The Transcription Factors Tbx18 and Wt1 Control the Epicardial Epithelial-Mesenchymal Transition through Bi-Directional Regulation of Slug in Murine Primary Epicardial Cells

<div><p>During cardiac development, a subpopulation of epicardial cells migrates into the heart as part of the epicardial epithelial-mesenchymal transition (EMT) and differentiates into smooth muscle cells and fibroblasts. However, the roles of transcription factors in the epicardial EMT are poorly understood. Here, we show that two transcription factors expressed in the developing epicardium, T-box18 (<i>Tbx18</i>) and Wilms’ tumor 1 homolog (<i>Wt1</i>), bi-directionally control the epicardial EMT through their effects on Slug expression in murine primary epicardial cells. Knockdown of Wt1 induced the epicardial EMT, which was accompanied by an increase in the migration and expression of N-cadherin and a decrease in the expression of ZO-1 as an epithelial marker. By contrast, knockdown of Tbx18 inhibited the mesenchymal transition induced by TGFβ1 treatment and Wt1 knockdown. The expression of Slug but not Snail decreased as a result of Tbx18 knockdown, but Slug expression increased following knockdown of Wt1. Knockdown of Slug also attenuated the epicardial EMT induced by TGFβ1 treatment and Wt1 knockdown. Furthermore, in normal murine mammary gland-C7 (NMuMG-C7) cells, Tbx18 acted to increase Slug expression, while Wt1 acted to decrease Slug expression. Chromatin immunoprecipitation and promoter assay revealed that Tbx18 and Wt1 directly bound to the <i>Slug</i> promoter region and regulated <i>Slug</i> expression. These results provide new insights into the regulatory mechanisms that control the epicardial EMT.</p> </div

The epicardial EMT induced by Wt1 knockdown is inhibited by knockdown of Tbx18 or Slug.

<p>(A) Representative images of Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug. Epicardial cells transfected with siControl or siWt1 were used as controls. (B) Immunostaining for ZO-1 (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNA. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on cellular morphology. (D) The relative mRNA expression of <i>Tbx18</i>, <i>Wt1</i> and <i>Slug</i> by real-time PCR in Wt1-knockdown epicardial cells co-transfected with siTbx18 or siSlug, in comparison to siControl-transfected epicardial cells (n = 3; *<i>P</i><0.05 vs. siControl, ^†<i>P</i><0.05 vs. siWt1). The results were normalized to <i>Gapdh</i> expression and the relative expression level is provided as a ratio to the siControl. The data are presented as the mean ± SD. Scale bars: 100 µm.</p

Knockdown of Wt1 and Tbx18 in primary epicardial cells.

<p>(A) Representative images of primary epicardial cells transfected with control siRNA (siControl) or siRNA directed against Tbx18 (siTbx18) or Wt1 (siWt1). Scale bar: 200 µm. (B) Immunostaining for ZO-1 or N-cadherin (green) and DAPI nuclear staining (blue) of primary epicardial cells transfected with siRNAs. Scale bars: 100 µm. (C) Percentage of cells categorized as “Enlarged” or “Cobblestone-like,” based on the cellular morphology of primary epicardial cells transfected with siRNAs. (D) Representative images of primary epicardial cells transfected with siRNAs at 0 and 14 hr after the scratch was made. Scale bar: 200 µm. (E) Quantification of migration distance, given as a ratio to the siControl (n = 4; *<i>P</i><0.01 vs. siControl). (F) The relative mRNA expression of <i>Tbx18</i>, <i>Wt1</i>, <i>Snail</i> and <i>Slug</i> by real-time PCR analysis (n = 3; *<i>P</i><0.05 vs. siControl). The results were normalized to <i>Gapdh</i> expression, and the relative expression level is given as a ratio to the siControl. (G) Western blot performed with antibodies against Tbx18, Wt1, adhesion molecules (E-cadherin and N-cadherin) and EMT regulators (Snail and Slug). β-actin and histone H3 were used as loading controls. The data are presented as the mean ± SD.</p

Hoxc8 is down-regulated during brown adipogenesis in vivo.

<p>(A) Immunofluorescence analysis of Hoxc8 in the mouse SVF cells derived from inguinal WAT. The cells were untreated (Undifferentiated) or induced to undergo differentiation (Adipogenic induction). The lipid droplets and nuclei were counterstained with Bodipy and DAPI, respectively. Scale bars indicate 30 µm. (B) Immunoblots of Hoxc8 in the mouse SVF cells left untreated or induced to undergo differentiation. β-actin served as a loading control. (C) Upper, the UCP1 expression in the differentiated mouse SVF cells. Scale bars indicate 30 µm. Lower, the fold increase of mRNA expression levels of <i>Ucp1</i> and <i>Ucp2</i> in the mouse WAT-SVF cells induced to undergo differentiation. The results were normalized to <i>β-actin</i>. (D) The expression of <i>Pgc-1α</i> and <i>C/EBPβ</i> induced during the differentiation of mouse SVF cells. The results were normalized to <i>β-actin</i>. The data are presented as means ± SEM; * <i>p</i><0.05. (E) Western blot analysis in different fat depots from mice treated with or without CL-316,243, a β3-adrenergic receptor agonist. ingWAT, epiWAT, and iBAT denote inguinal WAT, epididymal WAT, and interscapular BAT, respectively. (F) Western blot analysis of Hoxc8 in SVF cells and ingWAT of mice treated with CL-316,243 (CL) or saline.</p

Tbx18 and Wt1 are bound to the Slug promoter region and regulate the activity of the Slug promoter.

<p>(A) Immunoblot performed with an anti-Tbx18 antibody and an anti-Wt1 antibody. Tbx18 was immunoprecipitated with an anti-FLAG antibody (FLAG) or control IgG (IgG) in NMuMG-C7 cells expressing 3×FLAG-tagged Tbx18, and Wt1 was immunoprecipitated with an anti-Wt1 antibody (Wt1) or control IgG (IgG) in NMuMG-C7 cells expressing Wt1. (B) Direct binding of Tbx18 or Wt1 near the transcription start site (TSS) of the Slug gene in NMuMG-C7 cells, as assessed by ChIP. DNA fragments co-precipitated with Tbx18 or Wt1 were quantified by real-time PCR. The data are presented as the mean ± SD; n = 3; *<i>P</i><0.05 vs. control IgG. (C) The relative luciferase activity of a reporter construct carrying the <i>Slug</i> promoter in primary epicardial cells. The data are provided as ratios to siControl and are presented as the mean ± SD; n = 4; *<i>P</i><0.01 vs. siControl. (D) The relative luciferase activity of a reporter construct carrying the <i>Slug</i> promoter (Full) or the <i>Slug</i> promoter lacking the region around −200 from the TSS (−200 del) or +350 from the TSS (+350 del) in primary epicardial cells. The data are provided as ratios to Full and are presented as the mean ± SD; n = 4; *<i>P</i><0.05 vs. Full.</p

Primary culture of epicardial cells from E12.5 mouse embryos.

<p>(A) Representative image of primary epicardial cells generated from E12.5 mouse hearts, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057829#s2" target="_blank">Materials and Methods</a> section. (B) The relative mRNA expression levels of epicardial markers (<i>Tbx18 and Wt1</i>) and a cardiomyocyte marker (<i>Nkx2-5</i>) in primary epicardial cells and cardiomyocytes, as determined by quantitative real-time PCR (n = 3; *<i>P</i><0.0001 vs. primary epicardial cells). The results are normalized to <i>Gapdh</i> expression, and the relative expression level is given as a ratio to primary epicardial cells. (C) Immunostaining for Wt1 (green) and DAPI nuclear staining (blue) of primary epicardial cells after 4 days in culture. The data are presented as the mean ± SD. Scale bars: 200 µm.</p

Tbx18 and Wt1 regulate Slug expression in NMuMG-C7 cells.

<p>(A) Representative images of NMuMG-C7 cells expressing EGFP, Tbx18, Wt1 or a combination of Tbx18 and Wt1. Scale bar: 100 µm. (B) Western blot analysis of transduced NMuMG-C7 cell lines with antibodies against Tbx18, Wt1, Snail and Slug. β-actin was used as a loading control. (C) The relative mRNA expression of <i>Slug</i> in transduced NMuMG-C7 cell lines. The results were normalized to <i>Gapdh</i> expression, and the relative expression is provided as a ratio to EGFP transduced cells. The data are presented as the mean ± SD; n = 3; *<i>P</i><0.05 vs. EGFP transduced cells, ^†<i>P</i><0.0001 vs. Tbx18 transduced cells.</p

<i>HOXC8</i> is most highly expressed among clustered HOX genes in human WAT-progenitor cells.

<p>(A) Strand-specific RNA-seq results showing the expression levels of clustered HOX genes in undifferentiated human white fat (WAT) progenitor cells. The results with the clusters of HOXA, HOXB, HOXC, and HOXD are shown. The position of RefSeq genes are shown below. (B) The expression levels of clustered Hox genes from two biological replicates. FPKM, fragments per kilobase of exon per million mapped fragments.</p

A schematic of miR-196a-regulated brown adipogenesis of WAT-progenitor cells.

<p>Cold temperatures or β3-adrenergic stimulations induce miR-196a in the WAT-resident progenitor cells in mice. miR-196a post-transcriptionally suppress <i>Hoxc8</i>, which is one of the white-fat genes. The direct target of Hoxc8 is <i>C/EBPβ</i>, a master switch of brown adipogenesis that provokes brown fat gene program in the WAT-progenitor cells.</p

The miR-196a mice show resistance to obesity and improved glucose metabolism.

<p>(A) The appearance of the WT and miR-196a mice fed a high-fat diet for 16 wk. (B) The body weights of the WT and miR-196a mice (<i>n</i> = 8) fed a high-fat diet (HFD) or normal diet (ND) after 8 wk old. (C) The daily food intake of the WT and miR-196a mice (<i>n</i> = 8). (D) Oxygen consumption rates (V˙O2) in the WT and miR-196a mice fed a normal diet (<i>n</i> = 6). Measurements were performed on 3- to 4-mo-old mice with similar body weight that were given a standard diet. (E) The energy expenditure in the WT and miR-196a mice fed a normal diet (<i>n</i> = 6) calculated based on V˙O2 and V˙CO2 values and averaged separately for the light and dark phases. Measurements were performed on 3- to 4-mo–old mice with similar body weight that were given a standard diet. (F) The core body temperatures of the WT and miR-196a mice (<i>n</i> = 6). (G) The glucose tolerance test results for the WT and miR-196a mice (<i>n</i> = 10). (H) The plasma insulin concentrations after glucose injection in the WT (<i>n</i> = 8) and miR-196a (<i>n</i> = 10) mice. (I) The insulin tolerance test for the WT and miR-196a mice (<i>n</i> = 10). All data are presented as means ± SEM; * <i>p</i><0.05.</p