Biochemical and Biophysical Research Communications
Integrin-linked kinase function is required for transforming growth factor β-mediated epithelial to mesenchymal transition☆
Section snippets
Materials and methods
Antibodies and reagents. Mouse monoclonal antibodies against human E-cadherin and N-cadherin were purchased from Transduction Laboratories (Lexington, KY). Texas red-conjugated anti-mouse IgG and horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Sigma Chemical (St. Louis, MI). Akt and phospho-Ser-473 Akt polyclonal antibodies were from New England Biolabs (Beverly, MA). Smad2, phospho-Smad2, and ILK antibodies were purchased from Upstate Biotechnology (Lake Placid, NY).
TGFβ1 induces a morphological change in HaCat
Upon treatment with TGFβ (10 ng/ml), responsive HaCat cells acquired a spindle-type cell morphology and that the number of cell–cell contacts was reduced (Fig. 1A). In addition, the delocalization of E-cadherin, formation of the actin stress fibers, and the appearance of a fibroblastic marker, N-cadherin was observed by using immunohistochemistry and confirmed TGFβ activity on the EMT (Fig. 1A). The amount of E-cadherin was not changed under these experimental conditions (Fig. 1B), suggesting
Discussion
TGFβ has been known to induce epithelial transformation. Several molecules, such as Smad2/3 [13], AP-1 family [14], [15], Rho-A [16], and PI3K [17], are known to be involved in TGFβ-mediated EMT. However, signaling pathways involved in TGFβ-mediated EMT are not well defined. ILK is stimulated by extracellular matrix interactions and by certain growth factors [18]. Especially binding of PI 3,4,5-triphosphate, the product of PI 3-kinase, to the PH-like domain of ILK and autophosphorylation in the
Acknowledgements
This work was supported in part by 2000-N-NL-01-C-121 from the National Research Lab Program.
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Abbreviations: ILK, integrin-linked kinase; TGFβ, transforming growth factor-β; PI3K, phosphatidylinositol 3-kinase, Akt/PKB, protein kinase B; EMT, epithelial to mesenchymal transition; DN, dominant-negative; WT, wild-type; EV, empty-vector.