Journal of Biological Chemistry
Volume 275, Issue 47, 24 November 2000, Pages 36803-36810
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MECHANISMS OF SIGNAL TRANSDUCTION
Phosphatidylinositol 3-Kinase Function Is Required for Transforming Growth Factor β-mediated Epithelial to Mesenchymal Transition and Cell Migration*

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We have studied the role of phosphatidylinositol 3-OH kinase (PI3K)-Akt signaling in transforming growth factor β (TGFβ)-mediated epithelial to mesenchymal transition (EMT). In NMuMG mammary epithelial cells, exogenous TGFβ1 induced phosphorylation of Akt at Ser-473 and Akt in vitro kinase activity against GSK-3β within 30 min. These responses were temporally correlated with delocalization of E-cadherin, ZO-1, and integrin β1 from cell junctions and the acquisition of spindle cell morphology. LY294002, an inhibitor of the p110 catalytic subunit of PI3K, and a dominant-negative mutant of Akt blocked the delocalization of ZO-1 induced by TGFβ1, whereas transfection of constitutively active p110 induced loss of ZO-1 from tight junctions. In addition, LY294002 blocked TGFβ-mediated C-terminal phosphorylation of Smad2. Consistent with these data, TGFβ-induced p3TP-Lux and p(CAGA)12-Lux reporter activities were inhibited by LY294002 and transiently expressed dominant-negative p85 and Akt mutants in NMuMG and 4T1 cells. Dominant-negative RhoA inhibited TGFβ-induced phosphorylation of Akt at Ser-473, whereas constitutively active RhoA increased the basal phosphorylation of Akt, suggesting that RhoA in involved in TGFβ-induced EMT. Finally, LY294002 and neutralizing TGFβ1 antibodies inhibited ligand-independent constitutively active Akt as well as basal and TGFβ-stimulated migration in 4T1 and EMT6 breast tumor cells. Taken together, these data suggest that PI3K-Akt signaling is required for TGFβ-induced transcriptional responses, EMT, and cell migration.

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Published, JBC Papers in Press, August 31, 2000, DOI 10.1074/jbc.M005912200

*

This work was supported by Public Health Service (PHS) Grant R01 CA62212, U. S. Department of Defense, U. S. Army Medical Research Material Command Grant DAMD17-98-1-8262, a Clinical Investigator Award from the Department of Veterans Affairs (to C. L. A.), PHS Grant R35 CA42572 (to H. L. M.), National Institutes of Health Training Grant CA09592 (to N. A. B.), and Vanderbilt-Ingram Cancer Center NCI National Institutes of Health Support Grant CA68485.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.