Molecular control of endothelial lumen formation by Rho GTPases in three dimensional collagen matrices

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] 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 in 3D collagen matrices, and that their regulation is mediated by various downstream effectors including Pak2, Pak4, Par3, and Par6b. 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 PKC[epsilon] and PKC[zeta] as regulators of EC lumenogenesis in 3D collagen matrices. We further show that Src family kinases (SFKs) are involved in this PKC [epsilon]-mediated EC lumen formation process. SFKs also interact with Cdc42 during EC lumen formation in a PKC [epsilon] dependent manner. Our analysis indicates that two members of SFKs, Src and Yes, are differentially expressed during EC lumen formation. We identify Pak2 and Pak4 as key downstream molecular targets, where signaling pathways of Cdc42, PKC [epsilon], and SFKs converge to regulate EC lumen formation in 3D collagen matrices. Pak2 and Pak4 activate two Raf kinases, B-raf and C-raf, to induce EC lumen formation. Further examination of Raf kinase signaling reveals that regulation of EC lumen formation in 3D collagen matrices depends on ERK signaling. This data reveals a coordinated kinase signaling pathway downstream of Cdc42 activation involving PKC [epsilon], Src, Yes, Pak2, Pak4, Raf kinase and ERK1/2 that controls EC lumen formation in 3D collagen matrices.