Activation of the Epidermal Growth Factor Receptor (EGFR) by a Novel Metalloprotease Pathway
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m803732200
ISSN1083-351X
AutoresDavid A. Bergin, Catherine M. Greene, Erwin E. Sterchi, Cliona Kenna, Patrick Geraghty, Abderazzaq Belaaouaj, Clifford C. Taggart, Shane O’Neill, Noel G. McElvaney,
Tópico(s)Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
ResumoNeutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFα and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFκB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production. Neutrophil Elastase (NE) is a pro-inflammatory protease present at higher than normal levels in the lung during inflammatory disease. NE regulates IL-8 production from airway epithelial cells and can activate both EGFR and TLR4. TACE/ADAM17 has been reported to trans-activate EGFR in response to NE. Here, using 16HBE14o-human bronchial epithelial cells we demonstrate a new mechanism by which NE regulates both of these events. A high molecular weight soluble metalloprotease activity detectable only in supernatants from NE-treated cells by gelatin and casein zymography was confirmed to be meprin alpha by Western immunoblotting. In vitro studies demonstrated the ability of NE to activate meprin alpha, which in turn could release soluble TGFα and induce IL-8 production from 16HBE14o- cells. These effects were abrogated by actinonin, a specific meprin inhibitor. NE-induced IL-8 expression was also inhibited by meprin alpha siRNA. Immunoprecipitation studies detected EGFR/TLR4 complexes in NE-stimulated cells overexpressing these receptors. Confocal studies confirmed colocalization of EGFR and TLR4 in 16HBE14o- cells stimulated with meprin alpha. NFκB was also activated via MyD88 in these cells by meprin alpha. In bronchoalveolar lavage fluid from NE knock-out mice infected intra-tracheally with Pseudomonas aeruginosa meprin alpha was significantly decreased compared with control mice, and was significantly increased and correlated with NE activity, in bronchoalveolar lavage fluid from individuals with cystic fibrosis but not healthy controls. The data describe a previously unidentified lung metalloprotease meprin alpha, and its role in NE-induced EGFR and TLR4 activation and IL-8 production. Neutrophil elastase (NE) 4The abbreviations used are: NE, neutrophil elastase; BALF, bronchoalveolar lavage fluid; CF, cystic fibrosis; EGF, epidermal growth factor; EGFR, EGF receptor; HBEGF, heparin-binding epidermal growth factor; MMP, matrix metalloprotease; TACE, TNF-α-converting enzyme; TGF, transforming growth factor; TLR, toll-like receptor; IL, interleukin; FCS, fetal calf serum; PBS, phosphate-buffered saline; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide. 4The abbreviations used are: NE, neutrophil elastase; BALF, bronchoalveolar lavage fluid; CF, cystic fibrosis; EGF, epidermal growth factor; EGFR, EGF receptor; HBEGF, heparin-binding epidermal growth factor; MMP, matrix metalloprotease; TACE, TNF-α-converting enzyme; TGF, transforming growth factor; TLR, toll-like receptor; IL, interleukin; FCS, fetal calf serum; PBS, phosphate-buffered saline; HA, hemagglutinin; ELISA, enzyme-linked immunosorbent assay; LPS, lipopolysaccharide. is a 29-kDa serine protease that is stored in the azurophilic granules of neutrophils (1Pham C.T. Nat. Rev. Immunol. 2006; 6: 541-550Crossref PubMed Scopus (707) Google Scholar). The main intracellular physiological function of NE is the degradation of foreign organic molecules phagocytosed by neutrophils. NE is well documented as one of numerous neutrophil proteases responsible for destroying microbes (2Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (534) Google Scholar, 3Reeves E.P. Lu H. Jacobs H.L. Messina C.G. Bolsover S. Gabella G. Potma E.O. Warley A. Roes J. Segal A.W. Nature. 2002; 416: 291-297Crossref PubMed Scopus (879) Google Scholar). Its principal target is elastin (4Janoff A. Scherer J. J. Exp. Med. 1968; 128: 1137-1155Crossref PubMed Scopus (314) Google Scholar); however, it can also degrade extracellular matrix proteins, collagen types I-IV, proteoglycan, fibronectin, platelet IIb/IIIa receptor, complement receptor, thrombomodulin (5Abe H. Okajima K. Okabe H. Takatsuki K. Binder B.R. J. Lab. Clin. Med. 1994; 123: 874-881PubMed Google Scholar), lung surfactant proteins (6Liau D.F. Yin N.X. Huang J. Ryan S.F. Biochim. Biophys. Acta. 1996; 1302: 117-128Crossref PubMed Scopus (51) Google Scholar), cadherin (7Carden D. Xiao F. Moak C. Willis B.H. Robinson-Jackson S. Alexander S. Am. J. Physiol. 1998; 275: H385-H392PubMed Google Scholar), and cleave complement factors, immunoglobulin, protease inhibitors, and several proteases (8Klingemann H.G. Egbring R. Holst F. Gramse M. Havemann K. Thromb. Res. 1982; 28: 793-801Abstract Full Text PDF PubMed Scopus (22) Google Scholar). Neutrophils are the primary cell lineage that produces NE and are a key immune cell involved in pro inflammatory lung disorders. A high neutrophil burden has been linked to an increase in inflammation within the lung (9Rouhani F. Paone G. Smith N.K. Krein P. Barnes P. Brantly M.L. Chest. 2000; 117: 250S-251SAbstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and NE has been implicated in several diverse inflammatory lungs disorders including cystic fibrosis (10Doring G. Am. J Respir. Crit. Care Med. 1994; 150: S114-S117Crossref PubMed Google Scholar). Previous studies have identified in part the intracellular signaling pathway regulated by NE (11Walsh D.E. Greene C.M. Carroll T.P. Taggart C.C. Gallagher P.M. O'Neill S.J. McElvaney N.G. J. Biol. Chem. 2001; 276: 35494-35499Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) and established that NE can induce IL-8 via TLR4 (12Carroll T.P. Greene C.M. Taggart C.C. Bowie A.G. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 175: 7594-7601Crossref PubMed Scopus (27) Google Scholar). Other studies have demonstrated partial inhibition of NE-induced IL-8 production by using specific inhibitors of TLR signaling in airway epithelial cells (12Carroll T.P. Greene C.M. Taggart C.C. Bowie A.G. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 175: 7594-7601Crossref PubMed Scopus (27) Google Scholar, 13Greene C.M. Carroll T.P. Smith S.G. Taggart C.C. Devaney J. Griffin S. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 174: 1638-1646Crossref PubMed Scopus (180) Google Scholar). NE is also likely to regulate expression of IL-8 and other genes by alternative mechanisms. In addition to TLR4, another receptor of interest which NE has been shown to trans-activate, is EGFR (14Kohri K. Ueki I.F. Nadel J.A. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L531-L540Crossref PubMed Scopus (162) Google Scholar). EGFR plays a key role in many cellular processes (15Prenzel N. Fischer O.M. Streit S. Hart S. Ullrich A. Endocr. Relat. Cancer. 2001; 8: 11-31Crossref PubMed Scopus (549) Google Scholar) and is important for mucin production from airway epithelial cells (16Shao M.X. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 767-772Crossref PubMed Scopus (226) Google Scholar) and in sustaining neutrophil inflammation (17Hamilton L.M. Torres-Lozano C. Puddicombe S.M. Richter A. Kimber I. Dearman R.J. Vrugt B. Aalbers R. Holgate S.T. Djukanovic R. Wilson S.J. Davies D.E. Clin. Exp. Allergy. 2003; 33: 233-240Crossref PubMed Scopus (133) Google Scholar). EGFR is a transmembrane protein consisting of an extracellular ligand-binding domain to which EGFR ligands such as transforming growth factor α (TGFα) and epidermal growth factor (EGF) bind for activation. Its intracellular domain consists of a tyrosine kinase domain. Upon activation via ligand binding, EGFR is phosphorylated on key tyrosine residues and docking of signal transducers can occur (18Herbst R.S. Int. J. Radiat. Oncol. Biol. Phys. 2004; 59: 21-26Abstract Full Text Full Text PDF PubMed Scopus (979) Google Scholar). In addition to direct activation by EGFR ligands, EGFR can also be trans-activated by other stimuli such as lipoteichoic acid (19Lemjabbar H. Basbaum C. Nat. Med. 2002; 8: 41-46Crossref PubMed Scopus (291) Google Scholar), other TLR agonists (20Koff J.L. Shao M.X. Ueki I.F. Nadel J.A. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 294: L 1068-L 1075Crossref Scopus (184) Google Scholar) and NE (16Shao M.X. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 767-772Crossref PubMed Scopus (226) Google Scholar, 21DiCamillo S.J. Carreras I. Panchenko M.V. Stone P.J. Nugent M.A. Foster J.A. Panchenko M.P. J. Biol. Chem. 2002; 277: 18938-18946Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The mechanism of NE-induced Mucin (MUC5AC) production via EGFR in NCI-H292 cells, an epithelial line derived from a mucoepidermoid pulmonary carcinoma has been documented (16Shao M.X. Nadel J.A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 767-772Crossref PubMed Scopus (226) Google Scholar) with TNF-α-converting enzyme (TACE/ADAM17) shown to be involved in EGFR ligand generation. EGFR has also been implicated in NE-induced IL-8 production in lung epithelial cells (22Kuwahara I. Lillehoj E.P. Lu W. Singh I.S. Isohama Y. Miyata T. Kim K.C. Am. J. Physiol. Lung Cell Mol. Physiol. 2006; 291: L407-L416Crossref PubMed Scopus (95) Google Scholar); however, the in vivo biological significance of TACE in these events remains to be elucidated. Interestingly other metalloproteases in addition to TACE are reported to have a role in activation of EGFR (23Merlos-Suarez A. Ruiz-Paz S. Baselga J. Arribas J. J. Biol. Chem. 2001; 276: 48510-48517Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). We set out to understand in greater detail how NE trans-activates EGFR in human bronchial epithelial cells and to integrate this new knowledge with our current understanding of the NE mechanism of action on IL-8 expression. We postulated that a novel metalloprotease that shares redundancy with TACE is activated by NE in bronchial epithelial cells. Our aim was to identify such a protease, determine its biological activity against EGFR and TLR and elucidate its role in NE-induced activation of NFκB and regulation of IL-8 gene expression. Here we demonstrate the involvement of meprin alpha, a previously unidentified target of NE, in the NE-induced EGFR/TLR4 inflammatory pathway in human bronchial epithelial cells. Chemicals—Chemicals and reagents were purchased from Sigma unless indicated otherwise. Cell Culture and Treatment—16HBE14o- cells are an SV40-transformed human bronchial epithelial cell line and were obtained as a gift from D. Gruenert (University of Vermont). The cells were cultured in minimal essential medium (MEM) supplemented with 10% FCS, 1% l-glutamine, 1% penicillin/streptomycin, (Invitrogen). The human embryonic kidney cell line, HEK293, (ECACC-85120602) was cultured in Eagle's Minimal Essential Medium (EMEM, Invitrogen) supplemented with 10% FCS, 1% l-glutamine, 1% penicillin/streptomycin, 1% NEAA (Invitrogen). 293-hTLR4-HA cells, an isolated clone of HEK293 cells stably transfected with human HA-tagged TLR4 (InvivoGen, San Diego), were cultured in DMEM, 10% FCS, 10 μg/ml blastocidin S (InvivoGen), and 1% penicillin/streptomycin. NE Activity Assay—NE activity was quantified prior to all experiments using the NE substrate (0.5 mmN-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide). Δ absorbance units (2 min) at 405 nm were recorded and compared with an NE standard of known activity. Zymography—Zymography was performed on 150 μl serum-free medium from untreated, EGF- or NE-treated cells (1 × 106). Samples of human or murine BALF containing 400 ng or 2 μg of protein, respectively, were treated for 10 min with sample buffer (0.25% bromphenol blue, 50 mm Tris, pH 7.5, 40% glycerol, and 1% SDS) and were electrophoresed on a 7% SDS-polyacrylamide gel containing gelatin or casein (1 mg/ml). After electrophoresis, the gels were incubated in 50 mm Tris, pH 7.5, 5 mm CaCl2, 1 μm ZnCl2, and 2.5% (v/v) Triton X-100 for 30 min, washed in the same buffer without Triton X-100 for 5 min and incubated at 37 °C overnight in the same buffer supplemented with 1% (v/v) Triton X-100. The gels were stained with 0.125% Coomassie Blue and washed with 10% acetic acid and 40% methanol in water. RT PCR Analysis for Meprin Gene Expression—RNA was isolated from 16HBE14o- and HEK293 cells in TRI reagent, and RNA was extracted. RNA (2 μg) was reverse-transcribed into cDNA, and 2 μl was amplified with 1.25 units of TaqDNA polymerase, 1× PCR buffer, and 10 mm dNTPs (Promega) in a 50-μl volume containing 100 pmol each of the following primers: Meprin alpha: 5′-GAAATCCAGAAATGGCCTGA-3′, 5′-TGGAAATGTTCTGTCCCACA-3′; β-actin: 5′-GGGTACATGGTGGTGCCG-3′, 5′-GCCGGGAAATCGTGCGTG-3′. After a hot start, the amplification profile was 45 cycles of 1 min denaturation at 95 °C, 1 min annealing at 50 °C, and 1 min extension at 72 °C. RT-PCR amplification of meprin A and β-actin generated products of 242 bp and 402 bp, respectively. PCR products were resolved on a 1% agarose gel containing 0.5 μg/ml ethidium bromide. Microscopy—Immunofluorescence microscopy was performed as described previously (24Grogan A. Reeves E. Keep N. Wientjes F. Totty N.F. Burlingame A.L. Hsuan J.J. Segal A.W. J. Cell Sci. 1997; 110: 3071-3081Crossref PubMed Google Scholar) with some alterations. 16HBE14o- cells (1 × 105) were cultured overnight in chamber slides (Lab-Tek). Cells were washed and fixed with 4% (w/v) paraformaldehyde for 10 min, permeabilized with 0.2% (v/v) Triton X-100 in PBS for 10 min and blocked with 10 mm NaBH4 for 1 h. Cells were incubated with rabbit anti-meprin alpha (25Rosmann S. Hahn D. Lottaz D. Kruse M.N. Stocker W. Sterchi E.E. J. Biol. Chem. 2002; 277: 40650-40658Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) overnight at 4 °C, or mouse anti TLR4 FITC labeled (Abcam) and EGFR (SCBT) antibody for 1 h, washed, and incubated with goat anti-rabbit IgG secondary antibody Rhodamine labeled (Abcam) at 5 μg/ml for 1 h. Controls for this experiment included cells alone, Rabbit IgG isotype control (R&D Systems) and those exposed to secondary antibody only. Meprin Activity Assay—Activity assays were carried out using the fluorogenic peptide substrate Mca-YVADAPK-(Dnp)-OH according to the manufacturer's direction (R&D Systems). Briefly, to activate meprin alpha, the enzyme (100 ng) was incubated with 0.1 ng of trypsin or 100 nm of NE in TCNB (50 nm Tris, 10 mm CaCl2, 0.15 m NaCl, 0.05% Brij 35, pH 7.5) for 3 h at 37 °C. Trypsin and NE were inactivated with AEBSF (1 mm) or CMK (5 μm), respectively, to prevent turnover of the meprin substrate, and fluorescence was quantified at 320 nm excitation and 450 nm emission after 60 min. IL-8 ELISA—16HBE14o- cells (1 × 105/well) were left untreated or treated for 1 h with 5 μg/ml EGFR ligand neutralizing antibody (R&D Systems), 500 nm AG1478 (Calbiochem), 2 μg of EGFR neutralizing antibody (Oncogene Ab-3), or 100 μm actinonin followed by EGF, TGFα, HBEGF, or NE for 4 h. Medium was removed and used in an IL-8 ELISA (R&D Systems). 5 μm MetOSuc-Ala-Ala-Pro-Val-chloromethyl ketone (CMK) was added to NE-treated media to prevent active NE from interfering with IL-8 measurement. TGFα ELISA—Cells were untreated or treated with vehicle (DMSO) or actinonin (100 μm) for 1 h followed by NE, activated recombinant meprin alpha, or media for 4 h. For all experiments EGFR was blocked for 30 min with 2 μg/ml anti-EGFR neutralizing antibody (Calbiochem) prior to agonist treatment. Supernatants were collected, and TGFα was measured via ELISA (R&D Systems) (14Kohri K. Ueki I.F. Nadel J.A. Am. J. Physiol. Lung Cell Mol. Physiol. 2002; 283: L531-L540Crossref PubMed Scopus (162) Google Scholar). Bronchoalveolar Lavage Fluid (BALF)—NE gene-targeted mice were generated, as previously described (2Belaaouaj A. McCarthy R. Baumann M. Gao Z. Ley T.J. Abraham S.N. Shapiro S.D. Nat. Med. 1998; 4: 615-618Crossref PubMed Scopus (534) Google Scholar). NE knock-out mice (n = 3) and their wild-type littermates (n = 3) were intranasally challenged with PBS (50 μl) or PBS containing Pseudomonas aeruginosa H103 (4.8 × 106 CFUs). Twenty-four hours later, BALF was collected (26Hirche T.O. Atkinson J.J. Bahr S. Belaaouaj A. Am. J. Respir. Cell Mol. Biol. 2004; 30: 576-584Crossref PubMed Scopus (62) Google Scholar). BALF from individuals with CF (n = 11) or healthy controls (n = 10) was collected following informed consent using a protocol approved by Beaumont Hospital Ethics Committee (13Greene C.M. Carroll T.P. Smith S.G. Taggart C.C. Devaney J. Griffin S. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 174: 1638-1646Crossref PubMed Scopus (180) Google Scholar). Samples were filtered through gauze, centrifuged at 1,000 × g for 10 min, and cell-free supernatants were aliquoted and stored at -80 °C. EGFR trans-Activation—16HBE14o- cells were seeded at 1 × 106 on a 6-well plate for 24 h in supplemented MEM. Cells were serum-starved in serum-free MEM for 2 h prior to stimulation and then left untreated (control) or stimulated with EGF (R&D systems) (10 ng/ml) or 100 nm NE (Elastin Products, MO) for 10 min. Medium was removed, centrifuged, and stored at -20 °C. Cells were washed with PBS and lysed using a radioimmune precipitation assay buffer (10 mm Tris, pH 7.4, 100 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 20 mm Na4P2O7, 2 mm Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate) containing protease inhibitor tablets (Roche UK) and 1 mm phenylmethylsulfonyl fluoride. Cell lysates were clarified by centrifugation and were used in EGFR and EGFR [pY1173] ELISAs (BIOSOURCE CA) according to the manufacturer's instructions. Transfections—16HBE14o- cells were uniformly co-transfected for 24 h with a constitutive luciferase reporter gene construct, pRLSV40 (100 ng, Promega) and an empty vector pCDNA3.1 (150 ng, Invitrogen) or the same vector carrying a gene encoding a dominant negative functionally inactive MyD88 (150 ng) as previously reported (12Carroll T.P. Greene C.M. Taggart C.C. Bowie A.G. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 175: 7594-7601Crossref PubMed Scopus (27) Google Scholar, 13Greene C.M. Carroll T.P. Smith S.G. Taggart C.C. Devaney J. Griffin S. O'Neill S.J. McElvaney N.G. J. Immunol. 2005; 174: 1638-1646Crossref PubMed Scopus (180) Google Scholar) using Gene Juice (Novagen). Cells were left untreated or stimulated as indicated. NFκB activation was assessed (see below) or supernatants were retained after 24 h for IL-8 ELISA, and cells were lysed with Reporter Lysis buffer (Promega) and quantified by luminometry to determine transfection efficiencies (Wallac Victor2, 1420 multilabel counter). IL-8 protein production was calculated as pg per light unit (L.U.). 293-hTLR4-HA cells (5 × 106) were transfected for 48 h with an empty vector or a plasmid expressing human EGFR (1 μg, Upstate Cat. 21–176) and left untreated or treated with agonists (as described) for 10 min prior to immunoprecipitation with an anti-HA tag (InvivoGen) or an anti-EGFR monoclonal antibody (sc-120, Santa Cruz Biotechnology) and immunodetection with either anti-HA tag or anti-EGFR monoclonal antibodies. NFκB Assays—NFκB activation was assessed by three methods. (i) Cells (5 × 105) were co-transfected as described above with an (NFκB)5-luciferase reporter plasmid and pRLSV40 for 24 h then stimulated with meprin alpha for 6 h. Relative luciferase production was quantified by luminometry. (ii and iii) Cells were co-transfected with pCDNA3 or the ΔMyD88 expression plasmid for 24 h, then stimulated with meprin for 1 h. Nuclear extracts were prepared and examined by Western blotting using p65 and TBP antibodies (Santa Cruz Biotechnology), and p65/TBP ratios were quantified by densitometry or assessed 1 h post-stimulation with meprin using TransAM NFκB p65 (Actif Motif) as recommended by the manufacturer. RNAi—16HBE14o- cells (5 × 105) were transfected with 50 nm of a siRNA-targeting Meprin alpha (siRNA ID 104082, Ambion) or a scrambled control duplex (Silencer GAPDH siRNA Control Ambion) for 24 h. Cells were placed in serum-free medium and stimulated with NE (100 nm for 24h). Total RNA was extracted for qRT-PCR using the Roche LC480 and SYBR Green I Master with Meprin alpha or GAPDH (Forward: 5′-CATGAGAAGTATGACAACAGCCT-3′, Reverse: 5′-AGTCCTTCCACGATACCAAAGT-3′) primers (MWG Biotech) to quantify knockdown. Cell supernatants were retained for IL-8 ELISA. Statistical Analysis—Data were analyzed with the PRISM 3.0 software package (GraphPad, San Diego, CA). Results are expressed as the mean ± S.E. and were compared by Student's t test or analysis of variance. Differences were considered significant at p ≤ 0.05. Identification of Novel Protease Activity from 16HBE14o- Cells Treated with NE—In our search for novel metalloproteases activated by NE, we analyzed supernatants from untreated, EGFR ligand- and NE-treated 16HBE14o- cells by zymography. Fig. 1 shows that a high molecular weight gelatinase activity, above 175 kDa, was detectable in the NE-treated supernatants but was absent from the control and EGF-treated samples (Fig. 1A). Examination of protease activity using a casein zymogram showed this high molecular weight NE-induced gelatinase also had caseinase activity of the same size, which was not present in the control, or EGF-treated samples (Fig. 1B). Based on these properties and its migration in non-reducing PAGE, this indicated that it may be meprin; therefore, we performed immunoblot analysis using an anti-meprin alpha antibody. An immunoreactive band of the same size was detected in the NE-treated samples but was not detectable in the control or EGF-treated samples (Fig. 1C). No signals were detected in any of the samples at >175 kDa using MMP2 or MMP9 antibodies (data not shown). Detection of Meprin Alpha Expression in 16HBE14o- Cells—To verify meprin alpha expression by 16HBE14o- cells RT-PCR, Western blotting, and immunofluorescence confocal microscopy was performed (Fig. 2). Meprin alpha mRNA was detected in 16HBE14o- cells and HEK293 cells, a human embryonic kidney cell line known to express meprin alpha (Fig. 2A). Meprin alpha has a Mr of 84.4 kDa but because of glycosylation migrates with a molecular weight of ∼90 kDa in reducing SDS-PAGE (27Becker-Pauly C. Howel M. Walker T. Vlad A. Aufenvenne K. Oji V. Lottaz D. Sterchi E.E. Debela M. Magdolen V. Traupe H. Stocker W. J. Invest. Dermatol. 2006; 127: 1115-1125Abstract Full Text Full Text PDF Scopus (90) Google Scholar). Fig. 2B shows a Coomassie Blue-stained gel of meprin alpha immunoprecipitated from 16HBE14o- cells using a monoclonal antibody (lane 3). Recombinant meprin alpha was used as a control (lane 1) and molecular weight markers are in lane 2. A duplicate gel was transferred to a nitrocellulose membrane and probed with a rabbit polyclonal anti-meprin alpha antibody and an immunoreactive signal of the correct molecular weight was evident in the immunoprecipitate (Fig. 2C). Immunofluorescence and confocal microscopy were performed to visualize the location of meprin alpha in 16HBE14o- cells using a goat polyclonal anti-human meprin alpha antibody. As shown in Fig. 2D, meprin alpha expression was detected peri-nuclear and also on the cell membrane. Meprin Alpha Activated by NE Can Induce IL-8 Expression and Release TGFα from 16HBE14o- Cells—Trypsin is a potent activator of meprin alpha. We examined whether NE, like trypsin, could activate meprin alpha using a fluorimetric activity assay. A recombinant inactive form of meprin alpha was incubated with trypsin or NE, the serine proteases were inactivated, and meprin activity was measured using a substrate that had meprin specificity. Fig. 3A shows that both trypsin and NE can activate meprin to similar levels (p = 0.01 and p = 0.02, respectively). Having established that NE can activate meprin alpha we next evaluated whether meprin alpha, like NE, can induce IL-8 production from 16HBE14o- cells. Here cells were either untreated, treated with NE or active meprin, and IL-8 levels were quantified in cell supernatants. Fig. 3B shows that meprin alpha induced greater than 2-fold increase in IL-8 secretion compared with control cells (p < 0.001) similar to NE (p < 0.001 versus control). NE can generate soluble TGFα from 16HBE14o- cells (Fig. 3C, p = 0.03). We investigated the effect of meprin alpha on proTGFα. Cells were left untreated or treated with NE, meprin alpha, or supernatants from NE-treated 16HBE14o- cells (from which TGFα had been depleted and that had also been treated with a specific NE inhibitor). These supernatants had gelatinase activity (data not shown). TGFα levels in the cell supernatants were quantified by ELISA. Fig. 3C shows that like NE, meprin alpha or NE-treated supernatants leads to release of soluble TGFα (p = 0.01 and 0.01, respectively). Inhibition of Meprin Alpha Can Impair IL-8 Production in NE-stimulated Bronchial Epithelial Cells—NE-induced IL-8 expression is TGFα-dependent and can be inhibited using a TGFα-neutralizing antibody (Fig. 4A). Neutralizing antibodies to EGF and HBEGF have no effect. EGFR is also involved in NE-induced IL-8 expression. NE can trans-activate EGFR (Fig. 4B) and inhibition of EGFR using AG1478 or an EGFR antibody impairs NE-induced IL-8 expression (Fig. 4C). Actinonin is a naturally occurring hydroxamate that is the most effective inhibitor of meprin alpha (35Wolz R.L. Arch Biochem. Biophys. 1994; 310: 144-151Crossref PubMed Scopus (18) Google Scholar, 36Becker C. Kruse M.N. Slotty K.A. Kohler D. Harris J.R. Rosmann S. Sterchi E.E. Stocker W. Biol. Chem. 2003; 384: 825-831Crossref PubMed Scopus (81) Google Scholar, 37Kruse M.N. Becker C. Lottaz D. Köhler D. Yiallouros I. Krell H.W. Sterchi E.E. Stöcker W. Biochem. J. 2004; 378: 383-389Crossref PubMed Scopus (139) Google Scholar, 38Takayama J. Takaoka M. Sugino Y. Yamamoto Y. Ohkita M. Matsumura Y. Biol. Pharm. Bull. 2007; 30: 1905-1912Crossref PubMed Scopus (22) Google Scholar). We investigated its effects on NE-induced IL-8 and TGFα production. We performed a dose response of actinonin (0–100 μm) investigating its effect on NE-induced IL-8 expression in 16HBE14o- cells (data not shown), and from this selected 100 μm as the most effective inhibitory dose. Actinonin effectively inhibited meprin alpha activity induced by either trypsin or NE (p < 0.001 for both) (Fig. 5A) and also significantly inhibited NE-induced IL-8 (Fig. 5B) and TGFα (Fig. 5C) production from 16HBE14o- cells (#, p < 0.001). To confirm that inhibition of NE-induced IL-8 expression by actinonin is mediated via blocking meprin alpha, we next performed siRNA experiments knocking down meprin alpha (98.6% knockdown). Fig. 5D shows that NE-induced IL-8 expression is abrogated by a meprin alpha siRNA. Presence of Meprin Alpha in Bronchoalveolar Lavage Fluid (BALF) in Lung Inflammation Correlates with NE—We next assessed the role of meprin alpha in vivo. Meprin activity was examined in BALF of NE knock-out and wild-type mice infected with Pseudomonas aeruginosa. Gelatin zymography detected a high molecular weight gelatinase activity above 175 kDa that was present in higher quantities in wild type (Fig. 6A, lanes 1–3) versus NE knock-out mice (lanes 4–6). We also evaluated bronchoalveolar lavage fluid samples from individuals with cystic fibrosis (CF) and healthy controls for meprin activity. CF is an inflammatory lung condition associated with high levels of active NE in the lung. Fig. 6B shows evidence of a high molecular weight gelatinase activity indicative of meprin alpha in 11 of 11 BALF samples from CF individuals. No gelatinase activity was detectable in 10 control BALF samples under the same conditions. Fig. 6C shows the corresponding NE activities per μg of protein in each of the BALF samples (p = 0.0002 for normal versus CF BALF). The gelatinase activity in 2 representative CF BALF samples was confirmed to be meprin alpha by Western immunoblotting (Fig. 6D). Meprin Induces Co-localization of EGFR and TLR4—We investigated how the NE-Meprin-TGFα-EGFR-IL-8 pathway reported here integrates with our previous reports demonstrating the NE-TLR4-MyD88-IL-8 pathway (11Walsh D.E. Greene C.M. Carroll T.P. Taggart C.C. Gallagher P.M. O'Neill S.J. McElvaney N.G. J. Biol. Chem. 2001; 276: 35494-35499Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 28Devaney J.M. Greene C.M. Taggart C.C. Carroll T.P. O'Neill S.J. McElvaney N.G. FEBS Lett. 2003; 544: 129-132Crossref PubMed Scopus (208) Google Scholar). In Fig. 7A we evaluated the effect of ΔMyD88, an inhibitor of TLR4 signaling, on NE- and TGFα-induced IL-8 production. 16HBE14o- cells were transfected with
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