Published online before print April 28, 2009
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* San Raffaele Research Institute and
San Raffaele University, Faculty of Medicine, Milan, Italy
1. Correspondence: San Raffaele Research Institute, Genetics and Cell Biology, via Olgettina 58, 20132 Milano, Italy. E-mail: palumbo.roberta{at}hsr.it
ABSTRACT
HMGB1 is a nuclear protein that signals tissue damage, as it is released by cells dying traumatically or secreted by activated innate immunity cells. Extracellular HMGB1 elicits the migration to the site of tissue damage of several cell types, including inflammatory cells and stem cells. The identity of the signaling pathways activated by extracellular HMGB1 is not known completely: We reported previously that ERK and NF-
B pathways are involved, and we report here that Src is also activated. The ablation of Src or inhibition with the kinase inhibitor PP2 blocks migration toward HMGB1. Src associates to and mediates the phosphorylation of FAK and the formation of focal adhesions.
Key Words: chemokines • DAMP • danger signals • stem cells • tissue repair
Introduction
The HMGB1 protein was first identified as a key component of chromatin (reviewed in ref. [1
]). Later, it was realized that HMGB1 also acts as an extracellular signal that affects several cell functions in a variety of cell types [2
] (reviewed in ref. [3
]). HMGB1 is secreted by macrophages and other myeloid cells stimulated with LPS, TNF-
, and IL-1β and is released by cells dying in an unprogrammed way. It stimulates the motility of endothelial cells, fibroblasts, dendritic cells, macrophages, smooth muscle cells, and tumor cells. It has a mitogenic effect on vessel-associated stem cells (mesoangioblasts) and smooth muscle cells. Interestingly, HMGB1-induced angiogenesis may also occur through recruitment of endothelial progenitor cells. Finally, the overexpression of HMGB1 in a number of tumors might contribute to tumor development and metastasis, possibly by affecting the cell microenvironment.
RAGE has been identified as the receptor for HMGB1 [4 ]. However, evidence points to the existence of additional receptors, including possibly TLR-2, -4, and -9 [5 6 7 ].
RAGE is a member of the Ig superfamily, with three extracellular Ig domains, one transmembrane helix, and a short intracellular domain that does not contain a kinase domain or tyrosine residues [8
]. HMGB1 binding to RAGE leads to activation of multiple signaling molecules, including ERK1/2, p38, and stress-activated protein kinase/JNK, the small GTPases Rac and Cdc42, and NF-B [9
, 10
]. The organization of the signaling pathway between RAGE and the downstream signal transducers is not known completely. Previous reports indicated that the cytoplasmic domain of RAGE may be associated directly to ERKs [11
] and with the formin homology 1 domain of Dia-1 [12
].
Src is a non-receptor tyrosine kinase playing a role in a wide rage of processes, including cell differentiation, proliferation, and motility (reviewed in ref. [13 ]). It consists of a unique N-terminal segment, SH3 and SH2 domains, a catalytic domain, and a short C-terminal domain. The SH2 domain is available for binding with pTyr residues of other molecules, including FAK, which is a cytoplasmic tyrosine kinase that is implicated in signaling pathways that control cell cycle, cell spreading, and migration (reviewed in refs. [14 , 15 ]). Fibroblasts lacking FAK cannot migrate in response to normal stimuli. Both Src and FAK bind, phosphorylate, and activate exchange factors (guanine nucleotide exchange factors) of Rho-family small GTPases, which in turn, regulate cytoskeleton organization and initiate cell migration.
SFKs include a number of proteins that share structural and functional similarity to Src and are expressed ubiquitously or in a restricted number of cell types [13 ]. To some extent, SFKs can substitute for each other.
In this study, we have verified the involvement of SFKs and FAK in HMGB1-mediated cell migration. We show that the SFK-specific inhibitor PP2 abolishes HMGB1-elicited cell migration. Genetic ablation of the three ubiquitous SFKs, Src, Yes, and Fyn, also results in a loss of cellular migration in response to HMGB1; cell migration is restored by reintroducing Src in stable transfectants. Furthermore, our results also show that HMGB1 induces phosphorylation of FAK and paxillin, a scaffolding protein in focal adhesions, in a Src-dependent manner.
MATERIALS AND METHODS
HMGB1 and reagents
Full-length, LPS-free HMGB1 was provided by HMGBiotech (Milan, Italy). Anti-pTyr mAb PY99, rabbit polyclonal antibodies directed against FAK, paxillin, and Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and anti-Src mAb (clone GD11) from Upstate Biotechnology (Lake Placid, NY, USA). Rabbit polyclonal antibodies directed against pSrcTyr416, pSrcTyr527, pp44/42, and p44/42 were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin mAb and FITC-conjugated phalloidin were from Sigma-Aldrich (St. Louis, MO, USA). rhPDGFBB was purchased from R&D Systems (Minneapolis, MN, USA) and hSDF-1 from PeproTech (Rocky Hill, NJ, USA). PP2 was obtained from Calbiochem (San Diego, CA, USA), and fibronectin was from Roche (Nutley, NJ, USA).
Cell culture
3T3 mouse fibroblasts, SYF (Src–/– Yes–/– Fyn–/–) cells, and c-Src cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% FCS, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. PBMC from healthy volunteers were isolated by centrifugation using Ficoll reagent (Sigma-Aldrich) and kept in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Immunoblotting and immunoprecipitation
Cells were serum-starved in DMEM for 16 h (or when indicated for 6 h) and then stimulated with HMGB1 (100 ng/ml) or PDGFBB (20 ng/ml) for the indicated times. The incubation medium was then discarded, and cell monolayers were washed twice with ice-cold PBS. Cells were lysed (30 min at 4°C) with cell lysis buffer consisting of 1% Triton X-100, 10% glycerol, 150 mM NaCl, 50 mM Tris, pH 7.4, 5 mM EDTA, 1 mM PMSF, 1 mM NaF, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Sigma-Aldrich). The protein concentration in the lysates was measured using a Bradford assay (Sigma-Aldirch), and total proteins (25–50 µg) were separated on 12% SDS-PA gels and transferred to nitrocellulose membranes, which were blocked with 5% milk and incubated overnight with specific primary antibodies and for 1 h with secondary antibodies conjugated to HRP. Membranes were washed extensively, and detection was performed with an ECL kit according to the manufacturer’s instructions (GE Healthcare, UK). When necessary, membranes were stripped and reprobed. For paxillin and FAK immunoprecipitation, aliquots (1 mg) of cell lysate were mixed with various antibodies overnight at 4°C with gentle rocking. Protein A-Sepharose beads were added and incubated for 4 h at 4°C with gentle rocking. Immune complexes were precipitated by centrifugation, washed 10 times, boiled in SDS-PAGE loading buffer, separated by electrophoresis, and immunoblotted with the indicated antibodies.
Coimmunoprecipitation of Src and FAK
Whole cell lysates were obtained and processed as described in the preceding section and immunoprecipitated with rabbit polyclonal anti-Src antibody. Immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose. The membrane was incubated with rabbit polyclonal anti-FAK antibody followed by HRP-conjugated goat anti-rabbit IgG. The same membrane was stripped and reprobed with anti-Src antibody to confirm the amount of total Src used in each sample.
2DE
Cells treated with or without 100 ng/ml HMGB1 for 15 min were detached and lysed. Cell lysates were precipitated with 50% acetone for 2 h at –20°C. The precipitates were washed, dried, and solubilized for 1 h with 2DE solubilization buffer (8 M urea, 4% CHAPS, 20 mM DTT, 0.8% IPG buffer ampholine, pH 3–10, and 0.005% bromophenol blue). Protein samples (100 µg) were applied by in-gel rehydration to 7 cm IPG strips (pH 3–10 nonlinear; GE Healthcare) and separated by isoelectrofocusing on an IPGphor system (GE Healthcare) following a standard protocol [16
]. Strips were equilibrated in 50 mM Tris-HCl buffer, pH 8.8, containing 6 M urea, 30% glycerol, 3% SDS, and 2% DTT, followed by an incubation in the same buffer, where DTT was replaced with 3% iodoacetamide. The strips were loaded on top of 10% SDS-PA gels for separation on the second dimension. Proteins were electrontransferred onto nitrocellulose membranes, and Western blots were performed as described [16
].
Immunofluorescence microscopy
Mouse 3T3 fibroblasts (105), cultured on glass coverslips in DMEM, supplemented with 10% FCS, were serum-starved for 16 h and then stimulated with 100 ng/ml HMGB1 for 5, 20, and 60 min. The cells were washed, fixed in 3% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 and 0.2% BSA, and blocked with serum 1% for 2 h. Fixed cells were incubated overnight with rabbit polyclonal anti-paxillin antibody (1:200 dilution) at 4°C, washed, and incubated with fluorescent secondary antibody (Alexa Fluor 594-conjugated goat anti-rabbit IgG, Molecular Probes, Eugene, OR, USA) for 4 h at room temperature. The distribution of actin was visualized using FITC-conjugated phalloidin. Samples were analyzed with the DeltaVision restoration microscopy system (Applied Precision, Issaquah, WA, USA) built around an Olympus IX70 microscope equipped with mercury-arc illumination.
Proliferation assay
MCF-7 cells were seeded in six-well plates (5x105 cells/well) and grown in complete DMEM plus 10% FCS. After 24 h, the medium was replaced with serum-free DMEM medium for 16 h. MCF-7 cells were starved with DMEM without phenol red (Gibco, Grand Island, NY, USA). Subsequently, the cells were grown with serum-free medium, medium with the addition of 10% FBS, serum-free medium with 100 ng/ml HMGB1 with or without 10 µM PP2 (added 30 min before treatment), or serum-free medium with 10 µM PP2 alone. After 24 h, cells were detached and counted, and trypan blue dye exclusion was used as an indicator of cell viability.
Fibroblast chemotaxis
Modified Boyden chambers were used with filters (8 µm pores, Neuro Probe, Gaithersburg, MD, USA) coated with fibronectin (50 µg/ml). One million cells were seeded in 100 mm culture dishes the day before the experiment, pretreated where indicated with PP2 (3–30 µM) for 30 min at 37°C, and resuspended in serum-free DMEM. Fifty thousand cells in 200 µl were added to the upper chamber; HMGB1 (30 ng/ml) or PDGFBB (10 ng/ml) was added to the lower chamber, and then cells were left to migrate for 3 h at 37°C. Nonmigrating cells were removed with a cotton swab, and migrated cells were fixed with ethanol and stained with Giemsa (Sigma-Aldrich). Cells were then counted in 10 random fields/filter.
PBMC chemotaxis
PBMC (2x105 cells) were pretreated with PP2 (10 µM) for 30 min at 37°C, resuspended in serum-free RPMI, and seeded in the upper chamber of 24 transwell plates (5 µm pore size, Costar, Corning, NY, USA); 600 µl medium containing HMGB1 (30 ng/ml), PDGFBB (10 ng/ml), or SDF-1 (300 ng/ml) was placed in the lower chamber. After 2 h of incubation at 37°C, the transwell insert was removed, and the cells migrated to the lower chamber were resuspended and counted by using the Countess system (Invitrogen, Carlsbad, CA, USA).
Digital images
Digital Images were elaborated using Adobe Photoshop 8 and included in figures using Adobe Illustrator 11.0.
Statistical analysis
Statistical analysis was done using unpaired Student’s t-test with two-tailed P value for the comparison of sets of data with Gaussian distribution. Statistical analysis was performed using GraphPad Prism 4, V4.03 software (GraphPad Inc., San Diego, CA, USA).
RESULTS
Identification of proteins phosphorylated after HMGB1 stimulation
The earliest events in cell migration include the recruitment and phosphorylation of proteins such as FAK, paxillin p130cas, and the SFKs [13
, 15
, 17
]. We thus obtained a profile of pTyr-containing proteins in 3T3 mouse fibroblasts challenged with HMGB1. Fibroblasts were stimulated with 100 ng/ml HMGB1 for 15 min (the optimal dose and timing for biochemical assays; data not shown), and total cellular proteins were separated by 2DE, blotted, and stained with anti-pTyr antibodies. The comparison of immunoblots of control and treated cells revealed that some proteins between 55 and 65 kDa, the range including SFKs, changed their tyrosine phosphorylation status (Fig. 1
, upper panels).
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Figure 1. Characterization of pTyr proteins in cells stimulated with HMGB1. Fibroblast whole cell extracts (100 µg) were subjected to gel 2DE. (A) An anti-pTyr antibody was used to identify all of the proteins containing pTyr in extracts from untreated (Control; left panels) and HMGB1-treated cells (right panels). Both membranes were then stripped and reprobed with an antibody against total Src (anti-cSrc). The brackets indicate the position of Src isoforms in cell extracts. The dashed line (at a calculated pI of 4.86) separates different isoforms of Src. The position of the dashed line was set inductively based on the pattern of the signal detected by anti-Src under control conditions. These blots are representative of two independent experiments with similar results.
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SFKs are required for HMGB1-mediated cell migration
Fibroblasts express predominantly Src and lower amounts of other SFKs. All SFKs, however, are preferentially inhibited with the inhibitor PP2. To verify that SFKs are required for cell migration induced by HMGB1, we pretreated mouse 3T3 fibroblasts for 30 min with increasing concentrations of PP2 (3 µM, 10 µM, and 30 µM; Fig. 2A
). HMGB1 (1 nM, the optimal dose for cell migration assays; data not shown) induced cell migration, which was potently inhibited by PP2 in a dose-dependent manner. In the presence of 10 µM PP2, there was no significant difference between spontaneous or HMGB1-induced cell migration. In contrast, 3T3 cells pretreated with 3 and 10 µM PP2 maintain their ability to respond to PDGFBB, a chemoattractant that does not require the activity of SFKs [19
]. Only the higher dose of PP2 (30 µM) partially reduced PDGFBB-induced chemotaxis, possibly as a result of off-target effects. We conclude that at moderate doses, the SFK inhibitor PP2 interferes with fibroblast migration toward HMGB1, but not PDFGBB.
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Figure 2. SFKs mediate HMGB1-dependent cell migration. (A) Fibroblasts were pretreated with increasing concentrations of the SFK inhibitor PP2 for 30 min at 37°C and were allowed to migrate for 3 h at 37°C in response to DMEM containing HMGB1 (30 ng/ml), PDGFBB (10 ng/ml), or no chemoattractant as control. Bars represent the average of four replicates ± SD. One-way ANOVA analysis indicates that treatment with PP2 significantly reduces cell migration in response to HMGB1 (P<0.0001) and that each dose is significant when compared with the control (Dunnet test, P<0.01). Only the highest dose of PP2 (30 µM) reduces cell migration significantly in response to PDGFBB (P<0.01). At doses of 10 and 30 µM PP2, there is no significant difference between cells migrating toward HMGB1 and control (unpaired two-way t-test). This experiment was repeated three times. (B) c-Src cells or with control plasmid (SYF cells) were allowed to migrate in the presence of HMGB1, control stimulus, PDGFBB, or without any stimulus. Results are representative of at least three independent experiments and represent the mean of duplicate samples ± SD (*, P<0.05, unpaired t-test). (C) hPMBC were pretreated where indicated with PP2 (10 µM) and were allowed to migrate for 2 h in response to HMGB1 (30 ng/ml), SDF-1 (300 ng/ml) as positive control, or no chemoattractant as negative control. Results are representative of at least two independent experiments and represent the mean of duplicate samples ± SD (*, P<0.05, unpaired t-test; ***, P<0.001). (D) Proliferation of breast cancer MCF-7 cells treated with serum-free medium, serum-free medium containing 100 ng/ml HMGB1 with or without 10 uM PP2, or serum-free medium containing 10 uM PP2. The graph represents the number of cells after 24 h of stimulation, normalized to the cells at Time 0. Error bars represent SD; the experiment was repeated three times. ns, Not significant; *, P<0.05.
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We extended these observations to hPBMC (Fig. 2C) , which were tested in a PBMC-specific migration assay [20 ]. HMGB1 (1 nM) elicited significant cell migration, similar to that induced by the well-known chemoattractant SDF-1. PP2 (10 µM) interfered with HMGB1-induced cell migration, although a significant difference remained compared with PBMC not exposed to any chemoattractant. PP2 alone had no significant effect. PBMC do not respond to PDGFBB (data not shown), and thus, we could not test the effect of PP2 on PDGFBB-induced migration.
We also tested whether the role of SFKs was limited to cell migration or also extended to other responses elicited by HMGB1. We and others have shown previously that HMGB1 promotes the proliferation of stem cells and tumor cells [3 ]; the proliferative effect on fibroblasts is modest. We thus used MCF-7 tumor cells, which respond to HMGB1 by proliferating in serum-free medium (Fig. 2D , and T. Pusterla and M. E. Bianchi, unpublished). The effect of 10 µM PP2 on HMGB1-induced proliferation is small (P=0.06, not significant); 3 µM PP2 has no effect at all, and 30 µM causes cell death (data not shown).
Taken together, the proteomic, pharmacological, and genetic approaches indicate that SFKs (including Src but not necessarily limited to it) are important for HMGB1-stimulated cell migration, and their role in cell proliferation appears minor at most.
Stimulation with HMGB1 induces the transient phosphorylation of tyrosine 416 in Src
Src activity is regulated by tyrosine phosphorylation at two sites with opposite effects. Autophosphorylation of Src at Y416 induces an active conformation, whereas phosphorylation at Y527 represses its kinase activity [21
, 22
]. We evaluated the effects of HMGB1 on the two relevant tyrosines of Src. Growth-arrested fibroblasts were treated with 100 ng/ml HMGB1 for up to 60 min (Fig. 3
). Src Y416 was phosphorylated modestly but significantly, reaching a peak 30 min after stimulation and declining within 60 min (Fig. 3A)
. Src Y416 was also phosphorylated after stimulation with PDGFBB, with a similar time course (Fig. 3A)
. Neither HMGB1 nor PDGFBB affected the phosphorylation of Y527 (Fig. 3B)
.
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Figure 3. HMGB1 activates Src in fibroblasts. Serum-starved cells were stimulated with 100 ng/ml HMGB1 or 20 ng/ml PDGFBB as control for 5, 15, 30, or 60 min. Whole cell lysates were prepared and subjected to Western blot analysis using antibodies directed against phosphorylated sites of Src. β-Actin blotting is shown as a loading control. Blots are representative of three independent experiments performed with anti-Src416 (A) and anti-Src527 (B). Bar graphs represent quantitations of Src phosphorylation at 5,15, 30, and 60 min from three independent experiments (*, P<0.05, unpaired t-test).
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Figure 4. HMGB1 promotes cytoskeleton reorganization. (A) Fibroblasts were plated onto gelatin-coated coverslips and stimulated or not with 100 ng/ml HMGB1 for 20 min prior to being processed for indirect immunofluorescence with FITC-labeled phalloidin (green) and anti-paxillin (red) as described in Materials and Methods. The arrowheads indicate focal adhesions (high local concentrations of paxillin in low magnification images and paxillin-stress fiber colocalization in the enlarged, merged image). (B) Focal adhesions in 15 cells/group were tracked and counted with the ImageJ particle tracker function. The difference is statistically significant (P<10–4, unpaired t-test).
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Figure 5. HMGB1 induces Src-dependent phosphorylation of FAK. Fibroblasts (A, B, and C) or SYF cells (D) were plated, starved, and then stimulated with 100 ng/ml HMGB1 for 10, 30, or 60 min. Cell lysates were prepared and immunoprecipitated (IP) with anti-paxillin (A), anti-FAK (B and D), or Src (C). Membranes were immunoblotted with anti-pTyr, anti-paxillin, anti-FAK, or anti-Src, as indicated. Bar graphs show the intensities of proteins determined by densitometry from two independent experiments (*, P<0.05).
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We also found that in SYF cells, FAK phosphorylation was reduced dramatically, implicating that SFKs act upstream of FAK and paxillin (Fig. 5D) .
DISCUSSION
The intracellular signaling pathways mediating the biological effects of extracellular HMGB1 are poorly understood. In particular, although RAGE has been identified as the receptor for HMGB1, we do not know how RAGE itself transfers signals to the effectors required for cytoskeletal remodeling and cell migration, to the transcriptional machinery, and to the control of cell cycle. We showed previously that ERK and NF-B activation is required for HMGB1-elicited migration; moreover, inflammatory cells, stem cells, and fibroblasts appear to use the same signaling pathways to respond to extracellular HMGB1 [10
].
In this report, we provide evidence that Src and FAK play a crucial role in HMGB1-induced cell migration. First, cell migration was abrogated in fibroblasts lacking the three SFKs, Src, Yes, and Fyn. Conversely, cell migration was rescued in triple KO cells stably re-expressing Src (known as c-Src cells). Migration was also inhibited in a dose-dependent manner in wild-type cells pretreated with the SFK inhibitor PP2. HMGB1 stimulation induces Src activation via the phosphorylation of tyrosine 416, and phosphorylation of tyrosine 527 appears unaffected. Second, HMGB1 treatment promotes the phosphorylation of FAK and paxillin; FAK phosphorylation is not induced in SYF cells lacking SFKs. Furthermore, Src and FAK coimmunoprecipitate from lysates of cells stimulated with HMGB1. After stimulation of cells with HMGB1, paxillin relocalizes to the ends of actin stress fibers.
The SFKs are often activated during cytoskeletal remodeling events required for cell adhesion and cell migration [13 ]. Src in particular is the major kinase responsible for fibroblast growth factor 1-induced tyrosine phosphorylation of cortactin [29 ]. Src is also essential for the migration of endothelial cells in response to vascular endothelial growth fctor [30 ]. TNF regulates intestinal epithelial cell migration in a concentration-dependent manner through a Src-regulated pathway [31 ]. SFK-dependent activaction of p38 is a key component of epidermal growth factor-induced intestinal epithelial cell migration [32 ]. EphB1, angiotensin II, as well as E-selectin mediate chemotaxis through Src and the subsequent Erk1/2 activaction [33 34 35 ]. Remarkably, however, although Src is activated during PDFBB-induced cell migration, it is not necessary for it [19 ]. Thus, Src is not strictly necessary for cell migration, but still, it is involved in HMGB1-elicited cell migration.
Our experiments do not exclude the possibility that other SFKs might function similarly to Src and partially or totally substitute its function, especially in cells that express high levels of cell-specific SFKs. We have also shown that PP2 inteferes with HMGB1-induced chemotaxis of PBMC, but we were not able to distinguish between different SFKs for the lack of genetically modified cells.
A more specific question regards the connection between RAGE and Src. We did ask whether RAGE interacts directly with Src after HMGB1 stimulation. We were unable to detect this interaction by coimmunoprecipitation experiments, using Src or RAGE immunoprecitation (data not shown), even in c-Src cells where the signaling must necessarily involve Src and not other SFKs. Src activation after RAGE binding to its ligand S100B has been shown [36 ], but again, no direct interaction between RAGE and Src was reported. This suggests that the interaction between RAGE and Src may be indirect. RAGE and Src have been proposed to associate to lipid rafts [37 ]. Importantly, a recent study identified Dia-1 as a direct interactor of the RAGE cytosolic tail [12 ]. The interaction between RAGE and Dia-1 is necessary for cell migration through the activation of Rac1 and CDC42. Dia-1 might connect RAGE and Src physically, or alternatively, an indirect connection via other adaptors or effectors might occur; this will be the object of future studies.
Based on our data, we propose that HMGB1 induces, directly or indirectly, phosphorylation of Src and possibly other SFKs as well as FAK, leading through a series of steps to Erk1/2 and NF-B activation. Although additional work will be needed to fully analyze the overall response to HMGB1, the full picture may now be emerging.
ACKNOWLEDGMENTS
R. P. is supported by a contract from Ministero dell’Università e Ricerca. This work has been supported by grants from the Associazione Italiana Ricerca sul Cancro (to M. E. B.) and from the Association for International Cancer Research (to M. E. B. and R. P.). We thank Chiara Francavilla for initial experiments, Luca Cassetta for help on PBMC experiments, and all members of our lab for suggestions and discussions.
DISCLOSURES
The authors declare no direct financial interest. However, M. E. B. is founder and part owner of HMGBiotech, a company that provides goods and services related to HMGB proteins.
FOOTNOTES
Abbreviations: 2DE=two-dimensional electrophoresis, c-Src=SYF cells stably transfected with Src, Dia-1=diaphanous-1, FAK=focal adhesion kinase, h=human, HMGB1=high mobility group box 1, IPG=immobilized pH gradient, KO=knockout, p=phosphorylated, PA=polyacrylamide, PDGFBB=platelet-derived growth factor BB peptide, pI=isoelectric point, pTyr=phosphotyrosine, RAGE=receptor for advanced glycation end-products, SDF-1=stromal cell-derived factor 1, SFK=Src family kinase, SH3=Src homology 3
Received September 29, 2008; revised March 10, 2009; accepted March 19, 2009.
REFERENCES
B require the cytoplasmic domain of the receptor but different downstream signaling pathways J. Biol. Chem. 274,19919-19924B activation J. Cell Biol. 179,33-40This article has been cited by other articles:
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  M. Penzo, R. Molteni, T. Suda, S. Samaniego, A. Raucci, D. M. Habiel, F. Miller, H. p. Jiang, J. Li, R. Pardi, et al. Inhibitor of NF-{kappa}B Kinases {alpha} and {beta} Are Both Essential for High Mobility Group Box 1-Mediated Chemotaxis J. Immunol., April 15, 2010; 184(8): 4497 - 4509. [Abstract] [Full Text] [PDF]
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