Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling.

Rho GTPases regulate a diverse spectrum of cellular functions involved in vascular morphogenesis. Here, we show that Cdc42 and Rac1 play a key role in endothelial cell (EC) lumen and tube formation as well as in EC invasion in three-dimensional (3D) collagen matrices and that their regulation is mediated by various downstream effectors, including Pak2, Pak4, Par3 and Par6. RNAi-mediated or dominant-negative suppression of Pak2 or Pak4, two major regulators of cytoskeletal signaling downstream of Cdc42 or Rac1, markedly inhibits EC lumen and tube formation. Both Pak2 and Pak4 phosphorylation strongly correlate with the lumen formation process in a manner that depends on protein kinase C (PKC)-mediated signaling. We identify PKCepsilon and PKCzeta as regulators of EC lumenogenesis in 3D collagen matrices. Two polarity proteins, Par3 and Par6, are also required for EC lumen and tube formation, as they establish EC polarity through their association with Cdc42 and atypical PKC. In our model, disruption of any member in the Cdc42-Par3-Par6-PKCzeta polarity complex impairs EC lumen and tube formation in 3D collagen matrices. This work reveals novel regulators that control the signaling events mediating the crucial lumen formation step in vascular morphogenesis.