Epithelial to Mesenchymal Transition (EMT) is an extreme form of cellular plasticity defined by loss of epithelial cell morphology, dissociation of cell-cell contacts, reduction in proteins mediating cell-cell contacts, remodeling of the actin cytoskeleton, de novo expression of α-smooth muscle actin (α-SMA), and acquisition of mesenchymal cell shape [1–4]. During EMT, cells diminish epithelial gene expression and acquire mesenchymal gene expression . Cortical actins, the actin filament bundles below the plasma membrane, reorganize or are lost, while stress fibers comprising F-actin are gained. In normal development, EMT has been associated with processes in gastrulation, heart formation, palate formation, and Mullerian tract regression . In disease states, EMT has been exploited in both cancer and organ fibrosis. The mortality in human cancers is caused by primary tumor cells that have undergone oncogenic EMT and metastasized to other organs. Other diseases, such as end-state organ failure by fibrosis, are caused by repeated and sustained infliction of EMT. Thus, understanding the cellular mechanisms to reverse EMT is of great importance.
The TGF-β signaling pathway is considered a good target for EMT reversal because it is a key mediator of fibrosis and facilitator of metastasis [3, 6]. TGF-β induces EMT by both Smad-dependent and -independent signaling events [4, 7, 8]. TGF-β1 ligand exerts its signaling effects by activating a heteromeric receptor of two transmembrane serine/threonine kinases, type I and type II receptors (TβRI and TβRII) [7, 9]. TβRII transphosphorylates TβRI, activating its kinase function. Activated TβRI then phosphorylates the intracellular proteins Smad2 and Smad3. The phosphorylated Smad2 and Smad3 associate with Smad4, with the activated complex translocating to the nucleus where it interacts with other transcriptional co-activators and co-repressors to regulate expression of numerous genes . This Smad-dependent signaling up-regulates expression of several transcription factors important for EMT induction, including Snail, Slug, Twist, and members of the ZFH family, ZEB1 (also called EF1, TCF8, AREB6, ZFHEP, NIL-2A, ZFHX1A, and BZP) and ZEB2 (also called SIP1, SMADIP1, ZFHX1B, and KIAA0569) [11–13].
Of particular importance are ZEB1 and ZEB2 because they are crucial regulators of EMT during embryonic development and cancer [14, 15]. These transcription factors activate EMT by binding to E-box elements present in the E-cadherin promoter, suppressing synthesis of this cell-cell adhesion protein [16, 17]. ZEB1 also promotes EMT by repressing expression of basement membrane components and cell polarity proteins [13, 14, 18, 19]. ZEB2 has also been implicated in the induction of EMT . The loss of E-cadherin and other epithelial structural components is a major event during EMT. Mutations in the TCF8 gene (GenBank accession number NM 030751) result in a mesenchymal to epithelial transition (MET) in mouse embryos by reprogramming gene expression, leading to developmental defects by diminishing progenitor cell proliferation and cell migration . Thus, it is crucial to understand the role of ZEB1 and ZEB2 in the reversal of TGF-β-induced EMT.
Multiple signaling proteins in addition to Smads have been implicated in the induction of EMT by TGF-β1. These include Ras/MAPK , integrin β-1, integrin-linked kinase , p38 mitogen-activated protein kinase (p38 MAPK) , RhoA Kinase (ROCK) , phosphatidylinositol 3-OH kinase (PI3K) , Jagged1/Notch , SARA , nuclear factor kappa-B (NF-B) , Par6 [8, 30], and ERK . However, much less is known about how these signaling pathways and transcription factors maintain the mesenchymal program. Studies examining the reversal of EMT by perturbing one component of a signaling pathway with inhibitors or shRNAs demonstrate partial reversal of the mesenchymal state [32, 33].
Here, we report full reversal of EMT morphology and patterns of gene expression by concurrently inhibiting TβRI kinase and ROCK. We show that inhibition of TβRI kinase blocks mesenchymal gene expression, an effect mediated by down-regulation of ZEB1 and ZEB2 levels, while the ROCK inhibitor stabilizes the epithelial structure. These findings demonstrate that combined use of TβRI kinase and ROCK inhibitors is important to decrease TGF-β signaling to enable full reversal of EMT.