Interleukin-31 and Oncostatin-M Mediate Distinct Signaling Reactions and Response Patterns in Lung Epithelial Cells
2006; Elsevier BV; Volume: 282; Issue: 5 Linguagem: Inglês
10.1074/jbc.m609655200
ISSN1083-351X
AutoresSouvik Chattopadhyay, Erin Tracy, Ping Liang, Olivier Robledo, Stefan Rose‐John, Heinz Baumann,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoLung epithelial cells are primary targets of oncostatin M (OSM) and, to a lower degree, of interleukin (IL)-6 and IL-31, all members of the IL-6 cytokine family. The OSM receptor (OSMR) signals through activation of STAT and mitogen-activated protein kinase pathways to induce genes encoding differentiated cell functions, reduce cell-cell interaction, and suppress cell proliferation. IL-31 functions through the heteromeric IL-31 receptor, which shares with OSMR the OSMRβ subunit, but does not engage gp130, the common subunit of all other IL-6 cytokine receptors. Because the response of epithelial cells to IL-31 is unknown, the action of IL-31 was characterized in the human alveolar epithelial cell line A549 in which the expression of the ligand-binding IL-31Rα subunit was increased. IL-31 initiated signaling that differed from other IL-6 cytokines by the particularly strong recruitment of the STAT3, ERK, JNK, and Akt pathways. IL-31 was highly effective in suppressing proliferation by altering expression of cell cycle proteins, including up-regulation of p27Kip1 and down-regulation of cyclin B1, CDC2, CDK6, MCM4, and retinoblastoma. A single STAT3 recruitment site (Tyr-721) in the cytoplasmic domain of IL-31Rα exerts a dominant function in the entire receptor complex and is critical for gene induction, morphological changes, and growth inhibition. The data suggest that inflammatory and immune reactions involving activated T-cells regulate functions of epithelial cells by IL-6 cytokines through receptor-defined signaling reactions. Lung epithelial cells are primary targets of oncostatin M (OSM) and, to a lower degree, of interleukin (IL)-6 and IL-31, all members of the IL-6 cytokine family. The OSM receptor (OSMR) signals through activation of STAT and mitogen-activated protein kinase pathways to induce genes encoding differentiated cell functions, reduce cell-cell interaction, and suppress cell proliferation. IL-31 functions through the heteromeric IL-31 receptor, which shares with OSMR the OSMRβ subunit, but does not engage gp130, the common subunit of all other IL-6 cytokine receptors. Because the response of epithelial cells to IL-31 is unknown, the action of IL-31 was characterized in the human alveolar epithelial cell line A549 in which the expression of the ligand-binding IL-31Rα subunit was increased. IL-31 initiated signaling that differed from other IL-6 cytokines by the particularly strong recruitment of the STAT3, ERK, JNK, and Akt pathways. IL-31 was highly effective in suppressing proliferation by altering expression of cell cycle proteins, including up-regulation of p27Kip1 and down-regulation of cyclin B1, CDC2, CDK6, MCM4, and retinoblastoma. A single STAT3 recruitment site (Tyr-721) in the cytoplasmic domain of IL-31Rα exerts a dominant function in the entire receptor complex and is critical for gene induction, morphological changes, and growth inhibition. The data suggest that inflammatory and immune reactions involving activated T-cells regulate functions of epithelial cells by IL-6 cytokines through receptor-defined signaling reactions. Many mammalian cell types co-express several receptors for the evolutionarily related hematopoietic cytokines of the interleukin (IL) 4The abbreviations used are: IL, interleukin; CAT, chloramphenicol acetyltransferase; CRP, C-reactive protein; ERK, extracellular signal-regulated kinase; JAK, Janus kinase; JNK, Jun N-terminal kinase; GM-CSF, granulocyte macrophage-colony stimulating factor; LIF, leukemia inhibitory factor; OSM, oncostatin M; OSMR, oncostatin M receptor; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; RB, retinoblastoma; MAPK, mitogen-activated protein kinase. -6 group (1Huising M.O. Kruiswijk C.P. Flik G. J. Endocrinol. 2006; 189: 1-25Crossref PubMed Scopus (173) Google Scholar). Moreover, all cell types express gp130, the common signal-transducing subunit of this receptor family. A major unresolved issue is whether the receptors for the individual IL-6 cytokines exert redundant functions because of the involvement of shared subunits, or whether they exert specific actions because of the formation of heteromeric subunit combinations. Gross analyses of cell responses to IL-6 cytokines indicated similar signaling reactions, supporting the notion of redundancy (2Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1298) Google Scholar, 3Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Muller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2518) Google Scholar). However, the individual receptor forms determine not only the ability of the cells to respond to specific IL-6 cytokines by mediating ligand binding but also the quantitative signaling by the relative expression levels of receptor subunits (4Viswanathan S. Benatar T. Rose-John S. Lauffenburger D.A. Zandstra P.W. 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Leung D.Y. Maurer M. Howell M. Boguniewicz M. Yao L. Storey H. LeCiel C. Harder B. Gross J.A. J. Allergy Clin. Immunol. 2006; 117: 418-425Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 18Sonkoly E. Muller A. Lauerma A.I. Pivarcsi A. Soto H. Kemeny L. Alenius H. Dieu-Nosjean M.C. Meller S. Rieker J. Steinhoff M. Hoffmann T.K. Ruzicka T. Zlotnik A. Homey B. J. Allergy Clin. Immunol. 2006; 117: 411-417Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 19Bando T. Morikawa Y. Komori T. Senba E. Neuroscience. 2006; 142: 1263-1271Crossref PubMed Scopus (84) Google Scholar). Because antigenic challenge increased levels of IL-31 in a mouse model of airway hyper-responsiveness, this cytokine has also been proposed to play a role in airway hypersensitivity (15Dillon S.R. Sprecher C. Hammond A. Bilsborough J. Rosenfeld-Franklin M. Presnell S.R. Haugen H.S. Maurer M. Harder B. Johnston J. Bort S. Mudri S. Kuijper J.L. Bukowski T. Shea P. Dong D.L. Dasovich M. Grant F.J. Lockwood L. Levin S.D. LeCiel C. Waggie K. Day H. Topouzis S. Kramer J. Kuestner R. Chen Z. Foster D. Parrish-Novak J. Gross J.A. Nat. Immun. 2004; 5: 752-760Crossref Scopus (747) Google Scholar). The fact that keratinocytes and lung epithelial cells abundantly express OSMRβ and gp130 and respond to OSM by profound changes in gene transcription and proliferation (20Finelt N. Gazel A. Gorelick S. Blumenberg M. Cytokine. 2005; 31: 305-313Crossref PubMed Scopus (31) Google Scholar, 21Sallenave J.M. Tremblay G.M. Gauldie J. Richards C.D. J. Interferon Cytokine Res. 1997; 17: 337-346Crossref PubMed Scopus (34) Google Scholar, 22Loewen G.M. Tracy E. Blanchard F. Tan D. Yu J. Raza S. Matsui S. Baumann H. BMC Cancer. 2005; 5: 145Crossref PubMed Scopus (32) Google Scholar, 23Boutten A. Venembre P. Seta N. Hamelin J. Aubier M. Durand G. Dehoux M.S. Am. J. Respir. Cell Mol. 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Transient transfection experiments with native and chimeric IL-31Rα constructs indicated the recruitment of those signaling pathways known to be common to IL-6 cytokine receptors, including STAT1, -3, and -5, phosphoinositide 3-kinase, and ERK (16Diveu C. Lak-Hal A.H. Froger J. Ravon E. Grimaud L. Barbier F. Hermann J. Gascan H. Chevalier S. Eur. Cytokine Netw. 2004; 15: 291-302PubMed Google Scholar, 25Dreuw A. Radtke S. Pflanz S. Lippok B.E. Heinrich P.C. Hermanns H.M. J. Biol. Chem. 2004; 279: 36112-36120Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The studies demonstrated the requirement of the cytoplasmic domain of IL-31Rα, as present in the full-length receptor subunit, to enable signaling and determined tyrosine residues 652 and 721 (Tyr-652 and Tyr-721) to direct activation of STAT1/STAT5 and STAT1/STAT3, respectively. Considering the limitations of transient expression systems, the precise function of the IL-31R complex beyond the immediate signaling reactions and quantitative comparison to the action by the other IL-6 cytokine receptors remains to be determined. Despite the prediction of IL-31-responsive epidermal keratinocytes and other epithelial cells (15Dillon S.R. Sprecher C. Hammond A. Bilsborough J. Rosenfeld-Franklin M. Presnell S.R. Haugen H.S. Maurer M. Harder B. Johnston J. Bort S. Mudri S. Kuijper J.L. Bukowski T. Shea P. Dong D.L. Dasovich M. Grant F.J. Lockwood L. Levin S.D. LeCiel C. Waggie K. Day H. Topouzis S. Kramer J. Kuestner R. Chen Z. Foster D. Parrish-Novak J. Gross J.A. Nat. Immun. 2004; 5: 752-760Crossref Scopus (747) Google Scholar, 17Bilsborough J. Leung D.Y. Maurer M. Howell M. Boguniewicz M. Yao L. Storey H. LeCiel C. Harder B. Gross J.A. J. Allergy Clin. Immunol. 2006; 117: 418-425Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 18Sonkoly E. Muller A. Lauerma A.I. Pivarcsi A. Soto H. Kemeny L. Alenius H. Dieu-Nosjean M.C. Meller S. Rieker J. Steinhoff M. Hoffmann T.K. Ruzicka T. Zlotnik A. Homey B. J. Allergy Clin. Immunol. 2006; 117: 411-417Abstract Full Text Full Text PDF PubMed Scopus (728) Google Scholar, 19Bando T. Morikawa Y. Komori T. Senba E. Neuroscience. 2006; 142: 1263-1271Crossref PubMed Scopus (84) Google Scholar), the study of the receptor signaling in these cells proved to be challenging because the level of IL-31Rα is low relative to that of OSMRβ. We hypothesize that sequence elements, which include the kinase-modified tyrosine residues within the cytoplasmic domains of the IL-6 family of receptors, define not only the recruitment of signal-transducing pathways (16Diveu C. Lak-Hal A.H. Froger J. Ravon E. Grimaud L. Barbier F. Hermann J. Gascan H. Chevalier S. Eur. Cytokine Netw. 2004; 15: 291-302PubMed Google Scholar, 25Dreuw A. Radtke S. Pflanz S. Lippok B.E. Heinrich P.C. Hermanns H.M. J. Biol. Chem. 2004; 279: 36112-36120Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) but also the specificity of the cell response to the cytokines. This hypothesis was tested for IL-31Rα in lung epithelial cells. We identified that normal human lung epithelial cells and the transformed line A549 express low levels of functional IL-31Rα. To better visualize IL-31 action, expression of IL-31Rα was enhanced in A549 cells by stable integration of a retroviral expression vector. These cells permitted the functional analysis of the receptor combinations IL-31Rα·OSMRβ, OSMRβ·gp130, and gp130·gp130 and indicated a striking specificity of the signaling by IL-31 as compared with OSM and hyper-IL-6. Cells—Clonal lines of the human glioma CCF-3, -4, -124, U118, ROS-9, oligodendrocytoma HS683, neuroblastoma IMR5 (provided by Drs. J. Cowell and R. Fenstermaker, Roswell Park Cancer Institute), and alveolar carcinoma A549 (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Primary cultures of normal human bronchial and alveolar type II epithelial cells were generated from residual bronchoscopic brushing and tumor-free lung tissue, respectively, as described (22Loewen G.M. Tracy E. Blanchard F. Tan D. Yu J. Raza S. Matsui S. Baumann H. BMC Cancer. 2005; 5: 145Crossref PubMed Scopus (32) Google Scholar). For identification of cytokine signaling, cells from the first passage were used. Normal human skin keratinocytes (passage 2, provided by Dr. S. Sinha, State University of New York, Buffalo) and immortalized human epidermal keratinocytes (HEKα; ATCC) were cultured in serum-free hormonally defined keratinocyte medium (Invitrogen). COS-1 cells were used to express recombinant human cytokines and Hep3B cells (26Knowles B.B. Howe C.C. Aden D.P. Science. 1980; 209: 497-499Crossref PubMed Scopus (1506) Google Scholar) for functional reconstitution of the IL-31R complex by transient transfection of receptor subunits. Cytokine and Cytokine Receptor Constructs—The protein-coding region for human IL-31 and the long form (745 amino acids) IL-31Rα was identified in the genome data bank based on the published sequence (14Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 15Dillon S.R. Sprecher C. Hammond A. Bilsborough J. Rosenfeld-Franklin M. Presnell S.R. Haugen H.S. Maurer M. Harder B. Johnston J. Bort S. Mudri S. Kuijper J.L. Bukowski T. Shea P. Dong D.L. Dasovich M. Grant F.J. Lockwood L. Levin S.D. LeCiel C. Waggie K. Day H. Topouzis S. Kramer J. Kuestner R. Chen Z. Foster D. Parrish-Novak J. Gross J.A. Nat. Immun. 2004; 5: 752-760Crossref Scopus (747) Google Scholar). The IL-31 cDNA (encoding amino acid residues 24–164, NCBI accession number NP001014358.1) was generated by reverse transcription-PCR using RNA from mitogen-activated human peripheral T-cells. Because the first exon could not be unambiguously identified in the human genomic data base, the cDNA encoding the signal peptide of human IL-6 (amino acid residues 1–29) was added to the 5′ end of the cloned IL-31 cDNA under inclusion of an extra triplet encoding glycine. The resulting construct was inserted into the pcDNA3.1 vector (Invitrogen) and verified by sequencing. The IL-31Rα cDNA was amplified from RNA extracted from human glioma cell line U87 and cloned into pCMV-Tag 4A (Stratagene, La Jolla, CA) vector between the HindIII and XhoI sites upstream and in-frame with the DYKDDDDK (FLAG) sequence. The IL-31Rα-FLAG sequence was transferred into pLHCX retroviral delivery and expression vector (Clontech). The three tyrosine residues in the intracellular domain, Tyr-652, Tyr-683, and Tyr-721, were mutated to phenylalanine (YF), singly and in combinations, by QuickChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). IL-31Rα forms with the mutations of both Tyr-652 and Tyr-683, or Tyr-652 and Tyr-721 are labeled Y652F, Y683F and Y652F, Y721F, respectively. IL-31Rα with all three tyrosine mutations is labeled 3YF. Retroviruses were generated by a 2-day transient transfection procedure in PT67 packaging cells. The chimeric receptor GM-CSFRβ-IL-31Rα was constructed by inserting the EcoRI-NotI fragment encoding the IL-31Rα (amino acid residues 515–745) into the EcoRI-NotI restriction-digested pLNCX2-GM-CSFRβ, encoding the extracellular domain of GM-CSFRβ (amino acid residues 1–437). The chimeric receptor GM-CSFRα-OSMRβ was constructed by inserting the PCR-generated EcoRI-NotI fragment encoding the OSMRβ (amino acid residues 723–979) into the EcoRI-NotI restriction-digested pSVSPORT-GM-CSFRα, encoding the extracellular domain of GM-CSFRα (amino acid residues 1–315) (27Gearing D.P. King J.A. Gough N.M. Nicola N.A. EMBO J. 1989; 8: 3667-3676Crossref PubMed Scopus (524) Google Scholar). The chimeric receptor cDNA was excised using HindIII and PmeI restriction enzymes, a NotI site added 3′ to the PmeI site, and re-cloned into the HindIII and NotI sites of the retroviral pLPCX vector. The tyrosine residues 917 and 945 in the cytoplasmic domain of OSMRβ were mutated to phenylalanine and verified by sequencing. Expression vectors for human OSMRβ, OSM (Amgen Corp., Seattle (28Mosley B. De Imus C. Friend N. Boiani N. Thoma B. Park L.S. Cosman D. J. Biol. 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COS-1 cells in 10-cm diameter dishes (3 × 106 cells) were transfected with 4 μg of cytokine expression vector and 0.2 μg of pEGFP using Mirus TransIT-LT1 transfection reagent (Mirus Bio Corp., Madison, WI). After 24 h, the cultures with >50% transfected (GFP+) cells were changed to fresh Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 4.5 mg/ml glucose. The presence of 10% fetal calf serum was important to preserve the bioactivity of the synthesized cytokines. After 72 h of incubation, during which the cultures reached 100% confluence, the conditioned medium was collected and filtered through a 0.2-μm membrane. The cytokine concentrations were determined by Luminex multiplex assay (30duPont N.C. Wang K. Wadhwa P.D. Culhane J.F. Nelson E.L. J. Reprod. Immunol. 2005; 66: 175-191Crossref PubMed Scopus (240) Google Scholar, 31de Jager W. te Velthuis H. Prakken B.J. Kuis W. Rijkers G.T. Clin. Diagn. Lab Immunol. 2003; 10: 133-139Crossref PubMed Scopus (498) Google Scholar) using commercial antibodies and recombinant cytokines as standards (R&D Systems, Minneapolis, MN). The bioactivity of every cytokine preparation was independently determined by an in-house bioassay in H-35 cells (32Baumann H. Prowse K.R. Marinkovic S. Won K.A. Jahreis G.P. Ann. N. Y. Acad. Sci. 1989; 557: 280-296Crossref PubMed Scopus (199) Google Scholar), which were stably transduced with human OSMRβ (5Wang Y. Robledo O. Kinzie E. Blanchard F. Richards C. Miyajima A. Baumann H. J. Biol. Chem. 2000; 275: 25273-25285Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and human IL-31Rα. These cells have no measurable production of endogenous IL-6 cytokines and permit the measurement of the bioactivity of all human IL-6 cytokines by the dose-dependent induction of type II acute phase proteins, such as fibrinogen and thiostatin (5Wang Y. Robledo O. Kinzie E. Blanchard F. Richards C. Miyajima A. Baumann H. J. Biol. Chem. 2000; 275: 25273-25285Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 33Fuller G.M. Otto J.M. Woloski B.M. McGary C.T. Adams M.A. J. Cell Biol. 1985; 101: 1481-1486Crossref PubMed Scopus (61) Google Scholar). Half-maximal expression of the plasma proteins defines one hepatocyte-stimulating factor unit and is obtained with ∼1 ng/ml of IL-6 cytokines. The accuracy of the bioassay was confirmed by using purified recombinant IL-6 cytokines (R&D Systems). Generally, the cytokine concentrations in the COS-1 media ranged from 10 to 50 μg/ml. From these stocks, an initial dilution to 100 ng/ml cytokines was prepared that has been demonstrated to produce in all cell types optimal signaling reactions and cell responses. These preparations, along with further 10-fold serial dilutions, were applied to the experimental cell cultures (see “Results”). Conditioned medium from COS-1 cells transfected with control vector served as background reference (contained <0.1 ng/ml IL-6 equivalent bioactivity). This medium was diluted like the cytokine stocks and was included in the experimental applications as a no-cytokine control. Receptor expression vectors along with the reporter gene constructs pCRP(219)CAT and pEGFP (Invitrogen) were transfected into Hep3B cells, and cytokine-regulated CAT activity was determined and normalized as described (5Wang Y. Robledo O. Kinzie E. Blanchard F. Richards C. Miyajima A. Baumann H. J. Biol. Chem. 2000; 275: 25273-25285Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). A549 cells were transduced with receptor-encoding retroviruses, cloned by limited dilution within 4 days following transduction, and selected in hygromycin (1 mg/ml). Positive clones were identified by the expression of a FLAG epitope. All selected clones (at least three independently derived clones per receptor construct) were subjected to a second round of subcloning. Homogeneity of the selected clonal lines was verified by immunostaining for the FLAG epitope. Cell Treatments—The cell culture conditions and duration of cytokine treatments depended on the type of analysis. Generally, cells were plated into 6-well or 24-well culture plates. When the cultures reached a density of 50–100% (100% = 2 × 105 cells/cm2 culture area), the cells were incubated first in serum-free medium for 2 h and then changed to medium containing cytokines (0–100 ng/ml). For determining receptor-mediated signaling, treatments were limited to 15 min; for measuring induction of genes, altered cell morphology, or change in cell cycle markers, the treatments ranged from 24 to 48 h. To determine cell proliferation, the cells were seeded at 1% density into 24-well culture plates. After 24 h, the medium was replaced by fresh, serum-containing medium that also included 10-fold serially diluted cytokines or dexamethasone (34Greenberg A.K. Hu J. Basu S. Hay J. Reibman J. Yie T.A. Tchou-Wong K.M. Rom W.N. Lee T.C. Am. J. Respir. Cell Mol. Biol. 2002; 27: 320-328Crossref PubMed Scopus (91) Google Scholar). After 3 days, the culture medium was replaced, and another 3 days later, the number of viable cells in each well was counted in a hematocytometer. Values from quadruplicate cultures were expressed as mean ± S.D. Incorporation of [3H]thymidine was determined as described previously (22Loewen G.M. Tracy E. Blanchard F. Tan D. Yu J. Raza S. Matsui S. Baumann H. BMC Cancer. 2005; 5: 145Crossref PubMed Scopus (32) Google Scholar). Morphology of the cell cultures was recorded by inverted phase microscopy at ×10 magnification (TE2000; Nikon, Melville, NY). Immunoblot Analysis—Cells were washed with phosphate-buffered saline and lysed within the culture plate (10 μl/1 × 105 cells) with RIPA buffer (50 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm NaF, 1 mm sodium orthovanadate, 1 μg of leupeptin, 1 μg of aprotinin/ml, 1 mm phenylmethylsulfonyl fluoride, 10% glycerol). Replicate aliquots of lysates containing 1–50 μg of proteins were separated on 6–10% SDS-polyacrylamide gels (Bio-Rad) with series of 15 samples per gel. Proteins were transferred to nitrocellulose membrane (Schleicher & Schuell), and equal loading and transfer were confirmed in all cases by staining with Ponceau S red. Subsections of the membrane containing the proteins of interest were reacted with antibodies. The following phospho-specific antibodies were used: phospho-STAT1-Tyr-701 (9171), phospho-STAT3-Tyr-705 (9131), phospho-STAT5-Tyr-694 (9351), phospho-p44/42 MAPK-Thr-202/Tyr-204 (phospho-ERK1/2; 9106), phospho-SAPK/JNK-Thr-183/Tyr-185 (9251), phospho-Akt-Ser-473 (9271), or phospho-Hsp27-Ser-82 (2401) (Cell Signaling Technology Inc., Beverly, MA). The antibodies against total proteins included the following: STAT3 (C-20), ERK (K-23), JUNB (C-11), p27Kip1 (C-19), p53 (DO-1), cyclin B1 (H-433), cyclin D1 (M-20), cyclin E (HE-12), CDK2 (M-2), CDK4 (sc-260), CDK6 (C21), CDC2 (sc-54), MCM2 (H-126), MCM4 (H-300), MCL-1 (S-19), and SOCS3 (M-20) from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody against FLAG (026K4848) was obtained from Sigma; antibody against Rb (554136) was from Pharmingen; antibody against human IL-31Rα was from R&D Systems; and antibody against human α1-antichymotrypsin, α1-antitrypsin, and fibrinogen were from Dako Immunoglobins, Glostrup, Denmark. The membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (ICN Biomedical, Aurora, OH), and the antibody binding was visualized by enhanced chemiluminescence reaction (Pierce). In each experimental series, immunoblots were exposed to x-ray films for various lengths of time (1 s to 2 h) to obtain images that are in the linear range of signal detection (22Loewen G.M. Tracy E. Blanchard F. Tan D. Yu J. Raza S. Matsui S. Baumann H. BMC Cancer. 2005; 5: 145Crossref PubMed Scopus (32) Google Scholar). For pictorial presentation of immunoblot data, the signals for either total STAT3 or ERK1/2 served as loading controls. Immunoblots were digitalized and quantified with ImageQuant TL software (Amersham Biosciences). The net pixel value for each protein band that lies within the linear range of detection was compared with co-analyzed control cultures. Immunostaining—Cells were fixed with 4% paraformaldehyde, permeabilized with phosphate-buffered saline containing 0.05% Triton X-100, and reacted with anti-FLAG and fluoresceinated secondary antibody and 4,6-diamidino-2-phenylindole. The subcellular distribution of the labels was recorded on a Leica TCS SP2 confocal fluorescence microscope (Leica Microsystems, Exton, PA). Human Lung Epithelial Cells Respond to IL-31—As reported previously (14Diveu C. Lelievr
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