Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling - PubMed (original) (raw)

Transforming growth factor-β2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling

Damian Medici et al. Biochem J. 2011.

Abstract

EndMT (endothelial-mesenchymal transition) is a critical process of cardiac development and disease progression. However, little is know about the signalling mechanisms that cause endothelial cells to transform into mesenchymal cells. In the present paper we show that TGF-β2 (transforming growth factor-β2) stimulates EndMT through the Smad, MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase], PI3K (phosphinositide 3-kinase) and p38 MAPK signalling pathways. Inhibitors of these pathways prevent TGF-β2-induced EndMT. Furthermore, we show that all of these pathways are essential for increasing expression of the cell-adhesion-suppressing transcription factor Snail. Inhibition of Snail with siRNA (small interfering RNA) prevents TGF-β2-induced EndMT. However, overexpression of Snail is not sufficient to cause EndMT. Chemical inhibition of GSK-3β (glycogen synthase kinase-3β) allows EndMT to be induced by Snail overexpression. Expression of a mutant Snail protein that is resistant to GSK-3β-dependent inactivation also promotes EndMT. These results provide the foundation for understanding the roles of specific signalling pathways in mediating EndMT.

© The Authors Journal compilation © 2011 Biochemical Society

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Figures

Figure 1

Figure 1. TGF-β2 activates Smad, MEK, PI3K, and p38 MAPK signaling pathways

(A) p3TP-Lux reporter gene assay showing increased Smad activity upon treatment of HCMECs with TGF-β2. Expression of a dominant negative Smad4 (DN-Smad4) inhibited this increased activity. Data represent mean (n=3) ± SD; *P<0.01 for TGF-β2 compared to vehicle; **P<0.05 for TGF-β2 + DN-Smad4 compared to TGF-β2. (B–D) Immunoblotting for phosphorylation levels of ERK1/2 (B), AKT (C), and p38 MAPK (D) showing that TGF-β2 increases phosphorylation of these kinases. Chemical inhibitors against MEK1/2 (U0126; 10μM), PI3K (LY294002; 50μM), and p38 (SB202190; 25μM) inhibit the increases in ERK1/2, AKT, and p38 MAPK phosphorylation, respectively.

Figure 2

Figure 2. TGF-β2 promotes EndMT through Smad-dependent and Smad-independent signaling

(A) DIC imaging showing a change in cell morphology consistent with EndMT in HCMEC cultures treated with TGF-β2. Inhibitors against Smad4 (DN-Smad4), MEK1/2 (U0126; 10μM), PI3K (LY294002; 50μM), or p38 MAPK (SB202190; 25μM) prevented the TGF-β2-induced change in morphology. Scale bar, 20μm. (B) Real-time quantitative PCR analysis showing that TGF-β2 increases Snail gene expression, which is prevented by inhibitors of Smad4, MEK1/2, PI3K, or p38 MAPK. Data represent mean (n=3) ± SD; *P<0.01 for TGF-β2 compared to vehicle; **P<0.01 for all TGF-β2 + inhibitors compared to TGF-β2. (C) Immunoblotting showing that TGF-β2 decreases expression of VE-cadherin and CD31, and increases expression of FSP-1, α-SMA, and Snail. Inhibitors of Smad4, MEK1/2, PI3K, or p38 MAPK prevent these expression changes.

Figure 3

Figure 3. Snail activity is essential for TGF-β2-induced EndMT

(A) DIC imaging showing change in cell morphology in cultures transfected with control siRNA treated with TGF-β2. No EndMT was observed in cultures transfected with Snail siRNA. Scale bar, 20μm. (B) Immunoblotting showing that Snail siRNA inhibits TGF-β2-induced expression changes in VE-cadherin, CD31, FSP-1, and α-SMA.

Figure 4

Figure 4. Snail expression is not sufficient to induce EndMT

(A) DIC imaging showing no effect of snail over-expression on cell morphology. Scale bar, 20μm. (B) Immunoblotting confirming a dramatic increase in Snail gene expression in cells transfected with the Snail expression construct. No significant changes in expression of the endothelial markers VE-cadherin and CD31 or the mesenchymal markers FSP-1 and α-SMA were observed.

Figure 5

Figure 5. Inhibition of GSK-3β allows Snail-induced EndMT

(A) Immunoblotting showing increased phosphorylation of GSK-3β in endothelial cells treated with TGF-β2. Inhibition of PI3K with LY294002 (50μM) is sufficient to block GSK-3β phosphorylation induced by TGF-β1. (B) Immunoblotting demonstrating no phosphorylation of GSK-3β when over-expressing Snail. Lithium chloride (LiCl) is sufficient to induce phosphorylation of GSK-3β in cells transfected with pcDNA3 or pcDNA3-Snail plasmids. Snail expression is increased in cells transfected with pcDNA3-Snail and treated with LiCl. (C) DIC imaging showing that the GSK-3β inhibitor lithium chloride (LiCl) is sufficient to transform endothelial cells transfected with pcDNA3-Snail to mesenchyme. Scale bar, 20μm. (D) Immunoblotting confirming expression of Snail, decreased expression of endothelial markers VE-cadherin and CD31, and increased expression of mesenchymal markers FSP-1 and α-SMA in cells containing pcDNA3-Snail and treated with LiCl.

Figure 6

Figure 6. Induction of EndMT by a GSK-3β-resistant mutant form of Snail

(A) DIC imaging demonstrating EndMT of cells transfected with a mutant GSK-3β-resistant Snail (Snail-6SA) construct. Scale bar, 20μm. (B) Immunoblotting showing decreased expression of endothelial markers (VE-cadherin, CD31) and increased expression of mesenchymal markers (FSP-1, α-SMA) in cells expressing mutant Snail, but not wild-type Snail (Snail-WT).

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