Mol. important for TGF-Cinduced MAPK activation. Requirement of lipid rafts for MAPK activation was further confirmed by specific targeting of the intracellular domain name of TGF- type I receptor to different membrane locations. Together, our findings establish a novel link between cholesterol and EMT and cell migration, that is, cholesterol-rich lipid rafts are required for TGF-Cmediated MAPK activation, an event necessary for TGF-Cdirected epithelial plasticity. INTRODUCTION Transforming growth factor (TGF)- is usually a polypeptide that regulates a variety of cell events, including cell growth, death, differentiation, and migration. Two transmembrane serine/threonine kinase receptors, known as type I (TRI) and type II receptors (TRII) are required for TGF- signal transduction. Ligand binding promotes the formation of receptor complex where TRII phosphorylates TRI. The activated TRI in turn activates R-Smads, Smad2 and Smad3, via phosphorylation at their C-terminal serine residues. As a result, activated R-Smads form a heterocomplex with Smad4 and are accumulated in the nucleus to regulate gene expression (Massague and Chen, 2000 ; Feng and Derynck, MGCD0103 (Mocetinostat) 2005 Rabbit Polyclonal to JAB1 ). In addition to this canonical Smad2/3 pathway, TGF- has been reported to activate other signaling molecules, such as mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]), p38 and c-Jun N-terminal kinases [JNKs]), and phosphatidylinositol 3-kinase (PI3K)/Akt and p21-activated kinase at a cell-specific manner (Derynck and Zhang, 2003 ; Moustakas and Heldin, 2005 ). Despite activated at a relatively low level, these non-Smad pathways could make great contribution to total TGF- signal output (Moustakas and Heldin, 2005 ). Unlike the Smad pathway, TGF- activates non-Smad pathways in a cell-type- and context-dependent manner. TGF- signaling is usually regulated at multiple layers including MGCD0103 (Mocetinostat) TGF- receptor trafficking. Around the plasma membrane, TGF- receptors bind to clathrin-associated adaptor complex AP2 and are constitutively internalized via clathrin-coated pits (Lu centrifugation for 10 min, the lysates were immunoprecipitated with specific antibody and protein A-Sepharose (Zymed Laboratories, South San Francisco, CA). The precipitants were analyzed by immunoblotting. Immunofluorescence and reporter assay were performed as described previously (Zhang for 16 h at 4C. Twelve 1-ml fractions were collected from the top of the tube, and a portion of each fraction was analyzed by immunoblotting. RESULTS Cholesterol Depletion Inhibits TGF–induced EpithelialCMesenchymal Transition and Cell Migration When stimulated with TGF-, several cell lines, including HaCaT cells, undergo EMT, a process characterized by loss of E-cadherin from the plasma membrane, replacement of cortical actin filaments by actin stress fibers, and acquirement of spindle-like cell morphology (Zavadil and Bottinger, 2005 ; Thiery and Sleeman, 2006 ). In the absence of TGF-, HaCaT cells occurred as common epithelial cells with the epithelial marker E-cadherin and actin cytoskeleton arranged in a cortical pattern at cellCcell junctions; after 36 h stimulation with TGF-, cells acquired spindle-shaped fibroblast-like morphology with down-regulation/delocalization of E-cadherin as well as formation of actin stress fibers, as reported previously (Physique 1, A and B). Furthermore, when HaCaT cells were treated with nystatin, a cholesterol sequestrating agent (Simons and Toomre, 2000 ), the TGF-Cinduced EMT was blocked. Similar effect was observed when HaCaT cells were treated with filipin, another cholesterol sequestrating agent (data not shown). To extend our study to other cell types, we assessed the effect of nystatin on TGF-Cinduced EMT in murine mammary gland MGCD0103 (Mocetinostat) epithelial NMuMG cells and obtained similar results (Supplemental Physique S1). To test whether the inhibitory effect of nystatin was due to its cholesterol-sequestrating activity, we supplemented HaCaT cells with cholesterol and found cholesterol addition could restore TGF-Cinduced EMT (Physique 1C). We also examined E-cadherin protein levels in the EMT process, and in agreement with the above-mentioned immunofluorescence data, nystatin inhibited the TGF-Cinduced down-regulation of E-cadherin (Physique 1D). Open in a separate window Physique 1. TGF-Cinduced EMT is usually sensitive to cholesterol depletion. (A and B) HaCaT cells were cultured in 0.5% serum and incubated with 25 g/ml nystatin.