RhoA-mediated inhibition of vascular endothelial cell mobility: positive feedback through reduced cytosolic p21 and p27.

We previously identified that activation of the aryl hydrocarbon receptor (AhR) by 3-methylcholanthrene (3MC) exerts antiproliferative and antimigratory effects on human umbilical vein endothelial cells (HUVECs) through the upregulation of p21/p27 transcription and RhoA activation. In this study, we investigated the mechanisms of 3MC-mediated downregulation of cytosolic p21/ p27, and the effects of 3MC on RhoA activation and cell migration, in mouse cerebral vascular endothelial cells (MCVECs). Our results indicated that 3MC reduced the phosphorylation of p21/p27 through AhR/RhoA/PTEN-mediated PI3K/Akt inactivation, which reduced cytosolic p21/p27 retention, causing RhoA activation through positive feedback. Downregulation of p21/p27 by siRNA, and cytosolic p21/p27 by the nuclear export blocker leptomycin B, further reduced cell migration in the 3MC-treated cells. Reduced cytosolic p21/p27 expression led to reduced interaction between RhoA and the RhoA inhibitor p190RhoGAP, causing RhoA activation. Treatment with YS-49 activated PI3K/Akt, a downstream target of RhoA, to reduce RhoA/PTEN activation in the 3MC-treated cells, whereas treatment with wortmannin, a PI3K inhibitor, activated RhoA/PTEN. Gain- and loss-of-function analyses revealed that constitutively active (CA) Akt1, but not CA Akt2, inactivated RhoA and stimulated migratory activity. Considering the essential role of RhoA activation in cell migration, we evaluated the potential use of simvastatin, a RhoA inhibitor, as a therapeutic intervention in vivo using matrigel plug formation assays. Our results provide a molecular basis for the therapeutic application of simvastatin to reduce RhoA/PTEN activation, restore cytosolic levels of phosphorylated p21/p27, and induce angiogenic processes.