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  • Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling.

Pitx2a expression alters actin-myosin cytoskeleton and migration of HeLa cells through Rho GTPase signaling.

Molecular biology of the cell (2002-02-21)
Qize Wei, Robert S Adelstein
ABSTRACT

We ectopically expressed the transcription factor Pitx2a, one of the Pitx2 isoforms, in HeLa cells by using a tetracycline-inducible expression system and examined whether Pitx2a was capable of modulating Rho GTPase signaling and altering the cell's cytoskeleton. Ectopic expression of Pitx2a induced actin-myosin reorganization, leading to increased cell spreading, suppression of cell migration, and the strengthening of cell-cell adhesion, marked by the accumulation and localization of beta-catenin and N-cadherin to the sites of cell-cell contacts. Moreover, Pitx2a expression resulted in activation of the Rho GTPases Rac1 and RhoA, and the dominant negative Rac1 mutant N17Rac1 inhibited cell spreading and disrupted localization of beta-catenin to the sites of cell-cell contacts. Both reorganization of actin-myosin and cell spreading require phosphatidylinositol 3-kinase activity, which is also necessary for activation of the Rho GTPase proteins. Pitx2a induced the expression of Trio, a guanine nucleotide exchange factor for Rac1 and RhoA, which preceded cell spreading, and the expression of Trio protein was down-regulated after the changes in cell spreading and cell morphology were initiated. In addition, Pitx2a also induces cell cycle arrest at G0/G1, most likely due to the accumulation of the tumor suppressor proteins p53 and p21. Our data indicate that the transcriptional activities initiated in the nucleus by Pitx2a result in profound changes in HeLa cell morphology, migration, and proliferation.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
LY 294002
Sigma-Aldrich
Rho Activation Assay Kit, PP1/PP2A Toolbox for the selective in vitro dephosphorylation of proteins.
Sigma-Aldrich
Rac1 Activation Assay Kit, Non-radioactive Rac1 Activation Assay Kit can be used to precipitate Rac1-GTP from cell lysates & detection by a Rac1 specific monoclonal antibody.
Sigma-Aldrich
PD 98059