Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a "cuboidal" epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-beta-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.     

Requirement of podocalyxin in TGF-beta induced epithelial mesenchymal transition

Epithelial mesenchymal transition (EMT) is characterized by the development of mesenchymal properties such as a fibroblast-like morphology with altered cytoskeletal organization and enhanced migratory potential. We report that the expression of podocalyxin (PODXL), a member of the CD34 family, is markedly increased during TGF-β induced EMT. PODXL is enriched on the leading edges of migrating A549 cells. Silencing of podocalyxin expression reduced cell ruffle formation, spreading, migration and affected the expression patterns of several proteins that normally change during EMT (e.g., vimentin, E-cadherin). Cytoskeleton assembly in EMT was also found to be dependent on the production of podocalyin. Compositional analysis of podocalyxin containing immunoprecipitates revealed that collagen type 1 was consistently associated with these isolates. Collagen type 1 was also found to co-localize with podocalyxin on the leading edges of migrating cells. The interactions with collagen may be a critical aspect of podocalyxin function. Podocalyxin is an important regulator of the EMT like process as it regulates the loss of epithelial features and the acquisition of a motile phenotype.     

MMP-2 expression by fibroblasts is suppressed by the myofibroblast phenotype

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-β, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-β, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.     

The myofibroblast, cadherin, alpha smooth muscle actin and the collagen effect

In granulation tissue the myofibroblast, a specialized fibroblast characterized by cytoplasmic stress fibres with alpha smooth muscle actin (SMA), develops from mechano-tension between cells. In vitro the myofibroblast phenotype can be induced in the absence of obvious tension by plating human dermal fibroblasts at low density (LD). Upon reaching confluence the LD-plated cells express alpha SMA within stress fibres. In contrast, few cells express alpha SMA when those same fibroblasts are plated at high density (HD). Cadherins, trans-membrane proteins, and link cells tie the cytoskeletal stress fibres of neighbouring cells together. By immunohistology myofibroblasts (LD-plated fibroblasts) are shown to express cadherin-11 on their surface and between cells, while HD-plated fibroblasts expressed less cadherin-11 on their surface. Western blot analysis revealed elevated concentrations of cadherin-11 and alpha SMA in confluent LD-plated cell lysates. Reduced amounts of both proteins were found in confluent HD-plated cell lysates. Plating fibroblasts on collagen inhibits alpha SMA synthesis. When confluent LD-plated myofibroblasts were covered with a collagen lattice for 24 h, both the expression of cadherin-11 and alpha SMA were reduced by 50%. There is the possibility that direct linkage of the cytoskeleton stress fibres by cell surface cadherins maintains tension between neighbouring cells, which induces alpha SMA expression in stress fibres. Cell contact with collagen reduces cadherin expression, which may eliminate the generation of mechano-tension between fibroblasts. The other possibility is that the myofibroblast phenotype may be induced by factors other than mechano-tension.     

Proteome analysis of human mesothelial cells during epithelial to mesenchymal transitions induced by shed menstrual effluent

Peritoneal endometriosis is the result of ectopic implantation and growth of endometrium tissue that has been regurgitated into the abdominal cavity during menstruation. We have previously shown that menstrual effluent induces epithelial to mesenchymal transitions (EMT) in mesothelial cells, which results in cell retraction and exposure of submesothelial extracellular matrix. Since endometrial tissue preferentially adheres to the extracellular matrix, adhesion of endometrial tissue to the peritoneum is facilitated. The EMT were shown to be associated with differential expression and phosphorylation of mesothelial proteins. Using radiolabeling and proteomics we detected changes in protein expression and phosphorylation that occur in mesothelial cells during the EMT process. The identity of 73 proteins, which were obtained from 324 analyzed spots, was confirmed. The expression of 35 proteins involved in organization of the cytoskeleton, signal transduction, regulation of the redox state, and production of ATP, was altered during the EMT process. Four of the identified proteins were differentially phosphorylated: annexin-1, an actin-binding protein and a substrate for receptor tyrosine kinases; tropomyosin-alpha, a regulator of actin filament stability and cell shape; elongation factor 1 delta; ATP synthase beta-chain. In conclusion, factors from menstrual effluent induce specific changes in the expression and phosphorylation status of structural, regulatory and metabolic proteins relevant to the complex process of EMT in mesothelial cells.     

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Proteinase-activated receptors (PARs; PAR_1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR₄, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR₄ stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population.

Methods

EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells).

Results

Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR₄ agonist peptide (AYPGKF-NH₂, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR₄ stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR₄-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR₄-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR₄-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR₄ stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor.

Conclusion

PAR₄ stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.     

The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition

The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.     

DISRUPTION OF CELL—CELL ADHESION IN THE PRESENCE OF SODIUM BUTYRATE ACTIVATES EXPRESSION OF THE 92kDa TYPE IV COLLAGENASE IN MDCK CELLS

We have investigated the effects of altered cell shape on the regulation of the 92kDa type IV collagenase. In MDCK cells, anti-E-cadherin antibodies alter cell shape by disrupting normal cell—cell contacts, while sodium butyrate causes a marked flattening and spreading of cells. The disruption of cell—cell contacts led to a faint expression of the 92kDa collagenase. This effect was enhanced by sodium butyrate, which by itself did not induce collagenase expression. In contrast, stromelysin expression was not regulated in these conditions. Although mRNA expression was enhanced, the secreted collagenase activity was not altered in these conditions in either cell line. Examination of cytoskeletal and extracellular matrix proteins and cell—cell and cell—matrix adhesion proteins by immunofluorescence and Western blot revealed a disruption of the actin network, tight junctions, and fibronectin deposition by anti E-cadherin antibodies, and alterations in actin, cytokeratin 8, cytokeratin 14, laminin and β1 integrin induced by sodium butyrate. Thus, the induction of collagenase expression in epithelial cells by disrupted cell—cell adhesion and sodium butyrate is associated with changes in cell shape and structure.     

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity.     

Mechanical stretch induces epithelial-mesenchymal transition in alveolar epithelia via hyaluronan activation of innate immunity

Epithelial injury is a central event in the pathogenesis of many inflammatory and fibrotic lung diseases like acute respiratory distress syndrome, pulmonary fibrosis, and iatrogenic lung injury. Mechanical stress is an often underappreciated contributor to lung epithelial injury. Following injury, differentiated epithelia can assume a myofibroblast phenotype in a process termed epithelial to mesenchymal transition (EMT), which contributes to aberrant wound healing and fibrosis. We demonstrate that cyclic mechanical stretch induces EMT in alveolar type II epithelial cells, associated with increased expression of low molecular mass hyaluronan (sHA). We show that sHA is sufficient for induction of EMT in statically cultured alveolar type II epithelial cells and necessary for EMT during cell stretch. Furthermore, stretch-induced EMT requires the innate immune adaptor molecule MyD88. We examined the Wnt/β-catenin pathway, which is known to mediate EMT. The Wnt target gene Wnt-inducible signaling protein 1 (wisp-1) is significantly up-regulated in stretched cells in hyaluronan- and MyD88-dependent fashion, and blockade of WISP-1 prevents EMT in stretched cells. In conclusion, we show for the first time that innate immunity transduces mechanical stress responses through the matrix component hyaluronan, and activation of the Wnt/β-catenin pathway.     

Bone marrow mesenchymal/fibroblastic stromal cells induce a distinctive EMT-like phenotype in AML cells

The development of epithelial-to-mesenchymal transition (EMT) like features is emerging as a critical factor involved in the pathogenesis of acute myeloid leukaemia (AML). However, the extracellular signals and the signalling pathways in AML that may regulate EMT remain largely unstudied. We found that the bone marrow (BM) mesenchymal/fibroblastic cell line HS5 induces an EMT-like migratory phenotype in AML cells. AML cells underwent a strong increase of vimentin (VIM) levels that was not mirrored to the same extent by changes of expression of the other EMT core proteins SNAI1 and SNAI2. We validated these particular pattern of co-expression of core-EMT markers in AML cells by performing an in silico analysis using datasets of human tumours. Our data showed that in AML the expression levels of VIM does not completely correlate with the co-expression of core EMT markers observed in epithelial tumours. We also found that vs epithelial tumours, AML cells display a distinct patterns of co-expression of VIM and the actin binding and adhesion regulatory proteins that regulate F-actin dynamics and integrin-mediated adhesions involved in the invasive migration in cells undergoing EMT. We conclude that the BM stroma induces an EMT related pattern of migration in AML cells in a process involving a distinctive regulation of EMT markers and of regulators of cell adhesion and actin dynamics that should be further investigated. Understanding the tumour specific signalling pathways associated with the EMT process may contribute to the development of new tailored therapies for AML as well as in different types of cancers.     

Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus

Transforming growth factor-beta(1) (TGF-beta(1)) induces alpha-smooth muscle actin (alpha-SMA) and collagen synthesis in fibroblast both in vivo and in vitro and plays a significant role in tissue repair and the development of fibrosis. During these processes the fibroblasts differentiate into activated fibroblasts (so called myofibroblasts), characterized by increased alpha-SMA expression. Because TGF-beta(1) is considered the main inducer of the myofibroblast phenotype and cytoskeletal changes accompany this differentiation, the main objective of this investigation was to study how TGF-beta(1) alters protein expression of cytoskeletal-associated proteins. Metabolic labeling of cell cultures by [(35)S]methionine, followed by protein separation on two-dimensional gel electrophoresis, displayed approximately 2500 proteins in the pI interval of 3-10. Treatment of TGF-beta(1) led to specific spot pattern changes that were identified by mass spectrometry and represent specific induction of several members of the contractile apparatus such as calgizzarin, cofilin, and profilin. These proteins have not previously been shown to be regulated by TGF-beta(1), and the functional role of these proteins is to participate in the depolymerization and stabilization of the microfilaments. These results show that TGF-beta(1) induces not only alpha-SMA but a whole set of actin-associated proteins that may contribute to the increased contractile properties of the myofibroblast. These proteins accompany the induced expression of alpha-SMA and may participate in the formation of stress fibers, cell contractility, and cell spreading characterizing the myofibroblasts phenotype.     

Role of Complement 3 in TNF-α-Induced Mesenchymal Transition of Renal Tubular Epithelial Cells In Vitro

Injured renal tubular epithelial cells (RTECs) have been recently thought to directly contribute to the accumulation of myofibroblasts in renal tubulointerstitial fibrosis through a process of epithelial to mesenchymal transition (EMT). However, the factors inducing RTECs to undergo EMT and the underlying mechanisms need to be further elucidated. This study aimed to determine the EMT-inducing activity of proinflammatory cytokine TNF-α and the role for complement 3 (C3) in this activity in an in vitro model of human RTECs (HK-2 cells). Wild type HK-2 cells were treated with TNF-α, IFN-γ or C3a; C3 siRNA- or control siRNA-carrying HK-2 cells were treated with TNF-α. Changes in the cell morphology and phenotype were assessed by microscopy, RT-PCR, western blotting, and immunostaining. TNF-α effectively induced HK-2 cells to express C3 and to transform into morphologically myofibroblast-like cells that lost E-cadherin (a classical epithelial cell marker) expression but acquired alpha-smooth muscle actin (α-SMA, a classical myofibroblast differentiation marker) expression. C3 siRNA robustly attenuated all the morphologic and phenotypic changes induced by TNF-α but the control siRNA showed no effect. Our preliminary observations suggest that TNF-α may induce EMT in RTECs through inducing C3 expression.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.