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Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.

ABSTRACT: Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the cationic colloidal silica isolation technique to purify PM fractions from epithelial Madin-Darby canine kidney cells during Ras/TGF-?-mediated EMT. Proteins in the isolated membrane fractions were separated by one-dimensional SDS-PAGE and subjected to nano-LC-MS/MS-based protein identification. In this study, the first membrane protein analysis of an EMT model, we identified 805 proteins and determined their differential expression using label-free spectral counting. These data reveal that Madin-Darby canine kidney cells switch from cadherin-mediated to integrin-mediated adhesion following Ras/TGF-?-mediated EMT. Thus, during the EMT process, E-cadherin, claudin 4, desmoplakin, desmoglein-2, and junctional adhesion molecule A were down-regulated, whereas integrins ?6?1, ?3?1, ?2?1, ?5?1, ?V?1, and ?V?3 along with their extracellular ligands collagens I and V and fibronectin had increased expression levels. Conspicuously, Wnt-5a expression was elevated in cells undergoing EMT, and transient Wnt-5a siRNA silencing attenuated both cell migration and invasion in these cells. Furthermore, Wnt-5a expression suppressed canonical Wnt signaling induced by Wnt-3a. Wnt-5a may act through the planar cell polarity pathway of the non-canonical Wnt signaling pathway as several of the components and modulators (Wnt-5a, -5b, frizzled 6, collagen triple helix repeat-containing protein 1, tyrosine-protein kinase 7, RhoA, Rac, and JNK) were found to be up-regulated during Ras/TGF-?-mediated EMT.

SUBMITTER: Chen YS

PROVIDER: S-EPMC3033669 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.

Chen Yuan-Shou YS Mathias Rommel A RA Mathivanan Suresh S Kapp Eugene A EA Moritz Robert L RL Zhu Hong-Jian HJ Simpson Richard J RJ

Molecular & cellular proteomics : MCP 20100528 2

Epithelial-mesenchymal transition (EMT) describes a process whereby polarized epithelial cells with restricted migration transform into elongated spindle-shaped mesenchymal cells with enhanced motility and invasiveness. Although there are some molecular markers for this process, including the down-regulation of E-cadherin, our understanding of plasma membrane (PM) and associated proteins involved in EMT is limited. To specifically explore molecular alterations occurring at the PM, we used the ca ...[more]

PMID: 20511395

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Project description:Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40-100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies and are released from cells on fusion of multivesicular bodies with the plasma membrane. Exosomes from MDCK cells (MDCK-Exos) and 21D1 cells (21D1-Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep™), and protein content identified by GeLC-MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryo-electron microscopy and contained stereotypical exosome marker proteins such as TSG101, Alix, and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCK- and 21D1-Exos, i.e. reduction of characteristic inhibitor of angiogenesis, thrombospondin-1, and epithelial markers E-cadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1-Exos are enriched with several proteases (e.g. MMP-1, -14, -19, ADAM-10, and ADAMTS1), and integrins (e.g. ITGB1, ITGA3, and ITGA6) that have been recently implicated in regulating the tumor microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g. the master transcriptional regulator YBX1) and core splicing complex components (e.g. SF3B1, SF3B3, and SFRS1) in mesenchymal 21D1-Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.

Project description:Madin-Darby canine kidney (MDCK) cells were cultured on polycarbonate filters to study the synthesis and sorting of proteoglycans in polarized epithelial cells. Two strains of MDCK cells were used. MDCK I cells resemble distal tubule epithelial cells, and MDCK II cells share some characteristics with proximal tubule cells. Both strains were grown to confluency and labelled with [35S]sulphate for 24 h. The apical and basolateral media and the cell fractions were harvested and analysed by DEAE ion-exchange chromatography. A large portion of the [35S]sulphate-labelled macromolecules bound strongly to the ion-exchange columns, and could be eluted in three distinct peaks. The latest eluting peak was demonstrated to contain almost exclusively chondroitin sulphate, whereas peak 2 contained mostly heparan sulphate, demonstrated by using chondroitinase ABC and nitrous acid (pH 1.5) respectively to depolymerize the [35S]glycosaminoglycan chains. Peak 1 contained negligible amounts of proteoglycans. Large differences could be observed in proteoglycan sorting in MDCK I and II cells. Strain I secreted approx. 67% of the proteoglycans to the apical side and 17% to the basolateral side. The cell fraction contained 17% of the proteoglycans after 24 h of labelling. In contrast, 19% of the proteoglycans were sorted to the apical side of MDCK II cells and 61% to the basolateral side, whereas the cell fraction contained 20%. Furthermore, the level of [35S]proteoglycan biosynthesis (apical and basolateral media and cell fraction total) was higher in MDCK I cells than in strain II. Based on the amount of material degraded by chondroitinase ABC and nitrous acid respectively, and the total amounts of [35S]proteoglycans recovered from the cells, it was calculated that the MDCK I strain synthesized approx. 56% chondroitin sulphate and 44% heparan sulphate. In contrast, the MDCK II strain synthesized 69% heparan sulphate and 31% chondroitin sulphate. To further identify the [35S]proteoglycans synthesized by MDCK I and II cells, antibodies against perlecan, versican and syndecan were used. The antibody against mouse syndecan did not cross-react with any of the proteoglycans produced in MDCK I or II cells. Both MDCK I and II cells expressed perlecan; 57-61% could be recovered from the basolateral fractions and 18-34% from the apical medium. Versican was also found in both MDCK I and II cells. Compared with perlecan, a larger percentage of versican (43-53%) was found in the cell fractions.

Dataset Information

Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.

Publications

Proteomics profiling of Madin-Darby canine kidney plasma membranes reveals Wnt-5a involvement during oncogenic H-Ras/TGF-beta-mediated epithelial-mesenchymal transition.

Similar Datasets

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