Tobacco Smoke Control of Mucin Production in Lung Cells Requires Oxygen Radicals AP-1 and JNK

Abstract

In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFR-independent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK. In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFR-independent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK. The primary cause of morbidity in chronic bronchitis is mucin overproduction, a phenomenon for which the molecular pathogenesis is unknown. Inflammatory cells are abundant in smokers' airways (1Elliot J.G. 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Petitprez D. Porchet N. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2001; 276: 30923-30933Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), suggesting that at least some of the excessive mucin in smokers' lungs is secondary to inflammation. In addition, however, smoke itself can induce mucin synthesis in lung cells (8Basbaum C. Lemjabbar H. Longphre M. Li D. Gensch E. Mc-Namara N. Am. J. Respir. Crit. Care Med. 1999; 160: S44-S48Crossref PubMed Scopus (118) Google Scholar, 9Takeyama K. Jung B. Shim J.J. Burgel P.R. Dao-Pick T. Ueki I.F. Protin U. Kroschel P. Nadel J.A. Am. J. Physiol. 2001; 280: L165-L172Crossref PubMed Google Scholar). The question of how this occurs is complex in that smoke, a composite of irritant molecules including acetaldehyde, hydroquinone, formaldehyde, benzo-[a]pyrene, cresol, nicotine, catechol, acrolein, coumarin, anthracene, nitrogen oxides, and heavy metals (10Smith C.J. Fischer T.H. Atherosclerosis. 2001; 158: 257-267Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 11Smith C.J. Hansch C. Food Chem. Toxicol. 2000; 38: 637-646Crossref PubMed Scopus (187) Google Scholar) may act on lung epithelial cells in diverse ways. For example, the induction of cytochrome P450 by tobacco smoke (12Gebremichael A. Tullis K. Denison M. Cheek J. Pinkerton K. Toxicol. Appl. Pharmacol. 1996; 141: 76-83Crossref PubMed Google Scholar) is mediated by binding of the aryl hydrocarbon nuclear receptor to a dioxin response element in the 5′-flank of the gene, but the induction of the γ-glutamylcysteine synthetase heavy subunit (γ-GCSHS) gene is mediated by the binding of a c-Jun/c-Jun homodimer to an AP-1-like response element (13Rahman I. Smith C.A. Lawson M.F. Harrison D.J. MacNee W. FEBS Lett. 1996; 396: 21-25Crossref PubMed Scopus (147) Google Scholar). Previous reports have implicated the receptor for epidermal growth factor (EGFR) 1The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; SFM, serum-free medium; ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; RT, reverse transcriptase; PBS, phosphate-buffered saline; RXR, retinoid X receptor; PTP, protein tyrosine phosphatase.1The abbreviations used are: EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; SFM, serum-free medium; ROS, reactive oxygen species; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; ERK, extracellular signal-regulated kinase; RT, reverse transcriptase; PBS, phosphate-buffered saline; RXR, retinoid X receptor; PTP, protein tyrosine phosphatase. in the induction of mucin gene MUC5AC by smoke (9Takeyama K. Jung B. Shim J.J. Burgel P.R. Dao-Pick T. Ueki I.F. Protin U. Kroschel P. Nadel J.A. Am. J. Physiol. 2001; 280: L165-L172Crossref PubMed Google Scholar). Consistent with a role for EGFR in mucin induction, an EGF response element has been identified 200 bp upstream of the MUC5AC gene (14Perrais M. Pigny P. Copin M.C. Aubert J.P. Van Seuningen I. J. Biol. Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The response of this element to EGFR ligands EGF and transforming growth factor-α is mediated by Sp1. One might predict from these data that the induction of MUC5AC by smoke would depend on interaction between the EGF response element at -200 bp and Sp1. In contrast, in the present study we show that MUC5AC is controlled principally by a smoke response element ∼3 kb upstream of the EGF response element. This element is AP-1-dependent and is bound by JunD/Fra-2 heterodimers. Such heterodimers typically require both JNK and ERK kinases for transcriptional activity. Our results indicate that the ability of smoke to stimulate MUC5AC through AP-1 strongly depends on both JNK and ERK. Although ERK activation was a consequence of EGFR activation, JNK activation was not. Instead, JNK activation by smoke was mediated by Src in an EGFR-independent manner triggered by reactive oxygen species (ROS). The results indicate that both EGFR-dependent and independent mechanisms cooperate to stimulate mucin production in response to tobacco smoke. Reagents—The chemical inhibitors AG1478 and PP2 were obtained from Calbiochem (EMD Biosciences, Inc. La Jolla, CA). SP600125 was purchased from Biomol (Plymouth Meeting, PA). Recombinant human EGF was from Invitrogen (Carlsbad, CA). All other chemicals were from Sigma. Antibodies specific to JunD, Fra-2, and Src were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to phospho-JNK and phospho-ERK were from New England Biolabs (Beverly, MA). The antibody specific to phospho-Src was from BIOSOURCE (Camarillo, CA). The JNK1 dominant negative mutant was a gift from Dr. Roger Davis (Howard Hughes Medical Institute, Worcester, MA). In Situ Hybridization—Human smoker or non-smoker bronchial tissue was obtained at autopsy from pathologist Dr. Walt Finkbeiner (University of California San Francisco). The tissues were rinsed in sterile PBS to remove secretions and fixed in 4% paraformaldehyde in PBS overnight at 4 °C. Fixed tissue was embedded in paraffin, sectioned, and placed on prepared slides. Labeled RNA probes were prepared by incorporating [35S]UTP into MUC5AC-specific sense and antisense transcripts using T3 and T7 polymerases. Hybridizations were performed as described previously (15Dohrman A. Tsuda T. Escudier E. Cardone M. Jany B. Gum J. Kim Y. Basbaum C. Exp. Lung Res. 1994; 20: 367-380Crossref PubMed Scopus (51) Google Scholar). Briefly, sections were dewaxed, rehydrated, and rinsed in PBS for 5 min and then fixed in 4% paraformaldehyde for 20 min. After being rinsed twice in PBS, they were treated with 20 μg/ml Pronase in TE (1 mm Tris HCl, pH 8.0, 0.1 mm EDTA) for 7.5 min. The fixation step was repeated for 5 min, and the slides were rinsed twice in PBS. The sections were acetylated in acetic anhydride in 0.1 m triethanolamine-HCl (pH 7.5), rinsed, and dehydrated through ethanol. Sections were hybridized in 50% deionized formamide, 0.3 m NaCl, 20 mm Tris HCl, pH 8.0, 5 mm EDTA, mm pH 8.0, and a of overnight at °C. After sections were rinsed in 50% deionized formamide, 0.1 m for min at and then for min in m NaCl, mm pH 8.0, 5 mm at °C. After with A for min at the sections were rinsed in min at and min at and was performed using nuclear PA). cells and cells bronchial epithelial were in with and cells were in with and as experiments were in The are in the of were in by Dr. Pinkerton at the of Davis S. Pinkerton M. A. S. Toxicol. 1994; Scopus Google Scholar). were in the of the smoke on were by and smoke were to our they were at 4 to we for at in a of serum-free to a of of smoke of SFM, as cells were at in and to were performed using of and at a of for in were in and for For smoke cells were in at the for and then in for The were rinsed in PBS, in of and at in a and of of the human MUC5AC gene was as described previously D. M. N. Basbaum J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Briefly, the of the human mucin G. J. PubMed Scopus Google Scholar) was used to the transcription of the gene using of of from the was from in the human to by the kb of the of the human MUC5AC gene was and into The of the MUC5AC is in the data were obtained by with using were by using the mutant The were into All were by from cells were prepared using the nuclear to the to cells were to or The protein of the cell was using a protein with as probes as in were on the of the results of experiments described were by and and on a were at for min with nuclear of in a mm (pH 7.5), mm NaCl, mm mm EDTA, mm mm and 4% For a of was to the and for min on For of the antibody of (Santa Cruz was to the as described were to in The was to at for with by bronchial epithelial cells were on and in The cells were for 20 min in the or of The cells were then with PBS, fixed in and with antibody specific to phospho-JNK The phospho-JNK was with to to with a using an in of MUC5AC by human lung epithelial cells was with serum-free or for by for were used as the and were in the at the and the as the MUC5AC with and reverse with of RNA for was using the synthesis for as described by the with A of of RNA as a for with and reverse MUC5AC to on for a of were used as The were in the at the and the as the MUC5AC After a of were and using for to for to and for to were by and with to and were with of and were and by the of the Institute, were obtained at of were placed into and with for and The were at at a of and of were by to to smoke for was the were smoke was from and Institute, by as described previously B. 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Chem. 2002; 277: 32258-32267Abstract Full Text Full Text PDF PubMed Scopus (229) Google the response element was at The this element was for the MUC5AC response to smoke but not for response to EGF to data transcription factor binding in this AP-1 and of these of MUC5AC to smoke, with the from of the AP-1 JunD and to AP-1 the role of of these we performed using probes to in the and of smoke. was by the of smoke protein binding to the AP-1 of the of the and AP-1 and was to Instead, we a both This smoke-induced protein binding of that of the AP-1 with binding of the This was with the data in these the that both AP-1 a role in the response to smoke. AP-1 as response or heterodimers. Although the and of for and JunD, and to the of specific our that smoke both JunD and to to the AP-1 not of JunD and but synthesis of these response as by of protein in lung epithelial cells to protein binding to the Although the is by smoke to the of of these as by of the in of JNK and JunD and upstream signaling In are in a manner via phosphorylation of or in N-terminal J. E. PubMed Scopus Google Scholar, T. B. M. M. PubMed Scopus Google Scholar), are by phosphorylation of or in M. M. 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