p21-Activated Kinase 2 Regulates Endothelial Development and Function through the Bmk1/Erk5 Pathway

Abstract

c p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment, and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently, there is no consensus on the endothelial function of individual Pak isoforms, in particular p21-activated kinase 2 (Pak2), the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice, endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and they also pinpoint the Bmk1/ Erk5 pathway as a critical mediator of endothelial Pak2 signaling.T he p21-activated kinases (Paks) are evolutionarily conserved serine/threonine kinases that act downstream of the Rho family GTPases, Rac1 and Cdc42, to regulate numerous cellular processes. p21-activated kinase 1 (Pak1) to Pak3, the group I Paks, are expressed in numerous tissues with Pak1 being predominantly expressed in brain, muscle, gastrointestinal tract, and thyroid and Pak3 being predominantly expressed in brain (1, 2). Compared to the more restricted expression of Pak1 and Pak3, Pak2 is ubiquitously expressed (3-9). Group I Pak family members share a high degree of homology (3), but they may play distinct roles as observed by the significantly different phenotypes of knockout (KO) animal models. While Pak1 KO and Pak3 KO mice are viable (10, 11), Pak2 KO mice are embryonic lethal at embryonic day 8.5 (E8.5) due to multiple developmental abnormalities, including cardiovascular defects (12).The role of Pak in endothelial cell (EC) signaling has been studied in animal models (5, 13) and cultured cells (6,10,14,15). Pak signaling is critical in regulating EC attachment, migration, and lumen formation (4-9). Furthermore, Paks have been implicated in maintaining the integrity of the endothelial barrier, but conflicting data implicate various Pak isoforms as both positive and negative regulators of maintaining barrier function (5, 13, 14, 16).A few in vivo studies have specifically implicated Pak2 in vascular pathways. A study in Danio rerio showed that pak2a, one of two Pak2 orthologs in this organism, plays an important role in cerebral vascular maintenance (5). In this study, chemical mutagenesis of the pak2a gene and pak2a-targeting morpholinos caused cerebral hemorrhage, implicating this...

Optimization of Highly Kinase Selective Bis-anilino Pyrimidine PAK1 Inhibitors

“…While ethyl substitution (compare 15 with 11) gave no advantage, benzyl compound 16 improved PAK4 selectivity 15-fold. Addition of F (17) or CN (18) to the ortho position of the benzyl group gave a 4-fold PAK1 potency increase with improved selectivity against all antitarget kinases measured, including Src. Throughout the optimization process, cellular pPAK1 inhibition was measured in an MCF10A cell line.…”

Expansion of BCR/ABL1+ cells requires PAK2 but not PAK1

“…PAKs have been implicated in angiogenesis (Radu et al , 2014, 2015), and we tested whether PAK1 or PAK2 in leukaemic cells influences surrounding endothelial tissue. We performed an in vitro wound‐healing assay using human endothelial cells (HUVEC).…”

“…Upon loss of PAK1, mice display reduced mast cell degranulation and disturbed glucose homeostasis (Allen et al , 2009; Wang et al , 2011; Rane & Minden, 2014). Mice deficient for PAK2 die on embryonic day E8.5 due to defects in endothelial development (Hofmann et al , 2004; Radu et al , 2015). In adult mice, conditional deletion of PAK2 led to increased vascular permeability (Radu et al , 2015).…”

“…Mice deficient for PAK2 die on embryonic day E8.5 due to defects in endothelial development (Hofmann et al , 2004; Radu et al , 2015). In adult mice, conditional deletion of PAK2 led to increased vascular permeability (Radu et al , 2015). …”

Etiology of intracerebral hemorrhage (ICH): novel insights from Zebrafish embryos

“…2). Whereas depletion of PAK2 function in embryonic mice induces developmental defects and mortality, loss of PAK2 function in adult mice disrupts vascular permeability (Radu et al, 2015). In zebrafish, both ENU-mediated truncation of pak2a and morpholino-induced (Stainier et al, 1996;Buchner et al, 2007).…”