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Division of Respirology, the Department of Medicine, The Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario M5S 1A8, Canada
Division of Respirology, the Department of Medicine, The Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario M5S 1A8, Canada
Division of Respirology, the Department of Medicine, The Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario M5S 1A8, Canada
Division of Respirology, the Department of Medicine, The Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario M5S 1A8, Canada
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
Division of Respirology, the Department of Medicine, The Toronto General Hospital Research Institute of the University Health Network, Toronto, Ontario M5S 1A8, Canada
* This work was supported by the Canadian Institutes of Health Research (CIHR) (to G. D. and C. M.), the National Sanitarium Association, and the Canadian Arthritis Network. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** Recipient of a senior scientist award from the CIHR.
Focal adhesion complexes are actin-rich, cytoskeletal structures that mediate cell adhesion to the substratum and also selectively regulate signal transduction pathways required for interleukin (IL)-1β signaling to the MAP kinase, ERK. IL-1-induced ERK activation is markedly diminished in fibroblasts deprived of focal adhesions whereas activation of p38 and JNK is unaffected. While IL-1 signaling is known to involve the activity of protein and lipid kinases including MAP kinases, FAK, and PI3K, little is known about the role of phosphatases in the regulation of IL-1 signal generation and attenuation. Here we demonstrate that SHP-2, a protein tyrosine phosphatase present in focal adhesions, modulates IL-1-induced ERK activation and the transient actin stress fiber disorganization that occurs following IL-1 treatment in human gingival fibroblasts. Using a combination of immunoblotting, immunoprecipitation, and immunostaining we show that SHP-2 is present in nascent focal adhesions and undergoes phosphorylation on tyrosine 542 in response to IL-1 stimulation. Blocking anti-SHP-2 antibodies, electoporated into the cytosol of fibroblasts, inhibited IL-1-induced ERK activation, actin filament assembly, and cell contraction, indicating a role for SHP-2 in these processes. In summary, our data indicate that SHP-2, a focal adhesion-associated protein, participates in IL-1-induced ERK activation likely via an adaptor function.
) capable of stimulating multiple signaling cascades and inducing the expression of a variety of immune and inflammatory factors including c-Fos and c-Jun (
). Consequently, the unregulated production of IL-1 is associated with significant cellular and tissue damage observed in inflammatory diseases such as rheumatoid arthritis and periodontal diseases (
). Following IL-1 binding, the IL-1 receptor-associated protein is recruited to the type I IL-1 receptor thereby increasing the avidity of the receptor for its ligand (
), which initiate downstream signaling. Engagement of the IL-1 receptor initiates multiple signaling events implicated in the pathogenesis of chronic inflammatory diseases, including: (i) phosphorylation of multiple kinases and IL-1R1-associated proteins (
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 (
). 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 α-actinin (
). 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 (
). 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-κB response to IL-1 is enhanced by FAC (
). 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 (
). 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 (
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 (
). 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 (
). 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
Materials—Bovine fibronectin, poly-l-lysine, BSA, aprotinin, leupeptin, Nonidet P-40, mouse monoclonal antibodies to vinculin, β-actin, rabbit polyclonal anti-SHP-2, FITC-conjugated goat anti-mouse, magnetite beads, phenylmethylsulfonyl fluoride, Triton X-100, and Tween-20 were obtained from Sigma. Rabbit polyclonal antibodies to p38, phospho-p38, ERK1/2, JNK(SAPK), phospho-JNK(SAPK), mouse monoclonal anti-phospho-ERK1/2 antibodies, antibodies to phosphothreonine (rabbit polyclonal), and phospho-SHP-2 (Tyr-580 and Tyr-542) were purchased from Cell Signaling (Beverly, MA). Horseradish peroxidase-conjugated goat anti-mouse (H+L), goat anti-rabbit (H+L), and Latrunculin B were purchased from Cedarlane Laboratories (Hornby ON). The ECL chemiluminescence kit was purchased from Amersham Biosciences. Acidified bovine type I collagen (Vitrogen) was purchased from Cohesion Technologies Inc. (Palo Alto, CA). A magnetic separation stand was purchased from Promega (Madison, WI). The IL-1β was obtained from Cedarlane Laboratories (Mississauga, ON). Protein A Sepharose was obtained from Zymed Laboratories Inc. (San Francisco, CA). Swinholide A and herbimycin A were obtained from Calbiochem (La Jolla, CA). Rabbit polyclonal anti-SHP-2 (C terminus), anti-IRAK, and horseradish peroxidase-conjugated anti-SHP-2 (N terminus) were obtained form Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies to phosphoserine were from Zymed Laboratoratories (South San Francisco, CA). Platelet-derived growth factor (PDGF) BB was from R&D Systems (Minneapolis, MN).
Cell Culture and Bead Preparations—Human gingival fibroblasts were grown in minimal essential medium (α-MEM). Murine SHP-2 -/-(SHP-2(Δ46–110)) 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 (
). 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(Δ46–110), and SHP-2(Δ46–110) reconstituted with wild-type murine SHP-2) were generated and grown as previously described (
Isolation of Focal Adhesions—Cells were grown to 80–90% 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 (
). 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.
Immunoblotting—The 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).
Immunofluorescence—Chamber 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).
Immunoprecipitation—Cells that had attained 80–90% 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 Assay—Complexes 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 β-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 Assay—Human 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-1β, 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.
Electroporation—Cells were harvested by trypsinization, pelleted, and resuspended in serum-free α-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 α-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 Analysis—Data 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
SHP-2 Is Associated with Focal Adhesion Complexes—To determine which factors regulate IL-1 signaling to ERK in the context of FAC, we first determined if SHP-2 was localized to FAC in fibroblasts under our experimental conditions. Collagen-coated latex beads were added to cells to induce FAC assembly at sites of cell-bead contact on the dorsal cell surface. Immunofluorescence microscopy with anti-vinculin or anti-SHP-2 antibodies demonstrated that by 30 min after the addition of beads, vinculin and SHP-2 co-localized to the sites of bead-cell contact (Fig. 1), indicating that SHP-2 is associated with nascent focal adhesions. Proteins associated with FAC were purified using magnetic beads and immunoblot analysis of bead-associated proteins revealed a time-dependent recruitment of the focal adhesion protein vinculin and SHP-2 into nascent FAC (Fig. 1D). IRAK, a receptor-associated kinase known to be absent in FAC from unstimulated cells (
), was not present in these preparations and served here as a negative control (Fig. 1C).
Fig. 1SHP-2 is present in focal adhesion complexes. Human gingival fibroblasts were plated on fibronectin (10 μg/ml) for 48 h in normal growth medium (α-MEM/FBS). Fibroblasts were incubated with collagen-coated latex microbeads for 30 min at 37 °C to induce focal adhesion formation and immunostained for vinculin, SHP-2 or with a control antibody (A–C). Fibroblasts were plated overnight in normal growth medium (α-MEM/10% FBS). Cells were incubated with collagen-coated magnetic microbeads for 10 min at 4 °C or 10 min at 4 °C plus 10 min at 37 °C (lanes 1 and 2, respectively). Untreated or Swinholide A-treated cells were incubated with collagen-coated magnetic microbeads for 10 min at 4 °C or 10 min at 4 °C plus 10 min at 37 °C (lanes 3 and 4). FAC were isolated using the rapid focal adhesion isolation protocol (see “Experimental Procedures”), and the resulting proteins were resolved by SDS-PAGE and blots were probed with mouse monoclonal anti-vinculin or SHP-2 (D).
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 (
). 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-1—There 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).
Table ITyrosine phosphatase activity in SHP-2 immunoprecipitates (normalized for immunoreactive SHP-2 protein)
SHP-2 Association with FAC Does Not Require IL-1 Stimulation—SHP-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.
Fig. 2The level of SHP-2 associated with focal adhesions is not affected by IL-1 stimulation.A, human gingival fibroblasts were plated overnight in normal growth medium (α-MEM/10% FBS) then incubated with collagen-coated magnetic microbeads for 10, 20, 40, 60 min with or without IL-1β treatment (20 ng/ml). FACs were isolated as described (see “Experimental Procedures”), and the resulting proteins were resolved by SDS-PAGE and blots were probed with rabbit polyclonal anti-SHP-2. B, human gingival fibroblasts were plated on tissue culture plastic (overnight), left untreated or stimulated with IL-Iβ (20 ng/ml) for 10 min at room temperature. Lysates were immunoprecipitated with rabbit polyclonal anti-SHP-2 antibodies. The resulting immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies to SHP-2, vinculin, or talin.
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 (
). 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 (
). 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).
Fig. 3SHP-2 is phosphorylated on tyrosine 542 in response to IL-1.A, human gingival fibroblasts were labeled with [33P]orthophosphate and stimulated with IL-1 (20 ng/ml for 10 min), PDGF BB (50 ng/ml for 15 min), or vehicle control as indicated. SHP-2 was purified by immunoprecipitation, the proteins separated by SDS-PAGE, transferred to nitrocellulose and analyzed by a STORM phosphorimager. The nitrocellulose membrane was also blotted with antibodies to SHP-2 and equal loading of SHP-2 protein in each lane confirmed (not illustrated). B, human gingival fibroblasts grown on fibronectin were stimulated with IL-1 (20 ng/ml for 5, 10, or 15 min), PDGF BB (50 ng/ml for 15 min), or vehicle control as indicated. Equal amounts of total protein were loaded in each lane, separated by SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies to phospho-SHP-2 (tyrosine 580 and 542), and total SHP-2. C, human gingival fibroblasts grown on fibronectin were pretreated with herbimycin A (1 μg/ml for 90 min) or vehicle control. Subsequently, cells were exposed to IL-1 (20 ng/ml for 10 min) or vehicle control as indicated. Equal amounts of total protein were loaded in each lane, separated by SDS-PAGE, transferred to nitrocellulose, and blotted with antibodies to phospho-SHP-2 (tyrosine 542). D and E, human gingival fibroblasts grown on fibronectin were stimulated with IL-1, PDGF, or vehicle control as indicated. SHP-2 was purified by immunoprecipitation, the bead-associated proteins separated by SDS-PAGE, transferred to nitrocellulose and blotted with antibodies to phospho-SHP-2 (tyrosine 580; Fig. 3D) and phospho-SHP-2 (542; Fig. 3E), and total SHP-2 (Fig. 3, D and E). Lane 2 represents a control with beads alone without primary antibody incubated with cell lysates.
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-/- (Δ46–110) Murine Embryonic Fibroblasts Are Not a Suitable Model System to Study FAC-dependent IL-1 Signaling—We 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 (
), 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 (
) 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(Δ46–110)), an alteration that abrogates phosphopeptide recognition (
). Published experiments using these embryonic fibroblasts have demonstrated that while SHP-2(Δ46–110) prohibits receptor-mediated ERK activation in many signaling cascades, IL-1-induced ERK activation is relatively unaffected (
). 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-1β 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 (
). 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.
Fig. 4Focal adhesion complexes are required for IL-1β activation of ERK. Human gingival fibroblasts and murine embryonic fibroblasts from SHP-2(Δ46–110) embryos reconstituted with wild-type murine SHP-2 were plated on fibronectin or poly-l-lysine-coated tissue culture plastic in normal growth medium for 6 h and stimulated with 20 ng/ml IL-Iβ for 10 min. ERK1/2 activity was assessed by separating lysates via SDS-PAGE, and probing blots with mouse monoclonal anti-phospho-ERK1/2. Total ERK1/2 was quantified by probing the blots with rabbit polyclonal anti-ERK1/2.
The second issue relates to the potential functional importance of residual mutant SHP-2. As discussed above, the SHP-2(Δ46–110) 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(Δ46–110) and wild-type embryonic fibroblasts; the latter cells were reconstituted with wild-type SHP-2 as previously described (
). Both the 57-kDa mutant (Δ46–110) SHP-2 and the wild-type SHP-2 associated with FAC (Fig. 5). This indicates that the mutant SHP-2(Δ46–110) 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.
Fig. 5SHP-2(Δ46–110) is recruited to nascent focal adhesion complexes. SHP-2-reconstituted murine embryonic fibroblasts (lanes 1 and 3) and SHP-2(Δ46–110) (lanes 2 and 4) murine embryonic fibroblasts were plated on tissue culture plastic for 48 h in normal growth medium. Cells were incubated with collagen-coated (lanes 1 and 2) or BSA-coated (lanes 3 and 4) magnetic microbeads for 30 min at 37 °C. FACs were isolated as described under “Experimental Procedures,” the resulting proteins resolved by SDS-PAGE, and immunoblots were probed with mouse monoclonal anti-vinculin or anti-SHP-2 (recognizing the C terminus) antibodies.
SHP-2 Is Required for IL-1-induced ERK Activation—As 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 (
). 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 (α-MEM/10% FBS) for 4 h., and then stimulated with IL-1β 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 (
). 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.
Fig. 6SHP-2 is required for IL-1-induced ERK phosphorylation.A, human gingival fibroblasts were electroinjected with 150-kDa FITC-dextran, plated on fibronectin (10 mg/ml), incubated at 37 °C for 4 h, fixed with 3.7% paraformaldehyde, mounted, and viewed with a fluorescence microscope. Digital images were captures as described under “Experimental Procedures.” B, human gingival fibroblasts were electroinjected with rabbit anti-SHP-2 antibody (left panel) or buffer control (right panel), cultured on fibronectin (10 mg/ml) for 4 h at 37 °C, fixed with 1.5% paraformaldehyde, stained with Texas Red-labeled goat-anti rabbit F(ab′)2 antibodies, washed, and mounted and viewed with a fluorescence microscope. Digital images were captured as described under “Experimental Procedures.” The inset in the upper right corner is the corresponding Nomarski (transmitted light) image of the field. C, human gingival fibroblasts were electroinjected with rabbit-anti-mouse IgG (control) or rabbit polyclonal anti-SHP-2 antibody and allowed to recover for 4 h. Cells were incubated with collagen-coated magnetic microbeads for 20 min at room temperature. FACs were isolated as described (see “Experimental Procedures”), and the resulting proteins were resolved by SDS-PAGE. Blots were probed with mouse monoclonal anti-vinculin or SHP-2. D, cells were treated with IL-1β (20 ng/ml) for 10 or 60 min. Whole cell lysates were separated by SDS-PAGE (10% acrylamide), transferred to nitrocellulose membranes and probed with mouse monoclonal anti-phospho-ERK1/2 or rabbit polyclonal anti-ERK1/2. This is representative of n = 6 separate experiments.
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.
; 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 (
) 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).
Fig. 7SHP-2 is localized to actin filaments and is required for IL-1-induced cell contraction.A, human gingival fibroblasts were treated for 15 min with 20 ng/ml IL-1β, washed with PBS, and lysed with cytoskeletal buffer (1% Triton X-100, 5 mm EDTA, 50 μm VO4, 10 mm NaF, 2 mm phenylmethylsufonyl fluoride, 10 μg/ml aprotinin, 1 μg/ml leupeptin in PBS). The Triton X-100 insoluble and soluble fractions were isolated and separated by SDS-PAGE. Blots were probed with anti-β-actin (Sigma) and anti-SHP-2 (Santa Cruz). B–D, human gingival fibroblasts were plated on fibronectin (10 mg/ml) for 48 h and left untreated (B), treated with IL-Iβ (20 ng/ml, 37 °C) for 10 or 60 min (C and D). Cells were fixed with 3.7% formaldehyde and stained with rhodamine phalloidin to reveal actin stress fibers. E–G, human gingival fibroblasts were electroinjected with rabbit polyclonal anti-SHP-2 antibody, plated on fibronectin (10 mg/ml) for 4 h then treated with IL-1β (20 ng/ml) for 0, 10, or 60 min (E, F, G, respectively). Electroporated cells were plated in normal growth medium on fibronectin-coated glass slides 4 h and stimulated with IL-1β 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. H, image analysis of cell area (Bioquant, R&D, Nashville, TN) is represented graphically. The data were analyzed by ANOVA with correction for multiple comparisons (Dunnett's test).
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-1β (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 (α-MEM/10% FBS) on fibronectin-coated glass slides for 4 h and stimulated with IL-1β 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
IL-1 is a potent, pro-inflammatory cytokine capable of stimulating diverse signaling cascades which, if improperly regulated, can result in severe cellular and tissue damage (
). Currently, the role of SHP-2 in IL-1 signaling is not defined. Our current observations expand the putative functions of SHP-2 in cell signaling by demonstrating a role for this protein tyrosine phosphatase in selective IL-1-induced ERK activation and cytoskeletal reorganization in the context of FAC in fibroblasts.
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 (
). 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 (
), 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 (
), 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 (
). 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 (
), 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 (
). 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 (
). 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 (
), 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 (
) 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 (
). 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(Δ46–110)) 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 (
, 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 (
). 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.
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