Phosphatidylinositol-4-phosphate 5-Kinase and GEP100/Brag2 Protein Mediate Antiangiogenic Signaling by Semaphorin 3E-Plexin-D1 through Arf6 Protein

Abstract

The semaphorins are a family of secreted or membranebound proteins that are known to guide axons in the developing nervous system. Genetic evidence revealed that a class III semaphorin, semaphorin 3E (Sema3E), and its receptor Plexin-D1 also control the vascular patterning during development. At the molecular level, we have recently shown that Sema3E acts on Plexin-D1 expressed in endothelial cells, thus initiating a novel antiangiogenic signaling pathway that results in the retraction of filopodia in endothelial tip cells. Sema3E induces the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix. This process requires the activation of small GTPase Arf6 (ADPribosylation factor 6), which regulates intracellular trafficking of ␤1 integrin. However, the molecular mechanisms by which Sema3E-Plexin-D1 activates Arf6 remained to be identified. Here we show that GEP100 (guanine nucleotide exchange protein 100)/Brag2, a guanine nucleotide exchange factor for Arf6, mediates Sema3E-induced Arf6 activation in endothelial cells. We provide evidence that upon activation by Sema3E, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase, and its enzymatic lipid product, phosphatidylinositol 4,5-bisphosphate, binds to the pleckstrin homology domain of GEP100. Phosphatidylinositol 4,5-bisphosphate binding to GEP100 enhances its guanine nucleotide exchange factor activity toward Arf6, thus resulting in the disassembly of integrinmediated focal adhesions and endothelial cell collapse. Our present study reveals a novel phospholipid-regulated antiangiogenic signaling pathway whereby Sema3E activates Arf6 through Plexin-D1 and consequently controls integrin-mediated endothelial cell attachment to the extracellular matrix and migration.Angiogenesis, the formation of new blood capillaries from pre-existing ones, is an essential process during embryonic development. In adults, angiogenesis is required for tissue repair during physiological wound healing, whereas dysregulated angiogenesis contributes to a variety of pathological conditions, such as diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and cancer (1). Hence, the identification of molecular mechanisms controlling normal and aberrant angiogenesis may afford new therapeutic opportunities for multiple human diseases (2). In this regard, recent studies revealed that axon guidance molecules, including netrin, slit, eph, and semaphorins, play a key role in developmental and postnatal angiogenesis (3). Among them, multiple secreted class III semaphorins (Sema3s) 4 are reported to control endothelial cell migration in vitro and in vivo (4 -7). Sema3s signal through A-type and D-type Plexin family proteins (Plexin-A1, -A2, and -A3 and Plexin-D1) and utilize their coreceptor neuropilins (Nrp1 and Nrp2) to tightly control proand antiangiogenic responses (8). However, the downstream signaling pathways initiated by these semaphorin receptors are complex and not fully understood,...

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

“…Consistent with previous studies [20,45] HUVEC and human microvascular endothelial cells express Brag2a and Brag2b protein as assessed by Western blot (data not shown). Moreover, we confirmed that VEGF stimulation rapidly increases the activity of the small GTPase Arf6 in EC (supplemental Figures S1A and S1B) as previously reported [18,20,27].…”

“…Furthermore, the pharmacological inhibition of Arf6-GEFs of the cytohesin family with SecinH3 reduced angiogenesis [27]. Intriguingly, not only inhibition but also an over-activation of the Brag2/Arf6 pathway, as achieved by stimulation with Semaphorin 3E, blocks angiogenesis [44,45]. In conclusion, a stringent and balanced activation of Arf-GTPases by Brag2 is mandatory for efficient angiogenesis.…”

“…In EC VEGF stimulation induces the association of VEGFR2 with Brag2 and induces the activation of Arf6 [20]. Interestingly, Semaphorin 3E-induced over-activation of the Brag2/Arf6 pathway results in collapse of EC and angiogenesis inhibition [45]. On the other side, it was demonstrated that silencing of Brag2 reduces endothelial tube formation in vitro and angiogenesis in a model of Angioreactor-induced angiogenesis [20].…”

“…Therefore, we overexpressed following constructs in COS7 cells: WTBrag2a-GFP, Brag2a-DSec-GFP lacking the catalytic domain Sec7, which is essential for the activation of GTPases of the Arf family, Brag2a-DPH-GFP lacking the pleckstrin homology domain, which is required for the activation of the enzymatic activity of Brag2 by binding PI(4,5)P2 on membranes [26,45], or EGFP control vector and assessed b1-integrin endocytosis in a pulse-chased endocytosis assay by FACS in GFP ? cells.…”

“…Arf6 can be activated by several guanine exchange nucleotide factors (GEFs) [7]. Brag2 (GEP100/IQSEC1) is a GEF activating the small ArfGTPases Arf4, Arf5 and Arf6 [33,48] and is expressed in EC [20,45]. Brag2 was found to promote the invasive activity of breast cancer cells [34] and myoblast fusion [11,37].…”