Redox Regulation of β-Actin during Integrin-mediated Cell Adhesion

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion. Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion. Actin is the main component of the cytoskeleton and exists as monomeric G-actin, able upon extracellular stimuli to polymerize into filamentous F-actin. Different structures of F-actin are produced by elongation of preformed filaments by accessory proteins, such as profilin, actin-related protein 2/3, actin-depolymerizing factor/cofilin complexes, and many others. These actin-interacting proteins may act through different mechanisms, as forming transversal branching filaments or blocking the extensive elongation at the cell margins (1Winder S.J. Curr. Opin. Cell Biol. 2003; 15: 14-22Crossref PubMed Scopus (42) Google Scholar, 2Mullins R.D. Curr. Opin. Cell Biol. 2000; 12: 91-96Crossref PubMed Scopus (140) Google Scholar). Actin cytoskeleton is modified by integrin signal in response to extracellular matrix (ECM) 3The abbreviations used are: ECM, extracellular matrix; BIAM, N-(biotinoyl)-N′-(iodoaceyl)ethylenediamine; BSO, buthionine sulfoximine; LOX, lypoxygenase; FN, fibronectin; PL, polilysine; nmMHC, non-muscle myosin heavy chain; NDGA, nordihydroguaretic acid; PTP, protein-tyrosine phosphatases; ROS, reactive oxygen species; RIPA, radioimmune precipitation assay; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; wt, wild-type; TRITC, tetramethylrhodamine isothiocyanate. transforming actin architecture from a cortical ring to a plain structure of stress fibers in complete adherent cells. Integrin-mediated cell adhesion proceeds in a step-like program: cells contact the substratum, integrins ligate to the ECM proteins and clusterize, focal contacts form, the cell spreads and finally organizes the actin filaments in stress fibers for mechanical resistance. The control of cytoskeleton organization is mainly due by the action of the members of the family of the small GTPases Rho like Cdc42, Rho, and Rac. In response to growth or chemotactic factors and cell-cell or cell-matrix interaction, Rho is activated thus inducing the formation of actin stress fibers. In contrast, activated Rac provokes actin-dependent membrane ruffling and Cdc42 causes protrusion of actin-rich microspikes from the cell surface (1Winder S.J. Curr. Opin. Cell Biol. 2003; 15: 14-22Crossref PubMed Scopus (42) Google Scholar). Much evidence supports the idea that reactive oxygen species (ROS) act as second messengers thus modulating the activity of signaling proteins upon growth factors and cytokines stimulation. The downstream effect of ROS production is the reversible oxidation of redox-sensible proteins through a direct modification of the thiol group of reactive cysteines (3Finkel T. FEBS Lett. 2000; 476: 52-54Crossref PubMed Scopus (494) Google Scholar). Redox-regulated proteins include tyrosine phosphatases, several transcription factors, p53, and the p21Ras family of proto-oncogenes (4Chiarugi P. Cirri P. Trends Biochem. Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Recently, Chiarugi et al. (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar) proposed a redox regulation of integrin signaling during cell adhesion. In this new model, integrin activation is joined to generation of ROS specifically acting on cytoplasmic target proteins as low molecular weight-phosphotyrosine phosphatase (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar) and the tyrosine kinase Src (6Giannoni E. Buricchi F. Raugei G. Ramponi G. Chiarugi P. Mol. Cell. Biol. 2005; 25: 6391-6403Crossref PubMed Scopus (367) Google Scholar). The reversibile oxidation of both these proteins has profound effect on cell spreading onto ECM, through indirect activation of both Focal Adhesion Kinase and the small GTPase Rho (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar, 7Nimnual A.S. Taylor L.J. Bar-Sagi D. Nat. Cell Biol. 2003; 5: 236-241Crossref PubMed Scopus (429) Google Scholar), thus suggesting that ROS act as second messengers in the organization of cytoskeleton in response to integrin engagement. Herein, we report evidence that ROS produced by integrins upon ECM contact are essential mediators for actin fibers formation during cell spreading. In particular, ROS acts on mixed disulfide with glutathione which is a key step for actin stress fibers. Assay of Intracellular H2O2—Three minutes before assaying 2′,7′-dichlorofluorescein diacetate (Molecular Probes) was added to a final concentration of 5 μm. Cells were lysed in 1 ml of RIPA buffer containing 1% Triton X-100 and analyzed immediately by fluorescence spectrofotometric analysis using a Perkin Elmer Fluorescence Sprectrofotometer 650-10S equipped with a xenon power supply (excitation wavelength: 488 nm, emission wavelength: 510 nm). Cell Culture and Transfection—Murine fibroblasts NIH-3T3 (from American Type Culture Collection) were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum at 37 °C in a 5% CO2 humidified atmosphere. For transfection experiments, confluent cells were transfected with 4 μg of plasmid using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer's instruction. The pSG5-FLAG-β-actin and pSG5-FLAG-β-actin C374A was a generous gift of Prof. C. Stournaras. Cell Adhesion Assay—Cells were serum-starved for 24 h and then detached with 0.25% trypsin for 1 min. Trypsin digestion was then blocked by the use of 0.5 mg/ml soybean trypsin inhibitor (Sigma). Cells were centrifuged, diluted in fresh culture medium, incubated for 30 min in gentle agitation at 37 °C, and finally seeded for 45 min on polylysine (PL)-treated or fibronectin (FN)-treated dishes for the period indicated. For N-(biotinoyl)-N′-(iodoaceyl)ethylenediamine (BIAM) (Molecular Probes) labeling of proteins, cells were lysed in RIPA lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton, 2 mm EGTA) supplemented with BIAM (100 μm final concentration) and protease inhibitors mixture (Sigma). Lysates were then maintained on ice for 15 min and then centrifuged at 13,000 rpm for 15 min. For the binding of BIAM-labeled proteins with immobilized avidin (Pierce), 30 μl of resin were added to the clarified samples and maintained overnight at 4 °C in gentle agitation. The resin was first washed four times with RIPA buffer and then resuspended in Laemmli sample buffer. The pattern of BIAM-labeled proteins were visualized by a Western blot using horseradish peroxidase-avidin conjugate (Bio-Rad), washed, and developed with the enhanced chemiluminescence kit. Immunoprecipitation—PL- or FN-plated cells were lysed for 20 min on ice in 500 μl with RIPA lysis buffer. Lysates were clarified by centrifugation and immunoprecipitation was performed for 4 h at 4°C with 1-2 μg of the specific antibodies. Immunocomplexes were collected on protein A-Sepharose, separated by SDS-PAGE, and transferred onto polyvinylidene difluoride membrane. Immunoblots were incubated in 2% milk and 0.1% Tween 20 in PBS buffer for 1 h at room temperature, probed first with specific antibodies and then with secondary antibodies. Quantity-One software (Bio-Rad) was used to perform quantitative analysis. MALDI-TOF Analysis—BIAM-labeled lysates were run on SDS-PAGE. The gel was then stained by Coomassie Blue solution, subjected to destain solution for 24 h and finally washed in water until completely equilibrated. The bands of interest were excised, transferred to an Eppendorf tube, and then washed twice with 50 mm NH4HCO3/acetonitrile (1:1) and shrunk with acetonitrile. After drying, samples were subjected to a reduction reaction in a buffer containing 10 mm dithiothreitol, 25 mm NH4HCO3 for 45 min at 56 °C followed by an alkylation step in a buffer containing 55 mm Iodoacetic acid, 25 mm NH4HCO3 for 30 min at room temperature in the dark. After a final washing step, samples were dried up and trypsin digested for 24 h at 37 °C. The peptides were then extracted from gel bands by sonification and by supplementing 50% acetonitrile and 1% trifluoroacetic acid (1:1 proportion with sample), and the supernatants were recovered and then dried. Spectrometric analysis was conducted on an Ultraflex MALDI-TOF (Bruker Daltonics) using a Scout ion source and operating in a positive reflectron mode. Samples were mixed with α-cyano-4-hydroxycinnamic acid (1:1). An 0.8 picomol/μl sample was deposed with the dry droplet technique on an AnchorChip target. Peptides were identified within an error of 120 parts/million. Confocal Microscopy—Presuspended NIH-3T3 were seeded onto coverslips, washed with phosphate-buffered saline and then fixed in 3% paraformaldehyde for 20 min at 4 °C. Fixed cells were permeabilized with three washes with TBST (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.1% Triton X-100) and then blocked with 5.5% horse serum in TBST for 1 h at room temperature. Cells were then incubated with specific primary antibodies or diluted in (50 mm Tris-HCl, pH 7.4, 150 mm overnight at 4 °C. cells were incubated with secondary antibodies for 1 h at room temperature in TBST with 3% serum After extensive washes in TBST cells were with and a laser equipped with a laser source for fluorescence and with The and laser were used to the and the fluorescence were collected with a of were through the of the cells with a of 1 μm at of μm. NIH-3T3 cells were serum-starved for 24 and dishes were with a and cells were with 30 growth After h transfected cells were visualized by a reaction using the according to the manufacturer's the were to cells. Actin by ROS by during Cell of low molecular weight-phosphotyrosine phosphatase and the tyrosine kinase as target proteins of the ROS (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar, E. Buricchi F. Raugei G. Ramponi G. Chiarugi P. Mol. Cell. Biol. 2005; 25: 6391-6403Crossref PubMed Scopus (367) Google Scholar), we with a for proteins during integrin using BIAM, which is a reagent that the of reactive cysteine Biochem. 2000; PubMed Scopus Google Scholar). ECM adhesion of NIH-3T3 fibroblasts was 45 min onto in with the of ROS during cell spreading (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar) Western blot analysis of BIAM-labeled lysates of and cells an redox sensitivity of a protein of which was identified by MALDI-TOF analysis as and was by analysis to actin as a protein during oxidative stress T. F. T. G. E. P. Sci. S. 2003; PubMed Scopus Google Scholar), we to the of ROS on the organization of the actin of integrin the small GTPase plays a key role in ROS production the activation of (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar). We that the depletion of using specific inhibitor for LOX, nordihydroguaretic acid greatly affects actin organization during cell spreading cells with this inhibitor a to with a actin ring and a of this the spreading is the actin architecture suggesting that ROS a role in actin organization during the of cell spreading in response to binding to ECM Cells and Actin oxidation in the formation of mixed between protein and glutathione to actin has been as a protein during growth J. E. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the to during cell adhesion actin glutathionylation upon we binding to GSH during a analysis of actin that is a in actin glutathionylation upon of integrin receptors we that GSH is greatly in actin regulation during cytoskeleton due to cell spreading. depletion of GSH causes a of actin cytoskeleton organization leading to a cell to the effect in cells with In the effect of buthionine on actin organization and cell by of GSH and thus the of the inhibition of actin organization upon GSH we that affects the glutathionylation of proteins, we an effect on We the of actin glutathionylation in the actin with a cell and complete of actin glutathionylation is to an cell of 374 of Actin in during Cell protein by the specific of the redox of reactive cysteine (3Finkel T. FEBS Lett. 2000; 476: 52-54Crossref PubMed Scopus (494) Google Scholar). actin cysteine the structure that the thiol of cysteine in 374 is on the surface of the The in of et al. J. E. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) that cysteine 374 is a that upon growth stimulation. We at the role of cysteine 374 in GSH disulfide formation during cell using and the cysteine 374 to of We that the C374A glutathionylation is greatly upon integrin thus C374A as the main in the role of actin redox regulation during cell a labeling analysis of cells was to actin organization and actin The that cell spreading or F-actin C374A expression to a to the cytoskeleton and to in response to ECM cells the redox-insensitive a and a to These suggest that in vivo plays a key role in GSH disulfide formation upon integrin and in cell spreading. the we were in cysteine 374 oxidation is in actin cytoskeleton growth cell was analyzed by a in NIH-3T3 cells or C374A We that and C374A cells a growth suggesting that cysteine 374 cell Redox of Actin for of both and non-muscle cells is by the actinomyosin complex in response to extracellular or to cell Curr. Opin. Cell Biol. 2005; PubMed Scopus Google Scholar). We analyzed the redox sensitivity of the between actin and non-muscle myosin heavy in cell spreading due to ECM that and cell adhesion is with the disassembly of the actinomyosin as by the of the between myosin and actin upon GSH depletion and ROS both the between actin and nmMHC, a role for ROS production and GSH in the of actinomyosin complex. this is greatly enhanced in suggesting that glutathionylation of cysteine 374 is in the disassembly of the actinomyosin complex upon of cell spreading. The between actin and is by analysis performed on actin the used for or actin glutathionylation GSH or of actin glutathionylation the effect on actinomyosin complex is the inhibition of We report here during cell adhesion actin is a protein and a direct redox regulation by a physiological source of inhibition of completely actin formation during cell adhesion leading to a actin actin glutathionylation on during cell adhesion and the of this redox modification to the of cells to and to the cytoskeleton in response to of actin glutathionylation to inhibition of the disassembly of the actinomyosin suggesting that this redox modification is a key step in the of cytoskeleton during cell spreading. The an in which signal by oxygen species, through reversible regulation of protein-tyrosine and protein-tyrosine and proteins, a and component of the by several extracellular stimuli to (3Finkel T. FEBS Lett. 2000; 476: 52-54Crossref PubMed Scopus (494) Google Scholar, P. Cirri P. Trends Biochem. Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Integrin-mediated adhesion causes the of low molecular and phosphatase L. for the in tyrosine of the focal adhesion kinase and protein P. Cirri P. Taddei L. Giannoni E. G. G. Raugei G. Ramponi G. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). the regulation of ROS protein tyrosine through a direct action on protein-tyrosine We recently a direct redox regulation of the tyrosine kinase which a activation by an disulfide formation in response to integrin (6Giannoni E. Buricchi F. Raugei G. Ramponi G. Chiarugi P. Mol. Cell. Biol. 2005; 25: 6391-6403Crossref PubMed Scopus (367) Google Scholar). The the idea that ROS cytoskeleton through both indirect and direct regulation is through as low molecular weight-phosphotyrosine phosphatase or oxidation to a signal in cytoskeleton (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar, E. Buricchi F. Raugei G. Ramponi G. Chiarugi P. Mol. Cell. Biol. 2005; 25: 6391-6403Crossref PubMed Scopus (367) Google Scholar). this indirect ROS act on actin cytoskeleton through a the direct oxidation of a protein as of integrin the small GTPase plays a key role in ROS production the activation of (5Chiarugi P. Pani G. Giannoni E. Taddei L. Colavitti R. Raugei G. Symons M. Borrello S. Galeotti T. Ramponi G. J. Cell Biol. 2003; 161: 933-944Crossref PubMed Scopus (366) Google Scholar). We that the depletion of greatly affects actin organization and cell spreading. cells with this inhibitor a to cells a we report that ROS produced during cell adhesion the to of cells the actin cells actin fibers and a actin We that ROS are in actin oxidation as in with role as second messengers of integrin In of this cell spreading is the that ROS in actin during the of cell spreading. We that ROS are of during and may primary of this on several produced by as key in cell adhesion regulation R. D. Cell 2003; PubMed Scopus Google Scholar, M. J. Full Text Full Text PDF PubMed Scopus Google Scholar), we that both and in the regulation of cell adhesion. Intracellular oxidation in the formation of mixed between protein and GSH in Actin glutathionylation has been during growth J. E. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and in and cells to oxidative stress M. M. S. M. M. F. E. J. E. P. Sci. S. PubMed Scopus (494) Google Scholar). The an in actin glutathionylation upon of integrin suggesting that GSH is in actin regulation during cytoskeleton due to cell spreading. cells spreading and of cytoskeleton to cells with In particular, the effect of on organization and cell spreading by of suggesting that the inhibition of cell spreading in response to GSH and to the oxidative is we actin glutathionylation with cell spreading and the of this glutathionylation, due to GSH to a and a actin is in with the of et al. J. E. S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), that cell upon growth is with actin In vivo actin glutathionylation is a in which role in protein has been in vivo and in that the oxidation of a group to acid and to oxidation such as and acid and J. D. PubMed Scopus Google Scholar). These are on which reduction leading to modified In with low oxidation to proteins, the formation of either between or protein cysteines or with a mixed disulfide with a key role J. D. 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