Crosstalk between ERK and MRTF-A signaling regulates TGFß1-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell-cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast-like properties with enhanced expression of the mesenchymal protein marker α-smooth muscle actin and contractile activity. Transforming growth factor (TGF)-ß1 is a well-known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal-regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFß1-induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin-related transcription factor-A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes.

Matrix Rigidity Mediates TGFß1-Induced Epithelial-Myofibroblast Transition by Controlling Cytoskeletal Organization and MRTF-A Localization.

Myofibroblasts mediate normal wound healing and upon chronic activation can contribute to the development of pathological conditions including organ fibrosis and cancer. Myofibroblasts can develop from epithelial cells through an epithelial-mesenchymal transition (EMT) during which epithelial cells exhibit drastic morphological changes and upregulate cytoskeletal associated proteins that enable exertion of large contractile forces and remodeling of the surrounding microenvironment. Increased matrix rigidity is a hallmark of fibrosis and tumor progression and mechanical tension has been identified as a regulator of EMT; however, the mechanisms governing the mechanical regulation of EMT are not completely understood. Here, we find that matrix rigidity regulates transforming growth factor (TGF)-ß1-induced EMT, with rigid substrata enabling increased myofibroblast marker expression, cell morphology changes, and cytoskeletal reorganization while soft matrices block these changes. Furthermore, we find that matrix rigidity controls the subcellular localization of myocardin related transcription factor (MRTF)-A, a regulator of cytoskeletal protein expression that contributes to the acquisition of myogenic features during EMT. Results from these studies provide insight into how biophysical cues contribute to myofibroblast development from epithelial cells and may suggest ways to enhance wound healing or to engineer therapeutic solutions for fibrosis and cancer.

TGFß1-induced expression of caldesmon mediates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important process that mediates organ development and wound healing, and in pathological contexts, it can contribute to the progression of fibrosis and cancer. During EMT, cells exhibit marked changes in cytoskeletal organization and increased expression of a variety of actin associated proteins. Here, we sought to determine the role of caldesmon in mediating EMT in response to transforming growth factor (TGF)-ß1. We find that the expression level and phosphorylation state of caldesmon increase as a function of time following induction of EMT by TGFß1 and these changes in caldesmon correlate with increased focal adhesion number and size and increased cell contractility. Knockdown and forced expression of caldesmon in epithelial cells reveals that caldesmon expression plays an important role in regulating the expression of the myofibroblast marker alpha smooth muscle actin. Results from these studies provide insight into the role of cytoskeletal associated proteins in the regulation of EMT and may suggest ways to target the cell cytoskeleton for regulating EMT processes.

Cell-cell contact and matrix adhesion promote αSMA expression during TGFß1-induced epithelial-myofibroblast transition via Notch and MRTF-A.

During epithelial-mesenchymal transition (EMT) epithelial cells lose cell-cell adhesion, exhibit morphological changes, and upregulate the expression of cytoskeletal proteins. Previous studies have demonstrated that complete disruption of cell-cell contact can promote transforming growth factor (TGF)-ß1-induced EMT and the expression of the myofibroblast marker alpha smooth muscle actin (αSMA). Furthermore, increased cell spreading mediates TGFß1-induced αSMA expression during EMT. Here, we sought to examine how the presence of partial cell-cell contacts impacts EMT. A microfabrication approach was employed to decouple the effects of cell-cell contact and cell-matrix adhesion in TGFß1-induced EMT. When cell spreading is controlled, the presence of partial cell-cell contacts enhances expression of αSMA. Moreover, cell spreading and intercellular contacts together control the subcellular localization of activated Notch1 and myocardin related transcription factor (MRTF)-A. Knockdown of Notch1 or MRTF-A as well as pharmacological inhibition of these pathways abates the cell-cell contact mediated expression of αSMA. These data suggest that the interplay between cell-matrix adhesion and intercellular adhesion is an important determinant for some aspects of TGFß1-induced EMT.

Cytoskeletal signaling in TGFß-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a physiological process that plays an important role in embryonic development and wound healing and is appropriated during pathological conditions including fibrosis and cancer metastasis. EMT can be initiated by a variety of factors, including transforming growth factor (TGF)-ß, and is characterized by loss of epithelial features including cell-cell contacts and apicobasal polarity and acquisition of a motile, mesenchymal phenotype. A key feature of EMT is reorganization of the cytoskeleton and recent studies have elucidated regulation mechanisms governing this process. This review describes changes in gene expression patterns of cytoskeletal associated proteins during TGFß-induced EMT. It further reports TGFß-induced intracellular signaling cascades that regulate cytoskeletal reorganization during EMT. Finally, it highlights how changes in cytoskeletal architecture during EMT can regulate gene expression, thus further promoting EMT progression.

Biomechanics of TGFß-induced epithelial-mesenchymal transition: implications for fibrosis and cancer.

Fibrosis, a disease that results in loss of organ function, contributes to a significant number of deaths worldwide and sustained fibrotic activation has been suggested to increase the risk of developing cancer in a variety of tissues. Fibrogenesis and tumor progression are regulated in part through the activation and activity of myofibroblasts. Increasing evidence links myofibroblasts found within fibrotic lesions and the tumor microenvironment to a process termed epithelial-mesenchymal transition (EMT), a phenotypic change in which epithelial cells acquire mesenchymal characteristics. EMT can be stimulated by soluble signals, including transforming growth factor (TGF)-ß, and recent studies have identified a role for mechanical cues in directing EMT. In this review, we describe the role that EMT plays in fibrogenesis and in the progression of cancer, with particular emphasis placed on biophysical signaling mechanisms that control the EMT program. We further describe specific TGFß-induced intracellular signaling cascades that are affected by cell- and tissue-level mechanics. Finally, we highlight the implications of mechanical induction of EMT on the development of treatments and targeted intervention strategies for fibrosis and cancer.

Cell adhesion and shape regulate TGF-beta1-induced epithelial-myofibroblast transition via MRTF-A signaling.

Myofibroblasts, specialized cells that play important roles in wound healing and fibrosis, can develop from epithelial cells through an epithelial-mesenchymal transition (EMT). During EMT, epithelial cells detach from neighboring cells and acquire an elongated, mesenchymal-like morphology. These phenotypic changes are accompanied by changes in gene expression patterns including upregulation of a variety of cytoskeletal associated proteins which contribute to the ability of myofibroblasts to exert large contractile forces. Here, the relationship between cell shape and cytoskeletal tension and the expression of cytoskeletal proteins in transforming growth factor (TGF)-ß1-induced EMT is determined. We find that culturing cells in conditions which permit cell spreading and increased contractility promotes the increased expression of myofibroblast markers and cytoskeletal associated proteins. In contrast, blocking cell spreading prevents transdifferentiation to the myofibroblast phenotype. Furthermore, we find that cell shape regulates the expression of cytoskeletal proteins by controlling the subcellular localization of myocardin related transcription factor (MRTF)-A. Pharmacological inhibition of cytoskeletal tension or MRTF-A signaling blocks the acquisition of a myofibroblast phenotype in spread cells while overexpression of MRTF-A promotes the expression of cytoskeletal proteins for all cell shapes. These data suggest that cell shape is a critical determinant of myofibroblast development from epithelial cells.

Lipid membranes with a majority of cholesterol: applications to the ocular lens and aquaporin 0.

Using molecular dynamics (MD) simulations, we studied the structure and dynamics of two dimyristoylphosphatidylcholine (DMPC):cholesterol bilayers at concentrations representative of the ocular lens (ratios of 1:1 and 1:2). These MD simulations agree well with experimental deuterium order parameters and bilayer peak-to-peak distances. Although it is known that the average surface area per lipid rapidly decreases from low to moderate levels of cholesterol, our simulations indicate that there is a relatively small change in the average lipid area from 50 to 66.7% cholesterol (40.5 ± 0.2 and 39.5 ± 0.1 Å(2)/lipid, respectively). Radial distribution functions for the hydroxyl group on cholesterol indicate the formation of cholesterol-only nanoscale domains for the membrane with 66.7% cholesterol but a uniform distribution of cholesterol and DMPC for the membrane with 50% cholesterol. These small domains form a single shell of hexagonally packed cholesterols that are interconnected in a web-like structure of cholesterol. Calculations of internal DMPC dynamics show that the relaxation times for carbon-hydrogen reorientation of choline decrease with an increase in cholesterol, but the main body (carbonyl-glycerol to C11) is independent of cholesterol concentration. MD simulations of the aquaporin 0 tetramer show stabilization in its interactions with lipid membranes containing cholesterol by forming ring-ring stacking between surface aromatic residues of the protein and the rings of cholesterol. Moreover, there is an increase in hydrogen bonds with longer lifetimes in a mixed bilayer of DMPC and cholesterol.

Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types.

A significant modification to the additive all-atom CHARMM lipid force field (FF) is developed and applied to phospholipid bilayers with both choline and ethanolamine containing head groups and with both saturated and unsaturated aliphatic chains. Motivated by the current CHARMM lipid FF (C27 and C27r) systematically yielding values of the surface area per lipid that are smaller than experimental estimates and gel-like structures of bilayers well above the gel transition temperature, selected torsional, Lennard-Jones and partial atomic charge parameters were modified by targeting both quantum mechanical (QM) and experimental data. QM calculations ranging from high-level ab initio calculations on small molecules to semiempirical QM studies on a 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) bilayer in combination with experimental thermodynamic data were used as target data for parameter optimization. These changes were tested with simulations of pure bilayers at high hydration of the following six lipids: DPPC, 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1,2-dilauroyl-sn-phosphatidylcholine (DLPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE); simulations of a low hydration DOPC bilayer were also performed. Agreement with experimental surface area is on average within 2%, and the density profiles agree well with neutron and X-ray diffraction experiments. NMR deuterium order parameters (S(CD)) are well predicted with the new FF, including proper splitting of the S(CD) for the aliphatic carbon adjacent to the carbonyl for DPPC, POPE, and POPC bilayers. The area compressibility modulus and frequency dependence of (13)C NMR relaxation rates of DPPC and the water distribution of low hydration DOPC bilayers also agree well with experiment. Accordingly, the presented lipid FF, referred to as C36, allows for molecular dynamics simulations to be run in the tensionless ensemble (NPT), and is anticipated to be of utility for simulations of pure lipid systems as well as heterogeneous systems including membrane proteins.