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. 2015 Mar;16(3):321-31.
doi: 10.15252/embr.201439333. Epub 2015 Feb 2.

Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism

Daniel Mathow  1 , Federica Chessa  1 , Mariona Rabionet  2 , Sylvia Kaden  1 , Richard Jennemann  1 , Roger Sandhoff  2 , Hermann-Josef Gröne  3 , Alexander Feuerborn  4
Affiliations

Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism

Daniel Mathow et al. EMBO Rep. 2015 Mar.

Abstract

This study proposes that the transcription factor Zeb1 modulates epithelial cell adhesion by diverting glycosphingolipid metabolism. Zeb1 promotes expression of a-series glycosphingolipids via regulating expression of GM3 synthase (St3gal5), which mechanistically involves Zeb1 binding to the St3gal5 promoter as well as suppressing microRNA-mediated repression of St3gal5. Functionally, the repression of St3gal5 suffices to elevate intercellular adhesion and expression of distinct junction-associated proteins, reminiscent of knockdown of Zeb1. Conversely, overexpressing St3gal5 sensitizes cells towards TGF-β1-induced disruption of cell-cell interaction and partially antagonizes elevation of intercellular adhesion imposed by Zeb1 knockdown. These results highlight a direct connection of glycosphingolipid metabolism and epithelial cell adhesion via Zeb1.

Keywords: EMT; TGF‐β1; Zeb1; adhesion; glycosphingolipids.

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Figures

Figure 1
Figure 1
Suppression of St3gal5/GM3 synthase recapitulates changes in GSL expression of Zeb1 knockdown cells and repression of Zeb1 decreases St3gal5 expression

  1. A Schematic representation of enzymes (black boxes) and metabolites (grey) related to (G)SL metabolism. GM3 synthase (GM3S, red box) converts LacCer into GM3, the precursor for all higher gangliosides of a-, b- and c-series. Gangliosides of the b- and c-series were not detected in NM18 cells.

  2. B TLC-based comparison of GSLs. Cells repressed in Zeb1 or St3gal5 (72 h) reveal similar alterations in GSL expression as indicated by elevated levels of LacCer (†) and GM1b (º) and reduced levels of GM2 (-) and GM1a (•). * indicates no GSL.

  3. C qRT–PCR-based analysis of St3gal5 mRNA expression in Zeb1- and St3gal5-repressed cells. Cells were transfected for 72 h with indicated siRNAs (n + 3). Mean ± SD, n + 3, *P ≤ 0.05, **P ≤ 0.01, paired t-test.

  4. D Acidic GSLs after treatment with sialidase reveal elevated levels of GA1 in the neutral fraction of Zeb1- and St3gal5-repressed cells (left panel). Validation of GA1 via immune overlay (right panel).

  5. E Acidic GSLs of Zeb1- and St3gal5-silenced cells after treatment with Vibrio cholera sialidase. Sialidase cleavage leads to conversion of GM1b to GA1, detectable in the TLC of neutral GSLs (see D). GM1a levels are decreased in Zeb1- and St3gal5-knockdown cells (•).

Figure 2
Figure 2
Zeb1 regulates expression of St3gal5 in a direct and indirect manner

  1. A Luciferase assays employing St3gal5 promoter fragments of different lengths. Cells were transfected as indicated. Activity readings of cells transfected with Ctrl-siRNA and ‘full-length’ promoter (2668-LUC) were set to 1 and used as reference. Mean ± SD, n + 4, **P ≤ 0.01, one-way ANOVA with Tukey's Multiple Comparison Test.

  2. B qRT–PCR-based analysis of mRNA expression of Zeb1 and St3gal5 in transfected cells (24 h) in conjunction with TGF-β1 stimulation (24 h). Significance refers to untreated cells transfected with Ctrl-siRNA (n + 3). Mean ± upper/lower limit, n + 3, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, one-way ANOVA with Tukey's Multiple Comparison Test.

  3. C ChIP experiments of endogenous St3gal5 promoter after immunoprecipitation of Zeb1. Promoter regions (upper panel) were detectable by PCR after cells were treated with TGF-β1 (24 h). In contrast, a control region within intron 1 of St3gal5 (lower panel) was not amplified. ChIP experiments were independently performed twice with similar outcome.

  4. D Schematic representation of St3gal5 3′-UTR constructs. Predicted binding sites for miR-141/200a were mutated as indicated. Controls included empty luciferase plasmid (Luc-empty) and a construct with St3gal5 3′-UTR cloned in reversed orientation (Luc-reverse) (left panel). Cells were transfected with luciferase reporters in conjunction with Ctrl- or Zeb1-siRNA. Activity readings of cells transfected with wild-type plasmid (WT) and Ctrl-siRNAs were arbitrarily set to 1 and used as reference. The plot displays the loss of reporter activity of respective reporter plasmids upon Zeb1 knockdown. Independently, cells were also transfected with Ctrl- or Zeb1-siRNA together with a Ctrl or miRNA inhibitor. Activity readings of cells transfected with Ctrl-siRNA and Ctrl-inhibitor were arbitrarily set to 1 and used as reference. Mean ± SD, n + 3, ***P ≤ 0.001, one-way ANOVA with Tukey's Multiple Comparison Test.

Figure 3
Figure 3
St3gal5, like Zeb1, modulates epithelial cell adhesion

  1. A Western blot of E-cadherin, plakoglobin, desmoplakin 1 and 2 and plakophilin-2 in cells transfected with Zeb1- or St3gal5-siRNA (72 h).

  2. B Hanging-drop assays of Zeb1- and St3gal5-repressed cells. Cells were transfected with siRNAs (48 h) and stimulated with TGF-β1 for another 24 h. Mean ± SD, number of drops analysed per condition + 5, ***P ≤ 0.001, one-way ANOVA with Tukey's Multiple Comparison Test. The assay was independently repeated at least twice with similar outcome.

  3. C Giemsa staining of cells transfected as in (B). Scale bar: 200 μm.

  4. D Electron microscopy of cell–cell contacts in Zeb1- and St3gal5-suppressed cells (72 h). Scale bars: 1,000 nm (left panel), 250 nm (right panel). Total lengths of junctions between adjacent cells were measured in 20 randomly selected areas per condition. **P ≤ 0.01, ***P ≤ 0.001.

Figure 4
Figure 4
Overexpression of St3gal5 mitigates Zeb1-mediated increase in cell adhesion

  1. A qRT–PCR-based analysis of mRNA expression of total St3gal5 in cells stably overexpressing wild-type St3gal5 (pMSCV-St3gal5 wt, upper panel) and inactive St3gal5 (pMSCV-St3gal5 mut, lower panel) compared to control cells (pMSCV) (n + 4). Mean ± upper/lower limit, n + 4, ***P ≤ 0.001, unpaired t-test.

  2. B qRT–PCR-based analysis of mRNA expression of Zeb1 (left) and endogenous St3gal5 (right) upon Zeb1 repression in cells overexpressing wild-type (upper panel) or mutant St3gal5 (lower panel) (72 h). Mean ± upper/lower limit, n + 4, ***P ≤ 0.001, one-way ANOVA with Tukey's Multiple Comparison Test.

  3. C Western blot analysis of E-cadherin, plakoglobin, desmoplakin 1 and 2 and plakophilin-2 in wild-type (left panel) or mutant St3gal5-overexpressing cells (right panel) (72 h).

  4. D Relative quantification of intercellular adhesion by hanging-drop assays in cells overexpressing wild-type (left panel) or mutant St3gal5 (right panel) ± Zeb1 suppression (48 h) ± TGF-β1 stimulation (24 h). Mean ± SD, number of drops analysed per condition + 5, ***P ≤ 0.001, *P ≤ 0.05, one-way ANOVA with Tukey's Multiple Comparison Test. The assay was independently repeated at least twice with similar outcome.

Figure 5
Figure 5
The Zeb1–St3gal5 axis promotes expression of a-series GSLs during TGF-β1-induced EMT-like progression

  1. A Bright-field microscopy of cells treated with TGF-β1 for indicated times. Scale bar: 200 μm.

  2. B TLC-based analyses of neutral and acidic GSLs of NM18 cells treated with TGF-β1 as in (A). TGF-β1-treated cells reveal accumulation of LacCer (†) and decrease of HexCer in the neutral GSL fraction. *indicates no GSL. Expression of acidic a-series GSLs GM3 (′), GM2 (−), GM1a (•) and of 0-series lipid GM1b (º) increased upon TGF-β1 stimulation.

  3. C Repression of Zeb1 or St3gal5 partially impaired TGF-β1-induced accumulation of acidic GSLs of the a-series (GM1a (•), GM2 (−)). Cells were transfected with indicated siRNAs (48 h) and treated with TGF-β1 (24 h). Cells accumulate LacCer (†) and GM1b (º) upon Zeb1 and St3gal5 repression.

  4. D Bright-field microscopy of cells repressed for Zeb1 or St3gal5 (48 h) ± TGF-β1 (24 h). Scale bar: 200 μm.

  5. E Scheme summarizing the effects of Zeb1 on expression of GSLs. Repression of Zeb1 decreases expression of St3gal5 and results in accumulation of LacCer and 0-series GSLs (e.g. GM1b). Concomitantly, levels of a-series GSLs are reduced and cell–cell adhesion is increased. Conversely, Zeb1 promotes the expression of St3gal5 and a-series GSLs (GM3, GM2 and GM1a) during TGF-β1-induced EMT-like progression with alleviating effects on intercellular adhesion. Despite increased expression of St3gal5, TGF-β1-stimulated cells accumulate LacCer (and some GM1b), possibly as a consequence of elevated turnover of HexCer.

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