paxillin protein concentrations were detected in the belly
of the rooster ALD muscle.
Previous reports have indicated that FAK and paxillin proteins are
localized and concentrated at the myotendinous junction of skinned
skeletal muscle fibers in Xenopus
laevis (3, 49). We had hypothesized that FAK protein
would be found along the sarcolemma, so the spatial expression of FAK
protein in cross sections from the center of the rooster ALD muscle was
characterized. Immunohistochemical analysis indicated that FAK protein
was expressed in the belly of the ALD muscle and localized along the
sarcolemma (Fig.
2A).
Vinculin protein has previously been reported to localize to
cytoskeletal-membrane attachments (34) and to electron-dense subsarcolemmal densities in the ALD muscle that appear to be connected to the sarcolemma near I bands and Z lines, which are adjacent to
increased amounts of endomysial connective tissue found just outside
the basal lamina (43). We thus employed vinculin staining to determine
whether it colocalizes with FAK. Vinculin immunohistochemical staining
was also localized to sarcolemmal regions (Fig.
2C) (34, 43). Pardo et al. (34)
previously observed that vinculin defines a striking two-dimensional
lattice associated with the sarcolemma of skeletal muscle and termed
the lattice "costameres." At higher magnifications, FAK
immunoreactivity appeared irregular along the sarcolemma of ALD
myofibers (Fig. 2D). Weak FAK
immunoreactivity was detected in the interstitium and in the
cytoplasmic component of myofibers (Fig. 2,
A and
D).
|
FAK and paxillin protein concentrations and FAK autokinase activity increase during stretch-induced hypertrophy of the rooster ALD muscle. Loading one wing of roosters with 10% of their body weight stretches the ALD muscle and produces hypertrophy and hyperplasia (22). After 1.5, 7, and 13 days of stretch overload, total protein per stretched ALD muscle increased by 35 ± 12%, 122 ± 10%, and 191 ± 19%, respectively, relative to the contralateral control muscle. We have reported similar growth rates for this procedure in an earlier experiment (10).
FAK protein per total protein in the ALD muscle increased 77 ± 39% and 81 ± 19% after 1.5 and 7 days, respectively, of stretch overload (Fig. 3A). Paxillin protein per total protein in the ALD muscle increased by 206 ± 30% and 202 ± 48% after 1.5 and 7 days, respectively, of stretch overload (Fig. 3B). No significant change in FAK or paxillin proteins per unit of total protein was evident at 13 days of stretch overload.
|
|
FAK and paxillin protein concentrations increase during overload-induced hypertrophy of rat soleus muscle. The model of ablation of the gastrocnemius muscle in rats was selected to produce an overload enlargement of the synergistic soleus muscle by hypertrophy (46). The soleus muscle was chosen for comparison to the stretch response in rooster ALD muscle, inasmuch as both muscles are primarily slow twitch (23, 46).
In the ablated animals, soleus wet weight was significantly increased over sham-operated animals by 24.3% and 31.8% after 1 and 8 days of overload, respectively. Total protein per whole soleus muscle was not significantly elevated in ablated animals after 1 day of overload but was significantly (32.9%) higher in ablated than in sham-operated animals after 8 days of overload. In ablated vs. sham-operated animals, FAK protein content per total protein was significantly elevated (112%) within 1 day of overload and increased by 611% by 8 days of overload (Fig. 5A). There were no apparent differences between experimental groups in the distribution of FAK immunoreactivity among the doublet bands. Paxillin protein per total protein was also significantly elevated (87%) within 1 day of overload and remained elevated (431%) after 8 days of overload (Fig. 5B). Lastly, there was an apparent acute effect of the surgical procedure itself on paxillin, inasmuch as paxillin protein content was higher at 1 than at 8 days after surgery in the sham-operated animals (Fig. 5B).
|
FAK and paxillin protein concentrations and FAK autokinase activity
increased with fusion of myoblasts to myotubes.
This model was chosen to test for FAK and paxillin expression when new
myotubes were formed on removal of serum in cultured myoblasts. Our FAK
antiserum, but not its preimmune serum (data not shown), detected FAK
protein in total protein homogenate of primary chicken skeletal muscle
cells as a 125-kDa polypeptide (Fig.
6A). No
other protein bands were evident.
|
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DISCUSSION |
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Novel observations in the current study are that 1) FAK protein is immunolocalized to peripheral (sarcolemmal) regions of myofibers in the midbelly of the ALD muscle of roosters, 2) adaptive increases in the concentrations for FAK and paxillin proteins occur in hypertrophying-hyperplastic skeletal muscles, in hypertrophying skeletal muscle, and in new myotube formation in culture, and 3) adaptive increases in FAK autokinase activity were also present in hypertrophying-hyperplastic skeletal muscle.
The immunolocalization of FAK protein to the sarcolemma of the ALD muscle (Fig. 2, A and D) is similar to the previously reported localization of vinculin (Fig. 2C) (34, 43) and integrin (6). The latter two proteins have been localized to costameres that are found subsarcolemmally with focal adhesion sites in the ALD muscle. It has been postulated that costameres transmit forces initiated at the sarcomere during lengthening and shortening of muscle (34, 35). FAK protein has been shown to colocalize at FACs when integrin receptors cluster in cultured cells (7, 31). However, to our knowledge, this is the first observation of the immunolocalization of FAK protein to the sarcolemmal region of muscle fibers in living animals. Moreover, on higher magnifications, the pattern of FAK immunoreactivity along the sarcolemma was irregular in appearance, with some sarcolemmal segments devoid of FAK protein (Fig. 2D). These observations extend earlier reports that FAK protein is present in muscle fibers at myotendinous junctions (3).
Evidence points to FAK protein as a key regulator of FAC assembly (7). For example, a localized overexpression of FAK protein by somatic gene transfer between the tendon and tendon sheath has been shown to induce tendon adhesion formation (27). Lou et al. (27) interpreted their findings to mean that FAK and FAK-related signaling pathways may be involved in the formation of tendon adhesions. Furthermore, it is well established that FAK and paxillin proteins are localized to FACs within many cell types (for review see Ref. 7). We suggest that FAK and paxillin protein concentrations could serve as an index of FAC density. Thus our observation of transient increases in FAK and paxillin protein concentrations in the overloaded rooster ALD muscle permits the following speculation. Increased mechanical load per unit of cross-sectional area may signal an increased expression for FAK and paxillin proteins during the 1st wk of stretch. The loss of increased FAK and paxillin protein concentrations by 13 days of overload corresponds to the transition from fast to slow growth phases during chronic stretch (9). As a consequence of muscle enlargement, less of an increased mechanical load would now be borne per unit of cross-sectional area at 13 days of overload, which we speculate would diminish signaling for FAC formation. This scenario could explain the return of FAK and paxillin protein concentration to values found in control muscles at 13 days of overload.
An alternative explanation for the transient rise and fall in FAK and paxillin proteins in the overloaded rooster ALD muscle could be that these changes coincide with satellite cell activation. Kennedy et al. (22) showed that new fiber formation accompanies hypertrophy in this hypertrophy-hyperplasia model. During the first 2 wk of overloading the ALD muscle, satellite cells are activated, and both fuse into existing fibers (hypertrophy) and form new fibers (hyperplasia). Winchester and Gonyea (51) reported that the percentage of muscle fibers exhibiting incorporation of satellite cells as new myonuclei was significantly greater at 5 and 7 days of stretch of the ALD muscle, but they declined at 10 days, reaching near-control values by 14 days. The time course of satellite cell activation mirrors the increase and fall of FAK and paxillin protein densities in the ALD muscle. This parallelism with satellite cells led to two additional experiments that attempted to partition the effects of hypertrophy vs. hyperplasia for changes in FAK and paxillin protein concentration.
First, when the gastrocnemius muscle of rat is ablated, the soleus muscle enlarges by hypertrophy with no detectable hyperplasia (46). Our observation that FAK and paxillin protein concentrations were increased in the hypertrophying rat soleus muscle (Fig. 5) suggests that increased FAK and paxillin protein can occur independently of muscle fiber hyperplasia. Second, our observations in cultured cells that protein concentrations of FAK and paxillin increased with myoblast fusion into myotubes show that FAK and paxillin protein concentrations are also associated with new fiber formation. Accordingly, others have shown that focal adhesions must interact with the extracellular matrix to permit myogenesis (30, 32, 39).
FAK and paxillin proteins have previously been identified in fibroblast, smooth muscle, and endothelial cells (1, 8, 21, 40, 52), all of which are components of whole skeletal muscles. FAK autokinase activity and paxillin protein concentration, but not FAK protein concentration, were enhanced when myotubes were cocultured with replicating fibroblasts (Fig. 6C). However, immunohistochemistry of intact ALD muscle cross sections showed that the majority of FAK immunoreactivity was present in skeletal muscle cells (Fig. 2). Furthermore, skeletal muscle cells constitute the majority of volume in the ALD muscle. From this we speculate that the increase in FAK protein in stretched whole ALD muscle is at least partly attributable to an increase in FAK protein within the muscle cells themselves.
The increase in FAK activity in skeletal muscle undergoing hypertrophy-hyperplasia, although novel in skeletal muscle, has been previously noted in mechanically perturbed endothelial cells, vascular smooth muscle cells, and cardiomyocytes (47, 52). For example, shear stress in vascular endothelial cells, the tangential component of hemodynamic forces, induced the phosphorylation (and thus activation) of FAK, permitting the formation of FAK-Grb2-Sos ternary complex (25). This signaling pathway led to the activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK). On the upstream side, integrins, by interacting with FAK, may be involved in the mechanotransduction that transfers fluid shear stress to biochemical signals (25). Occupation of integrin receptors with extracellular matrix proteins is one mode to induce autophosphorylation of tyrosine-397 (26, 42). Models of FAK function have proposed that the phosphorylation of FAK generates a tyrosine phosphorylation cascade that correlates with the assembly of focal adhesions and the actin cytoskeleton (33). Phosphorylation of tyrosine-397 on FAK creates a high-affinity binding site for SH2 domains of Src-family kinases, which further phosphorylates FAK on additional tyrosine residues and leads to full activation of FAK (41). We speculate that some of the induction of FAK protein expression could be independent of ligand interaction with integrin receptors, because the autokinase activity of FAK remains elevated at 13 days of ALD muscle overload when FAK protein concentration is no longer elevated.
In summary, the adaptive elevations of FAK and paxillin proteins and of FAK autokinase activity to mechanical loading support an appealing hypothesis that they could be linked in a mechanochemical signaling pathway for enlargement of skeletal muscle. Future experiments should test the function of these changes with inducible muscle-specific promoters driving a dominant negative FAK to learn whether removing FAK lessens the growth of mechanically challenged muscles.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. C. Andres (Bern, Switzerland) for immunohistochemical assistance.
![]() |
FOOTNOTES |
---|
This research was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393.
Present address of M. Flück: M. E. Müller-Institute for Biomechanics, University of Bern, Murtenstrasse 35, Postfach 30, CH-3010 Bern, Switzerland.
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 and other correspondence: F. W. Booth, Dept. of Integrative Biology, University of Texas Medical School, 6431 Fannin St., Houston, TX 77030 (E-mail: fbooth{at}girch1.med.uth.tmc.edu).
Received 10 November 1998; accepted in final form 2 April 1999.
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