TGF-beta disruption of airway surface liquid (ASL) regulation in HAECs.

<p>Polarized non-CF HAECs had apical fluid removed, and the apical surface was then bolused with 20 µl of media. Cells were treated with vehicle (control) or TGF-beta (10 ng/ml), and the ASL volume was measured at 0, 24, 48, and 72 h post bolus (PB). TGF-beta or vehicle (diluted in water) was added to the to the apical bolus media at time  =  0, and to the basolateral media at time  =  0, 24 and 48 hours. N = 6. *<i>P</i> = 0.02 (control vs TGF-beta); **<i>P</i> = 0.00043 (control vs TGF-beta); ***<i>P</i> = 0.07 (control vs TGF-beta).</p

Rescue of TGF-beta-downregulated TMEM16A expression by a Smad3 inhibitor (SIS3) and CFTR expression by a p38 MAPK inhibitor (SB203580).

<p>T84 cells and HAECs were treated with either TGF-beta and SIS3 (5 µM, A and C) or TGF-beta and SB203580 (10 µM, B and D) for 48 h prior to lysis and immunoblot for TMEM16A (A and C) or CFTR (B and D). For each cell type, the upper gel panels show TMEM16A (A and C) or CFTR (B and D) detection from three replicate samples (with TGF-beta and either SIS3 or SB203580). SIS3 increased TMEM16A expression and SB203850 increased CFTR expression from TGF-beta-treated T84 cells and HAECs. The lower panels are summary densitometry data. T84 cells: *<i>P</i><0.0002 for TMEM16A and CFTR control vs TGF-beta. **<i>P</i><0.0007 for CFTR and TMEM16A + SB203580 and TGF-beta vs TGF-beta alone. HAECs: *<i>P</i><0.035 for TMEM16A and CFTR control vs TGF-beta. **<i>P</i><0.05 for CFTR and TMEM16A + SB203580 and TGF-beta vs TGF-beta alone.</p

Dose-response effects of TGF-beta on TMEM16A, CFTR, e-cadherin, and vimentin in T84 cells.

<p>T84 cells were treated with TGF-beta (vehicle control, 0.1, 1.0, and10 ng/ml) for 48 h, followed by Ussing chamber and immunoblot as described in Methods. (<b>A and B</b>) CaCC (*<i>P<</i>0.003, **P<0.01) and CFTR (*<i>P<</i>0.0001) currents were reduced by TGF-beta treatment at every dose used. (<b>C and D</b>) TMEM16A (*<i>P<</i>0.01, **P<0.005) and CFTR (*<i>P<</i>0.05, **P<0.0001) expression was suppressed by TGF-beta treatment at every dose used. E-cadherin expression (<b>E</b>) was decreased (*<i>P</i><0.011) and vimentin expression (<b>F</b>) was increased (*<i>P</i><0.05) with 1 ng/ml or greater TGF-beta treatment.</p

TGF-beta effects on TMEM16A and CFTR protein levels in T84 cells and HAECs.

<p>Lysates of T84 cells (<b>A and B</b>) or HAECs (<b>C and D</b>) were prepared and subjected to PAGE and immunoblot with either anti-TMEM16A or anti-CFTR antibody. For each cell type, the upper gel panel shows TMEM16A (<b>A and C</b>) or CFTR (<b>B and</b><b>D</b>) detection from three replicate samples (with or without 10 ng/ml TGF-beta exposure). The lower panels are summary densitometry data. <b>T84 cells:</b> *<i>P</i> = 0.01 for TMEM16A; *<i>P</i> = 0.01 for CFTR. <b>HAECs:</b> *<i>P</i> = 0.05 for TMEM16A; *<i>P</i> = 0.01 for CFTR.</p

Downregulation of both CaCC and CFTR currents in T84 cells and HAECs by TGF-beta.

<p>T84 (<b>A and B</b>) and HAECs (<b>C and D</b>) were treated with TGF-beta (10 ng/ml) for 48 h prior to I_sc measurement and studied as described in the Methods and as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106842#pone.0106842.s001" target="_blank">Figure S1</a>. T84 cells without nystatin were studied with symmetric apical and basolateral buffers. All other T84 and HAEC studies were completed with a basolateral-to-apical chloride secretory gradient <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106842#pone.0106842-VanGoor1" target="_blank">[27]</a>. Briefly, to activate CaCC-dependent chloride transport in T84 monolayers (<b>A</b>), cells were stimulated with ionomycin (Iono; 2 µM) to raise calcium and carbachol (CCh; 100 µM, basolateral) to activate basolateral potassium channels and drive apical chloride exit. Similar stimuli were used in HAECs (<b>C</b>) but carbachol had little effect on stimulated Isc. To activate CFTR-dependent chloride transport in both cell types (<b>B and D</b>), cells were stimulated with forskolin/IBMX (10 µM/100 µM) to raise cAMP. In T84 cells (<b>B</b>), carbachol (100 µM, basolateral) was used to increase apical chloride transport similar to A. In HAECs (<b>D</b>), genistein (50 µM, apical) was used to potentiate CFTR. The right two bars in each panel represent studies with a chloride secretory gradient following permeabilization of the basolateral membrane with nystatin (50 µg/ml). (<b>A</b>) *<i>P</i> = 0.001compared with control; **<i>P</i> = 0.007 compared with nystatin control. (<b>B</b>) *<i>P</i> = 0.003 compared with control; **<i>P</i> = 0.015 compared with nystatin control. (<b>C</b>) *<i>P</i> = 0.041 compared with control; **<i>P</i> = 0.016 compared with nystatin control. (<b>D</b>) *<i>P</i> = 0.006 compared with control; **<i>P</i> = 0.014 compared with nystatin control.</p

TGF-Beta Downregulation of Distinct Chloride Channels in Cystic Fibrosis-Affected Epithelia

<div><p>Rationale</p><p>The cystic fibrosis transmembrane conductance regulator (CFTR) and Calcium-activated Chloride Conductance (CaCC) each play critical roles in maintaining normal hydration of epithelial surfaces including the airways and colon. TGF-beta is a genetic modifier of cystic fibrosis (CF), but how it influences the CF phenotype is not understood.</p><p>Objectives</p><p>We tested the hypothesis that TGF-beta potently downregulates chloride-channel function and expression in two CF-affected epithelia (T84 colonocytes and primary human airway epithelia) compared with proteins known to be regulated by TGF-beta.</p><p>Measurements and Main Results</p><p>TGF-beta reduced CaCC and CFTR-dependent chloride currents in both epithelia accompanied by reduced levels of TMEM16A and CFTR protein and transcripts. TGF-beta treatment disrupted normal regulation of airway-surface liquid volume in polarized primary human airway epithelia, and reversed F508del CFTR correction produced by VX-809. TGF-beta effects on the expression and activity of TMEM16A, wtCFTR and corrected F508del CFTR were seen at 10-fold lower concentrations relative to TGF-beta effects on e-cadherin (epithelial marker) and vimentin (mesenchymal marker) expression. TGF-beta downregulation of TMEM16A and CFTR expression were partially reversed by Smad3 and p38 MAPK inhibition, respectively.</p><p>Conclusions</p><p>TGF-beta is sufficient to downregulate two critical chloride transporters in two CF-affected tissues that precedes expression changes of two distinct TGF-beta regulated proteins. Our results provide a plausible mechanism for CF-disease modification by TGF-beta through effects on CaCC.</p></div

TGF-beta downregulation of VX-809-corrected F508del CFTR in HAECs.

<p>F508del CFTR homozygous HAECs were grown at air-liquid interface culture for 6 weeks and treated with TGF-beta (10 ng/ml) and/or VX-809 (3 µM) for 48 h prior to I_sc measurement. (<b>A</b>) Stimulated I_sc under control (left), VX-809 alone (middle), or VX-809 + TGF-beta (right) treatment conditions. *<i>P</i><0.035, VX-809 compared with either control or VX-809 + TGF-beta. (<b>B</b>) Lysates of F508del HAECs from each filter were prepared and subjected to PAGE and immunoblot as described in Methods for detection of B Band and C Band under control, VX-809 alone, or VX-809 + TGF-beta treatment conditions. (<b>C</b>) All tested doses of TGF-beta downregulated VX-809-corrected F508del CFTR. *<i>P</i><0.00017, VX-809 compared with untreated control; **<i>P</i><0.0001, 0.1 ng TGF-beta + VX-809 compared with VX-809 alone. ***<i>P</i><0.0001, 1 ng TGF-beta + VX-809 compared with VX-809 alone. ****<i>P</i><0.0001, 10 ng TGF-beta + VX-809 compared with VX-809 alone. (<b>D</b>) Summary densitometry data for detection of F508del CFTR B Band (left) and C Band (right) with and without TGF-beta (10 ng/ml, similar to 5B). *<i>P</i>≤0.047, B Band VX-809 compared with either control or VX-809 + TGF-beta. *<i>P</i>≤0.05, C Band VX-809 compared with either control or VX-809 + TGF-beta.</p

Dose-response effects of TGF-beta on TMEM16A, CFTR, e-cadherin, and vimentin in HAECs.

<p>HAECs were treated with TGF-beta (vehicle control, 0.1, 1.0, and10 ng/ml) for 48 h, followed by Using chamber and immunoblot as described in Methods. (<b>A and B</b>) CaCC (*<i>P<</i>0.0016) and CFTR (*<i>P<</i>0.0001) currents were reduced by TGF-beta treatment at every dose used. (<b>C and D</b>) TMEM16A (*<i>P<</i>0.045, **P<0.02) and CFTR (*<i>P<</i>0.04, **P<0.006) expression was suppressed by TGF-beta treatment at every dose used. E-cadherin expression (<b>E</b>) was decreased (*<i>P</i><0.006, **<i>P</i><0.0001) and vimentin expression (<b>F</b>) was increased (*<i>P</i> = 0.001, **<i>P</i><0.0003) with 1 ng/ml or higher TGF-beta treatment.</p

Summary of TGF-beta inhibition of CaCC and CFTR in T84 cells and HAECs.

<p>Values are % reduction (±SD) produced by TGF-beta exposure (10 ng/ml, 48 h).</p><p>*TMEM16A protein and RNA levels are provided for comparative purposes.</p><p>Summary of TGF-beta inhibition of CaCC and CFTR in T84 cells and HAECs.</p

ROC curve analysis.

<p>Note that CF participants are coded as one, and non-CF participants are coded as zero. Average true-positive rate is the sensitivity of the current to detect CF participants, whereas the average-false positive rate (one-specificity) marks the cutoff whereby non-CF participants are falsely determined as CF. AUC values varied from 0.946-0.978 for the three CFTR-specific measurements (forskolin/IBMX (cAMP), carbachol (CCh), cAMP + CCh).</p