Functional cooperation between Snail1 and twist in the regulation of ZEB1 expression during epithelial to mesenchymal transition

Abstract

Snail1 and Zeb1 are E-cadherin-transcriptional repressors induced during epithelial mesenchymal transition (EMT). In this article we have analyzed the factors controlling Zeb1 expression during EMT. In NMuMG cells treated with TGF-β, Snail1 RNA and protein are induced 1 h after addition of the cytokine preceding Zeb1 up-regulation that requires 6-8 h. Zeb1 gene expression is caused by increased RNA levels but also by enhanced protein stability and is markedly dependent on Snail1 because depletion of this protein prevents Zeb1 protein and RNA up-regulation. In addition to Snail1, depletion of the Twist transcriptional factor retards Zeb1 stimulation by TGF-β or decreases Zeb1 expression in other cellular models indicating that this factor is also required for Zeb1 expression. Accordingly, Snail1 and Twist cooperate in the induction of Zeb1: co-transfection of both cDNAs is required for the maximal expression of ZEB1 mRNA. Unexpectedly, the expression of Snail1 and Twist shows a mutual dependence although to a different extent; whereas Twist depletion retards Snail1 up-regulation by TGF-β, Snail1 is necessary for the rapid increase in Twist protein and later up-regulation of Twist1 mRNA induced by the cytokine. Besides this effect on Twist, Snail1 also induces the nuclear translocation of Ets1, another factor required for Zeb1 expression. Both Twist and Ets1 bind to the ZEB1 promoter although to different elements: whereas Ets1 interacts with the proximal promoter, Twist does it with a 700-bp sequence upstream of the transcription start site. These results indicate that Snail1 controls Zeb1 expression at multiple levels and acts cooperatively with Twist in the ZEB1 gene transcription induction.

FIGURE 1.

Snail1 expression is required for Zeb1 induction by TGF-β in NMuMG cells. Panel A, NMuMG cells were incubated with TGF-β (5 ng/ml) for the indicated times; protein extracts were prepared or RNA was isolated and analyzed by Western blot or qRT-PCR. The left panel shows a representative Western blot of three experiments; the central panel, the average ± S.D. of the results of the densitometric analysis of the results of the three experiments performed. Values are referred to the values obtained at 1 h (Snail1) or 6 h (Zeb1). The right panel presents the average ± S.D. of three independent experiments. Panel B, NMuMG cells, transfected with shRNA specific for Snail1 (mshRNA, see supplemental “Methods”) or a scrambled control were incubated with TGF-β and expression of the indicated genes were analyzed by Western blot or RT-PCR. The figure shows a representative Western blot or the average ± S.D. of three experiments (B). Panel C, NMuMG cells were transfected with pcDNA3-Zeb1 and pcDNA3-Snail1-HA or an empty plasmid when indicated. Cells were also incubated with TGF-β for 24 h previously to the CHX addition. Cell medium was supplemented with CHX to block protein synthesis and protein extracts were prepared after the indicated times and analyzed by Western blot. A 65% inhibition of Zeb1 RNA induction was obtained at 12 h using a murine siRNA against Snail1 instead of the shRNA presented in this figure.

FIGURE 2.

Snail1 controls Zeb1 mRNA levels. Panels A and B, RNAs were prepared from the indicated cell lines stably transfected with Snail1-HA or infected with a retrovirus expressing human SNAIL1 shRNA (hshRNA1) or scrambled control shRNA. Expression of the indicated genes was determined by qRT-PCR. Values are presented as fold-stimulation by Snail1 with respect to control cells (transfected with the empty plasmid) (A) or as percentage of expression with respect to cells infected with the control shRNA (B). 45 and 55% inhibition of Zeb1 expression was obtained with another human SNAIL1 shRNA (hshRNA2) in MiaPaca-2 and SW-620, respectively. Panel C, activity of the 1004/+29 ZEB1 promoter was determined after transfection of the indicated cell lines stably expressing Snail1-HA or treated with TGF-β for 48 h. Triplicates were systematically included and experiments were repeated at least three times. The figure shows the average ± S.D. of 3–5 experiments. Values obtained for Snail1-expressing cells were different from the corresponding control with a p < 0.01 in RWP1 cells and p < 0.05 in SW-480 and NMuMG cells; differences between NMuMG and TGF-β-treated NMuMG cells were also statistically different (p < 0.05). Panels D and E, RNA was prepared from RWP1, RWP1-Snail1 (D), or NMuMG cells treated 1 or 24 h with TGF-β (E). When indicated, Snail1 expression was inhibited in NMuMG cells using a specific siRNA (msiRNA). Expression of miR-200 was determined as indicated under “Experimental Procedures.” Absence of amplification of DNA was verified as control. Analysis of Pumilio RNA was carried out to determine that equal amounts of RNA were used.

FIGURE 3.

Twist and Snail1 cooperate in Zeb1 expression. 5 μg of TWIST1 hshRNA1 or Non-Target Control Vectors were transfected to RWP-1 Snail1 (panel A), SW-620 or MiaPaca-2 cells (panel B). Transfected cell populations were selected with puromycin (2.5 μg/ml), and analyzed by quantitative (A) or semi-quantitative RT-PCR (B). Similar results were obtained with another TWIST1 shRNA (hshRNA2) in RWP-1 Snail1 cells that inhibited ZEB1 expression by 55%. Panel C, RWP-1 cells were transfected with expression plasmids containing Twist or Snail1 cDNAs; cell populations were selected with G418 and analyzed by RT-PCR for the expression of the indicated genes. Panel D, RWP-1 cells were transiently transfected with the indicated cDNAs and pGL3-ZEB1 promoter. Luciferase activity was determined after 48 h. The figure shows the average ± S.D. of the results (D) or a representative experiment of three performed (A, B, and C).

FIGURE 4.

Snail1 and Twist mutually regulate their expression in NMuMG cells. Panels A and B, NMuMG cells expressing scrambled, or shRNAs specific for Snail1 or Twist1 were incubated with TGF-β for the indicated times; protein extracts were prepared or RNA was isolated and analyzed by Western blot (WB) (A) or qRT-PCR (B). Panels C and D, Twist1 RNA (C) and protein (D) were analyzed in RWP-1 or HT-29 M6 cells transfected with Snail1-HA or control plasmid. In panel E, NMuMG cells treated with TGF-β for 24 h when indicated, or RWP1 cells ectopically expressing Snail1 or control plasmid were supplemented with CHX to block protein synthesis. Protein extracts were prepared after the indicated times and analyzed by Western blot. The figure shows representative results (A and D) or the average ± range (B, C, and E) of two experiments performed.

FIGURE 5.

Twist and Ets1 interact with different elements in Zeb1 promoter. Panels A and E, analysis of Twist (A) or Ets1 (E) binding to the indicated elements in the ZEB1 promoter was performed by ChIP as described under “Experimental Procedures.” Panel B, promoter activity of −1004/+29 or −147/+29 DNA fragments was determined as before in SW-620, RWP-1 control, or RWP-1 transfected with Snail1. Differences between Snail1-transfected and Snail1 cells were statistically significant with a p < 0.01 for the −1004/+29 promoter and p < 0.05 for the −147/+29 promoter. The difference between the activity of the two promoters was also significant (p < 0.05) in both cell lines. Panel C, SW-620 or RWP-1 Snail1 cells were transfected with control of ETS1-specific shRNA (hshRNA1). Similar effects were obtained with another ETS1 shRNA (hshRNA2) in SW-620 cells that caused a 70% decrease in ETS1 RNA levels. After selection, RNA was obtained and levels of ETS1 and ZEB1 were determined by qRT-PCR. Panel D, nuclear and cytoslic fractions were prepared from NMuMG treated with TGF-β for the indicated times or RWP-1 control and transfected with Snail1. Levels of the indicated proteins were determined in the nuclear (NMuMG) or nuclear and cytosolic fractions (RWP1 cells). Poly(ADP-ribose) polymerase (PARP)-1 and Lamin B were used as controls for the nuclear fraction; pyruvate kinase (PyrK), for the cytosolic fraction. Results presented in this figure correspond to representative results (panel C) or the average ± S.D. of three experiments.

FIGURE 6.

Scheme of the different regulation levels of Zeb1 expression by Snail1 and TGF-β. Up-regulation of Snail1 by TGF-β increases Zeb1 protein acting on different levels: 1) it decreases the expression of miRNA200 that destabilizes Zeb1 RNA; 2) it stimulates Zeb1 protein stability through the inhibition of the function of the still unidentified ubiquitin ligase involved in Zeb1 degradation; and 3) it activates Zeb1 gene transcription. This stimulation is mediated by the activation of two transcriptional factors, Twist and Ets1 that bind to different elements on the Zeb1 promoter. Snail1 also up-regulates Zeb1 transcription at several levels, because it promotes Ets1 translocation to the nucleus, increases Twist protein stability, and is required for the stimulation in Twist1 RNA caused by TGF-β. In addition, full Snail1 up-regulation by TGFβ also requires Twist expression (depicted by a dotted line).