Lysyl oxidase-like protein-2 regulates sprouting angiogenesis and type IV collagen assembly in the endothelial basement membrane.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.

S1P inhibits gap junctions in astrocytes: involvement of G and Rho GTPase/ROCK.

Sphingosine-1-phosphate (S1P) is a potent and pleiotropic bioactive lysophospholipid mostly released by activated platelets that acts on its target cells through its own G protein-coupled receptors. We have previously reported that mouse striatal astrocytes expressed mRNAs for S1P1 and S1P3 receptors and proliferate in response to S1P. Here, we investigated the effect of S1P on gap junctions. We show that a short-term exposure of astrocytes to S1P causes a robust inhibition of gap junctional communication, as demonstrated by dye coupling experiments and double voltage-clamp recordings of junctional currents. The inhibitory effect of S1P on dye coupling involves the activation of both Gi and Rho GTPases. Rho-associated kinase (ROCK) also plays a critical role. The capacity of S1P to activate a Rho/ROCK axis in astrocytes is demonstrated by the typical remodeling of actin cytoskeleton. Connexin43, the protein forming gap junction channels, is a target of the Gi- and Rho/ROCK-mediated signaling cascades. Indeed, as shown by Western blots and confocal immunofluorescence, its nonphosphorylated form increases following S1P treatment and this change does not occur when both cascades are disrupted. This novel effect of S1P may have an important physiopathological significance when considering the proposed roles for astrocyte gap junctions on neuronal survival.

Heterodimerization of endothelin-converting enzyme-1 isoforms regulates the subcellular distribution of this metalloprotease.

Endothelin-converting enzyme (ECE) is a membrane metalloprotease that generates endothelin from its direct precursor big endothelin. Four isoforms of ECE-1 are produced from a single gene through the use of alternate promoters. These isoforms share the same extracellular catalytic domain and contain unique cytosolic tails, which results in their specific subcellular targeting. We investigated the distribution of ECE-1 isoforms in transfected AtT-20 neuroendocrine cells. Whereas ECE-1a and 1c were present at the plasma membrane, ECE-1b and ECE-1d were retained inside the cells. We found that both intracellular isoforms were concentrated in the endosomal system: ECE-1d in recycling endosomes, and ECE-1b in late endosomes/multivesicular bodies. Leucine-based motifs were involved in the intracellular retention of these isoforms, and the targeting of ECE-1b to the degradation pathway required an additional signal in the N terminus. The concentration of ECE-1 isoforms in the endosomal system suggested new functions for these enzymes. Potential novel functions include redistribution of other isoforms through direct interaction. We have showed that ECE-1 isoforms could heterodimerize, and that in such heterodimers the ECE-1b targeting signal was dominant. Interaction of a plasma membrane isoform with ECE-1b resulted in its intracellular localization and decreased its extracellular activity. These data demonstrated that the targeting signals specific for ECE-1b constitute a regulatory domain per se that could modulate the localization and the activity of other isoforms.