(Received for publication, October 16, 1995; and in revised form, December 15, 1995)
From the
We have previously shown that mechanical strain-induced fetal
rat lung cell proliferation is transduced via the phospholipase
C--protein kinase C pathway. In the present study, we found that
protein-tyrosine kinase activity of fetal lung cells increased after a
short period of strain, which was accompanied by tyrosine
phosphorylation of proteins of
110-130 kDa. Several
components of this complex were identified as pp60
substrates. Strain increased pp60
activity
in the cytoskeletal fraction, which coincided with a shift in
subcellular distribution of pp60
from the
Triton-soluble to the cytoskeletal fraction. Strain-induced
pp60
translocation did not appear to be mediated
via the focal adhesion kinase-paxillin pathway. In contrast, strain
increased the association between pp60
and the
actin filament-associated protein of 110 kDa. Preincubation of cells
with herbimycin A, a tyrosine kinase inhibitor, abolished
strain-induced phospholipase C-
1 tyrosine phosphorylation and its
coimmunoprecipitation with pp60
. It also
inhibited strain-induced DNA synthesis. These results suggest that
activation of pp60
is an upstream event of the
phospholipase C-
-protein kinase C pathway that may represent an
important mechanism by which mechanical perturbations are converted to
biological reactions in fetal lung cells.
All tissues in the body are subjected to physical forces originating either from tension, created by cells themselves, or from the environment(1) . The role of mechanical force as an important regulator of structure and function of mammalian cells has recently been recognized for many tissues and cell types(2) . Although the biological responses of cells to physical forces vary, physical stimuli have to be sensed by cells and transmitted through intracellular signal transduction pathways. In response to mechanical stimulation, cells may generate a variety of second messengers and activate different phospholipases, protein kinases, and other signal transduction-related enzymes, depending upon the tissue and cell type, developmental stage, the manner and period of physical forces applied, and environmental conditions(2, 3) .
Respiration is
a unique feature of the lung. Physical forces, derived from breathing,
play an important role in regulating lung structure, function, and
metabolism(4) . ``Breathing movements'' can be
observed by sonography in the human fetus as early as 10 weeks of
gestation. There are several lines of evidence from physiological
studies and clinical observations to suggest that fetal breathing
movements play an important role in lung
growth(5, 6) . We have shown that an intermittent
strain regimen that simulates fetal breathing movements enhanced DNA
synthesis and cell division of fetal rat lung cells maintained in
organotypic culture(7, 8, 9) . We have also
observed a dramatic increase in the intracellular concentrations of two
second messengers, inositol 1,4,5-trisphosphate and diacylglycerol,
after a short period of strain. These increases in inositol
1,4,5-trisphosphate and diacylglycerol were accompanied by an increased
tyrosine phosphorylation of phospholipase C-1 (PLC-
1) (
)and protein kinase C (PKC) activity. Strain-induced PKC
activation and DNA synthesis were blocked by PLC and PKC
inhibitors(9) . These results suggest that mechanical
strain-induced fetal lung cell proliferation is mediated through the
PLC-
-PKC pathway. However, the mechanism by which strain-induced
cytoskeleton deformation is converted to biochemical reactions remains
unknown.
Herein, we report that mechanical strain of fetal rat lung
cells induced a rapid activation and translocation of pp60 from the Triton-soluble to the cytoskeletal fraction.
Strain-induced pp60
translocation appears to be
mediated via the actin filament-associated protein of 110 kDa
(AFAP-110). Preincubation of cells with a protein-tyrosine kinase (PTK)
inhibitor, herbimycin A, blocked strain-induced PLC-
1 tyrosine
phosphorylation and its association with pp60
as
well as strain-induced DNA synthesis. These results suggest that
strain-induced PTK activation is an upstream event of the PLC-
-PKC
pathway.
Triton-soluble and cytoskeletal
fractions were prepared according to a modified method of Clark and
Brugge(12) . Briefly, sponges were immersed into Triton buffer
(2% (v/v) Triton X-100, 2 mM EGTA, 100 mM Tris, 500
µg/ml leupeptin, 0.2 mM phenylmethylsulfonyl fluoride, 2.0
mM benzamidine, 2.0 mM NaVO
,
and 200 kallikrein inactivator units/ml aprotinin, pH 7.2) and
incubated overnight at 4 °C. The Triton-soluble fraction was
separated by centrifugation of sponges over glass-wool filters. The
sponges were then immersed in radioimmune precipitation assay buffer
for 4 h. The Triton-insoluble/radioimmune precipitation assay
buffer-soluble lysate (cytoskeletal fraction) was collected by
centrifugation at 300
g for 1 min through glass-wool
filters. Both fractions were stored as aliquots at -70 °C
until analyzed.
For measurement of Src family PTK activity, a
synthetic peptide,
[Lys]Cdc2-(6-20)-NH
, a
specific and efficient substrate of Src family PTKs(13) , was
used. Two pseudosubstrate peptides,
[Val
,Ser
,Lys
]Cdc2-(6-20)-NH
and
[Phe
,Lys
]Cdc2-(6-20)-NH
,
were used as negative controls. An aliquot (5 µl) of either
substrate peptide solution (1.5 mM) or water was mixed with 5
µl of assay buffer (250 mM Tris, 125 mM MgCl
, 25 mM MnCl
, and 0.25 mM Na
VO
, pH 7.0) and 10 µl of lysates of
either the Triton-soluble or cytoskeletal fraction. The reaction was
started by the addition of 5 µl of ATP (0.5 mM) containing
1000 cpm/pmol [
-
P]ATP at timed intervals
and incubated at 30 °C for 30 min. The reaction was stopped by the
addition of 10 µl of 50% (w/v) acetic acid and centrifugation at
3000
g for 5 min. The supernatant (25 µl) was
spotted on phosphocellulose filters. The filters were washed four times
with 0.75% (v/v) phosphoric acid and once with excess acetone. After
drying, filters were transferred to scintillation vials, and
radioactivity was measured. PTK activity was defined as the activity
measured in the presence of the substrate peptide minus the activity
measured in the absence of the peptide. To specifically measure
pp60
activity, pp60
was immunoprecipitated
from Triton-soluble and cytoskeletal fractions using polyclonal
anti-pp60
antibody. The immunoprecipitates were washed
twice with dilution buffer (200 mM HEPES, 10% (v/v) glycerol,
and 0.1% (v/v) Nonidet P-40, pH 7.0) and resuspended in dilution
buffer, and an aliquot of the suspension (10 µl) was then used for
the activity assay.
Figure 1: Mechanical strain induces protein-tyrosine kinase activation. Fetal rat lung cells were subjected to intermittent strain for various times (strain, 0-15 min; relaxation, 15-60 min; strain, 60-75 min) (strain periods are indicated by dashed lines) and then lysed, and total PTK activity was measured by phosphorylation of a PTK-specific peptide substrate, RR-SRC. Data are from a representative experiment (mean ± S.E. from three sponges). Similar results were confirmed in a separate experiment. Statistical analysis was by one-way analysis of variance followed by Duncan's multiple range test. *, p < 0.05 compared with static control.
Figure 2: Mechanical strain induces protein tyrosine phosphorylation. Fetal rat lung cells were subjected to strain for 5-15 min and then lysed, subjected to SDS-PAGE, and analyzed with antibodies to phosphotyrosine. An illustrative blot is shown in A, and results (mean ± S.E.) of densitometric analyses of four separate experiments are shown in B. Statistical analysis was by one-way analysis of variance followed by Duncan's multiple range test. *, p < 0.05 compared with static control.
Figure 3:
Mechanical strain induces tyrosine
phosphorylation of pp60 substrates. Fetal rat
lung cells were subjected to strain or static culture for 5 min and
then lysed. Aliquots of lysates, normalized for protein content, were
immunoprecipitated (ptp.) with antibodies to phosphotyrosine (p-tyr) or pp125
. Lysates and immunoprecipitates
were analyzed by SDS-PAGE. The blots were immunoblotted (blot)
as indicated with antibodies to phosphotyrosine, pp120, pp110
(AFAP-110), pp130, and cortactin. Molecular mass marker positions are
indicated. Similar results were obtained in two to four separate
experiments. C, control static cultures; S, strained
cultures.
Figure 4:
Mechanical strain induces
pp60activation in the cytoskeleton. Fetal rat
lung cells were preincubated for 3 h with or without either 1
µM cytochalasin B or 1 µg/ml herbimycin A and then
subjected to strain or static culture for 5 min. Triton-soluble and
cytoskeletal fractions were prepared, and aliquots, equalized for
protein content, were immunoprecipitated with polyclonal antibodies to
pp60
. PTK activity in the immunoprecipitates was
assayed using peptide
[Lys
]Cdc2-(6-20)-NH
as
substrate and is expressed as pmol/min/mg of protein. Data are mean
± S.E. from three sponges. A, Triton-soluble fraction; B, cytoskeletal fraction. Non, untreated cells; Cyto.B, cytochalasin B; Herb.A, herbimycin A. *, p < 0.05 compared with static
controls.
Figure 5:
Mechanical strain induces
pp60translocation to the cytoskeleton. Fetal
rat lung cells were preincubated for 3 h with or without either 1
µM cytochalasin B or 1 µg/ml herbimycin A and then
subjected to strain or static culture for 5 min. Triton-soluble (sol) and cytoskeletal (cysk) fractions were
prepared, and aliquots, equalized for protein content, were
immunoblotted with antibodies to pp60
(mAb
GD11). The position of pp60
is indicated.
Similar results were obtained in two separate experiments. C,
control static cultures; S, strained
cultures.
Figure 6:
Mechanical strain does not influence
pp60 tyrosine phosphorylation. Fetal rat lung
cells were subjected to strain or static culture for 5 min.
Triton-soluble (sol) and cytoskeletal (cysk)
fractions were prepared, and aliquots, equalized for protein content,
were immunoprecipitated (i.p.) with antibodies to
phosphotyrosine (p-tyr) and immunoblotted (blot) with
antibodies to pp60
(mAb GD11). A representative
blot is shown in A, and the mean and the range of
densitometric analysis of blots from two separate experiments are shown
in B.
Figure 7:
Mechanical strain induces association of
pp60 with AFAP-110. Fetal rat lung cells were
subjected to strain for 5 min and then lysed. Cell lysates, normalized
for protein content, were immunoprecipitated (ptp.) with
polyclonal anti-pp60
antibody. Lysates(-)
and immunoprecipitates (Src) were analyzed by SDS-PAGE. The
blots were immunoblotted (blot) as indicated with antibodies
to pp120, cortactin, and pp110 (AFAP-110). Positions are indicated by arrowheads. Similar results were obtained in two to four
separate experiments. C, control static cultures; S,
strained cultures.
Figure 8:
Mechanical strain does not activate the
pp125-paxillin pathway. Fetal rat lung cells were
subjected to strain for 5 min and then lysed. Cell lysates, equalized
for protein content, were immunoprecipitated (ptp.) with
polyclonal anti-pp60
or monoclonal
anti-pp125
antibody. Lysates(-) and
immunoprecipitates (Src or FAK) were analyzed by
SDS-PAGE. The blots were immunoblotted (blot) as indicated
with antibodies to pp130, paxillin, and Src. Positions are indicated by arrowheads. Similar results were obtained in two separate
experiments. C, control static cultures; S, strained
cultures.
Figure 9:
Strain-induced PLC-1 tyrosine
phosphorylation and PLC-
1-pp60
association
are blocked by the PTK inhibitor herbimycin A. Fetal rat lung cells
were preincubated for 3 h with or without 1 µg/ml herbimycin A and
then subjected to strain or static culture. Cells were lysed after 5
min of strain, and cell lysates, normalized for protein content, were
immunoprecipitated (ptp.) with antibodies to phosphotyrosine (p-tyr; left panel) or pp60
(right panel). The immunoprecipitates were
electrophoresed and immunoblotted (blot) with antibodies to
PLC-
1. The position of PLC-
1 is indicated. Similar results
were obtained in two separate experiments. C, control static
cultures; S, strained cultures.
Figure 10:
Strain-induced DNA synthesis is blocked
by the PTK inhibitor herbimycin A. Fetal rat lung cells were
preincubated with or without 1 µg/ml herbimycin A for 3 h and then
subjected to a 48-h intermittent strain or static culture.
[H]Thymidine incorporation into DNA was measured.
Data are mean ± S.E. from three sponges. Statistical analysis
was by one-way analysis of variance followed by Duncan's multiple
range test. *, p < 0.05 compared with the static control
group.
Mechanical strain-induced increases of inositol
1,4,5-trisphosphate and diacylglycerol have been observed in various
cell types, such as endothelial cells(27) , vascular smooth
muscle cells(28) , cardiac myocytes(29) , skeletal
muscle(30) , and bone cells (31) as well as fetal lung
cells(9) . The increase of inositol 1,4,5-trisphosphate and
diacylglycerol is followed by PKC activation, and blockage of PLC or
PKC activity can inhibit physical force-induced cell
proliferation(9, 28) . Therefore, PLC-PKC seems to be
a common pathway by which cells transduce physical signals. How this
pathway becomes activated is unknown. In this study, mechanical strain
of fetal lung cells rapidly activated PTKs and increased tyrosine
phosphorylation of several phosphotyrosine-containing proteins,
AFAP-110, pp120, and cortactin. The phosphorylation of these
pp60 substrates by mechanical strain coincided with
pp60
activation and translocation to the cytoskeletal
compartment. The PTK inhibitor herbimycin A blocked strain-induced
PLC-
1 tyrosine phosphorylation, PKC activation, and DNA synthesis,
suggesting that the rapid activation of cytoplasmic PTKs is an upstream
event of the PLC-
-PKC pathway. In vitro studies have
shown that PLC-
1 can be tyrosine-phosphorylated by pp60
and other Src family PTKs(32) . In platelets,
electrotransjection of monoclonal pp60
antibody inhibited activation of PLC-
1(33) .
Moreover, pp60
has been coimmunoprecipitated with
PLC-
1 in platelets(33) , and this association was
increased after treatment of platelets with platelet-activating
factor(33) . Similarly, we observed coimmunoprecipitation of
pp60
and PLC-
1 in fetal rat lung cells, and this
association was increased by mechanical strain and inhibited by
herbimycin A treatment. Thus, our present data are compatible with
strain-induced PLC-
1 tyrosine phosphorylation in fetal lung cells
being, at least in part, mediated via pp60
.
Phosphorylated PLC-
then activates the PKC pathway and downstream
signal cascades.
Increased tyrosine phosphorylation of proteins
ranging from 110 to 130 kDa is a common phenomenon in extracellular
matrix-integrin-cytoskeleton-mediated signal transduction initiated by
integrin clustering (15) or cell attachment(16) . An
integrin-related increase of tyrosine phosphorylation has been found
for proteins such as pp130 (15) and
pp125(16, 34) . Although tyrosine
phosphorylation of pp130 and pp125
was observed in fetal
lung cells, it was not affected by short periods of mechanical strain,
and the association between these two proteins and pp60
in fetal rat lung cells was not detected by
coimmunoprecipitation. Paxillin has been suggested to be a direct
substrate for pp125
(35) and has been found to
bind in vitro to the SH3 domain of c-Src(36) . In this
study, coimmunoprecipitation of pp60
and paxillin was
demonstrated in fetal rat lung cells. However, the tyrosine
phosphorylation and association of paxillin with pp60
were not altered by mechanical strain. The strain-induced
pp60
translocation therefore appears not to be mediated
through the pp125
-paxillin pathway. In addition,
mechanical strain-induced cell proliferation was not affected when
fetal lung cells were preincubated with an RGD peptide to block the
extracellular matrix-integrin interaction. (
)Taken together,
these results suggest that the mechanical strain-induced signals are
different from those initiated by integrins, although the cytoskeleton
system appears to be involved in the transmission of both signals.
Tyrosine phosphorylation of pp130, pp125, pp120, and
cortactin has been observed upon epidermal growth factor, PDGF, and
colony-stimulating factor-1 stimulation of several cell
types(35, 37, 38) . The effect of these
growth factors is likely mediated through intermediate PTKs such as Src
family members. PDGF stimulation of quiescent NIH 3T3 cells and human
fibroblasts activates Src family tyrosine kinases(39) .
Treatment of A172 glioblastoma cells with PDGF or epidermal growth
factor induces activation and translocation of c-Src to the
cytoskeleton(40) . We have recently demonstrated that
strain-enhanced proliferation is mediated via PDGF(8) .
However, it is unlikely that strain-induced pp60
activation is a result of an exocytosis of PDGF. We found no
measurable changes of PDGF in the culture medium within 4 h of
mechanical strain(8) . Tyrosine phosphorylation of RasGAP has
been observed for various cell types upon activation of epidermal
growth factor or PDGF receptors(35, 41, 42) .
We also observed tyrosine phosphorylation of RasGAP and PDGF
-receptors after PDGF-BB treatment of fetal rat lung cells. In
contrast, we did not observe tyrosine phosphorylation of RasGAP or PDGF
-receptors after 5 min of mechanical strain (data not shown).
One of the regulatory mechanisms for pp60 activation
is through its association with the cytoskeletal matrix(43) .
All oncogenic variants of pp60
have been shown to be
tightly associated with the Triton-insoluble cytoskeletal matrix, and
this association correlates with the elevated tyrosine kinase activity
of pp60
(44, 45, 46) . Normally,
pp60
does not associate with the detergent-insoluble
cellular matrix(44) . However, redistribution of activated
pp60
to the cytoskeletal matrix has been
reported for thrombin-stimulated platelets (12) and growth
factor-stimulated A172 glioblastoma cells(40) . The reason for
Src activation following its translocation to the cytoskeleton is
unknown. In the current model for Src regulation, the
tyrosine-phosphorylated C-terminal Tyr(P)-527 sequence binds by an
intramolecular interaction to Src's own SH2 domain to maintain
Src in an inactive state(47, 48) . The
tyrosine-phosphorylated Src C-terminal Tyr(P)-527 sequence does,
however, not resemble the consensus high affinity SH2-binding site and
therefore binds poorly to the SH2 domain of Src. In contrast, AFAP-110,
a distinctive cytoskeleton-associated
protein(24, 25) , contains four putative SH2-binding
sites including a consensus high affinity tyrosine-phosphorylated
SH2-binding site(25) . Mechanical strain-induced cytoskeleton
deformation may physically facilitate the approximation of these
binding sites to pp60
and activate pp60
by
competing with Src Tyr(P)-527 for Src SH2 binding. In this study, we
indeed observed an increased association between pp60
and
AFAP-110. The increased association between AFAP-110 and pp60
may activate pp60
, which results in an increased
tyrosine phosphorylation of AFAP-110 and other proteins, such as
PLC-
1.