Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Abstract

Background: Epithelial to Mesenchymal Transition (EMT) induced by Transforming Growth Factor-beta (TGF-beta) is an important cellular event in organogenesis, cancer, and organ fibrosis. The process to reverse EMT is not well established. Our purpose is to define signaling pathways and transcription factors that maintain the TGF-beta-induced mesenchymal state.

Results: Inhibitors of five kinases implicated in EMT, TGF-beta Type I receptor kinase (TbetaRI), p38 mitogen-activated protein kinase (p38 MAPK), MAP kinase kinase/extracellular signal-regulated kinase activator kinase (MEK1), c-Jun NH-terminal kinase (JNK), and Rho kinase (ROCK), were evaluated for reversal of the mesenchymal state induced in renal tubular epithelial cells. Single agents did not fully reverse EMT as determined by cellular morphology and gene expression. However, exposure to the TbetaRI inhibitor SB431542, combined with the ROCK inhibitor Y27632, eliminated detectable actin stress fibers and mesenchymal gene expression while restoring epithelial E-cadherin and Kidney-specific cadherin (Ksp-cadherin) expression. A second combination, the TbetaRI inhibitor SB431542 together with the p38 MAPK inhibitor SB203580, was partially effective in reversing EMT. Furthermore, JNK inhibitor SP600125 inhibits the effectiveness of the TbetaRI inhibitor SB431542 to reverse EMT. To explore the molecular basis underlying EMT reversal, we also targeted the transcriptional repressors ZEB1 and ZEB2/SIP1. Decreasing ZEB1 and ZEB2 expression in mouse mammary gland cells with shRNAs was sufficient to up-regulate expression of epithelial proteins such as E-cadherin and to re-establish epithelial features. However, complete restoration of cortical F-actin required incubation with the ROCK inhibitor Y27632 in combination with ZEB1/2 knockdown.

Conclusions: We demonstrate that reversal of EMT requires re-establishing both epithelial transcription and structural components by sustained and independent signaling through TbetaRI and ROCK. These findings indicate that combination small molecule therapy targeting multiple kinases may be necessary to reverse disease conditions.

Figure 1

TGF-β1 induces EMT in renal tubular epithelial cells. (A) Phase contrast (left) and Phalloidin staining (right) of mTEC-KO cells incubated for 72 hours as indicated without TGF-β1, with 100 pM TGF-β1, or with TGF-β1 and 10 μM SB431542. Cell morphology was observed by bright field phase microscopy at 100× magnification. Phalloidin was used to detect F-actin at 400× magnification, while DAPI was used to detect nuclei. White arrows point to stress fibers. (B) Immunoblot showing protein expression of E-cadherin, α-SMA, and β-tubulin following incubation of mTEC-KO cells with 100 pM TGF-β1 for the indicated times. A dash indicates non-detectable levels of protein, while a plus sign indicates detectable protein levels over background. (C-E) show quantitative RT-PCR analysis of (C) Ksp-cadherin, (D) MMP-9, and (E) SM22 RNA levels in mTEC-KO cells incubated with TGF-β1 for the indicated times. Asterisks (*) indicate significant differences (P < 0.05, n = 9).

Figure 2

Treatment with a TRβI inhibitor reverses PAI-1 RNA level in TGF-β1-induced mesenchymal renal tubular epithelial cells to levels present in epithelial cells. mTEC-KOs were incubated with 100 pM TGF-β1 ligand for 72 hours, followed by the addition of 5 μM SB431542 plus 100 pM TGF-β1 for 24 hours. Cell lysates were prepared and relative PAI-1 RNA levels were determined by quantitative RT-PCR. Significant differences are indicated with an asterisk (*) (P <0.05, n = 9).

Figure 3

Treatment of mTEC-KO cells with a TβRI inhibitor and a ROCK inhibitor together, but not individually, reverses the mesenchymal actin cytoskeleton stress fibers induced by TGF-β1. mTEC-KO cells were incubated with 100 pM TGF-β1 for 72 hours, kinase inhibitors were added, and incubation was continued for an additional 24 hours. F-actin was visualized by staining with Texas Red-phalloidin. mTEC-KO cells were (A) untreated or treated with (B) 100 pM TGF-β1 for 72 hours followed by (C-G) single kinase inhibitor or (H-K) SB431542 plus a second kinase inhibitor. Single kinase inhibitors and concentrations were as follows: (C) 5 μM SB431542, (D) 1 μM SB203580, (E) 1 μM Y27632, (F) 10 μM U0126, and (G) 15 μM SP600125. Combinations of kinase inhibitors were 5 μM SB431542 with (H) 1 μM SB203580, (I) 1 μM Y27632, (J) 10 μM U0126, or (K) 15 μM SP600125. (L) Combination of 5 μM Y27632 and 5 μM SB203580. White arrows point to stress fibers.

Figure 4

Restoration of epithelial gene expression patterns requires a combination of kinase inhibitors. mTEC-KO cells were incubated with 100 pM TGF-β1 for 72 hours to induce EMT. Afterward, they were incubated for an additional 24 hours with the indicated individual or combination of kinase inhibitors: 5 μM SB431542, 1 μM SB203580, and 1 μM Y27632. (A) Ksp-cadherin, (B) SM22, and (C) MMP-9 mRNA levels were determined by quantitative RT-PCR. Significant differences between the untreated cells (lane 1) versus cells treated with TGF-β1 (lane 2) or TGF-β1 followed by inhibitors (lanes 3-7) are indicated by asterisks (*) (P < 0.05, n = 9).

Figure 5

E-cadherin expression is restored by combined treatment with a TβRI kinase inhibitor and either a ROCK or p38 MAPK inhibitor. A-I Phase contrast microscopy of mTEC-KO cells incubated for 72 hours with 100 pM TGF-β1 followed by the indicated inhibitor(s) for an additional 36 hours prior to fixing and staining with DAPI. (A) untreated control cells; (B) treated only with TGF-β1; treated individually with the inhibitors (C) 5 μM SB431542, (D) 5 μM SB203580, (E) 5 μM Y27632, or (F) 15 μM SP600125; and treated together with 5 μM SB431542 plus (G) 5 μM SB203580, (H) 5 μM Y27632, or (I) 15 μM SP600125. (J) Immunoblot analysis of cell lysates obtained from the above-treated mTEC-KOs for the presence of E-cadherin. β-tubulin served as a control.

Figure 6

Restoration of E-cadherin expression in NMuMG cells is dependent on levels of ZEB1 and ZEB2. NMuMG cells were incubated for 48 hours with 100 pM TGF-β1 to induce EMT. Thereafter, the cells were incubated for an additional 24 hours with the indicated inhibitors: (lane 3), 1 μM SB431542; (lane 4), 1 μM Y27632; (lane 5), 1 μM SB431542 and 10 μM SP600125; and (lane 6), 1 μM SB431542 and 1 μM Y27632. NMuMG cells were also infected with shRNAmir-targeting ZEB1 and ZEB2 lentiviruses followed by incubation for an additional 24 hours without TGF-β1 (lane 7), with 100 pM TGF-β1 (lane 8), or with 100 pM TGF-β1 plus 1 μM Y27632 (lane 9). Levels of ZEB1, ZEB2, E-cadherin, and β-actin protein in the cells were assessed by immunoblot analysis. Dash indicates protein present at level below background; plus sign indicates protein present at clearly detectable level. Similar findings were observed in two other independent experiments.

Figure 7

ZEB1 and ZEB2 depletion by shRNAs in NMuMG cells attenuates F-actin stress fibers. Cells were treated as indicated with 100 pM TGF-β1 for 48 hours followed by an additional 72 hour incubation with a lentivirus encoding pLKO.1 (a control shRNA) or lentiviruses encoding shRNAs against ZEB1 and ZEB2, with 1 μM Y27632 or 10 μM SP600125 added during the last 24 hour incubation with the shRNAs. F-actin was visualized by phalloidin. Cells were viewed at a 400× magnification. White arrows point to stress fibers.

Figure 8

Model for reversal of EMT induced by TGF-β1. To re-express epithelial proteins such as E-cadherin, a TβRI kinase inhibitor is needed to decrease expression of mesenchymal genes (e.g., ZEB1 and ZEB2), while a Rho kinase inhibitor is required to stabilize the epithelial cortical actin. A p38 MAPK inhibitor may also be useful as this drug in conjunction with the TβRI kinase inhibitor can further EMT reversal by reducing stress fiber actin formation.