Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway.
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.
Das
Shreyas
S
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706, USA. shreyasidas@wisc.edu
Becker
Bryan N
BN
Hoffmann
F Michael
FM
Mertz
Janet E
JE
D000818:Animals; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D016601:RNA-Binding Proteins; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D000071799:Zinc Finger E-box-Binding Homeobox 1; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*
D015820:Cadherins; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051741:Kruppel-Like Transcription Factors; D047428:Protein Kinase Inhibitors; D016601:RNA-Binding Proteins; C485848:Snip1 protein, mouse; D016212:Transforming Growth Factor beta; C519422:ZEB1 protein, mouse; D000071799:Zinc Finger E-box-Binding Homeobox 1; D054460:rho-Associated Kinases