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• W1994697836 abstract "Interleukin-13 (IL-13), a Th2 cytokine, plays a pivotal role in pathogenesis of bronchial asthma via IL-13 receptor α1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). Recent studies show that a decoy receptor for IL-13, namely IL-13Rα2, mitigates IL-13 signaling and function. This study provides evidence for regulation of IL-13Rα2 production and release and IL-13-dependent signaling by lysophosphatidic acid (LPA) in primary cultures of human bronchial epithelial cells (HBEpCs). LPA treatment of HBEpCs in at imedependent fashion increased IL-13Rα2 gene expression without altering the mRNA levels of IL-13Rα1 and IL-4Rα. Pretreatment with pertussis toxin (100 ng/ml, 4 h) or transfection of c-Jun small interference RNA or an inhibitor of JNK attenuated LPA-induced IL-13Rα2 gene expression and secretion of soluble IL-13Rα2. Overexpression of catalytically inactive mutants of phospholipase D (PLD) 1 or 2 attenuated LPA-induced IL-13Rα2 gene expression and protein secretion as well as phosphorylation of JNK. Pretreatment of HBEpCs with 1 μm LPA for 6 h attenuated IL-13-but not IL-4-induced phosphorylation of STAT6. Transfection of HBEpCs with IL-13Rα2 small interference RNA blocked the effect of LPA on IL-13-induced phosphorylation of STAT6. Furthermore, pretreatment with LPA (1 μm, 6 h) attenuated IL-13-induced eotaxin-1 and SOCS-1 gene expression. These results demonstrate that LPA induces IL-13Rα2 expression and release via PLD and JNK/AP-1 signal transduction and that pretreatment with LPA down-regulates IL-13 signaling in HBEpCs. Our data suggest a novel mechanism of regulation of IL-13Rα2 and IL-13 signaling that may be of physiological relevance to airway inflammation and remodeling. Interleukin-13 (IL-13), a Th2 cytokine, plays a pivotal role in pathogenesis of bronchial asthma via IL-13 receptor α1 (IL-13Rα1) and IL-4 receptor α (IL-4Rα). Recent studies show that a decoy receptor for IL-13, namely IL-13Rα2, mitigates IL-13 signaling and function. This study provides evidence for regulation of IL-13Rα2 production and release and IL-13-dependent signaling by lysophosphatidic acid (LPA) in primary cultures of human bronchial epithelial cells (HBEpCs). LPA treatment of HBEpCs in at imedependent fashion increased IL-13Rα2 gene expression without altering the mRNA levels of IL-13Rα1 and IL-4Rα. Pretreatment with pertussis toxin (100 ng/ml, 4 h) or transfection of c-Jun small interference RNA or an inhibitor of JNK attenuated LPA-induced IL-13Rα2 gene expression and secretion of soluble IL-13Rα2. Overexpression of catalytically inactive mutants of phospholipase D (PLD) 1 or 2 attenuated LPA-induced IL-13Rα2 gene expression and protein secretion as well as phosphorylation of JNK. Pretreatment of HBEpCs with 1 μm LPA for 6 h attenuated IL-13-but not IL-4-induced phosphorylation of STAT6. Transfection of HBEpCs with IL-13Rα2 small interference RNA blocked the effect of LPA on IL-13-induced phosphorylation of STAT6. Furthermore, pretreatment with LPA (1 μm, 6 h) attenuated IL-13-induced eotaxin-1 and SOCS-1 gene expression. These results demonstrate that LPA induces IL-13Rα2 expression and release via PLD and JNK/AP-1 signal transduction and that pretreatment with LPA down-regulates IL-13 signaling in HBEpCs. Our data suggest a novel mechanism of regulation of IL-13Rα2 and IL-13 signaling that may be of physiological relevance to airway inflammation and remodeling. The Th2-type cytokine, interleukin-13 (IL-13), 2The abbreviations used are: IL, interleukin; IL-13R, IL-13 receptor; LPA, lysophosphatidic acid; HBEpCs, human bronchial epithelial primary cells; STAT6, signal transducer and activator of transcription 6; PLD, phospholipase D; hPLD1, human phospholipase D1; mPLD2, mouse phospholipase D2; siRNA, small interference RNA; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; AP-1, activator protein 1; EGF-R, epidermal growth factor receptor; RT, reverse transcription. has been shown to play a critical role in the pathogenesis of bronchial asthma (1Wills-Karp M. Luyimbazi J. Xu X. Schofield B. Neben T.Y. Karp C.L. Donaldson D.D. Science. 1998; 282: 2258-2261Crossref PubMed Scopus (2399) Google Scholar, 2Grunig G. Warnock M. Wakil A.E. Venkayya R. Brombacher F. Rennick D.M. Sheppard D. Mohrs M. Donaldson D.D. Locksiey R.M. Corry D.B. Science. 1998; 282: 2261-2263Crossref PubMed Scopus (1737) Google Scholar, 3Zhu Z. Homer R.J. 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Immunol. 2003; 21: 425-456Crossref PubMed Scopus (789) Google Scholar). IL-13 levels are increased in bronchoalveolar lavage of asthma patients (9Huang S.K. Xiao H.Q. Klein-Tebbe J. Paciotti G. Marsh D.G. Lichtenstein L.M. Liu M.C. J. Immunol. 1995; 55: 2688-2694Google Scholar, 10Bodey K.J. Semper A.E. Redington A.E. Madden J. Teran L.M. Holgate S.T. Frew A.J. Allergy (Cph.). 1999; 54: 1083-1093Crossref PubMed Scopus (103) Google Scholar) and ovalbumin-challenged mice (11Henderson Jr., W.R. Tang L.O. Chu S.J. Tsao S.-M. Chiang G.K. Jones F. Jonas M. Pae C. Wang H. Chi E.Y. Am. J. Respir. Crit. Care Med. 2002; 165: 108-116Crossref PubMed Scopus (407) Google Scholar). Experiments using IL-13 gene knock-out mice have shown that IL-13 regulates airway hyper-responsiveness and mucus hypersecretion (12Webb D.C. McKenzie A.N. Koskinen A.M. Yang M. Mattes J. Foster P.S. J. Immunol. 2000; 165: 108-113Crossref PubMed Scopus (288) Google Scholar). The biological activities of IL-13 are mediated through the IL-13 receptor α1(IL-13Rα1)·IL-4Rα heterodimer complex expressed on the surface of target cells (13Malabarba M.G. Rui H. Deutsch H.H. Chung J. Kalthoff F.S. Farrar W.L. Kirken R.A. Biochem. J. 1996; 319: 865-872Crossref PubMed Scopus (52) Google Scholar, 14Zurawski S.M. Chomarat P. Djossou O. Bidaud C. McKenzie A.N. Miossec P. Banchereau J. Zurawski G. J. Biol. Chem. 1995; 270: 13869-13878Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 15Kotowicz K. Callard R.E. Friedrich K. Matthews D.J. Klein N. Int. Immunol. 1996; 8: 1915-1925Crossref PubMed Scopus (74) Google Scholar, 16Miloux B. Laurent P. Bonnin O. Lupker J. Caput D. Vita N. Perrara P. FEBS Lett. 1997; 401: 163-166Crossref PubMed Scopus (192) Google Scholar). IL-13Rα1 binds to IL-13 at low affinity, whereas heterodimerization of the IL-13Rα1·IL-4Rα generates high-affinity receptor to IL-13, mediating IL-13-induced activation of Janus kinases and STAT6 (14Zurawski S.M. Chomarat P. Djossou O. Bidaud C. McKenzie A.N. Miossec P. Banchereau J. Zurawski G. J. Biol. Chem. 1995; 270: 13869-13878Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 15Kotowicz K. Callard R.E. Friedrich K. Matthews D.J. Klein N. Int. Immunol. 1996; 8: 1915-1925Crossref PubMed Scopus (74) Google Scholar, 16Miloux B. Laurent P. Bonnin O. Lupker J. Caput D. Vita N. Perrara P. FEBS Lett. 1997; 401: 163-166Crossref PubMed Scopus (192) Google Scholar). The human IL-13 decoy receptor, termed IL-13Rα2, has been cloned and shares ∼33% homology and ∼21% identity with human IL-13Rα1 (17Zhang J.G. Hilton D.J. Willson T.A. McFarlane C. Roberts B.A. Mortiz R.L. Simpson R.J. Alexander W.S. Metcalf D. Nicola N.A. J. Biol. Chem. 1997; 272: 9474-9480Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). IL-13Rα2 binds to IL-13 with much higher affinity (Kd = 0.25–1.2 nm), compared with IL-13Rα1 (Kd = 2–10 nm) (17Zhang J.G. Hilton D.J. Willson T.A. McFarlane C. Roberts B.A. Mortiz R.L. Simpson R.J. Alexander W.S. Metcalf D. Nicola N.A. J. Biol. Chem. 1997; 272: 9474-9480Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 18Donaldson D.D. Whitters M.J. Fitz L.J. Neben T.Y. Finnerty H. Henderson S.L. O'Hara Jr., R.M. Beier D.R. Turner K.J. Wood C.R. Collins M. J. Immunol. 1998; 161: 2317-2324PubMed Google Scholar, 19Kawakami K. Taguchi J. Murata T. Puri R.K. Blood. 2001; 97: 2673-2679Crossref PubMed Scopus (211) Google Scholar). Overexpression of IL-13Rα2 in nonexpressing heterologous cells inhibited IL-4- and IL-13-induced signal transduction in glioblastoma cells (20Rahaman S.O. Sharma P. Harbor P.C. Aman M.J. Vogelbaum M.A. Haque S.J. Cancer Res. 2002; 62: 1103-1109PubMed Google Scholar), whereas knock-out of IL-13Rα2 increased IL-13 function in vivo (21Wood N. Whitters M.J. Jacobson B.A. Witek J. Sypek J.P. Kasaian M. Eppihimer M.J. Unger M. Tanaka T. Goldman S.J. Collins M. Donaldson D.D. Grusby M.J. J. Exp. Med. 2003; 197: 703-709Crossref PubMed Scopus (179) Google Scholar). The effect of IL-13 inhibition by IL-13Rα2 has been attributed to: 1) its high affinity for IL-13 and preventing IL-13 from binding the IL-13Rα1/IL-4Rα complex on cell surface; and 2) its short cytoplasmic tail, which has no signaling motif (17Zhang J.G. Hilton D.J. Willson T.A. McFarlane C. Roberts B.A. Mortiz R.L. Simpson R.J. Alexander W.S. Metcalf D. Nicola N.A. J. Biol. Chem. 1997; 272: 9474-9480Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 18Donaldson D.D. Whitters M.J. Fitz L.J. Neben T.Y. Finnerty H. Henderson S.L. O'Hara Jr., R.M. Beier D.R. Turner K.J. Wood C.R. Collins M. J. Immunol. 1998; 161: 2317-2324PubMed Google Scholar, 20Rahaman S.O. Sharma P. Harbor P.C. Aman M.J. Vogelbaum M.A. Haque S.J. Cancer Res. 2002; 62: 1103-1109PubMed Google Scholar). In addition to the role of IL-13Rα2 in adaptive immunity, the role of IL-13Rα2 in cancer therapy has recently been demonstrated (22Kawakami K. Husain S.R. Bright R.K. Puri R.K. Cancer Gene Ther. 2001; 8: 861-868Crossref PubMed Scopus (36) Google Scholar, 23Kawakami K. Kawakami M. Snoy P.J. Husain S.R. Puri R.K. J. Exp. Med. 2001; 194: 1743-1754Crossref PubMed Scopus (75) Google Scholar, 24Kawakami K. Terabe M. Kawakami M. Berzofsky J.A. Puri R.K. Cancer Res. 2006; 66: 4434-4442Crossref PubMed Scopus (27) Google Scholar). Overexpression of IL-13Rα2 increased the sensitivity and antitumor activity of IL-13-PE38QQR toxin in cancer cells (22Kawakami K. Husain S.R. Bright R.K. Puri R.K. Cancer Gene Ther. 2001; 8: 861-868Crossref PubMed Scopus (36) Google Scholar, 23Kawakami K. Kawakami M. Snoy P.J. Husain S.R. Puri R.K. J. Exp. Med. 2001; 194: 1743-1754Crossref PubMed Scopus (75) Google Scholar, 24Kawakami K. Terabe M. Kawakami M. Berzofsky J.A. Puri R.K. Cancer Res. 2006; 66: 4434-4442Crossref PubMed Scopus (27) Google Scholar). The IL-13Rα2 promoter has been characterized, and several putative transcriptional factor binding sites for nuclear factor of activated T cells-1, AP-1 (c-Jun and c-Fos), AP2, GABP, OCT1, GATA3, PRE, and C-ETS1 were predicted in the promoter region (25Wu A.H. Low W.C. Neuro-Oncology. 2003; 5: 179-187Crossref PubMed Scopus (23) Google Scholar). Elevated IL-13Rα2 mRNA expression has been detected in Schistosoma egg-induced liver fibrosis (26Chiaramonte M.G. Mentink-Kane M. Jacobson B.A. Cheever A.W. Whitters M.J. Goad M.E. Wong A. Collins M. Donaldson D.D. Grusby M.J. Wynn T.A. J. Exp. Med. 2003; 197: 687-701Crossref PubMed Scopus (238) Google Scholar). In addition, interferon-γ or IL-13 treatment induced IL-13Rα2 mRNA expression in U937 (27Daines M.O. Hershey G.K. J. Biol. Chem. 2002; 277: 10387-10393Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) and bronchial epithelial cells (28Heller N.M. Matsukura S. Georas S.N. Boothby M.R. Rothman P.B. Stellato C. Schleimer R.P. Am. J. Respir. Cell Mol. Biol. 2004; 31: 573-582Crossref PubMed Scopus (59) Google Scholar, 29Yasunaga S. Yuyama N. Arima K. Tanaka H. Toda S. Maeda M. Matsi K. Goda C. Yang Q. Sugita Y. Nagai H. Izuhara K. Cytokine. 2003; 24: 293-303Crossref PubMed Scopus (62) Google Scholar). However, the molecular mechanisms that regulate IL-13Rα2 gene expression and production are poorly understood. Lysophosphatidic acid (LPA), a bioactive phospholipid, induces variety of biological activities on different cell types through G protein-coupled receptors (LPA1–3) (30Chun J. Contos J.J. Munroe D. Cell Biochem. Biophys. 1999; 30: 213-242Crossref PubMed Scopus (140) Google Scholar, 31Fukushima N. Chun J. 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Chem. 2006; 281: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). LPA exists in various biological fluids, including serum and bronchoalveolar lavage fluid (38Georas S.N. Berdyshev E. Hubbard W. Gorshkova I.A. Usatyuk P.V. Saatian B. Myers A.C. Williams M.A. Xiao H.Q. Liu M. Natarajan V. Clin. Exp. Allergy. 2007; (in press)PubMed Google Scholar, 39Siess W. Zangl K.J. Essler M. Bauer M. Brandl R. Corrinth C. Bittman R. Tigyi G. Aepfelbacher M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6931-6936Crossref PubMed Scopus (374) Google Scholar). Extracellular LPA is produced by the action of both secretory phospholipase A2 (40Budnik L.T. Mukhopadhyay A.K. Biol. Reprod. 2002; 66: 859-865Crossref PubMed Scopus (45) Google Scholar) and lysophospholipase D (also named autotoxin) (41Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (803) Google Scholar) by using phosphatidic acid or lysophosphatidylcholine as substrate, respectively. The role of LPA in regulating expression of Th1 and Th2 type cytokines has been studied in bronchial epithelial cells and T cells. We previously demonstrated that treatment of HBEpCs with LPA induces IL-8 expression and secretion via LPA receptors regulated by changes in intracellular calcium and protein kinase C δ, p38 MAPK-dependent transcriptional activation of NF-κB, and JNK-dependent activation of AP-1 (42Cummings R. Zhao Y. Jacoby D. Spannhake E.W. Ohba M. Garcia J.G. Watkins T. He D. Saatian B. Natarajan V. J. Biol. Chem. 2004; 279: 41085-41094Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 43Zhao Y. Usatyuk P.V. Cummings R. Saatian B. He D. Watkins T. Morris A. Spannhake E.W. Brindley D.N. Natarajan V. Biochem. J. 2005; 385: 493-502Crossref PubMed Scopus (62) Google Scholar, 44Saatian B. Zhao Y. He D. Georas S.N. Watkins T. Spannhake E.W. Natarajan V. Biochem. J. 2006; 393: 657-668Crossref PubMed Scopus (94) Google Scholar). In addition, LPA can activate cells via epidermal growth factor receptor (EGF-R) transactivation in HBEpCs (45Zhao Y. He D. Saatian B. Watkins T. Spannhake E.W. Pyne N.J. Natarajan V. J. Biol. Chem. 2006; 281: 19501-19511Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). IL-8 is a potent chemoattractant for neutrophils and plays a pivotal role in innate immunity (46Koch A.E. Polverini P.J. Kunkel E.J. Harlow L.A. DiPietro L.A. Elner V.M. Elner S.G. Strieter R.M. Science. 1992; 258: 1798-1801Crossref PubMed Scopus (1910) Google Scholar, 47Car B.D. Meloni F. Luisetti M. Semenzato G. Gialdroni-Grassi G. Walz A. Am. J. Respir. Crit. Care Med. 1994; 149: 655-659Crossref PubMed Scopus (256) Google Scholar, 48Folkard S.G. Westwick J. Millar A.B. Eur. Respir. J. 1997; 10: 2097-2104Crossref PubMed Scopus (70) Google Scholar). In T-cells, LPA enhanced IL-13 gene expression under conditions of submaximal activation (49Rubenfeld J. Guo J. Sookrung N. Chen R. Chaicumpa W. Casolaro V. Zhao Y. Natarajan V. Georas S. Am. J. Physiol. 2006; 290: L66-L74Crossref PubMed Scopus (43) Google Scholar). Taken together, these studies suggest that LPA plays a critical role in airway inflammation by regulating immunomodulation of bronchial and alveolar epithelial cells However, mechanisms of LPA-dependent modulation of Th2 type cytokine-induced signaling are unclear. To further determine the role of LPA in airway inflammation, we report here that LPA induces IL-13Rα2 mRNA expression through phospholipase D (PLD)-dependent JNK/AP-1 signaling in HBEpCs. This is the first report on LPA induced IL-13Rα2 gene expression and production in HBEpCs. Further, we found that elevated IL-13Rα2 expression attenuated IL-13-mediated phosphorylation of STAT6 and eotaxin and SOCS-1 gene expression in HBEpCs. These results demonstrate a novel role for LPA in attenuating Th2 cytokine signaling in airway inflammation. Materials—1-Oleoyl (18:1) LPA, 1-butanol, 3-butanol, and dimethyl sulfoxide were purchased from Sigma. Antibodies to phospho-STAT6, STAT6, RelA, c-Jun, phospho-JNK, and JNK1 were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies to human phosphatidylcholine-specific PLD1 and mouse PLD2 were purchased from BIOSOURCE International (Camarillo, CA). Antibodies to IL-13Rα2 and recombinant IL-13 were procured from Cell Signaling Technology (Beverly, MA). Scrambled and IL-13Rα2 small interfering RNA (siRNA) were from Dharmacon Inc. (Lafayette, CO). Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary antibodies were purchased from Molecular Probes (Eugene, OR). An ECL kit for detection of proteins by Western blotting was obtained from Amersham Biosciences. Real-time PCR reagents were from Bio-Rad Laboratories. Transfection reagents were from Qiagen (Valencia, CA). Bronchial epithelial cell basal medium and a supplement kit were purchased from Cambrex Bio Science Inc. (Walkersville, MD). A Millipore™ 10 kit was purchased from Millipore (Bedford, MA). All other reagents were of analytical grade. Cell Culture—HBEpCs were isolated from normal human lung obtained from lung transplant donors following previously described procedures (50Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. Davis C.W. Randell S.H. Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Crossref PubMed Scopus (173) Google Scholar, 51Wang L. Cummings R. Zhao Y. Kazlauskas A. Sham J.K. Morris A. Georas S. Brindley D.N. Natarajan V. J. Biol. Chem. 2003; 278: 39931-39940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The isolated passage 0 (P0) HBEpCs were cultured in serum-free basal essential growth medium supplemented with growth factors. Cells were incubated at 37 °C in 5% CO2 and 95% air to ∼80% confluence and subsequently were propagated in 35-mm or 6-well collagen-coated dishes. All experiments were carried out between passages 1 and 4. Preparation of Cell Lysates, Media, and Western Blotting—After the indicated treatments, media were collected and centrifuged at 5,000 × g for 10 min, and supernatants were concentrated by Millipore™ 10 kit according to manufacturer's instruction. Cells were rinsed twice with ice-cold phosphate-buffered saline and lysed in 200 μl of buffer containing 20 mm Tris-HCl (pH 7.4), 150 mm NaCl, 2 mm EGTA, 5 mm β-glycerophosphate, 1 mm MgCl2, 1% Triton X-100, 1 mm sodium orthovanadate, 10 μg/ml protease inhibitors, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin. Cell lysates were incubated at 4 °C for 10 min, sonicated on ice for 10 s, and centrifuged at 5000 × g for 5 min at 4 °C in a microcentrifuge. Protein concentrations were determined with a BCA protein assay kit (Pierce Chemical Co.) using bovine serum albumin as standard. Equal amounts of protein (20 μg) or concentrated media (20 μl) were subjected to 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, blocked with 5% (w/v) bovine serum albumin in TBST (25 mm Tris-HCl, pH 7.4, 137 mm NaCl, and 0.1% Tween 20) for 1 h, and incubated with primary antibodies in 5% (w/v) bovine serum albumin in TBST for 1–2 h at room temperature. The membranes were washed at least three times with TBST at 15-min intervals and then incubated with mouse, rabbit, or goat horseradish peroxidase-conjugated secondary antibody (1: 3,000) for 1 h at room temperature. The membranes were developed with an enhanced chemiluminescence detection system according to the manufacturer's instructions. RNA Isolation—Total RNA was isolated from cultured HBEpCs using TRIzol® reagent (Invitrogen) according to the manufacturer's instructions. RNA was quantified spectrophotometrically, and samples with an absorbance of ≥1.8 measured at 260/280 nm were analyzed by real-time RT-PCR. Real-time RT-PCR and Reverse Transcription-PCR—1 μgof RNA was reverse-transcribed using a cDNA synthesis kit (Bio-Rad), and real-time PCR and regular PCR were performed to assess expression of the IL-13Rα1, IL-13Rα2, IL-4Rα, eotaxin-1, and SOCS-1 genes using primers designed on the basis of human mRNA sequences (Table 1). For real-time RT-PCR, amplicon expression in each sample was normalized to its 18 S RNA content. The relative abundance of target mRNA in each sample was calculated as 2 raised to the negative of its threshold cycle value times 106 after being normalized to the abundance of its corresponding 18 S, e.g. 2–(IL-13Rα2 threshold cycle)/2–(18 S threshold cycle) × 106.TABLE 1Primers for RT-PCR and real-time RT-PCRPrimers for RT-PCR IL-13Rα1Forward,5′-GGAGAATACATCTTGTTTCATGG-3′Reverse,5′-GCGCTTCTTACCTATACTCATTTCTTGG-3′ IL-13Rα2Forward,5′-AATGGCTTTCGTTTGCTTGG-3′Reverse,5′-ACGCAATCCATATCCTGAAC-3′ IL-4RαForward,5′-GACCTGGAGCAACCCGTATC-3′Reverse,5′-CATAGCACAACAGGCAGACG-3′ GAPDHaGlyceraldehyde-3-phosphate dehydrogenase.Forward,5′-GGAGCCAAAAGGGTCATCATCT-3′Reverse,5′-AGTGGGTGTCGCTGTTGAAGT-3′Primers for real-time RT-PCR IL-13Rα2Forward,5′-ACTGGTATGAGGGCTTGGAT-3′Reverse,5′-TCTGATGCCTCCAAATAGGG-3′ SOCS-1Forward,5′-TTGGAGGGAGCGGATGGGTGTAG-3′Reverse,5′-AGAGGTAGGAGGTGCGAGTTCAGGTC-3′ Eotaxin-1Forward,5′-AACCACCTGCTGCTTTAACC-3′Reverse,5′-CACAGCTTTCTGGGGACATT-3′ 18SForward,5′-GTAACCCGTTGAACCCCATT-3′Reverse,5′-CCATCCAATCGGTAGTAGCG-3′a Glyceraldehyde-3-phosphate dehydrogenase. Open table in a new tab Infection with Adenoviral Constructs—Infection of HBEpCs (∼60% confluence) with purified adenoviral vectors of catalytically inactive mutants of human PLD1 (hPLD1) and mouse PLD2 (mPLD2) were carried out in 6-well plates as described previously (51Wang L. Cummings R. Zhao Y. Kazlauskas A. Sham J.K. Morris A. Georas S. Brindley D.N. Natarajan V. J. Biol. Chem. 2003; 278: 39931-39940Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Cells were infected with different multiplicities of infection in 1 ml of serum-free basal essential growth medium for 48 h. Transfection of siRNA for IL-13Rα2—HBEpCs (P1) cultured onto 6-well plates (40–50% confluence) were transiently transfected with SMARTpool RNA duplexes corresponding to human IL-13Rα2 and scrambled siRNA using Transmessenger transfection reagent. Briefly, IL-13Rα2 siRNA or scrambled siRNA (100 nm) was condensed with enhancer R and formulated with Transmessenger reagent according to the manufacturer's instructions. The transfection complex was diluted into 900 μl of bronchial epithelial cell basal medium and added directly to the cells. The medium was replaced with bronchial epithelial cell basal medium after 3 h, and cells were used for experiments at 72 h after transfection. Statistical Analyses—All results were subjected to statistical analysis using one-way analysis of variance and, whenever appropriate, were analyzed using the Student-Newman-Keuls test. Data are expressed as means ± S.D. of triplicate samples from at least three independent experiments, and the level of significance was taken to p < 0.05. LPA Induces IL-13Rα2 Gene Expression and Secretion through Gαi in HBEpCs—Using a DNA microarray, we found that LPA treatment of HBEpCs induced IL-13Rα2 gene expression after 24 h. 3L. Wang and V. Natarajan, unpublished data. To confirm this result, we examined the changes in IL-13Rα1, IL-13Rα2, and IL-4Rα gene levels with or without LPA treatment by RT-PCR and related real-time RT-PCR. As shown in Fig. 1, A and B, compared with IL-13Rα1 and IL-4Rα gene levels, the IL-13Rα2 mRNA level is very low in HBEpCs. The increase of IL-13Rα2 gene expression was detected at 6 h (∼3-fold) after LPA (1 μm) treatment, reaching ∼7-fold after 24 h (Fig. 1B), whereas LPA (1 μm) treatment had no effect on IL-13Rα1 and IL-4Rα gene expression (Fig. 1A). These results show that only IL-13Rα2, not IL-13Rα1 or IL-4Rα, is inducible by LPA treatment in HBEpCs. Similar to LPA treatment, IL-13 (10 ng/ml, 6 h) and IL-4 (10 ng/ml, 6 h) also increased IL-13Rα2 gene expression (Fig. 1B). To further confirm up-regulation of IL-13Rα2 by LPA, we next investigated IL-13Rα2 secretion after LPA treatment in HBEpCs. Using an anti-IL-13Rα2 extracellular domain antibody, we monitored soluble IL-13Rα2 protein levels in media after LPA (1 μm) treatment and observed a peak at 6 h (Fig. 1C). A 6-h incubation with LPA induced IL-13Rα2 secretion in a dose-dependent fashion (Fig. 1D). Pretreatment of HBEpCs with pertussis toxin (100 ng/ml) for 4 h attenuated LPA-induced IL-13Rα2 gene expression and soluble IL-13Rα2 secretion (Fig. 1, E and F). These results suggest that LPA-induced IL-13Rα2 gene expression and secretion is through Gi-coupled LPA receptors on the cell surface in HBEpCs. Role of JNK/AP-1 Signaling in LPA-induced IL-13Rα2 Gene Expression and Secretion—Our previous studies (41Umezu-Goto M. Kishi Y. Taira A. Hama K. Dohmae N. Takio K. Yamori T. Mills G.B. Inoue K. Aoki J. Arai H. J. Cell Biol. 2002; 158: 227-233Crossref PubMed Scopus (803) Google Scholar, 42Cummings R. Zhao Y. Jacoby D. Spannhake E.W. Ohba M. Garcia J.G. Watkins T. He D. Saatian B. Natarajan V. J. Biol. Chem. 2004; 279: 41085-41094Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 43Zhao Y. Usatyuk P.V. Cummings R. Saatian B. He D. Watkins T. Morris A. Spannhake E.W. Brindley D.N. Natarajan V. Biochem. J. 2005; 385: 493-502Crossref PubMed Scopus (62) Google Scholar) have shown that NF-κB and AP-1 transcriptional factors were involved in LPA-induced IL-8 secretion in HBEpCs. To determine the mechanisms of regulation of IL-13Rα2 gene expression and secretion by LPA, we inhibited NF-κB and AP-1 activities by transfection of siRNA or pharmacological inhibitors prior to LPA challenge. Transfection of NF-κB subunit RelA siRNA or pretreatment with the I-κB kinase inhibitor Bay11-7082 (5 and 10 μm) did not affect LPA-induced IL-13Rα2 gene expression and protein release (Fig. 2, A and C). In contrast, down-regulation of c-Jun protein expression (Fig. 2B) by transfection of c-Jun siRNA attenuated LPA-induced IL-13Rα2 secretion in HBEpCs (Fig. 2A). Further, pretreatment with the JNK inhibitor JNKiII for 1 h significantly attenuated LPA-mediated IL-13Rα2 mRNA expression (Fig. 2D). Fig. 2B shows that the transfection of RelA siRNA or c-Jun siRNA down-regulated expression of the respective transcription factors in HBEpCs as determined by Western blotting with specific antibodies to RelA or c-Jun. These results suggest that regulation of IL-13Rα2 gene expression and secretion by LPA is at least partly dependent on JNK/AP-1 signal transduction. Role of PLD in LPA-induced IL-13Rα2 Gene Expression and Secretion—Our previous studies have shown that LPA stimulates PLD1 and -2 in HBEpCs (50Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. Davis C.W. Randell S.H. Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Crossref PubMed Scopus (173) Google Scholar). Furthermore, LPA-induced PLD activation could be blocked by overexpression of hPLD1 or mPLD2 catalytically inactive mutants, or by pretreatment with 1-butanol but not 3-butanol (50Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. Davis C.W. Randell S.H. Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Crossref PubMed Scopus (173) Google Scholar). Therefore, we next investigated the role of PLD activation in LPA-induced IL-13Rα2 release. LPA-induced IL-13Rα2 release was attenuated by pretreatment with 1-butanol (0.5%, 15 min) not 3-butanol (0.5%, 15 min; Fig. 3A), suggesting the involvement of PLD. To confirm these data, inhibition of PLD activity by overexpression of hPLD1 or mPLD2 mutants was performed by transfection of adenoviral vectors encoding hPLD1 or mPLD2 mutants (50Bernacki S.H. Nelson A.L. Abdullah L. Sheehan J.K. Harris A. Davis C.W. Randell S.H. Am. J. Respir. Cell Mol. Biol. 1999; 20: 595-604Crossref PubMed Scopus (173) Google Scholar). Consistent with the data using 1-butanol, both hPLD1 and mPLD2 mutants attenuated LPA-induced IL-13Rα2 gene expression and secretion (Fig. 3, B and D). Fig. 3C confirms overexpression of hPLD1 and mPLD2 mutants by Western blotting with specific antibodies to PLD1 or PLD2. As JNK/AP-1 regulated LPA-mediated I" @default.
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