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Vol. 20, Issue 21, 4596-4610, November 1, 2009

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G12 Inhibits 2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Tianqing Kong^*, Daosong Xu, Wanfeng Yu^*, Ayumi Takakura^*, Ilene Boucher^*, Mei Tran^*, Jordan A. Kreidberg, Jagesh Shah^*, Jing Zhou^*, and Bradley M. Denker^*

*Renal Division, Brigham and Women's Hospital, Harvard Institutes of Medicine, Boston, MA 02115; Dana Farber Cancer Institute, Boston, MA 02115; and Children's Hospital, Boston, MA 02115

Submitted March 18, 2009; Revised August 19, 2009; Accepted September 15, 2009
Monitoring Editor: Asma Nusrat

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G12 inhibits interaction of MDCK cells with collagen-I, the major ligand for 2β1 integrin. Activating G12 (QL point mutation or stimulating endogenous G12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G12-regulated attachment to collagen-I, G12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of 2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.

Address correspondence to: Bradley M. Denker (bdenker{at}rics.bwh.harvard.edu).

Abbreviations used: dox, doxycycline; ADPKD, autosomal dominant polycystic kidney disease.