Tie1 deficiency induces endothelial-mesenchymal transition

Abstract

Endothelial-mesenchymal transition (EndMT) has a significant role in embryonic heart formation and in various pathologies. However, the molecular mechanisms that regulate EndMT induction remain to be elucidated. We show that suppression of receptor tyrosine kinase Tie1 but not Tie2 induces human endothelial cells to undergo EndMT and that Slug deficiency reverts this process. We find that Erk1/2, Erk5 and Akt cascades control Slug promoter activity induced by Tie1 deficiency. Interestingly, EndMT is present in human pancreatic tumour. We propose that EndMT associated with Tie1 downregulation participates in the pathological development of stroma observed in tumours.

Figure 1

Tie1 silencing in pHMVECs induces endothelial–mesenchymal transition. (A) Tie1 (left panel) or Tie2 (right panel) siRNA suppresses mRNA and protein expression. pHMVECs were transfected twice with Tie1A or Tie1B or Tie2 or Ctrl siRNA. mRNA and protein levels were quantified by real-time PCR and western blotting, respectively. (B) Tie1 silencing, but not Tie2, induces morphological changes. (C) Tie1 silencing decreases the mRNA (left panels) and protein (right panels) expressions of vascular endothelial markers (CD31, VE-cadherin, CD34, FVIII) and increases the mRNA (left panels) and protein (right panels) expressions of mesenchymal markers (αSMA, S100A4, Col1a1, SM22α, N-cadherin). (D) Tie2 silencing does not modify the expression of vascular endothelial and mesenchymal markers. TBP was used as internal Ctrl for real-time PCR and β-tubulin as loading Ctrl for western blotting analysis. Results are from triplicates of three different experiments. Significant differences are indicated by solid lines (^*P<0.03, ^**P<0.003 by t-test). αSMA, α-smooth muscle actin; Col1a1, collagen type I α1; Ctrl, control; FVIII, coagulation factor VIII; mRNA, messenger RNA; pHMVEC, primary human microvascular endothelial cell; siRNA, short-interfering RNA; TBP, TATA-box binding protein.

Figure 2

Tie1 silencing-induced endothelial–mesenchymal transition is Slug dependent. (A) Tie1 silencing increases Slug expression. HMVECs were transfected with Tie1 or Ctrl siRNA. Slug and Snail mRNA levels and protein expressions were quantified by real-time PCR or western blotting. (B) Slug deficiency prevented the morphological changes induced by Tie1 silencing. (C) Slug siRNA abrogated or partially reverted the modifications induced by Tie1 silencing on the expression of vascular endothelial markers (VE-cadherin, CD31) and mesenchymal markers (Col1a1, S100A4). HMVECs were transfected with Tie1 or/and Slug or Ctrl. mRNA level (left panels) and protein expression (right panels) were quantified by real-time PCR or western blotting. TBP was used as internal Ctrl for real-time PCR and β-tubulin as loading Ctrl for western blotting analysis. Results are from triplicates of three different experiments. (D) Tie1 silencing increases pHMVEC migration and Slug siRNA partially reverts this effect. pHMVECs were transfected with Tie1 siRNA or/and Slug siRNA or Ctrl and were allowed to migrate in the presence or absence of PDGF in a modified Boyden chamber assay. Migration scores were measured for three fields. Similar results were obtained in three different experiments. Significant modifications are indicated by solid lines (^*P<0.03, ^**P<0.003 by t-test). Col1a1, collagen type I α 1; Ctrl, control; HMVEC, human microvascular endothelial cell; mRNA, messenger RNA; pHMVEC, primary HMVEC; PDGF, platelet-derived growth factor β; siRNA, short-interfering RNA; TBP, TATA-box binding protein.

Figure 3

Tie1 silencing increases Erk1/2, Erk5, Akt phosphorylation and Slug expression. Effect of Tie1 silencing on Erk1/2, Erk5, Akt phosphorylation (A) and on Slug mRNA (B, upper panel) and protein (B, lower panel). HMVECs were transfected with Tie1 or control siRNA and were treated for 24 h with MAPK (U0126) or/and Akt (LY-294,002) inhibitors. Protein expression was quantified by western blotting. β-tubulin, Erk1/2, Erk5 and Akt were used as loading controls. TBP was used as internal control for real-time PCR. Results are from triplicates of three different experiments. (C,D) Effect of Tie1 deficiency on the human Slug promoter. HMVECs were treated for 24 h with MAPK (U0126) or/and Akt (LY-294,002) inhibitors (C) or with Erk1/2 and Erk5 siRNAs (D). Thereafter, cells were co-transfected with Tie1 or control siRNA and pGL3-human Slug promoter plasmid. After normalization, the luciferase activity was expressed as fold induction compared with the promoterless plasmid (pGL3-basic vector) expression. In D, as shown in the western blot insets, Erk1/2 or Erk5 siRNA induce decreases in Erk1/2 or Erk5 protein levels. Similar results were obtained in three different experiments. Significant modifications are indicated by solid lines (^**P<0.003 by t-test). Erk 1/2, 5, extracellular signal-regulated kinase 1/2, 5; HMVEC, human microvascular endothelial cell; MAPK, mitogen-activated protein kinase; mRNA, messenger RNA; siRNA, short-interfering RNA; TBP, TATA-box binding protein.

Figure 4

EndMT is present in human pancreatic tumours and not in human pancreas. (A) Confocal microscopy of immunofluorescence triple labelling to endothelial marker (CD31; blue), mesenchymal markers (αSMA, S100A4, SM22α, FAP, N-cadherin red) and Tie2 (green) were performed in human pancreatic adenocarcinoma tissues. Coexpression of CD31⁺/αSMA⁺ or CD31⁺/S100A4⁺ or CD31⁺/N-cadherin⁺ or CD31⁺/FAP⁺ or CD31⁺/SM22α⁺ are detectable with Tie2 (white arrow). (B) Confocal microscopy of immunofluorescence double labelling to endothelial marker (CD31; green), mesenchymal marker (αSMA, red) were performed on non-tumoural pancreas tissues. Pictures display representative photomicrographs. Scale bars, 10 μm (inset) or 20 μm. αSMA, α-smooth muscle actin; EndMT, endothelial–mesenchymal transition; FAP, fibroblast activation protein; N-CAD, N-cadherin.

Figure 5

EndMT is not associated with Tie1 expression in human pancreatic tumours. Confocal microscopy of immunofluorescence triple labelling to endothelial marker (CD31; blue), mesenchymal markers (αSMA, S100A4, SM22α, FAP, N-cadherin; red) and Tie1 (green) were performed on human pancreatic adenocarcinoma tissues. Coexpression of CD31⁺/Tie1⁺ is detectable but never in association with mesenchymal marker. Pictures display representative photomicrographs. Scale bars, 10 μm (inset) or 20 μm. αSMA, α-smooth muscle actin; EndMT, endothelial–mesenchymal-transition; FAP, fibroblast activation protein; N-CAD, N-cadherin.