NHE1-deficient PS120 fibroblasts derived from hamster lung CCL39 fibroblasts (Pouyssegur et al., 1984
) were obtained from J. Pouyssegur (INSERM, Nice, France). Wild-type and mutant NHE1 containing an hemagglutinin (HA)-epitope at the COOH-terminus and stably expressed in PS120 cells (WT, E266I, and KRA2 cells) were previously described (Denker et al., 2000
). An NHE1-E266I-Myc construct was generated in pCDNA3.1 Hygro (+) and stably expressed in KRA2 cells (named K2E cells) by using Lipofectamine 2000 for transfection (Invitrogen, Carlsbad, CA). Heterologous expression of the System N1 transporter in PS120 cells (SN1 cells) was previously described (Chaudry et al., 1999
). Fibroblasts were maintained in DME-H21 medium supplemented with 5% FBS and 1% Pen-Strep (growth medium) at 5% CO2
. Mink lung epithelial cells (MLEC) expressing a truncated promoter of plasminogen activator inhibitor-1 (PAI-1; provided by R. Derynck, University of California, San Francisco) were maintained in DME-H21 medium supplemented with 10% FBS, 1% Pen-Strep, and 250 μg/ml geneticin (G-418; Invitrogen, Grand Island, NY) at 5% CO2
. Where indicated, cells were treated with recombinant human (rh)TGF-β1 (PeproTech, Rocky Hill, NJ), and SB-431542 (Tocris Bioscience, Ellisville, MI) was used to inhibit activity of the TGF-β receptor type I (TGF-β-RI).
Unless otherwise indicated, cells grown for 48 h on glass coverslips were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, washed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked for 15 min with 10% FBS in PBS. Fixed cells were incubated with primary antibodies for 60 min, washed, and incubated with fluorescent-conjugated secondary antibodies for 45 min. For live-cell staining of extracellular FN, cells were incubated at 4°C with antibodies for FN for 1 h and fixed as described above without permeabilization. Primary antibodies were to paxillin (1:100; Zymed Laboratories, South San Francisco, CA), FN (1:100; Sigma, St. Louis, MO), β1 integrin (9EG7; 1:100; BD PharMingen, San Diego, CA), HA (12CA5; 1:100; Roche Molecular Biochemicals, Mannheim, Germany), and Myc (9B11; 1:200; Cell Signaling Technology, Danvers, MA). Secondary antibodies conjugated to FITC or Texas-red (Invitrogen) were used at 1:200 and nuclei were stained by incubating cells for 5 min with Hoechst 33342 (1:10,000; Invitrogen). Immunolabeling was visualized using a Zeiss Axiovert 35 (Thornwood, NY), and images were collected with a Spot RT cooled CCD (Diagnostic Instruments, Sterling Heights, MI).
For β1 integrin labeling, the Avidin/Biotin/Streptavidin Blocking Kit (Vector Laboratories, Burlingame, CA) was used. Cells were incubated with Avidin D for 15 min, rinsed with PBS, and then incubated with Biotin blocking solution for 15 min followed by incubating with anti-β1-integrin 9EG7 antibodies. The avidin conjugate was used for β1 integrin labeling, which was visualized as described above.
Unless otherwise indicated, immunoblotting used lysates from cells plated in 100-mm dishes and maintained in growth medium. Cells were washed three times in PBS and lysed in modified RIPA buffer (50 mM Tris-HCl, 135 mM NaCl, 3 mM KCl, 1% NP-40, protease inhibitors, 1 mM EGTA, 5 mM NaF, 10 mM sodium pyrophosphate, 1 mM glycerol phosphate, and 1 mM sodium vanadate, pH 7.2), and proteins in postnuclear supernatant fractions collected after centrifugation (850 × g for 5 min) were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were probed with antibodies to FN (1:1000, Sigma), Egr-1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), Smad3 (1:1000; Cell Signaling Technology), phosphorylated Smad3 (1:1000; Cell Signaling Technology), and β-actin (C4, 1:5000; Millipore, Temecula, CA). Bound antibody was detected by enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ).
Immunoblotting for secreted FN was performed after chloroform-methanol precipitation of cell culture medium from cells 48 h after plating. Briefly, the medium was mixed methanol:chloroform (3:1 vol), and the mix was vortexed for 2 min and centrifuged at 10,000 × g for 5 min. The upper phase was discarded, leaving the interface intact, and the remaining mixture was washed with 300 μl methanol and centrifuged for 5 min, and pellets were resuspended in Laemmli buffer according to the concentration of the corresponding cell lysate. Proteins were separated by SDS-PAGE and processed for immunoblotting using antibodies to FN and β-actin.
Immunoblotting for NHE1-HA and TGF-β-RI used HA immune-precipitated complexes. Postnuclear supernatants from cell lysates were prepared as described for immunoblotting and incubated overnight with Sepharose-conjugated anti-HA antibodies (Roche Molecular Biochemicals). Immune complexes were recovered by centrifugation, separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies to NHE1 (1:2000; Millipore), Myc (9B11, 1:1000), HA (12CA5; 1:1000), or TGF-β-RI (V-22, 1:500, Santa Cruz Biotechnology).
Immunoblotting for focal adhesion kinase (FAK) used lysates from cells maintained overnight in 0.2% FBS, trypsinized, resuspended in DMEM supplemented with 0.2% FBS, and plated in 100-mm dishes precoated with on FN (10 μg/ml). At the indicated times, postnuclear supernatants were collected as described above, and equal amounts of protein were precleared with protein G-Sepharose beads and incubated for 2 h with antibodies to FAK (2.5 μg; BD Biosciences, San Jose, CA), and for 1 h with protein G-Sepharose beads. Immune complexes were recovered by centrifugation, washed, separated by SDS-PAGE, and processed for immunoblotting with antibodies to phosphorylated FAK-pY397 (1:1000; Invitrogen). Membranes were stripped and probed with antibodies to FAK (1:1000; BD Biosciences).
Collagen expression was determined by reverse transcription-PCR (RT-PCR) analysis for hamster collagen α1 (V) chain. Total RNA extracted using the RNeasy kit (Qiagen, Valencia, CA), and 2 μg was reverse transcribed using random decamer primers and amplified with PCR Supermix High Fidelity (Invitrogen) for 30 cycles at 94, 56, and 72°C. Oliogs included were F2 forward primer, 5′TACCCTGGAAGACAAGGGCC3′, and R2 reverse primer, 5′TCCTGGAGGGCCAGTCTTGC3′, which yielded a 322-base pair product, and F3 forward primer, 5′ATGGTGAACCTGGACAGACG3′, and R3 reverse primer, 5′TCCTTTGAGTCCAGGGAGTC3′, which yielded a 329-base pair product. Primers against 18S RNA were used as a control.
Cells were plated in 100-mm culture dishes to achieve 95, 50, and 25% confluency after 48 h. Total RNA was extracted using the RNeasy kit (Qiagen). RNA (20 μg) was separated on 1.0% agarose gels containing formaldehyde and transferred to a nylon membrane (Hybond-N+, Amersham). An FN DNA fragment (FN N29; provided by C. Damsky, University of California, San Francisco) was used as a template to prepare radiolabeled probe by using the Rad Prime DNA system (Invitrogen) according to the manufacturer's directions. Hybridization was performed under stringent conditions with 1× SSC and 0.1% SDS at 65°C for 2 h. GAPDH was used to confirm equal sample loading.
The pGLFN105 promoter vector (Promega, Madison, WI) containing the FN promoter region between −105 and +14 was provided by D. Mercola (Sidney Kimmel Cancer Center, San Diego, CA) and previously described (Liu et al., 2000
). pRK5 containing β-galactosidase under control of a human CMV promoter was provided by R. Derynck (University of California, San Francisco) and previously described (Feng et al., 1995
). Cells were transfected using Transfast according to the manufacturer's recommendations (Promega). For each transfection the total quantity of transfected plasmid DNA pGLFN105 and pRK5 was kept constant. Forty-eight hours after transfection, cells were harvested and lysed in Reporter Lysis Buffer (Promega), and reporter activity was determined using a luciferase assay system (PharMingen) and β-galactosidase detection kit (Tropix, Bedford, MA) and measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA). Data were normalized for transfection efficiency by β-galactosidase activity.
Secreted TGF-β was determined using MLEC cells stably expressing a truncated promoter of PAI-1 fused to the firefly luciferase reporter gene as described (Abe et al., 1994
). MLEC cells were plated for 24 h in six-well tissue culture dishes, washed twice with PBS, and maintained for 4 h in DME-H21 medium without FBS. Conditioned medium collected from the indicated cells maintained for 18 h in DMEM supplemented with 0.2% FBS was directly applied to MLEC cells for determining active TGF-β or heated at 80°C for 10 min before applying for determining secreted latent TGF-β. In parallel cultures, MLEC cells were incubated in DMEM supplemented with (rh)TGF-β1 (0.1–5000 ng/ml; PeproTech). After 24 h MLEC cell extracts were prepared and assayed for luciferase activity using the Luciferase Assay System (Promega) according to the manufacturer's instructions, and luminescence was measured as described above. Luciferase units obtained from conditioned medium samples were normalized to cell number, and secreted TGF-β was quantified relative to luciferase units from samples supplemented with rhTGF-β1.
FITC-FN Labeling and Assembly Assays
Fibronectin assembly was determined by plating cells maintained in growth medium on glass coverslips coated with exogenous FITC-labeled bovine FN (15–50 μg/ml) or by adding exogenous FITC-labeled FN in the medium (McKeown-Longo and Mosher, 1995
). Bovine plasma FN (Calbiochem-Novabiochem, La Jolla, CA) was labeled using the FluoReporter FITC protein labeling kit (Molecular Probes Invitrogen, Eugene, OR) according to the manufacturer's directions. Cells were maintained for 48 h, unless otherwise indicated, washed with PBS, fixed, and visualized as described for immunolabeling.
Integrin Expression and FITC-FN Binding
To determine β1 integrin expression, cells were biotinylated on ice for 90 min and lysed in modified RIPA buffer, and lysates were incubated with β1 integrin antibodies (9EG7, 1:100) for 60 min, followed by incubation for 60 min with protein A-Sepharose beads. Eluted proteins were probed for biotin with streptavidin-HRP. Integrin expression was confirmed by flow cytometry analysis. Cells were incubated in PBS-based dissociation buffer, washed twice with PBS, and kept in suspension in PBS containing 2% FBS. After incubating cells with anti-β1 integrin antibodies in PBS at 4°C for 1 h, cells were washed with PBS and incubated with anti-rabbit-Alexa Fluor 488 (A488) F(ab)2 (Invitrogen) at 4°C for 30 min. Cells were washed with PBS and analyzed using a FACS Vantage SE cell sorter (Becton Dickinson, San Jose, CA). Flow cytometry data were analyzed using CellQuest Pro4.0.1 software (Becton Dickinson). For β1 integrin binding to FN cells in suspension were mixed with the indicated concentrations of FITC-labeled FN for 60 min, and FITC-positive cells were analyzed as described above.
Intracellular pH Measurements
NHE1 activity and intracellular pH were determined in cells plated at 25 × 105
per well in 12-well plates and loaded with the fluorescent pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF; Invitrogen, Carlsbad, CA) by modifications of previously described methods (Denker et al., 2000
). Ratios of BCECF fluorescence at Ex 490/Em 530 and Ex 440/Em 530 were acquired using a SpectraMax M5 plate reader (Molecular Dynamics). Fluorescence ratios were converted to pHi
by calibrating each experiment with 10 μM nigericin (Invitrogen) in 105 mM KCl.