Endothelial lumen signaling complexes control 3D matrix–specific tubulogenesis through interdependent Cdc42- and MT1-MMP–mediated events

Abstract

Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecule (Jam)-B and Jam-C, membrane type 1-matrix metalloproteinase (MT1-MMP), and integrin ␣ 2 ␤ 1 , which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant-negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP-dependent signal transduction, and blockade of MT1-MMP-dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling, which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces, whereas Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP-dependent proteolysis, a process that occurs selectively in 3D collagen matrices and that requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis. (Blood. 2010;115(25):5259-5269) IntroductionRecent work has lead to an increased understanding of how endothelial and epithelial cells make lumens and tubes in 3D extracellular matrices. [1][2][3][4][5][6][7][8][9] Key regulators of lumen formation include Cdc42, which was first shown to regulate this process in endothelial cells (ECs), [10][11][12] and later in epithelial cells. 13,14 Components of the cell polarity machinery including Par3, Par6, and PKC control lumen formation of both cell types. 6,11,[13][14][15] Furthermore, we recently reported that Cdc42 activates a signaling cascade involving PKC⑀, Pak2, Pak4, Src, Yes, B-Raf, C-Raf, and Erk1/2 to control this process. 12,16 In addition, EC-directed cellsurface proteolytic events through membrane type 1-matrix metalloproteinase (MT1-MMP) 17,18 controls EC lumen and vascular guidance tunnel formation in 3D collagen matrices. 19,20 A key question that has remained unresolved is how Cdc42-dependent signaling and MT1-MMP-dependent proteolysis are functionally coupled to regulate EC tube formation. 2 Recent studies have revealed that MT1-MMP directs 3D matrix-specific events in relationship to tumor motility, cellular differentiation, and morphogenesis. 2,21 We have shown that both tumor cell and EC invasion of 3D collagen matrices requires MT1-MMP, but not motility on 2D collagen substrates. 19,22 Interestingly, adipocyte differentiation occurs in an MT1-MMP-dependent manner in 3D matrices but not on 2D matrix surfaces, 23 and MT1-MMP controls the 3D-specific process of EC lumen and tube formation. 18,19 Thus, MT1-MMP is functionally linked to critical cellular events that specifically occur in 3D...

Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

“…Thus, it is tempting to speculate that S1P stimulation enhances interactions between Hic-5 and p130Cas in endothelial cells. Other potential interacting partners for Hic-5 include vimentin, which complexes with p130Cas (Wang et al, 2007), MT1-MMP (Kwak et al, 2012), caveolin (Labrecque et al, 2004), the α2β1 integrin (Sacharidou et al, 2010) and RACK1, which itself complexes with vimentin and FAK in invading endothelial cells (Dave et al, 2013). Taken together, these data suggest that Hic-5 might be a part of a larger signaling network involving other key molecules, whose assembly is initiated with endothelial cell activation.…”

“…S2A). Endothelial lumen formation is a crucial aspect of endothelial morphogenesis and tubulogenesis (Davis et al, 2011), and MT1-MMP is required for both successful lumen formation and endothelial cell sprouting into collagen matrices (Chun et al, 2004;Stratman et al, 2009;Sacharidou et al, 2010). S1P has been shown to stimulate phosphorylation of MT1-MMP at Y573 (Nyalendo et al, 2007).…”

Tubulogenesis

“…In particular, the polarized expression of specific membrane-tethered matrix metalloproteinases in vascular sprouts facilitates activation of Cdc42 and progressive invasion through the degradation of ECM molecules (Sacharidou et al, 2010).…”

Single cell analysis of endothelial morphogenesis in vivo

“…Adhesion to surrounding extracellular matrix (ECM), loss of some EC-EC contacts, or repulsive interaction between EC surfaces have all been suggested as important forces promoting expansion of nascent lumenal spaces (Eilken and Adams, 2010;Lampugnani et al, 2010;Strilic et al, 2010Strilic et al, , 2009Wang et al, 2010;Zovein et al, 2010). Other work, primarily in vitro, has uncovered molecular regulators crucial for lumenogenesis (Bayless and Davis, 2002;Bayless et al, 2000;Egginton and Gerritsen, 2003;Iruela-Arispe and Davis, 2009;Koh et al, 2009Koh et al, , 2008Popson et al, 2014;Sacharidou et al, 2010Sacharidou et al, , 2012.…”