From the
Division of Respirology, the Department of
Medicine, The Toronto General Hospital Research Institute of the University
Health Network, Toronto, Ontario M5S 1A8, Canada, the
¶Burnham Institute, La Jolla, California 92037,
and the ||Samuel Lunenfeld Research Institute, Mt.
Sinai Hospital and the
Canadian Institutes of
Health Research Group in Matrix Dynamics, Faculty of Dentistry, the University
of Toronto, Toronto, Ontario M5S 1A8, Canada
Received for publication, December 23, 2002 , and in revised form, March 20, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gingival fibroblasts and chondrocytes are cells involved in inflammatory
disorders that require focal adhesion complex (FAC) formation in
vitro for IL-1-induced ERK activation
(23,
28). FAC are adhesive domains
that comprise the termini of actin stress fibers in physical association with
a number of actin-binding proteins including vinculin, talin, filamin A,
paxillin, and -actinin
(29). Nascent focal adhesions
also contain a number of signaling molecules including p125 focal adhesion
kinase, PI3K, Ras, Raf-1, MEK1, ERK, and JNK in addition to several growth
factor and cytokine receptors, indicating a pivotal role for FAC in
receptor-mediated signaling
(3033).
Notably, in human gingival fibroblasts, the density of IL-1 receptors is
highest in the vicinity of FAC
(21,
34,
35). Further, IL-1-induced
phosphorylation of ERK and focal adhesion kinase, which are necessary for
IL-1-induced Ca2+ flux, do not occur in the absence of FACs and the
NF-
B response to IL-1 is enhanced by FAC
(24). Taken together, these
data indicate that FAC are critical for the propagation and regulation of
IL-RI-mediated ERK activation.
In addition to FAC, the organization of the actin cytoskeleton is critical for IL-1-induced ERK activation (28). This phenomenon may be linked causally as the increased density of actin filaments in FAC may provide a scaffold that directs precise interactions between actin filaments and receptor-associated signaling molecules (36). The interdependence between the actin cytoskeleton and IL-1 signaling is also supported by observations showing that IL-1 induces a transient cell contraction and disorganization of the actin filament network (24).
The regulation of chemical and mechanical signals, such as ERK activation and actin stress fiber organization, respectively, is achieved in part by a precise balance of kinase and phosphatase activities. SHP-2 is a Src homology 2 domain-containing protein tyrosine phosphatase involved in focal adhesion and stress fiber remodeling. Cells lacking functional SHP-2 have greater actin stress fiber density, exhibit increased numbers of FAC, have a stronger attachment to fibronectin and demonstrate reduced cell migration (3740). Although the catalytic domain of SHP-2 is required for actin binding, its phosphatase activity is apparently not, indicating a functionally important adaptor role for the catalytic domain (40). SHP-2 is an important mediator of integrin, growth factor, and cytokine receptor-mediated activation of Ras and in turn ERK (4143). Notably, SHP-2 is involved in PDGF-induced ERK activation in fibroblasts grown on fibronectin (44). Following binding to the phosphorylated PDGF receptor, SHP-2 acts as an adaptor by mediating the association of PDGF-R with the Grb-2-Sos complex via its SH2 domains, leading to the activation of Ras and ERK. This positive regulatory effect is potentiated in cells plated on fibronectin, conditions that promote focal adhesion formation.
Evidently, SHP-2 plays a crucial role in the regulation of growth factor and cytokine-induced ERK activation, in part by interacting with and perhaps modulating focal adhesions and associated proteins. However, the mechanisms by which SHP-2 regulates cytokine-induced signal generation and attenuation are incompletely understood. Here we demonstrate that SHP-2 is present in focal adhesions in fibroblasts from mechanically active environments and modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 receptor engagement. We propose that the adaptor functions of SHP-2 may be important in mediating these processes.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Bead PreparationsHuman gingival
fibroblasts were grown in minimal essential medium (-MEM). Murine SHP-2
-/-(SHP-2(
46110)) fibroblasts, and the NIH 3T3 cells were grown
in high glucose DME. All media contained 10% fetal bovine serum and
antibiotics (0.17% penicillin V, 0.1% gentamicin sulfate, and 0.01%
amphotericin). Cells were used between the 5th and 12th passages as previously
described (28). Magnetite
beads were added to acid-solubilized collagen (3 mg/ml, pH < 1.0) or
poly-L-lysine (1 mg/ml) and vortexed. NaOH was added to the
collagen solution to a final concentration of 0.1 M to equilibrate
pH to 7.4. The suspension was incubated at 37 °C for 20 min. The beads
were then washed several times, resuspended in PBS, and sonicated for 10 s
(output setting 3, power 15%). To coat beads with BSA, beads were added to a
solution of PBS containing 1 mg/ml BSA, vortexed for 30 s, incubated for 20
min at 37 °C, washed three times, and resuspended in PBS. Murine embryonic
fibroblasts (wild type, SHP-2(
46110), and
SHP-2(
46110) reconstituted with wild-type murine SHP-2) were
generated and grown as previously described
(50).
Isolation of Focal AdhesionsCells were grown to 8090% confluence on 60-mm tissue culture dishes and subsequently were cooled to 4 °C prior to the addition of collagen-coated or BSA-coated magnetite beads. FACs were isolated from dishes after various incubation time periods as described (45). In brief, cells were washed three times with ice-cold PBS to remove unbound beads and scraped into ice-cold cytoskeleton extraction buffer (CSKB; 0.5% Triton X-100, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 20 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride, 10 mM PIPES, pH 6.8). The cell-bead suspension was sonicated for 10 s (output setting 3, power 15% Branson), and the beads were isolated from the lysate using a magnetic separation stand. The beads were resuspended in fresh ice-cold CKSB, homogenized with a Dounce homogenizer (20 strokes) and re-isolated magnetically. The beads were washed in CSKB, sedimented with a microcentrifuge, resuspended in Laemmli sample buffer, and placed in a boiling water bath for 10 min to allow the collagen-associated complexes to dissociate from the beads. The beads were sedimented and the lysate collected for analysis.
ImmunoblottingThe protein concentrations of the cell lysates were determined by Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded onto an SDS-polyacrylamide gel (10% acrylamide), resolved by electrophoresis, and transferred to a nitrocellulose membrane. The membrane was incubated overnight at 4 °C in a Tris-buffered saline solution with 5% milk to block nonspecific binding sites. Membranes were incubated with the primary antibodies for a minimum of 1 h at room temperature in Tris-buffered saline with 0.1% Tween-20. Horseradish peroxidase secondary antibodies were incubated for 1 h at room temperature in Tris-buffered saline with 0.1% Tween-20 and 5% milk. Labeled proteins were visualized by chemiluminescence as per the manufacturer's instructions (Amersham Biosciences).
ImmunofluorescenceChamber slides (8-well; Labtek) were coated with fibronectin (10 µg/ml in PBS). Cells were plated and allowed to spread for 24 h prior to treatment. Following treatment cells were fixed in 3% paraformaldehyde in PBS for 10 min at room temperature, blocked and permeabilized in PBS with 0.2% Triton X-100 and 0.2% BSA for 15 min at room temperature. Antibodies were diluted in PBS with 0.2% Triton X-100 and 0.2% BSA. Immunofluorescence staining for vinculin, IRAK, and SHP-2 was performed with monoclonal anti-vinculin, rabbit anti-IRAK, or rabbit anti-SHP-2 antibody (1:50, 1:20, and 1:50 dilutions, respectively) for 1 h at room temperature or 3 h at 37 °C. Slides were washed with PBS, incubated with goat anti-mouse FITC-conjugated antibody (1:50 dilution) for 60 min at 4 °C, washed, and sealed with a coverslip. The slides were viewed with a Nikon 300 inverted fluorescence microscope equipped with Nomarski optics. Images were captured digitally using C-Imaging (Compix Imaging, Philadelphia, PA).
ImmunoprecipitationCells that had attained 8090% confluence on 100-mm tissue culture dishes in normal growth medium were washed three times in ice cold PBS and 1 ml of Nonidet P-40 Buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 mM NaF, 2 mM Na3VO4,) was added to each dish. The cell lysates were scraped into a microcentrifuge tube and rotated for 10 min at 4 °C. The tubes were then centrifuged for 15 min at 4 °C and the supernatant transferred to new tubes. 30 µl/ml agarose-conjugated rabbit polyclonal SHP-2 was added to the lysates, and the tubes were rotated for 2 h at 4 °C. The tubes were then centrifuged for 2 min at 4 °C. The pellet was washed three times with Nonidet P-40 buffer, and then the beads and associated proteins were resuspended in sample buffer, boiled for 5 min, and the eluted proteins analyzed by SDS-PAGE followed by immunoblotting.
In Vitro Phosphatase AssayComplexes of anti-SHP-2 antibody
prebound to protein A/G Plus-Agarose beads were added to the cleared
supernatant. The samples were rotated at 4 °C for 2 h, after which the
beads were separated and extensively washed. One-fifth of the bead volume was
removed and used for immunoblot analysis by resuspension in Laemmli sample
buffer and then subjected to SDS-PAGE and immunoblotting. The blots were
probed with monoclonal anti-SHP2 antibody followed by washing and then
incubation with horseradish peroxidase-labeled goat-anti mouse antibody. The
remaining beads were sedimented, washed, and resuspended in assay buffer
containing 0.1 mM phosphopeptide (RRLIEDAEpYAARG) and 60
mM -mercaptoethanol. Samples were shaken for 3 h at 37 °C
and centrifuged briefly. Malachite Green was used to detect free phosphate
released from the phosphopeptide by measurement of absorbance at 650 according
to the manufacturer's instructions (Upstate Biotechnologies Inc., Lake Placid,
NY).
33P Phosphorylation AssayHuman gingival
fibroblasts were cultured in phosphate-free media for 18 h in the presence of
5 µCi/ml of [33P]orthophosphate, washed three times with medium,
and then exposed to IL-1, PDGF, or vehicle control as indicated. SHP-2
was purified by immunoprecipitation as described above. Immunoprecipitates
were separated by SDS-PAGE and the protein transferred to nitrocellulose as
described above. The nitrocellulose membranes were analyzed using a STORM
phosphorimager. Radioactive 33P incorporation into SHP-2 was
quantified by densitometry based on the electrophoretic mobility of SHP-2
determined by Western blotting with anti-SHP-2 antibodies on the same
membrane.
ElectroporationCells were harvested by trypsinization,
pelleted, and resuspended in serum-free -MEM buffered with 12.5
mM HEPES. A 30-µl aliquot of cells was placed in a cuvette with
30 µg of rabbit polyclonal anti-SHP-2 in HEPES-buffered
-MEM at 4
°C. The cells were electroporated at 100 V/cm and capacitance 960 µF
using a Bio-Rad Gene Pulser with a capacitance expander and Gene Pulser
cuvettes (0.2-cm interelectrode distance). Cells were incubated at 4 °C
for 10 min and replated in normal growth medium. In selected experiments, to
determine the intracellular localization of the anti-SHP-2 antibody, cells
were cultured on fibronectin-coated glass coverslips for 4 h after
electroporation, fixed with 1.5% paraformaldehyde, permeabilized with 0.1%
Triton X-100, and stained with Texas Red-conjugated goat anti-rabbit
F(ab')2 antibodies. As a control, cells were subject to
electroporation under identical conditions but without the primary anti-SHP-2
antibody.
Data AnalysisData were analyzed by ANOVA with correction for multiple comparisons (Dunnett) or by paired or unpaired Student's t test, as indicated. Statistical significance was set at p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To ensure that the collagen-coated beads were able to specifically recruit focal adhesion proteins, we repeated the experiment with cells that had been pretreated with 50 nM swinholide A (SWA). This toxin severs actin filaments and prevents actin polymerization, thereby blocking the formation of FAC (46). Under these conditions, neither vinculin nor SHP-2 was recruited to the beads (Fig. 1D). BSA-coated beads also failed to recruit vinculin or SHP-2 (not shown) indicating receptor specificity. In concert, these data indicate that the proteins isolated in this manner were specifically recruited into bead-bound complexes by mechanisms that are dependent on the formation of integrin-associated FAC and an intact actin cytoskeleton.
Catalytic Activity of SHP-2 Is Not Modulated by IL-1There are several mechanisms by which SHP-2 could modulate IL-1-induced signal transduction leading to ERK activation including alterations of catalytic activity, recruitment to FAC leading to interaction with other signaling molecules, reversible phosphorylation, or by a combination of these events. To investigate possible alterations in its catalytic activity, SHP-2 was purified by immunoprecipitation from control and IL-1-stimulated cells and its catalytic activity analyzed by an in vitro phosphatase assay using a phosphopeptide as a substrate as described under "Experimental Procedures." This analysis revealed that IL-1 stimulation did not alter the tyrosine phosphatase activity of SHP-2 (Table I).
|
SHP-2 Association with FAC Does Not Require IL-1 StimulationSHP-2 might mediate FAC restriction of IL-1-induced ERK activation by differential recruitment to focal adhesions following IL-1 induction. To determine if IL-1 enhances the amount of SHP-2 recruited to nascent FAC, these complexes were isolated from cells using collagen-coated beads as described above. Beads were added to the dorsal surface of cells followed by treatment with IL-1 for 0, 10, 40, and 60 min. SHP-2 was recruited to nascent FAC in a time-dependent manner, but there was no additional increment in the relative abundance of SHP-2 in FAC following IL-1 treatment (Fig. 2). We next examined the effect of IL-1 on SHP-2-FAC association in mature adhesion complexes. For these experiments, cells were plated overnight on fibronectin to facilitate formation of mature FAC, stimulated with IL-1 or vehicle control, and SHP-2 was purified by immunoprecipitation with a polyclonal anti-SHP-2 antibody. The immunoprecipitates were immunoblotted with monoclonal antibodies against the C terminus of SHP-2 or against the focal adhesion proteins talin and vinculin. IL-1 stimulation had no effect on the levels of the focal adhesion proteins talin or vinculin (used here as a marker of FAC) that co-precipitated with SHP-2 (Fig. 2). We conclude that although a fraction of SHP-2 associates with focal adhesion proteins, IL-1 does not alter this association.
|
SHP-2 Is Phosphorylated on Tyrosine in Response to IL-1
SHP-2 could modulate IL-1-induced ERK activation by rapid post-translational
modifications such as reversible phosphorylation. There is precedent for this
type of modification; SHP-2 is phosphorylated on serine and threonine residues
in response to insulin or phorbol ester
(47) and on tyrosine residues
in response to IL-3 stimulation of fibroblasts
(48) or in response to
MIP1 stimulation of lymphocytes
(49). To determine if SHP-2
was phosphorylated under our experimental conditions in response to IL-1,
cells were labeled with [33P]orthophosphate and then exposed to
vehicle control, IL-1, or PDGF, the latter used here as a positive control
(44). SHP-2 was purified by
immunoprecipitation, separated by SDS-PAGE, transferred to nitrocellulose, and
analyzed with a phosphorimager. This analysis revealed that SHP-2 was
phosphorylated in response to IL-1 (Fig.
3A). In three separate experiments, IL-1 induced a 2.1
± 0.13 (mean ± S.E.)-fold increase in 33P
incorporation into SHP-2 compared with control (p = 0.0067). By
comparison, PDGF increased 33P incorporation into SHP-2 by 3.43
± 0.55-fold compared with control (p < 0.0001).
|
To determine the identity of the residues undergoing phosphorylation in response to IL-1, anti-SHP-2 immunoprecipitates from control, and IL-1-stimulated cells were immunoblotted with antibodies to phosphoserine and phosphothreonine. This analysis revealed no detectable phosphorylation on either serine or threonine residues (not illustrated).
To determine if SHP-2 was phosphorylated on tyrosine residues in response to IL-1, whole cell lysates (Fig. 3B) and anti-SHP-2 immunoprecipitates (Fig. 3, D and E) were immunoblotted with antibodies that recognize tyrosine phosphorylation of 2 specific residues of SHP-2, tyrosine 542 and tyrosine 580. These experiments demonstrated that in response to IL-1, SHP-2 is phosphorylated on tyrosine 542 but not on tyrosine 580. Additionally, preincubation of cells with 1 µM herbimycin A, a tyrosine kinase inhibitor, prevented IL-1 induced phosphorylation of tyrosine 542 (Fig. 3C). By contrast, stimulation with PDGF-induced tyrosine phosphorylation of both tyrosines 542 and 580 (Fig. 3, B, D, and E). Taken together, these data indicate that SHP-2 undergoes site-specific phosphorylation on tyrosine 542 in response to IL-1 by a herbimycin A-sensitive tyrosine kinase.
SHP-2-/- (46110) Murine Embryonic
Fibroblasts Are Not a Suitable Model System to Study FAC-dependent IL-1
SignalingWe endeavored to develop a model system that would allow
more direct analysis of the role of SHP-2 in IL-1-induced ERK activation
through FAC. As SHP-2 deficiency in mice is lethal during embryogenesis
(50), we used fibroblast cell
lines from these embryos that have previously been used as a model system to
study the role of SHP-2 in diverse signaling pathways
(38,
42,
51). The gene-targeting
strategy used in these experiments
(50) results in markedly
diminished levels of expression of a mutant 57-kDa SHP-2 protein in which 64
residues within the N-terminal SH2 domain have been deleted
(SHP-2(
46110)), an alteration that abrogates phosphopeptide
recognition (50,
52). Published experiments
using these embryonic fibroblasts have demonstrated that while
SHP-2(
46110) prohibits receptor-mediated ERK activation in many
signaling cascades, IL-1-induced ERK activation is relatively unaffected
(38,
41,
51). Although these
experiments might suggest no significant role for SHP-2 in IL-1-induced ERK
activation, two issues are relevant. First, it is not clear that these
embryonic fibroblasts recapitulate conditions present in mature fibroblasts
derived from mechanically active environments (e.g. gingival
fibroblasts and chondrocytes). Second, it is unknown if the expression of a
mutant (and possibly partially functional) SHP-2 complicates the
interpretation of the model system.
To examine the first issue, we compared the responsiveness of human
gingival fibroblasts and murine embryonic fibroblasts to IL-1 under conditions
where FAC complexes were present or absent. Gingival fibroblasts plated on
fibronectin (to promote FAC formation) demonstrated robust activation of ERK
in response to IL-1 while cells plated on poly-L-lysine (to
prevent FAC formation) failed to activate ERK in response to the cytokine
(Fig. 4), consistent with
previous results (23). In
contrast, wild type murine embryonic fibroblasts demonstrated a strong
increase in phospho-ERK in response to IL-1 in either the presence or absence
of intact FAC. Thus IL-1-induced ERK activation in murine embryonic cells is
not restricted by focal adhesions.
|
The second issue relates to the potential functional importance of residual
mutant SHP-2. As discussed above, the SHP-2(46110) murine
embryonic fibroblasts express small amounts of a mutant SHP-2 protein. We
considered the possibility that a partially functional mutant SHP-2 protein is
expressed in amounts sufficient to permit signal transduction to ERK after
IL-1 stimulation. To investigate this possibility, we isolated FAC from both
the SHP-2(
46110) and wild-type embryonic fibroblasts; the latter
cells were reconstituted with wild-type SHP-2 as previously described
(51). Both the 57-kDa mutant
(
46110) SHP-2 and the wild-type SHP-2 associated with FAC
(Fig. 5). This indicates that
the mutant SHP-2(
46110) possesses sufficient binding activity to
mediate protein-protein interactions that direct it to FAC. Further,
IL-1-induced ERK activation was comparable in the wild-type and mutant cells.
From these experiments we conclude that the murine embryonic fibroblasts do
not recapitulate FAC-restriction of IL-1-induced ERK activation and express
residual amounts of a partially functional mutant SHP-2 protein. These cells
therefore cannot be used to investigate focal adhesion-restricted IL-1
signaling.
|
SHP-2 Is Required for IL-1-induced ERK ActivationAs an
alternative strategy to functionally deplete SHP-2, we introduced anti-SHP-2
antibodies into the cytosol of human gingival fibroblasts using
electroporation, a strategy that we have used successfully in previous studies
to delineate the role of IRAK in IL-1-induced signaling to ERK
(28). A rabbit polyclonal
anti-SHP-2 antibody was used to sequester endogenous SHP-2 and prevent its
association with other signaling molecules. As a control, an irrelevant rabbit
polyclonal antibody was used under identical conditions. We first determined
if electroporation could be used to facilitate the entry of large (150
kDa) molecules into the fibroblast cytosol. The conditions used in these
experiments (field strength of 100 V/cm and 960 µF capacitance) resulted in
introduction of significant amounts of 150-kDa FITC-conjugated dextran into
>95% of cells (Fig.
6A). FITC-dextran of this molecular weight was used
because of its similar size to the anti-SHP-2 antibody. Human gingival
fibroblasts were electroporated in the presence of control (irrelevant) or
anti-SHP-2 antibody, plated in normal growth medium (
-MEM/10% FBS) for
4 h., and then stimulated with IL-1
for 0, 10, or 60 min. The successful
introduction of anti-SHP-2 antibody into the cytosol of the fibroblasts was
confirmed by staining fixed and permeabilized cells with Texas Red-labeled
goat anti-rabbit antibody and visualizing the cells with fluorescence
microscopy (Fig. 6B).
In both control and anti-SHP-2 antibody-treated cells, a small increase in the
basal ERK phosphorylation was noted (Fig.
6C), likely reflecting active cell spreading that occurs
at early times after plating and that is associated with ERK activation
(24). In cells electroporated
with an irrelevant (control) antibody, immunoblots of cell lysates
demonstrated the expected increase of IL-1-induced ERK phosphorylation
compared with unstimulated cells (Fig.
6C, left panel). In contrast, the IL-1-induced
increase in phospho-ERK was abrogated in cells that had been electroporated
with the anti-SHP-2 antibody (Fig.
6C, right panel), indicating that SHP-2 is
required for IL-1-induced activation of ERK.
|
To ensure that the electroporation procedure per se did not introduce potentially confounding effects such as prevention of the formation of FAC or the recruitment of SHP-2 into nascent FAC, cells were electroporated with an irrelevant control antibody or the anti-SHP-2 antibody and FAC isolated. Immunoblot analysis of FAC-associated proteins revealed normal vinculin recruitment into focal adhesions in both control and anti-SHP-2 antibody electroporated cells. By contrast, SHP-2 was detected in FAC preparations from the control (irrelevant antibody) but not from anti-SHP-2 antibody treated cells, presumably due to sequestration of SHP-2 by the antibody (Fig. 6D). Collectively, these results indicate that SHP-2 and its presence in focal adhesions are required for IL-1-induced ERK activation.
SHP-2 Is Associated with the Actin Cytoskeleton and Is Required for IL-1-induced Actin Assembly and Cell Contraction There is an apparent interdependent relationship between the assembly of actin filaments and IL-1 signal transduction. Specifically, IL-1 stimulation of fibroblasts grown on fibronectin results in reversible cell contraction and disorganization of actin stress fibers (Ref. 24; also see below). As SHP-2 is involved in actin stress fiber organization and cell motility in addition to receptor-mediated ERK activation, we asked if SHP-2 might regulate IL-1-mediated cell contraction. We first examined whether the association of SHP-2 with actin (40) was affected by IL-1 stimulation. Triton X-100 insoluble and soluble fractions were analyzed for the presence of SHP-2 following IL-1 stimulation. A fraction of SHP-2 was associated with the Triton X-100 insoluble (cytoskeletal) fraction but that there was no change in this proportion in response to IL-1 (Fig. 7A).
|
Next, we examined the effect of IL-1 stimulation on actin stress fiber
organization. Fluorescence microscopy revealed that treatment of cells plated
on fibronectin with IL-1 (20 ng/ml) for 10 min causes transient
disassembly of actin filaments, particularly those filaments crossing the cell
body (Fig. 7B). By 60
min after addition of IL-1, stress fibers had reformed. Cells were loaded with
rabbit polyclonal anti-SHP-2 antibody by electroporation as described above to
sequester endogenous SHP-2 and prevent its association with potential
interacting proteins involved in signaling. Electroporated cells were plated
in normal growth medium (
-MEM/10% FBS) on fibronectin-coated glass
slides for 4 h and stimulated with IL-1
for 0, 10, or 60 min. The cells
were fixed, permeabilized, and stained with TRITC phalloidin to label actin
filaments and obtain clearly demarcated outlines of the cell edge. Image
analysis of cell area showed that in control cells loaded with the irrelevant
antibody and stimulated with IL-1, there was a marked reduction in cell area
indicative of cell contraction. By contrast, cells loaded with anti-SHP-2
antibody failed to contract in response to IL-1. Moreover, these cells also
failed to reorganize the actin cytoskeleton in response to IL-1 as shown by
the density and pattern of stress fibers or the intensity of TRITC-phalloidin
staining. These results indicate that SHP-2 is critical for IL-1-induced
changes in cell morphology, reorganization of the actin cytoskeleton, and cell
contraction.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Engagement and clustering of integrin receptors leads to recruitment of both structural and signaling molecules into FAC, events that modulate cellular responses to environmental stimuli (29). In the present study, we used collagen-coated magnetite microbeads to stimulate temporally and spatially regulated assembly of FAC and to enable the specific isolation of proteins physically associated with the beads. The focal adhesion protein vinculin and SHP-2 were both physically associated with FAC isolates as determined by immunoblotting of collagen bead-associated proteins and in immunohistochemistry experiments showing that SHP-2 and vinculin spatially co-localized to FAC. These observations, in concert with previous studies showing that SHP-2 is a positive regulator of receptor-mediated ERK activation (55), provided the rationale to examine in more depth the potential role of SHP-2 in IL-1-induced ERK activation restricted by FAC.
There are several mechanisms by which SHP-2 could modulate IL-1 signaling pathways including alterations in catalytic (phosphatase) activity, protein-protein interactions, and posttranslational modifications such as reversible phosphorylation. In the current study, we were unable to detect alterations in the catalytic activity of SHP-2 in response to IL-1 stimulation. However, as the physiological substrates of SHP-2 remain unidentified (56), it remains possible that selective, substrate-dependent alterations in SHP-2 catalytic activity occur in response to IL-1 stimulation.
A second mechanism by which SHP-2 could modulate IL-1 signaling is via its functioning as an adaptor protein in mediating protein-protein interactions. Notably, SHP-2 provides a crucial adaptor role in PDGF (49, 57) and IL-3/IL-6 signaling (58). Further, protein interactions with SHP-2 can enhance its catalytic activity (59, 62). Although we observed that SHP-2 was recruited to nascent FAC, there was no further recruitment in response to IL-1 and no alteration of its catalytic activity. SHP-2 is also known to interact with structural proteins such as F-actin (40), but in our system, IL-1 did not alter this association.
A third possible mechanism of regulation of SHP-2 involves reversible phosphorylation as has been reported in other systems. Our data indicate that IL-1 induces site-specific phosphorylation of Tyr-542. This differs from the phosphorylation reported in response to PDGF that occurs on both Tyr-542 and Tyr-580 and is mediated by the PDGF receptor kinase (63). The identity of the tyrosine kinase(s) responsible for phosphorylation of SHP-2 in response to IL-1 is currently unknown. The IL-1 receptor possesses no intrinsic kinase activity and therefore cannot be responsible for SHP-2 phosphorylation. However, there are several other possible candidates including Src family kinases, an area that is the subject of current studies in our laboratory.
The putative functions of SHP-2 are diverse and include regulation of
integrin, growth factor and cytokine signaling pathways
(4143).
However, elucidation of the functional importance of SHP-2 in many pathways
has been complicated by several factors. First, as mentioned above, SHP-2 has
several distinct structural domains, the functions of which are not completely
understood and disruptions of different domains may result in different
phenotypes. Second, loss-of-function mutations in murine SHP-2 are lethal in
mid-embryonic development
(50), thereby preventing a
more comprehensive analysis of the function of SHP-2 in well differentiated
tissues. While fibroblast cell lines have been derived from these embryos,
they may not be suitable models to study the functions of SHP-2 in more
differentiated and or specialized cells. The primary purpose of the current
study was to examine the signaling pathway regulating IL-1-induced ERK
activation in primary cultures of fibroblasts derived from mechanically active
environments that are susceptible to inflammatory diseases (i.e.
human gingival fibroblasts). We have previously demonstrated that certain
functions of gingival fibroblasts
(21,
23) and chondrocytes,
specialized cell-types involved in clinically important chronic inflammatory
diseases, are differentially regulated by the formation of FAC. Specifically,
we have reported that IL-1 induced activation of ERK is critically dependent
on the formation of FAC. In turn, ERK activation under these conditions leads
to the expression of c-Fos and the synthesis and release of matrix
metalloproteinases that can mediate the pathological degradation of
extracellular matrix proteins
(3). This differential
regulation of ERK is present in primary cultures of fibroblasts and
chondrocytes but not in cultured fibroblast cell lines that we have examined.
This has particular relevance to the studies with murine embryonic fibroblasts
derived from SHP-2 deficient (SHP-2(46110)) embryos. As
discussed above, these embryonic cells did not demonstrate this selective
(FAC-restricted) activation of ERK by IL-1 and therefore could not be used to
delineate this pathway. It is currently not known what cellular alterations
result in this loss of selective IL-1-induced ERK activation. This requirement
for the differentiated phenotype of primary cultures of fibroblasts also
obviated our ability to use transfection of recombinant forms of SHP-2
(e.g. catalytically inactive) as an experimental strategy because
these primary cultures are intractable to high efficiency transfection.
An interdependent relationship exists between IL-1 signaling and the actin cytoskeletal network. While an organized network of actin filaments is required for IL-1-induced ERK activation, IL-1 itself causes a transient contraction of fibroblasts associated with disorganization of actin stress fibers. Moreover, SHP-2 is evidently involved in the regulation of actin stress fiber density, the abundance of focal adhesions, the strength of attachment to the extracellular matrix, cell spreading, and motility (3840, 67). These observations provide strong evidence that SHP-2 is involved in cytoskeletal remodeling processes, similar to those caused by IL-1 stimulation. Our data support and extend these observations. In the current study, we demonstrated that SHP-2 is critical for IL-1-induced cell contraction and stress fiber disorganization. Fibroblasts loaded with anti-SHP-2 antibodies that apparently prevented its association with F-actin and other binding partners, were unable to contract in response to IL-1 or to effect any changes in actin stress fiber density.
Conceivably, SHP-2 influences cellular events via modulation of ERK localization and activity. For example, SHP-2 may mediate ERK activation by recruiting large, macromolecular signaling complexes required to transmit the IL-1 signal from the FAC-localized receptors to ERK, similar to that of the activated PDGF receptor (44). This activation of ERK then initiates actin remodeling. Accordingly, sequestration of SHP-2 in the cytosol with antibody effectively blocks IL-1-induced ERK activation, thereby interfering with actin reorganization. Another possibility is that SHP-2 mediates IL-1-induced actin remodeling, which is required for activation of ERK. Consequently, sequestering SHP-2 with antibody blocks its association with actin filaments and consequently disrupts cytoskeletal reorganization. As actin remodeling is necessary for ERK translocation to the site of signal transduction, the inability of the cell to contract or remodel its actin cytoskeleton blocks ERK activation in response to IL-1. A third possibility is that the adaptor functions of SHP-2 modulate a pathway in parallel to IL-1-dependent ERK activation and that these are interdependent signaling processes, both of which must occur optimally for appropriate IL-1 signal generation.
In conclusion, the propagation and regulation of IL-1 signaling pathways are regulated by the formation of signaling complexes joined together in a spatially confined manner by scaffolding proteins including actin filaments and associated binding proteins. A complete knowledge of the molecules present in the FAC that mediate the exchange of signals between the activated IL-1 receptor and the downstream MAPK cascade has yet to be achieved. In this manuscript, we provide evidence that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function. This has important implications for regulation of cellular activation in inflammatory disorders.
![]() |
FOOTNOTES |
---|
** Recipient of a senior scientist award from the CIHR.
Holds the R. Fraser Elliott Chair in Transplantation Research from the Toronto
General Hospital of the University Health Network and a Canada Research Chair
in Respiration from the CIHR. To whom correspondence should be addressed:
Clinical Sciences Division, Rm. 6264, Medical Sciences Building, The
University of Toronto, 1 Kings College Circle, Toronto, Ontario M5S 1A8,
Canada. Tel.: 416-978-8923; Fax: 416-971-2112; E-mail:
gregory.downey{at}utoronto.ca.
1 The abbreviations used are: IL-1, interleukin 1; MAP, mitogen-activated
protein; JNK, Jun N-terminal kinase; ERK, extracellular signal-regulated
kinase; FAC, focal adhesion complex; BSA, bovine serum albumin; PBS,
phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PDGF,
platelet-derived growth factor; FITC, fluorescein isothiocyanate; FBS, fetal
bovine serum; TRITC, tetramethylrhodamine isothiocyanate.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|