Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMPindependent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly. (Blood. 2009;114: 237-247) IntroductionMuch progress has occurred in our understanding of the molecular events controlling the processes underlying vascularization of tissues in the context of development and disease. [1][2][3][4][5][6][7] Work that is receiving increasing attention focuses on identifying specific steps required for vascular morphogenesis, including those involving endothelial cell (EC) lumen formation. [8][9][10][11][12] In addition to the identification of specific molecules required for these events, it is important to determine how different cell types such as endothelial cells, pericytes, and vascular smooth muscle cells interact and assemble to form the different characteristic blood vessel types. 1,6,13,14 Recent work from our laboratory reveals that ECs form lumens in 3-dimensional (3D) collagen matrices through a signaling cascade involving integrins, Rho GTPases, and membrane-type matrix metalloproteinases (MT-MMPs). [8][9][10][11][12] These signaling events stimulate EC intracellular vacuole formation and coalescence that controls EC lumen formation in vitro and in vivo. 8,10,12 A variety of integrins have been described to be relevant in regulating angiogenesis and tube formation including both ␤1 and ␣v integrins. The relevance of any particular integrin appears to be primarily dependent on the matrix environment (eg, adult, embryonic, wound, tumor) where the EC tube morphogenic process takes place. 3,9,[15][16][17][18][19] Extracellular matrix (ECM) proteolysis is thought to be an important step in how cells move through 3D matrix environments [20][21][22][23][24][25][26][27] and has been implicated in vessel for...

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...

Skeletal muscle repair and regeneration requires the activity of satellite cells, a population of myogenic stem cells scattered throughout the tissue and activated to proliferate and differentiate in response to myotrauma or disease. While it seems likely that satellite cells would need to navigate local muscle tissue to reach damaged areas, relatively little data on such motility exist, and most studies have been with immortalized cell lines. We find that primary satellite cells are significantly more motile than myoblast cell lines, and that adhesion to laminin promotes primary cell motility more than fourfold over other substrates. Using timelapse videomicroscopy to assess satellite cell motility on single living myofibers, we have identified a requirement for the laminin-binding integrin α7β1 in satellite cell motility, as well as a role for hepatocyte growth factor in promoting directional persistence. The extensive migratory behavior of satellite cells resident on muscle fibers suggests caution when determining, based on fixed specimens, whether adjacent cells are daughters from the same mother cell. We also observed more persistent long-term contact between individual satellite cells than has been previously supposed, potential cell-cell attractive and repulsive interactions, and migration between host myofibers. Based on such activity, we assayed for expression of “pathfinding” cues, and found that satellite cells express multiple guidance ligands and receptors. Together, these data suggest that satellite cell migration in vivo may be more extensive than currently thought, and could be regulated by combinations of signals, including adhesive haptotaxis, soluble factors, and guidance cues. Stem Cells 2009;27:2527–2538

SUMMARY Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a “synthetic lethal” interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E “rewires” metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.

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