A Novel Signaling Pathway
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.010249
ISSN1083-351X
AutoresGuoqiang Zhang, Kelly Kernan, Alison Thomas, Sarah Collins, Yumei Song, Ling Li, Weizhong Zhu, Renee Leboeuf, Allison A. Eddy,
Tópico(s)Renin-Angiotensin System Studies
ResumoThe nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRα1 during fibrogenesis. Silencing of the nAChRα1 gene led to significantly fewer interstitial αSMA+ myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRα1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRα1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. 35S-Labeled uPA bound to nAChRα1 with a Kd of 1.6 × 10−8 m, which was displaced by the specific nAChRα1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [3H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRα1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRα1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease. The nicotinic acetylcholine receptor α1 (nAChRα1) was investigated as a potential fibrogenic molecule in the kidney, given reports that it may be an alternative urokinase (urokinase plasminogen activator; uPA) receptor in addition to the classical receptor uPAR. In a mouse obstructive uropathy model of chronic kidney disease, interstitial fibroblasts were identified as the primary cell type that bears nAChRα1 during fibrogenesis. Silencing of the nAChRα1 gene led to significantly fewer interstitial αSMA+ myofibroblasts (2.8 times decreased), reduced interstitial cell proliferation (2.6 times decreased), better tubular cell preservation (E-cadherin 14 times increased), and reduced fibrosis severity (24% decrease in total collagen). The myofibroblast-inhibiting effect of nAChRα1 silencing in uPA-sufficient mice disappeared in uPA-null mice, suggesting that a uPA-dependent fibroblastic nAChRα1 pathway promotes renal fibrosis. To further establish this possible ligand-receptor relationship and to identify downstream signaling pathways, in vitro studies were performed using primary cultures of renal fibroblasts. 35S-Labeled uPA bound to nAChRα1 with a Kd of 1.6 × 10−8 m, which was displaced by the specific nAChRα1 inhibitor d-tubocurarine in a dose-dependent manner. Pre-exposure of uPA to the fibroblasts inhibited [3H]nicotine binding. The uPA binding induced a cellular calcium influx and an inward membrane current that was entirely prevented by d-tubocurarine preincubation or nAChRα1 silencing. By mass spectrometry phosphoproteome analyses, uPA stimulation phosphorylated nAChRα1 and a complex of signaling proteins, including calcium-binding proteins, cytoskeletal proteins, and a nucleoprotein. This signaling pathway appears to regulate the expression of a group of genes that transform renal fibroblasts into more active myofibroblasts characterized by enhanced proliferation and contractility. This new fibrosis-promoting pathway may also be relevant to disorders that extend beyond chronic kidney disease. Urokinase was first isolated from human urine in 1955 and identified as an activator of plasminogen (urokinase plasminogen activator (uPA) 2The abbreviations used are: uPAurokinase plasminogen activatoruPARuPA receptornAChRnicotinic acetylcholine receptorUUOunilateral ureteral obstructionαSMAα-smooth muscle actinIPimmunoprecipitationPCNAproliferating cell nuclear antigenATFamino terminal uPA fragmentANOVAanalysis of varianceαBTxα-bungarotoxind-TCd-tubocurarineneoNneonicotinesiRNAsmall interfering RNAWTwild typeDMEMDulbecco's modified Eagle's mediumIBimmunoblotBrdUrdbromodeoxyuridineSFMserum-free mediumIHCimmunohistochemicalAChacetylcholine. 2The abbreviations used are: uPAurokinase plasminogen activatoruPARuPA receptornAChRnicotinic acetylcholine receptorUUOunilateral ureteral obstructionαSMAα-smooth muscle actinIPimmunoprecipitationPCNAproliferating cell nuclear antigenATFamino terminal uPA fragmentANOVAanalysis of varianceαBTxα-bungarotoxind-TCd-tubocurarineneoNneonicotinesiRNAsmall interfering RNAWTwild typeDMEMDulbecco's modified Eagle's mediumIBimmunoblotBrdUrdbromodeoxyuridineSFMserum-free mediumIHCimmunohistochemicalAChacetylcholine.) (1Astrup T. Sterndorff I. Scand. J. Clin. Lab. Invest. 1955; 7: 239-245Crossref PubMed Scopus (10) Google Scholar). This serine protease is abundantly produced by kidney tubular cells and secreted across the apical membrane into the urinary space. Other cellular sources include monocytes/macrophages, fibroblasts, and myofibroblasts (2Zhang G. Eddy A.A. Front. Biosci. 2008; 13: 5462-5478Crossref PubMed Scopus (31) Google Scholar). Despite high uPA levels, its primary physiological function in the kidney remains unknown. Suggested roles have been an inhibitor of kidney stone formation and urinary tract infections due to its proteolytic activity and endogenous antibiotic function, respectively (3du Toit P.J. Van Aswegen C.H. Steinmann C.M. Klue L. Du Plessis D.J. Med. Hypotheses. 1997; 49: 57-59Crossref PubMed Scopus (12) Google Scholar, 4Jin T. Bokarewa M. Tarkowski A. J. Infect. Dis. 2005; 192: 429-437Crossref PubMed Scopus (22) Google Scholar). Increased uPA activity has been reported in several pathological conditions, such as chronic kidney disease (2Zhang G. Eddy A.A. Front. Biosci. 2008; 13: 5462-5478Crossref PubMed Scopus (31) Google Scholar), atherosclerosis, and malignant tumors (5Steins M.B. Padró T. Schwaenen C. Ruiz S. Mesters R.M. Berdel W.E. Kienast J. Blood Coagul. Fibrinolysis. 2004; 15: 383-391Crossref PubMed Scopus (50) Google Scholar, 6Killeen S. Hennessey A. El Hassan Y. Waldron B. Drug News Perspect. 2008; 21: 107-116Crossref PubMed Scopus (11) Google Scholar). Endogenous plasma uPA levels may be elevated 2–4-fold in patients with chronic kidney disease due to increased uPA released from damaged kidneys (7Brommer E.J. Van den Wall Bake A.W. Dooijewaard G. van Loon B.J. Emeis J.J. Weening J.J. Thromb. Haemost. 1994; 71: 19-25Crossref PubMed Scopus (3) Google Scholar, 8Pawlak K. Pawlak D. Mysliwiec M. Thromb. Res. 2007; 120: 871-876Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). urokinase plasminogen activator uPA receptor nicotinic acetylcholine receptor unilateral ureteral obstruction α-smooth muscle actin immunoprecipitation proliferating cell nuclear antigen amino terminal uPA fragment analysis of variance α-bungarotoxin d-tubocurarine neonicotine small interfering RNA wild type Dulbecco's modified Eagle's medium immunoblot bromodeoxyuridine serum-free medium immunohistochemical acetylcholine. urokinase plasminogen activator uPA receptor nicotinic acetylcholine receptor unilateral ureteral obstruction α-smooth muscle actin immunoprecipitation proliferating cell nuclear antigen amino terminal uPA fragment analysis of variance α-bungarotoxin d-tubocurarine neonicotine small interfering RNA wild type Dulbecco's modified Eagle's medium immunoblot bromodeoxyuridine serum-free medium immunohistochemical acetylcholine. Since its identification as a mediator of fibrin/fibrinogen degradation, uPA has been used in clinical settings as a fibrinolytic agent. The classical cellular urokinase receptor (uPAR) was first discovered on the surface of monocytes in 1985. Since then, a diverse array of biological functions triggered by uPA-uPAR interactions has been elucidated and shown to have important effects on cellular behavior during embryogenesis, angiogenesis, wound healing, and metastases (9Vassalli J.D. Baccino D. Belin D. J. Cell Biol. 1985; 100: 86-92Crossref PubMed Scopus (590) Google Scholar, 10Mondino A. Blasi F. Trends Immunol. 2004; 25: 450-455Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 11Annecke K. Schmitt M. Euler U. Zerm M. Paepke D. Paepke S. von Minckwitz G. Thomssen C. Harbeck N. Adv. Clin. Chem. 2008; 45: 31-45Crossref PubMed Scopus (92) Google Scholar). The specific role of uPA in fibrotic disorders appears to be organ-specific. uPA deficiency worsened bleomycin-induced lung fibrosis and reduced fibrosis in hearts damaged by viral myocarditis or left ventricular pressure overload, whereas there was no net effect on the severity of renal fibrosis induced by ureteral obstruction (UUO), although uPAR deficiency worsened fibrosis in that model (2Zhang G. Eddy A.A. Front. Biosci. 2008; 13: 5462-5478Crossref PubMed Scopus (31) Google Scholar). Despite its association with a broad repertoire of activities, the uPA mechanism of action remains incompletely understood. In particular, there is accumulating evidence that uPA may have protease- and uPAR-independent cellular effects. For example, macrophage uPA overexpression causes cardiac inflammation and fibrosis. Of particular note, this effect is independent of the classic uPA receptor uPAR and can be abrogated using a calcium channel blocker (12Stempien-Otero A. Plawman A. Meznarich J. Dyamenahalli T. Otsuka G. Dichek D.A. J. Biol. Chem. 2006; 281: 15345-15351Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Recent evidence suggests that additional uPA receptor(s) may exist (13Koopman J.L. Slomp J. de Bart A.C. Quax P.H. Verheijen J.H. J. Biol. Chem. 1998; 273: 33267-33272Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 14Mukhina S. Stepanova V. Traktouev D. Poliakov A. Beabealashvilly R. Gursky Y. Minashkin M. Shevelev A. Tkachuk V. J. Biol. Chem. 2000; 275: 16450-16458Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 15Poliakov A.A. Mukhina S.A. Traktouev D.O. Bibilashvily R.S. Gursky Y.G. Minashkin M.M. Stepanova V.V. Tkachuk V.A. J. Recept. Signal Transduct. Res. 1999; 19: 939-951Crossref PubMed Scopus (26) Google Scholar). We reported that urokinase initiates renal fibroblast signaling via the MAPK/ERK pathway (16Zhang G. Cai X. López-Guisa J.M. Collins S.J. Eddy A.A. J. Am. Soc. Nephrol. 2004; 15: 2090-2102Crossref PubMed Scopus (14) Google Scholar). This response appears to be mediated, at least in part, by an alternative urokinase receptor, since uPA can initiate mitogenesis in uPAR−/− fibroblasts. Using phage display technology, Liang et al. (17Liang O.D. Chavakis T. Linder M. Bdeir K. Kuo A. Preissner K.T. Biol. Chem. 2003; 384: 229-236Crossref PubMed Scopus (15) Google Scholar) reported putative uPA-binding consensus sequences in 12 transmembrane receptors and suggested them as candidate alternative uPA receptor(s). Of these candidate receptors, several are already known as uPAR co-receptors: low density lipoprotein receptor-related protein, gp130, integrin αv, uPAR-associated protein (also known as Endo180 and Mrc2), and the insulin-like growth factor II/mannose 6-phosphate receptor. It is also possible that different uPA domains might simultaneously bind to uPAR and one of its co-receptors (18Franco P. Vocca I. Carriero M.V. Alfano D. Cito L. Longanesi-Cattani I. Grieco P. Ossowski L. Stoppelli M.P. J. Cell Sci. 2006; 119: 3424-3434Crossref PubMed Scopus (57) Google Scholar). The muscle type nicotinic receptor α1 (nAChRα1) was among the receptor candidates. The muscle type nAChR is a ligand-gated ion channel known to mediate signal transduction at the neuromuscular junction (19Kalamida D. Poulas K. Avramopoulou V. Fostieri E. Lagoumintzis G. Lazaridis K. Sideri A. Zouridakis M. Tzartos S.J. FEBS J. 2007; 274: 3799-3845Crossref PubMed Scopus (238) Google Scholar). This receptor is a pentametric glycoprotein comprising five membrane-spanning subunits (two α1, β1, γ, and δ) that form a ligand-gated ion channel. The currently known nAChRα1 ligands are nicotine and acetylcholine. The ligand-binding domain (interface of α1/γ or α1/δ) involves the two α1 chains, which form a specialized pocket of aromatic and hydrophobic residues structurally similar to uPAR (20Cauley K Agranoff B.W. Goldman D. J. Cell Biol. 1989; 108: 637-645Crossref PubMed Scopus (28) Google Scholar, 21Ploug M. Ellis V. FEBS Lett. 1994; 349: 163-168Crossref PubMed Scopus (243) Google Scholar). Upon ligation, the receptor changes its conformation and becomes permeable to sodium and calcium ions. Receptor function is regulated by tyrosine phosphorylation and dephosphorylation by kinases and phosphatases, respectively (22Miles K. Audigier S.S. Greengard P. Huganir R.L. J. Neurosci. 1994; 14: 3271-3279Crossref PubMed Google Scholar). The nAChRα1 is expressed and activated during muscle differentiation during embryonic development and following mature muscle denervation (23Wang X.M. Tsay H.J. Schmidt J. EMBO J. 1990; 9: 783-790Crossref PubMed Scopus (34) Google Scholar). Vascular endothelium, macrophages, and fibroblasts are also known to express certain nAChR subtypes (24Carlisle D.L. Hopkins T.M. Gaither-Davis A. Silhanek M.J. Luketich J.D. Christie N.A. Siegfried J.M. Respir. Res. 2004; 5: 27-43Crossref PubMed Scopus (85) Google Scholar). We observed that nAChRα1 expression was significantly higher in the kidneys of uPAR-deficient mice that develop worse scarring during UUO (supplemental Fig. S1a). Evidence that nAChRα1 might function as an alternative uPA receptor was suggested by our microarray data that compared uPAR−/− and uPAR+/+ renal fibroblasts. The nAChRα1 was the only one of the 12 receptor candidates identified by Liang et al. (17Liang O.D. Chavakis T. Linder M. Bdeir K. Kuo A. Preissner K.T. Biol. Chem. 2003; 384: 229-236Crossref PubMed Scopus (15) Google Scholar) that was significantly up-regulated on the uPAR−/− fibroblasts (n = 5, p < 0.01 by analysis of variance (ANOVA), >2-fold change) (supplemental Fig. S1b). Based on the assumption that the receptor may be up-regulated in damaged kidneys in the absence of uPAR and contribute to the development of more severe fibrosis, this study was designed to determine if a ligand-receptor relationship exists between uPA and nAChRα1 and to investigate its functional role in fibroblast growth and renal fibrosis. In vivo functional knockdown of the nAChRα1 was shown to significantly attenuate fibrosis after ureteral obstruction, an effect that was uPA-dependent. In vitro studies provided additional evidence that nAChRα1 was an uPA signaling receptor for fibroblasts, activating a complex of signaling proteins by tyrosine phosphorylation and calcium influx to stimulate proliferation and enhance contractility. Antibodies used in this study and their sources were as follows: rabbit anti-mouse uPA, Molecular Innovations Inc. (Novi, MI); antibodies to nAChRα1, -β1, or -γ and FGF-2, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies to uPAR1, phosphotyrosine, and E-cadherin (R&D Systems, Minneapolis, MN); rat monoclonal antibody to F4/80, Serotec Ltd. (Oxford, UK); EPOTM horseradish peroxidase-conjugated monoclonal antibodies to vimentin, αSMA, or PCNA and rat monoclonal antibody to Ki-67 (TEC-3), Dako Corp. (Carpinteria, CA); rabbit monoclonal antibody to Ki-67 (SP6), Novus Biologicals Inc. (Littleton, CO); rabbit antibody to receptor-conjugated choline glutaric acid (receptor-bound acetylcholine), Abcam Inc. (Cambridge, MA); fluorescein isothiocyanate-conjugated antibody to β-actin, Sigma; and rat anti-bromodeoxyuridine (BrdUrd) monoclonal antibody (Harlan SERA-LAB, Loughborough, UK). Protein reagents used in the in vitro intervention studies were as follows: active mouse urokinase (uPA) and amino-terminal fragment of mouse uPA (ATF), Molecular Innovations, Inc.; α-bungarotoxin (αBTx), d-tubocurarine (d-TC), and neonicotine (neoN), Sigma. High protein rat tail collagen, type 1 (12.29 mg/ml), used in forming a three-dimensional gel, was purchased from BD Biosciences. Several siRNA hairpin oligonucleotides to the target nAChRα1 mRNA sequence (NM-007389) were designed and synthesized using a strategy described previously (25Gou D. Jin N. Liu L. FEBS Lett. 2003; 548: 113-118Crossref PubMed Scopus (49) Google Scholar) (supplemental Fig. S2a). A scrambled RNA hairpin oligonucleotide was produced as a control. The hairpin inserts were cloned into the Promega psisTRIKETM vector (Promega Corp.) according to the manufacturer's instruction. Following transformation and amplification in Escherichia coli bacteria, plasmid DNA was isolated, digested with PstI, and confirmed by DNA sequencing (supplemental Fig. S2b). The silencing efficacy of these constructs was tested in renal fibroblast cultures (supplemental Fig. S2, c and d). The vector with the highest silencing effect against nAChRα1, which targets mouse nAChRα1 sequence at 1213–1238, was named psir2. The vector expressing the scrambled RNA sequence (GGCAUAAGAUUUAGCGGCAAGCAAU) was designated as pscr. All animal studies were approved by the Institutional Animal Care and Use Committee of the Seattle Children's Research Institute. All mice were on a C57BL/6 background. Wild-type (WT) mice were purchased from the Jackson Laboratory and underwent UUO surgery and cDNA intervention experiments at 12 weeks of age, as reported previously (26Zhang G. Kernan K.A. Collins S.J. Cai X. López-Guisa J.M. Degen J.L. Shvil Y. Eddy A.A. J. Am. Soc. Nephrol. 2007; 18: 846-859Crossref PubMed Scopus (84) Google Scholar). To functionally knock down nAChRα1 expression, the psir2 naked plasmid DNA was administered via tail veins (n = 10) using a hydrodynamic protocol as described previously (27Zhang G. Budker V. Wolff J.A. Hum. Gene Ther. 1999; 10: 1735-1737Crossref PubMed Scopus (817) Google Scholar, 28Dai C. Yang J. Liu Y. J. Am. Soc. Nephrol. 2002; 13: 411-422Crossref PubMed Google Scholar). Controls were injected with pscr plasmid DNA. Following rapid tail vein injection of 100 μg of DNA (psir2 or pscr) in 1 ml of normal saline, UUO surgery was performed in the left kidney. Kidneys and other tissues were harvested 7 days later. Additional mice were injected with pEGFP (a plasmid cDNA encoding enhanced green fluorescence protein obtained from Invitrogen using the same procedure. In order to delineate the functional relationship between nAChRα1 and uPA, urokinase-deficient Plau−/− mice (29Carmeliet P. Schoonjans L. Kieckens L. Ream B. Degen J. Bronson R. De Vos R. van den Oord J.J. Collen D. Mulligan R.C. Nature. 1994; 368: 419-424Crossref PubMed Scopus (910) Google Scholar) were obtained from the Jackson Laboratories (Bar Harbor, ME) and used in another set of RNA interference experiments, as described above for the WT mice. Primary renal fibroblast cultures were used as an in vitro model to further identify, characterize, and delineate this potential ligand-receptor relationship and to investigate its functional effects on renal fibroblasts. Wild-type and uPAR−/− mouse renal fibroblast primary cultures were established and characterized as previously described (16Zhang G. Cai X. López-Guisa J.M. Collins S.J. Eddy A.A. J. Am. Soc. Nephrol. 2004; 15: 2090-2102Crossref PubMed Scopus (14) Google Scholar). Cells were routinely cultured at 37 °C with 5% CO2 in DMEM/F-12 (1:1) supplemented with 5% (v/v) fetal calf serum (pH 7.35–7.45) and were passed after treatment with 0.05% trypsin, 0.53 mm EDTA digestion buffer (Mediatech Inc., Manassas, VA). The psir2 and pscr cDNAs were transfected into the uPAR−/− cells, using siPORTTM XP-1 transfection agent (Ambion Inc., Austin, TX) according to the manufacturer's instructions. Neomycin-resistant cell clones were selected and amplified. The cellular nAChRα1 silencing efficiency was confirmed at the mRNA and protein levels (supplemental Fig. S3). All in vitro experiments were performed, and results were compared with cells at 8–10 passages. Total RNA was isolated from cells or kidneys using TRIzolTM reagent (Invitrogen) according to the manufacturer's protocol. Northern blot analysis for αSMA (26Zhang G. Kernan K.A. Collins S.J. Cai X. López-Guisa J.M. Degen J.L. Shvil Y. Eddy A.A. J. Am. Soc. Nephrol. 2007; 18: 846-859Crossref PubMed Scopus (84) Google Scholar) and uPAR (30Zhang G. Kim H. Cai X. Lopez-Guisa J.M. Carmeliet P. Eddy A.A. J. Am. Soc. Nephrol. 2003; 14: 1234-1253Crossref PubMed Scopus (53) Google Scholar) and TaqMan real time reverse transcription-PCR for nAChRα1 were performed as described previously (24Carlisle D.L. Hopkins T.M. Gaither-Davis A. Silhanek M.J. Luketich J.D. Christie N.A. Siegfried J.M. Respir. Res. 2004; 5: 27-43Crossref PubMed Scopus (85) Google Scholar, 26Zhang G. Kernan K.A. Collins S.J. Cai X. López-Guisa J.M. Degen J.L. Shvil Y. Eddy A.A. J. Am. Soc. Nephrol. 2007; 18: 846-859Crossref PubMed Scopus (84) Google Scholar). The nAChRα1 and phosphotyrosine were immunolabeled in cultured renal fibroblasts using a standard ABC kit protocol (Vector Laboratories) (30Zhang G. Kim H. Cai X. Lopez-Guisa J.M. Carmeliet P. Eddy A.A. J. Am. Soc. Nephrol. 2003; 14: 1234-1253Crossref PubMed Scopus (53) Google Scholar, 31Zhang G. Kim H. Cai X. López-Guisa J.M. Alpers C.E. Liu Y. Carmeliet P. Eddy A.A. J. Am. Soc. Nephrol. 2003; 14: 1254-1271Crossref PubMed Scopus (98) Google Scholar). Paraffin-embedded kidney sections were stained with primary antibodies to nAChRα1, receptor-conjugated choline glutaric acid (receptor-bound acetylcholine), αSMA, Ki-67, and FGF-2. For IHC double staining, anti-αSMA antibody was directly labeled with horseradish peroxidase (developed with 3,3′-diaminobenzidine plus nickel, resulting in a black color), and nAChRα1 or Ki-67 was indirectly labeled with alkaline phosphatase-conjugated secondary antibodies (developed with Fast Red, resulting in a red color) (26Zhang G. Kernan K.A. Collins S.J. Cai X. López-Guisa J.M. Degen J.L. Shvil Y. Eddy A.A. J. Am. Soc. Nephrol. 2007; 18: 846-859Crossref PubMed Scopus (84) Google Scholar). For immunofluorescent double stains, uPA and nAChRα1 were identified with fluorescein isothiocyanate (green) and tetramethylrhodamine (red) fluorescence, respectively. Images were captured with a SPOT camera. For αSMA and receptor-bound acetylcholine, positive stain was quantified using Image-Pro Plus software. For nAChRα1, the staining in glomeruli, tubules, and the interstitium was separately quantified using the Image-Pro Plus software (for glomeruli) or a previously published point-counting method (for tubular or interstitial areas) (30Zhang G. Kim H. Cai X. Lopez-Guisa J.M. Carmeliet P. Eddy A.A. J. Am. Soc. Nephrol. 2003; 14: 1234-1253Crossref PubMed Scopus (53) Google Scholar). The results were expressed as positive percentage of area of interest (glomerular, tubular, or interstitial areas). Total renal collagen was measured biochemically, as previously described (26Zhang G. Kernan K.A. Collins S.J. Cai X. López-Guisa J.M. Degen J.L. Shvil Y. Eddy A.A. J. Am. Soc. Nephrol. 2007; 18: 846-859Crossref PubMed Scopus (84) Google Scholar). In brief, an accurately weighed portion of the kidney was homogenized in distilled water, hydrolyzed in 10 n HCl, and incubated at 110 °C for 18 h. The hydrolysate was dried by speed-vacuum centrifugation and redissolved in buffer (25 g of citric acid, 6 ml of glacial acetic acid, 60 g of sodium acetate, and 17 g of sodium hydroxide in 500 ml (pH 6.0)). Total hydroxyproline in the hydrolysate was determined according to the chemical method of Kivirikko et al. (32Kivirikko K.I. Laitinen O. Prockop D.J. Anal. Biochem. 1967; 19: 249-255Crossref PubMed Scopus (971) Google Scholar). Total collagen in the tissue was calculated on the assumption that collagen contains 12.7% hydroxyproline by weight. Final results were expressed as μg/mg kidney wet weight. Immunoblotting (IB) and co-immunoprecipitation (IP/IB) experiments were performed following standard protocols (33Asanuma K. Yanagida-Asanuma E. Faul C. Tomino Y. Kim K. Mundel P. Nat. Cell Biol. 2006; 8: 485-491Crossref PubMed Scopus (326) Google Scholar). Fibroblast nAChRα1 protein expression was evaluated using a standard flow cytometric approach. Renal fibroblasts were cultured in medium with or without uPA (2.4 × 10−8 m, a dose selected based on its binding Kd and our previous dose-response studies) (16Zhang G. Cai X. López-Guisa J.M. Collins S.J. Eddy A.A. J. Am. Soc. Nephrol. 2004; 15: 2090-2102Crossref PubMed Scopus (14) Google Scholar). At the end of the experiment (from 24 to 72 h), cells were harvested and subsequently incubated with ab1 specific for nAChRα1 and fluorescein isothiocyanate-conjugated ab2. Following stringent washes, cells were counted, and fluorescence intensities were evaluated using a flow cytometry (FACSCalibur; BD Biosciences). Fibroblast proliferation was evaluated using the CellTiter 96 Aqueous One Solution cell proliferation kit (Promega) according to the manufacturer's instructions (16Zhang G. Cai X. López-Guisa J.M. Collins S.J. Eddy A.A. J. Am. Soc. Nephrol. 2004; 15: 2090-2102Crossref PubMed Scopus (14) Google Scholar). Since uPAR+/+ fibroblasts did not survive for 72 h in serum-free culture conditions, uPA (2.4 × 10−8 m)-stimulated cell proliferation experiments were performed in DMEM/F-12 medium with 0.1% fetal calf serum. Similar stimulating experiments using uPAR−/− fibroblasts were performed under serum-free conditions. For each genotype, fibroblast proliferation was measured over several time periods in the presence or absence of uPA stimulation, comparing the responses of nAChRα1-expressing and -silenced fibroblasts. Each experiment was typically performed with n = 12 separate wells of fibroblasts in 96-well plates. Control experiments were performed with neoN and αBTx serving as an nAChRα1 agonist or antagonist, respectively. In additional experiments, nuclear mitotic activity was assessed by flow cytometric analysis of BrdUrd incorporation (34Lengronne A. Pasero P. Bensimon A. Schwob E. Nucleic Acids Res. 2001; 29: 1433-1442Crossref PubMed Scopus (136) Google Scholar). BrdUrd was added to the culture medium (final concentration 400 μg/ml) and incubated for 2 h prior to the experimental end point. Cells were then harvested, and their DNA was denatured with 2 n HCl at 37 °C for 30 min. BrdUrd was revealed with a rat anti-BrdUrd monoclonal antibody (1:10). Secondary antibody was goat anti-rat coupled to fluorescein isothiocyanate (Southern Biotechnology Associates, Birmingham, AL). Cell fluorescence intensity was evaluated using a flow cytometry (FACSCalibur; BD Biosciences), with the results expressed as a percentage of BrdUrd-positive cells (BrdUrd incorporation rate). The ability of the uPA-nAChRα1 interactions to modulate fibroblast-mediated collagen gel contraction was determined using the three-dimensional collagen gel slow contraction assay, as previously described (35Montesano R. Orci L. Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 4894-4897Crossref PubMed Scopus (487) Google Scholar). Briefly, fibroblasts were incubated in DMEM containing type I collagen (0.75 mg/ml) with or without uPA (2.4 × 10−8 m) to form a three-dimensional collagen lattice. The gels released from the plate wells were incubated in DMEM plus 1% fetal calf serum for 4 days. The gel area was measured at 0, 48, and 96 h. In an effort to determine if nAChRα1 mediates uPA binding to uPAR−/− fibroblasts, urokinase and nicotine competition binding studies were performed. Urokinase binding assays were carried out using two strategies. In the first, serial concentrations of the labeled uPA and a fixed concentration of the competitive inhibitor α-BTx were simultaneously added to the cells to measure binding competition using a classical assay described by Mazzieri et al. (36Mazzieri R. D'Alessio S. Kenmoe R.K. Ossowski L. Blasi F. Mol. Biol. Cell. 2006; 17: 367-378Crossref PubMed Scopus (68) Google Scholar). The uPA (American Diagnostic, Hauppauge, NY) was labeled using the DSB-X Biotin Protein Labeling Kit (Invitrogen) and streptavidin-35S (GE Healthcare) according to the manufacturers' instructions. In order to determine binding specificity, experiments were performed with or without a 100-fold excess of unlabeled uPA, in the presence of the nAChRα1 competitive antagonist α-BTx (0.1 μm) or in the absence of receptor nAChRα1 (receptor-silenced cells). After stringent washes, emitted β-activities were counted. In the second binding study strategy, the nAChRα1 expressed by uPAR−/− fibroblasts was preblocked with 0.1 μm α-BTx or a gradient concentration of d-TC for 30 min before the addition of biotinylated uPA (2.4 × 10−8 m). A previously published co-IP method (37Carlin B.E. Lawrence Jr., J.C. Lindstrom J.M. Merlie J.P. Proc. Natl. Acad. Sci. U.S.A. 1986; 83: 498-502Crossref PubMed Scopus (19) Google Scholar) was used to pull down the nAChRα1-bound uPA with an antibody specific for nAChRα1. The immunoprecipitated proteins were then run in an SDS gel and transferred to a membrane, and the biotinylated uPA was probed with ImmunoPure streptavidin-rhodamine (Pierce). Blots were scanned, and the fluorescence intensity was analyzed using a Typhoon 9410 variable mode imager (Amersham Biosciences). Nicotine binding assays were performed with serial concentrations (from 1.8 to 600 nm) of 3H-labeled nicotine (PerkinElmer Life Sciences) at room temperature for 45 min. Competition bin
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