Amel Mettouchi

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

The E3 Ubiquitin-Ligase HACE1 Catalyzes the Ubiquitylation of Active Rac1

Rac1 small GTPase controls essential aspects of cell biology and is a direct target of numerous bacterial virulence factors. The CNF1 toxin of pathogenic Escherichia coli addresses Rac1 to ubiquitin-proteasome system (UPS). We report the essential role of the tumor suppressor HACE1, a HECT-domain containing E3 ubiquitin-ligase, in the targeting of Rac1 to UPS. HACE1 binds preferentially GTP-bound Rac1 and catalyzes its polyubiquitylation. HACE1 expression increases the ubiquitylation of Rac1, when the GTPase is activated by point mutations or by the GEF-domain of Dbl. RNAi-mediated depletion of HACE1 blocks the ubiquitylation of active Rac1 and increases GTP-bound Rac1 cellular levels. HACE1 antagonizes cell isotropic spreading, a hallmark of Rac1 activation, and is required for endothelial cell monolayer invasion by bacteria. Together, these data establish the role of the HACE1 E3 ubiquitin-ligase in controlling Rac1 ubiquitylation and activity.

Laminin-Binding Integrins Induce Dll4 Expression and Notch Signaling in Endothelial Cells

Rationale: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through “lateral inhibition” in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. Objective: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. Methods and Results: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of &agr;2&bgr;1 and &agr;6&bgr;1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by &agr;2&bgr;1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin &ggr;1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins &agr;2 or &agr;6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. Conclusions: We show that, in endothelial cells, ligation of &agr;2&bgr;1 and &agr;6&bgr;1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection.

Ubiquitin‐Mediated Proteasomal Degradation of Rho Proteins by the CNF1 Toxin

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.

Hace1 controls ROS generation of vertebrate Rac1-dependent NADPH oxidase complexes

The Hace1-HECT E3 ligase is a tumor suppressor that ubiquitylates the activated GTP-bound form of the Rho family GTPase Rac1, leading to Rac1 proteasomal degradation. Here we show that, in vertebrates, Hace1 targets Rac1 for degradation when Rac1 is localized to the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme. This event blocks de novo reactive oxygen species generation by Rac1-dependent NADPH oxidases, and thereby confers cellular protection from reactive oxygen species-induced DNA damage and cyclin D1-driven hyper-proliferation. Genetic inactivation of Hace1 in mice or zebrafish, as well as Hace1 loss in human tumor cell lines or primary murine or human tumors, leads to chronic NADPH oxidase-dependent reactive oxygen species elevation, DNA damage responses and enhanced cyclin D1 expression. Our data reveal a conserved ubiquitin-dependent molecular mechanism that controls the activity of Rac1-dependent NADPH oxidase complexes, and thus constitutes the first known example of a tumor suppressor protein that directly regulates reactive oxygen species production in vertebrates.

Transcriptome dysregulation by anthrax lethal toxin plays a key role in induction of human endothelial cell cytotoxicity

We have investigated how Bacillus anthracis lethal toxin (LT) triggers caspase‐3 activation and the formation of thick actin cables in human endothelial cells. By DNA array analysis we show that LT has a major impact on the cell transcriptome and we identify key host genes involved in LT cytotoxic effects. Indeed, upregulation of TRAIL and downregulation of XIAP both participate in LT‐induced caspase‐3 activation. LT induces a downregulation of the immediate early gene and master regulator of transcription egr1. Importantly, its re‐expression in LT‐intoxicated cells blocks caspase‐3 activation. In parallel, we found that the formation of actin cables induced by LT occurs in the absence of direct activation of RhoA/ROCK signalling. We show that knock‐down of cortactin and rhophilin‐2 under conditions of calponin‐1 expression defines the minimal set of genes regulated by LT for actin cable formation. Together our data establish that the modulation of the cell transcriptome by LT plays a key role in triggering human endothelial cell toxicity.

alpha2beta1 integrin controls association of Rac with the membrane and triggers quiescence of endothelial cells.

Integrin receptors and their extracellular matrix ligands provide cues to cell proliferation, survival, differentiation and migration. Here, we show that α2β1 integrin, when ligated to the basement membrane component laminin-1, triggers a proliferation arrest in primary endothelial cells. Indeed, in the presence of strong growth signals supplied by growth factors and fibronectin, α2β1 engagement alters assembly of mature focal adhesions by α5β1 and leads to impairment of downstream signaling and cell-cycle arrest in the G1 phase. Although the capacity of α5β1 to signal for GTP loading of Rac is preserved, the joint engagement of α2β1 interferes with membrane anchorage of Rac. Adapting the ‘split-ubiquitin’ sensor to screen for membrane-proximal α2 integrin partners, we identified the CD9 tetraspanin and further establish its requirement for destabilization of focal adhesions, control of Rac subcellular localization and growth arrest induced by α2β1 integrin. Altogether, our data establish that α2β1 integrin controls endothelial cell commitment towards quiescence by triggering a CD9-dependent dominant signaling.

Ubiquitylation of active Rac1 by the E3 ubiquitin-ligase HACE1.

Rho GTPases undergo ubiquitylation and degradation via the ubiquitin-proteasome pathway. We now report in the November issue of Developmental Cell that the E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of GTP-bound Rac1. Depletion of HACE1 leads to an increase of Rac1 activity. We have proposed that HACE1 limits Rac1 activity in cells, a regulation that is usurped by some pathogenic bacteria for efficient invasion of host cell monolayers. We here review these findings in parallel with the regulation of RhoA by the ubiquitin and proteasome system (UPS) and discuss the impact of these regulations on the capacity of Rho GTPases to signal.

The role of extracellular matrix in vascular branching morphogenesis

Angiogenesis requires the development of a hierarchically branched network of vessels, which undergoes radial expansion and anastomosis to form a close circuit. Branching is achieved by coordinated behavior of endothelial cells that organize into leading “tip” cells and trailing “stalk” cells. Such organization is under control of the Dll4-Notch signaling pathway, which sets a hierarchy in receptiveness of cells to VEGF-A. Recent studies have shed light on a control of the Notch pathway by basement membrane proteins and integrin signaling, disclosing that extracellular matrix exerts active control on vascular branching morphogenesis. We will survey in the present review how extracellular matrix is a multifaceted substrate, which behind a classical structural role hides a powerful conductor function to shape the branching pattern of vessels.

Inhibition of the GTPase Rac1 Mediates the Antimigratory Effects of Metformin in Prostate Cancer Cells

Cell migration is a critical step in the progression of prostate cancer to the metastatic state, the lethal form of the disease. The antidiabetic drug metformin has been shown to display antitumoral properties in prostate cancer cell and animal models; however, its role in the formation of metastases remains poorly documented. Here, we show that metformin reduces the formation of metastases to fewer solid organs in an orthotopic metastatic prostate cancer cell model established in nude mice. As predicted, metformin hampers cell motility in PC3 and DU145 prostate cancer cells and triggers a radical reorganization of the cell cytoskeleton. The small GTPase Rac1 is a master regulator of cytoskeleton organization and cell migration. We report that metformin leads to a major inhibition of Rac1 GTPase activity by interfering with some of its multiple upstream signaling pathways, namely P-Rex1 (a Guanine nucleotide exchange factor and activator of Rac1), cAMP, and CXCL12/CXCR4, resulting in decreased migration of prostate cancer cells. Importantly, overexpression of a constitutively active form of Rac1, or P-Rex, as well as the inhibition of the adenylate cyclase, was able to reverse the antimigratory effects of metformin. These results establish a novel mechanism of action for metformin and highlight its potential antimetastatic properties in prostate cancer. Mol Cancer Ther; 14(2); 586–96. ©2014 AACR.