Troglitazone Attenuates TGF-β1-Induced EMT in Alveolar Epithelial Cells via a PPARγ-Independent Mechanism

<div><p>Peroxisome proliferator activated receptor γ (PPARγ) agonists are effective antifibrotic agents in a number of tissues. Effects of these agents on epithelial-mesenchymal transition (EMT) of primary alveolar epithelial cells (AEC) and potential mechanisms underlying effects on EMT have not been well delineated. We examined effects of troglitazone, a synthetic PPARγ agonist, on transforming growth factor (TGF)-β1-induced EMT in primary rat AEC and an alveolar epithelial type II (AT2) cell line (RLE-6TN). TGF-β1 (2.5 ng/mL) induced EMT in both cell types, as evidenced by acquisition of spindle-like morphology, increased expression of the mesenchymal marker α-smooth muscle actin (α-SMA) and downregulation of the tight junctional protein zonula occludens-1 (ZO-1). Concurrent treatment with troglitazone (or rosiglitazone), ameliorated effects of TGF-β1. Furthermore, following stimulation with TGF-β1 for 6 days, troglitazone reversed EMT-related morphological changes and restored both epithelial and mesenchymal markers to control levels. Treatment with GW9662 (an irreversible PPARγ antagonist), or overexpression of a PPARγ dominant negative construct, failed to inhibit these effects of troglitazone in AEC. Troglitazone not only attenuated TGF-β1-induced phosphorylation of Akt and glycogen synthase kinase (GSK)-3β, but also inhibited nuclear translocation of β-catenin, phosphorylation of Smad2 and Smad3 and upregulation of the EMT-associated transcription factor SNAI1. These results demonstrate inhibitory actions of troglitazone on TGF-β1-induced EMT in AEC via a PPARγ-independent mechanism likely through inhibition of β-catenin-dependent signaling downstream of TGF-β1, supporting a role for interactions between TGF-β and Wnt/β-catenin signaling pathways in EMT.</p> </div

AQP5 and AQP2 colocalize and partially reside in the ER/Golgi compartments in IMCD3 cells.

<p>(<b>A</b>–<b>C</b>) Representative confocal microscopy images showing colocalization of RFP-AQP5 and GFP-AQP2 (<b>A</b>), lack of colocalization when GFP-AQP2 (<b>B</b>) and RFP-AQP5 (<b>C</b>) was coexpressed with RFP and GFP alone, respectively. (<b>D</b>) Representative confocal microscopy images showing AQP5-AQP2 complex partially locates in the ER in IMCD3 cells. IMCD3 cells were co-transfected with the plasmids as indicated and examined by confocal microscopy. DsRed-ER was used as an endoplasmic reticulum (ER) marker <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053342#pone.0053342-Ganley1" target="_blank">[39]</a> and shown in red. GFP-AQP2 is shown in green. Myc-AQP5 was not directly detected, but can be inferred by its interaction and colocalization with AQP2 as large discrete foci seen in <b>A</b>. AQP2 alone seldom forms large discrete foci. In addition, more than 95% of transfected IMCD3 cells were always doubly transfected. Therefore, the presence of the large discrete foci is indicative of AQP2-AQP5 complex. (<b>E</b>) As in D except that DsRed-ER was omitted and cells were stained with an antibody specific for GM130, a cis-Golgi marker. Scale bar: 40 µM.</p

AQP5 coimmunoprecipitates with AQP2 and impairs its cell surface localization in IMCD3 cells.

<p>(<b>A–D</b>) Co-IP showing human AQP5 interacts specifically with human AQP2. AQP5 and AQP2 were expressed as FLAG- or GFP- fusions in 293T cells, and analyzed by IP-IB with antibodies as indicated. (<b>E</b>) Representative IBs of biotinylation assay showing that AQP5 impairs cell surface localization of AQP2. FLAG-AQP2 and Myc-AQP5 were expressed separately or in combination in IMCD3 cells, biotinylated, and analyzed by IB with antibodies as indicated. *: unknown protein.</p

Aqp5 is significantly upregulated and coexpressed with Aqp2 in the kidney of <i>Dot1l^AC</i> mice on the normal Na⁺ pellet diet.

<p>(<b>A</b>) Real-time RT-qPCR for expression of Aqp5 in kidney of mice fed the normal Na⁺ pellet diet, with β-actin as internal control. n = 6 mice/group. <i>+/+AC</i>: <i>Dot1l^+/+Aqp2:Cre</i> (<b>B</b>) As in <b>A</b>, agarose gel analysis of the final RT-qPCR products of Aqp5 and β-actin. (<b>C</b>) Sequencing of a regular RT-PCR product from a <i>Dot1l^f/f</i> mouse kidney. A part of the tracing file showing Aqp5 sequence encoding aa 47–55 (GenBank#: EDL04123.1) is given. (<b>D</b>) Representative IF images showing Aqp5 (green) expression in Aqp2⁺ (red) cells in mice as indicated. OM and IM: outer and inner medulla. *: An IC without Aqp5 expression, possibly due to lack of Aqp2Cre-mediated <i>Dot1l</i> ablation. Arrow: PC with strong Aqp2 and weak Aqp5, highlighting the lack of cross reactivity of the two antibodies. Aqp5⁺Aqp2⁻ cells are most likely the intercalated cells derived from the Aqp2-expressing progenitor cells or mature PC <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053342#pone.0053342-Wu2" target="_blank">[30]</a>. Arrowhead: Colocalization of Aqp5 with Aqp2, which is amplified in the inserts. Scale bar: 50 µM. For more images with lower magnification, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053342#pone.0053342.s003" target="_blank">Figure S3</a>.</p

AQP5 is pathologically expressed and colocalizes with AQP2 at the perinuclear region in patients with DN.

<p>(<b>A</b>–<b>D</b>) Representative IF images of kidney biopsies from a patient with MCD showing no detectable AQP5 (<b>A</b>), from two patients with DN showing AQP5 colocalization with AQP2 at perinuclear region (<b>B–C</b>), and from a patient with DN showing diffuse AQP5 without AQP2 in the same cells (<b>D</b>). There are 12 AQP2⁺AQP5⁺ tubules in <b>B</b>. Three detached cells are shown in the insert. Hematoxylin/eosin staining verified no significant pathological changes in the tubules of the MCD samples, but various tubular abnormalities in all of the DN samples (see text for details). (<b>E</b>) Summary of the relative abundance of the three types of tubules from 17 patients with DN. Scale bar: 50 µM.</p

Dot1a represses Aqp5 mRNA expression in mouse cortical collecting duct M1 and mouse lung epithelial MLE-15 cells.

<p>(<b>A–B</b>) Real-time RT-qPCR showing that siRNA-mediated depletion of Dot1la in M1 cells increases endogenous Aqp5 expression (<b>A</b>) and that overexpression of Dot1a decreases endogenous Aqp5 expression in MLE-15 cells (<b>B</b>). (<b>C</b>) Luciferase assays showing that overexpression of Dot1a reduces expression of a luciferase reporter driven by a 4.3-kb (−4300 in <b>E</b>) promoter of rat Aqp5 in MLE-15 cells. (<b>D</b>) As in <b>C</b>, except that various amounts of Dot1a plasmid were used for transfection. (<b>E</b>) Luciferase assay showing that Dot1a represses Aqp5 promoter-luciferase constructs as indicated. The relative positions of the Dot1l- and H3m2K79-binding subregions of mouse Aqp5 in ChIP assays were shown at the bottom. The percentages of identities between rat and mouse were also indicated. Note: Dot1a-mediated repression was eliminated in the −139 construct.</p

Troglitazone (Tro) prevents EMT-associated alterations in ZO-1 and α-SMA protein expression in primary AEC.

<p>Western analysis reveals inhibition of TGF-β1-mediated decreases in ZO-1 (A) and increases in α-SMA (B) by troglitazone in primary AEC. <i>*P</i><0.05 compared to TGF-β1; n = 3.</p

<i>Dot1l</i>^AC mice displayed polyuria without impaired Aqp2 expression.

<p>(<b>A–B</b>). <i>Dot1l^f/f</i> and <i>Dot1l^AC</i> mice deprived of water for 24 h (n = 14 mice/group) were subjected to 24-h urine analyses as indicated. For additional measurements, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053342#pone.0053342.s001" target="_blank">Figure S1</a>. *P<0.05 vs. <i>Dot1l^f/f</i>. (<b>C</b>) <i>Dot1l^AC</i> vs. <i>Dot1l^f/f</i> mice (n = 4 mice/group, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053342#s4" target="_blank">Materials and Methods</a>) have only subtle changes in the expression of all known Aqps as indicated except for Aqp5. Shown are the fold changes revealed by cDNA array anlalysis. (<b>D</b>) Real-time RT-qPCR for expression of Aqp2 and AQP3 in kidney of mice fed the normal Na⁺ pellet diet, with β-actin as internal control. n = 6 mice/group. (<b>D</b>) IB for Aqp2 expression, with β-actin as internal control. n = 4 mice/group.</p

Troglitazone (Tro) reverses TGF-β1-induced EMT in primary AEC.

<p>A. Following treatment with TGF-β1 starting on day 2 for 6 days, ZO-1 immunoreactivity was markedly decreased while α-SMA was robustly expressed, reflecting that cells are undergoing EMT (<i>ii,vi</i>). Following subsequent treatment with troglitazone for an additional 6 days (from day 8 through day 14), ZO-1 expression was restored and α-SMA returned to control levels (<i>iv, viii</i>). Nuclei are labeled with DAPI. Cells treated with TGF-β1 vehicle (<i>i,v</i>) serve as negative control. TGF-β1 removal (<i>iii, vi</i>) only shows partial reversal of EMT. B. Treatment with increasing amounts of TGF-β1 (2.5–10 ng/ml) in the presence of troglitazone (10 µM) does not prevent inhibitory effects of troglitazone on TGF-β1-induced α-SMA expression. These data are representative of three separate experiments. C. Treatment with increasing amounts of troglitazone (2.5–10 µM) in the presence of TGF-β1 (2.5 ng/ml) for 2 hours reduced phosphorylation of Smad2 and Smad3 induced by TGF-β1. These data are representative of two separate experiments.</p

Troglitazone (Tro) inhibits EMT in primary AEC.

<p>A. Under control conditions, cells exhibit cobblestone appearance typical of epithelial morphology. Following treatment with TGF-β1, loss of cell-cell contacts and acquisition of fibroblast-like morphology are seen. Troglitazone attenuates TGF-β1-induced changes and maintains epithelial morphology. Nuclei are labeled with 4',6-diamidino-2-phenylindole (DAPI). B. Primary AEC treated with TGF-β1± troglitazone were fixed and stained for ZO-1 and α-SMA. Control cells exhibit ZO-1 staining along intercellular surfaces with minimal α-SMA expression. Treatment with TGF-β1 gives rise to loss of cell membrane-associated ZO-1 with a marked increase in α-SMA. Cells treated with both TGF-β1 and troglitazone maintain normal ZO-1 immunoreactivity with an absence of α-SMA. Nuclei are labeled with DAPI. C. TGF-β1 (present from day 2 onward) induces a decrease in transepithelial resistance (<i>R_t</i>) of primary AEC monolayers. Decreases in <i>R_t</i> are prevented by concurrent treatment with both TGF-β1 and troglitazone. <i>*P</i><0.05 compared to vehicle; n = 3.</p