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Interaction of RAGE with focal adhesion molecules during alveolar epithelial‐mesenchymal transition
Author(s) -
Ehrhardt Carsten,
Medina Carlos,
Buckley Stephen Timothy
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.865.1
Subject(s) - ezrin , moesin , rage (emotion) , microbiology and biotechnology , radixin , chemistry , a549 cell , actin , colocalization , cytoskeleton , western blot , actin cytoskeleton , cd44 , focal adhesion , phosphorylation , epithelial–mesenchymal transition , biology , cell , downregulation and upregulation , biochemistry , neuroscience , gene
Ezrin/radixin/moesin (ERM) proteins provide a linkage between F‐actin and membrane proteins. RAGE also associates with components of the cytoskeleton. This study tested the hypothesis that RAGE‐ERM interaction plays a role in regulating EMT‐associated structural changes in alveolar epithelial cells. A549 cells were stimulated with TGF‐β1 for 72 h. The interaction of ERM proteins and F‐actin was investigated by immunofluorescence microscopy (IFM) and Western blot (WB). RAGE and sRAGE expression and localisation was determined by WB and IFM. MMP‐9 activity was evaluated using gelatin zymography. Co‐immunoprecipitation studies and IFM was performed to assess the association of ERM and RAGE, and ERM and CD44. Exposure to TGF‐β1 gave rise to re‐distribution of ERM to the cell periphery and co‐localisation with EMT‐associated actin stress fibres. Concurrently, ERM underwent phosphorylation. Expression of RAGE was diminished, coupled with release of its soluble isoform (sRAGE) in a MMP‐9 dependent manner. IFM and co‐IP identified strong association between ERM and RAGE under basal conditions, which was disrupted when challenged with TGF‐β1, with pERM forming a complex with membrane‐linked CD44. These data indicates that disruption of the ERM‐RAGE complex may be a crucial step in actin microfilament re‐arrangement during cytokine‐induced EMT of human alveolar epithelial cells.