TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses

Through the action of its membrane-bound type I receptors, transforming growth factor-beta (TGF-beta) elicits a wide range of cellular responses that regulate cell proliferation, differentiation and apoptosis. Many of the signaling responses induced by TGF-beta are mediated by Smad proteins, but certain evidence has suggested that TGF-beta can also signal independently of Smads. We found in mouse mammary epithelial (NMuMG) cells, which respond to TGF-beta treatment in multiple ways, that TGF-beta-induced activation of p38 MAP kinase is required for TGF-beta-induced apoptosis, epithelial-to-mesenchymal transition (EMT), but not growth arrest. We further demonstrated that activation of p38 is independent of Smads using a mutant type I receptor, which is incapable of activating Smads but still retains the kinase activity. This mutant receptor is sufficient to activate p38 and cause NMuMG cells to undergo apoptosis. However, it is not sufficient to induce EMT. These results indicate that TGF-beta receptor signals through multiple intracellular pathways and provide first-hand biochemical evidence for the existence of Smad-independent TGF-beta receptor signaling.     

TGF-beta induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors

The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E- cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta- mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.     

Downregulation of Par-3 expression and disruption of Par complex integrity by TGF-beta during the process of epithelial to mesenchymal transition in rat proximal epithelial cells

Epithelial to mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis and the initial step is disruption of cell junction and cell polarity. TGF-beta has been demonstrated as the most important mediator of EMT which is sufficient to initiate and complete the whole course of EMT, however, the detailed mechanism of TGF-beta in modulating the disruption of cell junction still remains unclear. Par-3 is a component of Par complex which plays a crucial role in the establishment and maintenance of epithelial polarity. In this study, we found that TGF-beta treatment resulted in a dose- and time-dependent downregulation of Par-3 protein together with the suppression of E-cadherin expression and induction of alpha-SMA. The decreased Par-3 subsequently resulted in the redistribution of Par-6-aPKC complex from cell membrane to cytoplasm. Forced expression of exogenous Par-3 into rat proximal epithelial cells (NRK52E) led to a drastic blockage of TGF-beta1-induced E-cadherin suppression and alpha-SMA induction. In contrast, knockdown Par-3 expression by siRNA significantly enhanced TGF-beta1-induced E-cadherin suppression and alpha-SMA induction. These data indicate that downregulation of Par-3 and subsequent disruption of Par complex integrity might be one mechanism that TGF-beta destroys cell polarity during EMT.     

Snail is required for transforming growth factor-beta-induced epithelial-mesenchymal transition by activating PI3 kinase/Akt signal pathway

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. We have previously shown that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and more specifically, on signaling via Smad3. In this report, we suggest phosphatidylinositol 3-OH kinase (PI3K)/Akt signaling is also necessary for TGF-beta-induced EMT in lens epithelial cells by showing that LY294002, an inhibitor of the p110 catalytic subunit of PI3K, blocked the expression of alpha-smooth muscle actin (alpha-SMA) and morphological changes. We also identify Snail as an effector of TGF-beta-induced EMT. Snail has been shown to be a mediator of EMT during metastasis of cancer. We show that Snail is an immediate-early response gene for TGF-beta and the proximal Snail promoter is activated by TGF-beta through the action of Smad2, 3, and 4. We show that antisense inhibition of Snail expression blocks TGF-beta-induced EMT and furthermore Akt activation. All of these findings suggest that Snail participates in TGF-beta-induced EMT by acting upstream of Akt activation.     

Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor beta and bone morphogenetic protein

Transforming growth factors beta (TGF-betas) inhibit growth of epithelial cells and induce differentiation changes, such as epithelial-mesenchymal transition (EMT). On the other hand, bone morphogenetic proteins (BMPs) weakly affect epithelial cell growth and do not induce EMT. Smad4 transmits signals from both TGF-beta and BMP pathways. Stimulation of Smad4-deficient epithelial cells with TGF-beta 1 or BMP-7 in the absence or presence of exogenous Smad4, followed by cDNA microarray analysis, revealed 173 mostly Smad4-dependent, TGF-beta-, or BMP-responsive genes. Among 25 genes coregulated by both factors, inhibitors of differentiation Id2 and Id3 showed long-term repression by TGF-beta and sustained induction by BMP. The opposing regulation of Id genes is critical for proliferative and differentiation responses. Hence, ectopic Id2 or Id3 expression renders epithelial cells refractory to growth inhibition and EMT induced by TGF-beta, phenocopying the BMP response. Knockdown of endogenous Id2 or Id3 sensitizes epithelial cells to BMP, leading to robust growth inhibition and induction of transdifferentiation. Thus, Id genes sense Smad signals and create a permissive or refractory nuclear environment that defines decisions of cell fate and proliferation.     

Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenotype

Transforming growth factor-beta (TGF-beta) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-beta induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC-MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-beta-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-beta1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.     

Rho activation is required for transforming growth factor-beta-induced epithelial-mesenchymal transition in lens epithelial cells

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. We have recently shown that TGF-beta-induced EMT in lens epithelial cells depends on PI3 kinase/Akt signal pathway. In this report, we suggest Smad3 is necessary for TGF-beta-induced EMT by showing that the expression of dominant-negative Smad3 blocks the expression of alpha-smooth muscle actin (alpha-SMA) and morphological changes. We also show that TGF-beta induces a biphasic change in Rho activity, and that Y27632, a selective inhibitor of Rho effector ROCK, inhibits TGF-beta-induced EMT in vitro and in vivo. We finally show that Smad3 activation and Rho signal activation is independent each other. All of these findings suggest that Rho/ROCK activation together with Smad3 is necessary for TGF-beta-induced EMT in lens epithelial cells.     

Cooperation between snail and LEF-1 transcription factors is essential for TGF-beta1-induced epithelial-mesenchymal transition

Transforming growth factor beta 1 (TGF-beta1) has been shown to induce epithelial-mesenchymal transition (EMT) during various stages of embryogenesis and progressive disease. This alteration in cellular morphology is typically characterized by changes in cell polarity and loss of adhesion proteins such as E-cadherin. Here we demonstrate that EMT is associated with loss of claudin-1, claudin-2, occludin, and E-cadherin expression within 72 h of exposure to TGF-beta1 in MDCKII cells. It has been suggested that this expression loss occurs through TGF-beta1 in a Smad-independent mechanism, involving MEK and PI3K pathways, which have previously been shown to induce expression of the Snail (SNAI-1) gene. Here we show that these pathways are responsible for loss of tight junctions and a partial loss of E-cadherin. However, our results also demonstrate that a complete loss of E-cadherin and transformation to the mesenchymal phenotype are dependent on Smad signaling, which subsequently stimulates formation of beta-catenin/LEF-1 complexes that induce EMT.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.