The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2 participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y2 receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal step in EGFR transactivation and downstream signaling.
References
[1]
Burgel PR, Nadel JA (2008) Epidermal growth factor receptor-mediated innate immune responses and their roles in airway diseases. Eur Respir J 32: 1068–1081.
[2]
Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, et al. (2000) Involvement of the epidermal growth factor receptor in epithelial repair in asthma. Faseb J 14: 1362–1374.
[3]
Kato A, Schleimer RP (2007) Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity. Curr Opin Immunol 19: 711–720.
[4]
Ohtsu H, Dempsey PJ, Eguchi S (2006) ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors. Am J Physiol Cell Physiol 291: C1–10.
[5]
Blobel CP (2005) ADAMs: key components in EGFR signalling and development. Nat Rev Mol Cell Biol 6: 32–43.
[6]
Gooz M (2010) ADAM-17: the enzyme that does it all. Crit Rev Biochem Mol Biol 45: 146–169.
[7]
Scheller J, Chalaris A, Garbers C, Rose-John S (2011) ADAM17: a molecular switch to control inflammation and tissue regeneration. Trends Immunol 32: 380–387.
[8]
Lazarowski ER, Boucher RC (2009) Purinergic receptors in airway epithelia. Curr Opin Pharmacol 9: 262–267.
[9]
Ahmad S, Ahmad A, White CW (2006) Purinergic signaling and kinase activation for survival in pulmonary oxidative stress and disease. Free Radic Biol Med 41: 29–40.
[10]
Wesley UV, Bove PF, Hristova M, McCarthy S, van der Vliet A (2007) Airway epithelial cell migration and wound repair by ATP-mediated activation of dual oxidase 1. J Biol Chem 282: 3213–3220.
[11]
Belete HA, Hubmayr RD, Wang S, Singh RD (2011) The role of purinergic signaling on deformation induced injury and repair responses of alveolar epithelial cells. PLoS One 6: e27469.
[12]
Davis CW, Dickey BF (2008) Regulated airway goblet cell mucin secretion. Annu Rev Physiol 70: 487–512.
[13]
Boucher I, Rich C, Lee A, Marcincin M, Trinkaus-Randall V (2010) The P2Y2 receptor mediates the epithelial injury response and cell migration. Am J Physiol Cell Physiol 299: C411–421.
[14]
Peterson TS, Camden JM, Wang Y, Seye CI, Wood WG, et al. (2010) P2Y2 nucleotide receptor-mediated responses in brain cells. Mol Neurobiol 41: 356–366.
[15]
Block ER, Tolino MA, Klarlund JK (2011) Extracellular ATP stimulates epithelial cell motility through Pyk2-mediated activation of the EGF receptor. Cell Signal 23: 2051–2055.
[16]
Meng TC, Fukada T, Tonks NK (2002) Reversible oxidation and inactivation of protein tyrosine phosphatases in vivo. Mol Cell 9: 387–399.
[17]
Chen K, Kirber MT, Xiao H, Yang Y, Keaney JF Jr (2008) Regulation of ROS signal transduction by NADPH oxidase 4 localization. J Cell Biol 181: 1129–1139.
[18]
Myers TJ, Brennaman LH, Stevenson M, Higashiyama S, Russell WE, et al. (2009) Mitochondrial reactive oxygen species mediate GPCR-induced TACE/ADAM17-dependent transforming growth factor-alpha shedding. Mol Biol Cell 20: 5236–5249.
[19]
Paulsen CE, Truong TH, Garcia FJ, Homann A, Gupta V, et al. (2012) Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat Chem Biol 8: 57–64.
[20]
Koff JL, Shao MX, Kim S, Ueki IF, Nadel JA (2006) Pseudomonas lipopolysaccharide accelerates wound repair via activation of a novel epithelial cell signaling cascade. J Immunol 177: 8693–8700.
[21]
Doedens JR, Mahimkar RM, Black RA (2003) TACE/ADAM-17 enzymatic activity is increased in response to cellular stimulation. Biochem Biophys Res Commun 308: 331–338.
[22]
Schlondorff J, Becherer JD, Blobel CP (2000) Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). Biochem J 347 Pt 1: 131–138.
[23]
McCarthy SM, Bove PF, Matthews DE, Akaike T, van der Vliet A (2008) Nitric oxide regulation of MMP-9 activation and its relationship to modifications of the cysteine switch. Biochemistry 47: 5832–5840.
[24]
Boots AW, Hristova M, Kasahara DI, Haenen GR, Bast A, et al. (2009) ATP-mediated activation of the NADPH oxidase DUOX1 mediates airway epithelial responses to bacterial stimuli. J Biol Chem 284: 17858–17867.
[25]
Willems SH, Tape CJ, Stanley PL, Taylor NA, Mills IG, et al. (2010) Thiol isomerases negatively regulate the cellular shedding activity of ADAM17. Biochem J 428: 439–450.
[26]
Wang Y, Herrera AH, Li Y, Belani KK, Walcheck B (2009) Regulation of mature ADAM17 by redox agents for L-selectin shedding. J Immunol 182: 2449–2457.
[27]
Zhuang S, Schnellmann RG (2004) H2O2-induced transactivation of EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells. Am J Physiol Renal Physiol 286: F858–865.
[28]
Khan EM, Heidinger JM, Levy M, Lisanti MP, Ravid T, et al. (2006) Epidermal growth factor receptor exposed to oxidative stress undergoes Src- and caveolin-1-dependent perinuclear trafficking. J Biol Chem 281: 14486–14493.
[29]
Moro L, Dolce L, Cabodi S, Bergatto E, Boeri Erba E, et al. (2002) Integrin-induced epidermal growth factor (EGF) receptor activation requires c-Src and p130Cas and leads to phosphorylation of specific EGF receptor tyrosines. J Biol Chem 277: 9405–9414.
[30]
Donepudi M, Resh MD (2008) c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling. Cell Signal 20: 1359–1367.
[31]
Giannoni E, Buricchi F, Raugei G, Ramponi G, Chiarugi P (2005) Intracellular reactive oxygen species activate Src tyrosine kinase during cell adhesion and anchorage-dependent cell growth. Mol Cell Biol 25: 6391–6403.
[32]
Yoo SK, Starnes TW, Deng Q, Huttenlocher A (2011) Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature 480: 109–112.
[33]
Senga T, Hasegawa H, Tanaka M, Rahman MA, Ito S, et al. (2008) The cysteine-cluster motif of c-Src: its role for the heavy metal-mediated activation of kinase. Cancer Sci 99: 571–575.
[34]
Yoo SK, Freisinger CM, Lebert DC, Huttenlocher A (2012) Early redox, Src family kinase, and calcium signaling integrate wound responses and tissue regeneration in zebrafish. J Cell Biol 199: 225–234.
[35]
Gattas MV, Forteza R, Fragoso MA, Fregien N, Salas P, et al. (2009) Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli. Free Radic Biol Med 47: 1450–1458.
[36]
Koff JL, Shao MX, Ueki IF, Nadel JA (2008) Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium. Am J Physiol Lung Cell Mol Physiol.
[37]
Puchelle E, Zahm JM, Tournier JM, Coraux C (2006) Airway epithelial repair, regeneration, and remodeling after injury in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3: 726–733.
[38]
Liu J, Liao Z, Camden J, Griffin KD, Garrad RC, et al. (2004) Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors. J Biol Chem 279: 8212–8218.
[39]
Ratchford AM, Baker OJ, Camden JM, Rikka S, Petris MJ, et al. (2010) P2Y2 nucleotide receptors mediate metalloprotease-dependent phosphorylation of epidermal growth factor receptor and ErbB3 in human salivary gland cells. J Biol Chem 285: 7545–7555.
[40]
Sahin U, Weskamp G, Kelly K, Zhou HM, Higashiyama S, et al. (2004) Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J Cell Biol 164: 769–779.
[41]
Maretzky T, Zhou W, Huang XY, Blobel CP (2011) A transforming Src mutant increases the bioavailability of EGFR ligands via stimulation of the cell-surface metalloproteinase ADAM17. Oncogene 30: 611–618.
[42]
Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA, et al. (2003) Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature 422: 322–326.
[43]
Vermeer PD, Panko L, Welsh MJ, Zabner J (2006) erbB1 functions as a sensor of airway epithelial integrity by regulation of protein phosphatase 2A activity. J Biol Chem 281: 1725–1730.
[44]
Joo JH, Ryu JH, Kim CH, Kim HJ, Suh MS, et al. (2012) Dual oxidase 2 is essential for the toll-like receptor 5-mediated inflammatory response in airway mucosa. Antioxid Redox Signal 16: 57–70.
[45]
Dauer DJ, Ferraro B, Song L, Yu B, Mora L, et al. (2005) Stat3 regulates genes common to both wound healing and cancer. Oncogene 24: 3397–3408.
[46]
Giannoni E, Taddei ML, Chiarugi P (2010) Src redox regulation: again in the front line. Free Radic Biol Med 49: 516–527.
[47]
Forteza R, Salathe M, Miot F, Forteza R, Conner GE (2005) Regulated hydrogen peroxide production by Duox in human airway epithelial cells. Am J Respir Cell Mol Biol 32: 462–469.
[48]
Douillet CD, Robinson WP 3rd, Milano PM, Boucher RC, Rich PB (2006) Nucleotides induce IL-6 release from human airway epithelia via P2Y2 and p38 MAPK-dependent pathways. Am J Physiol Lung Cell Mol Physiol 291: L734–746.
[49]
Boucher I, Yang L, Mayo C, Klepeis V, Trinkaus-Randall V (2007) Injury and nucleotides induce phosphorylation of epidermal growth factor receptor: MMP and HB-EGF dependent pathway. Exp Eye Res 85: 130–141.
[50]
Norambuena A, Palma F, Poblete MI, Donoso MV, Pardo E, et al. (2010) UTP controls cell surface distribution and vasomotor activity of the human P2Y2 receptor through an epidermal growth factor receptor-transregulated mechanism. J Biol Chem 285: 2940–2950.
[51]
Davis FM, Kenny PA, Soo ET, van Denderen BJ, Thompson EW, et al. (2011) Remodeling of purinergic receptor-mediated Ca2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells. PLoS One 6: e23464.
[52]
van der Vliet A (2008) NADPH oxidases in lung biology and pathology: host defense enzymes, and more. Free Radic Biol Med 44: 938–955.
[53]
Moskwa P, Lorentzen D, Excoffon KJ, Zabner J, McCray PB Jr, et al. (2007) A novel host defense system of airways is defective in cystic fibrosis. Am J Respir Crit Care Med 175: 174–183.
Hoeven R, McCallum KC, Cruz MR, Garsin DA (2011) Ce-Duox1/BLI-3 generated reactive oxygen species trigger protective SKN-1 activity via p38 MAPK signaling during infection in C. elegans. PLoS Pathog 7: e1002453.
[56]
Juarez MT, Patterson RA, Sandoval-Guillen E, McGinnis W (2011) Duox, Flotillin-2, and Src42A are required to activate or delimit the spread of the transcriptional response to epidermal wounds in Drosophila. PLoS Genet 7: e1002424.
[57]
Bae YS, Choi MK, Lee WJ (2010) Dual oxidase in mucosal immunity and host-microbe homeostasis. Trends Immunol 31: 278–287.
[58]
Harper RW, Xu C, Eiserich JP, Chen Y, Kao CY, et al. (2005) Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium. FEBS Lett 579: 4911–4917.
[59]
Luxen S, Noack D, Frausto M, Davanture S, Torbett BE, et al. (2009) Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells. J Cell Sci 122: 1238–1247.
[60]
Linderholm AL, Onitsuka J, Xu C, Chiu M, Lee WM, et al. (2010) All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium. Am J Physiol Lung Cell Mol Physiol 299: L215–221.
[61]
Yu M, Lam J, Rada B, Leto TL, Levine SJ (2011) Double-stranded RNA induces shedding of the 34-kDa soluble TNFR1 from human airway epithelial cells via TLR3-TRIF-RIP1-dependent signaling: roles for dual oxidase 2- and caspase-dependent pathways. J Immunol 186: 1180–1188.
[62]
Shao MX, Nadel JA (2005) Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells. Proc Natl Acad Sci U S A 102: 767–772.
[63]
Seong J, Lu S, Ouyang M, Huang H, Zhang J, et al. (2009) Visualization of Src activity at different compartments of the plasma membrane by FRET imaging. Chem Biol 16: 48–57.
[64]
Wang L, Zhen H, Yao W, Bian F, Zhou F, et al. (2011) Lipid raft-dependent activation of dual oxidase 1/H2O2/NF-kappaB pathway in bronchial epithelial cells. Am J Physiol Cell Physiol 301: C171–180.
[65]
Tschumperlin DJ, Dai G, Maly IV, Kikuchi T, Laiho LH, et al. (2004) Mechanotransduction through growth-factor shedding into the extracellular space. Nature 429: 83–86.
[66]
Avraham R, Yarden Y (2011) Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat Rev Mol Cell Biol 12: 104–117.
[67]
Filosto S, Khan EM, Tognon E, Becker C, Ashfaq M, et al. (2011) EGF receptor exposed to oxidative stress acquires abnormal phosphorylation and aberrant activated conformation that impairs canonical dimerization. PLoS One 6: e23240.
[68]
Bromann PA, Korkaya H, Courtneidge SA (2004) The interplay between Src family kinases and receptor tyrosine kinases. Oncogene 23: 7957–7968.
[69]
Kemble DJ, Sun G (2009) Direct and specific inactivation of protein tyrosine kinases in the Src and FGFR families by reversible cysteine oxidation. Proc Natl Acad Sci U S A 106: 5070–5075.
[70]
Okutani D, Lodyga M, Han B, Liu M (2006) Src protein tyrosine kinase family and acute inflammatory responses. Am J Physiol Lung Cell Mol Physiol 291: L129–141.
[71]
Rhee SG, Woo HA, Kil IS, Bae SH (2012) Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem 287: 4403–4410.
[72]
Gutscher M, Sobotta MC, Wabnitz GH, Ballikaya S, Meyer AJ, et al. (2009) Proximity-based protein thiol oxidation by H2O2-scavenging peroxidases. J Biol Chem 284: 31532–31540.
[73]
Fomenko DE, Koc A, Agisheva N, Jacobsen M, Kaya A, et al. (2011) Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide. Proc Natl Acad Sci U S A 108: 2729–2734.
[74]
De Deken X, Wang D, Many MC, Costagliola S, Libert F, et al. (2000) Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family. J Biol Chem 275: 23227–23233.
