Fibroblast growth factor–specific modulation of cellular response by syndecan-4

Proteoglycans participate in growth factor interaction with the cell surface through their heparan sulfate chains (HS), but it is not known if they are otherwise involved in growth factor signaling. It appears now that the syndecan-4 core protein, a transmembrane proteoglycan shown previously to bind phosphatidylinositol 4,5-bisphosphate (PIP2) and activate PKCα, participates in mediating the effects of fibroblast growth factor (FGF)2 on cell function. Mutations in the cytoplasmic tail of syndecan-4 that either reduced its affinity to PIP2 (PIP2−) or disrupted its postsynaptic density 95, disk large, zona occludens-1 (PDZ)-dependent binding (PDZ−) produced a FGF2-specific dominant negative phenotype in endothelial cells as evidenced by the marked decline of their migration and proliferation rates and the impairment of their capacity to form tubes. In both cases, the molecular mechanism was determined to consist of a decrease in the syndecan-4–dependent activation of PKCα. This decrease was caused either by inhibition of FGF2-induced syndecan-4 dephosphorylation in the case of the PDZ− mutation or by disruption of basolateral targeting of syndecan-4 and its associated PDZ-dependent complex in the case of the PIP2− mutation. These results suggest that PKCα activation and PDZ-mediated formation of a serine/threonine phosphatase-containing complex by syndecan-4 are downstream events of FGF2 signaling

Murine MPDZ-linked hydrocephalus is caused by hyperpermeability of the choroid plexus.

Though congenital hydrocephalus is heritable, it has been linked only to eight genes, one of which is MPDZ Humans and mice that carry a truncated version of MPDZ incur severe hydrocephalus resulting in acute morbidity and lethality. We show by magnetic resonance imaging that contrast medium penetrates into the brain ventricles of mice carrying a Mpdz loss-of-function mutation, whereas none is detected in the ventricles of normal mice, implying that the permeability of the choroid plexus epithelial cell monolayer is abnormally high. Comparative proteomic analysis of the cerebrospinal fluid of normal and hydrocephalic mice revealed up to a 53-fold increase in protein concentration, suggesting that transcytosis through the choroid plexus epithelial cells of Mpdz KO mice is substantially higher than in normal mice. These conclusions are supported by ultrastructural evidence, and by immunohistochemistry and cytology data. Our results provide a straightforward and concise explanation for the pathophysiology of Mpdz-linked hydrocephalus

The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

AbstractThough the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion regulator, Filamin A (FlnA). Endothelial cells (ECs) expressing a Nrp1 mutant devoid of the cytoplasmic domain (nrp1cytoΔ/Δ) migrated significantly slower in response to VEGF relative to the cells expressing wild-type Nrp1 (nrp1+/+ cells). The rate of FA turnover in VEGF-treated nrp1cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1+/+ ECs, thus accounting for the slower migration rate of the nrp1cytoΔ/Δ ECs

Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin

Receptor-type protein tyrosine phosphatases (RPTPs) of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, di-merization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC) in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascu-lar integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with con-trols. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a mem-brane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane do-mains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that re-moval of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and transmembrane domain of R3-PTPs in interaction with VE-cadherin

VEGF and Angiopoietin-1 Exert Opposing Effects on Cell Junctions by Regulating the Rho GEF Syx

Vascular endothelial growth factor (VEGF) and Ang1 (Angiopoietin-1) have opposing effects on vascular permeability, but the molecular basis of these effects is not fully known. We report in this paper that VEGF and Ang1 regulate endothelial cell (EC) junctions by determining the localization of the RhoA-specific guanine nucleotide exchange factor Syx. Syx was recruited to junctions by members of the Crumbs polarity complex and promoted junction integrity by activating Diaphanous. VEGF caused translocation of Syx from cell junctions, promoting junction disassembly, whereas Ang1 maintained Syx at the junctions, inducing junction stabilization. The VEGF-induced translocation of Syx from EC junctions was caused by PKD1 (protein kinase D1)-mediated phosphorylation of Syx at Ser806, which reduced Syx association to its junctional anchors. In support of the pivotal role of Syx in regulating EC junctions, syx−/− mice had defective junctions, resulting in vascular leakiness, edema, and impaired heart function

PLoS One

Quantitative analysis of the vascular network anatomy is critical for the understanding of the vasculature structure and function. In this study, we have combined microcomputed tomography (microCT) and computational analysis to provide quantitative three-dimensional geometrical and topological characterization of the normal kidney vasculature, and to investigate how 2 core genes of the Wnt/planar cell polarity, Frizzled4 and Frizzled6, affect vascular network morphogenesis. Experiments were performed on frizzled4 (Fzd4-/-) and frizzled6 (Fzd6-/-) deleted mice and littermate controls (WT) perfused with a contrast medium after euthanasia and exsanguination. The kidneys were scanned with a high-resolution (16 μm) microCT imaging system, followed by 3D reconstruction of the arterial vasculature. Computational treatment includes decomposition of 3D networks based on Diameter-Defined Strahler Order (DDSO). We have calculated quantitative (i) Global scale parameters, such as the volume of the vasculature and its fractal dimension (ii) Structural parameters depending on the DDSO hierarchical levels such as hierarchical ordering, diameter, length and branching angles of the vessel segments, and (iii) Functional parameters such as estimated resistance to blood flow alongside the vascular tree and average density of terminal arterioles. In normal kidneys, fractal dimension was 2.07±0.11 (n = 7), and was significantly lower in Fzd4-/- (1.71±0.04; n = 4), and Fzd6-/- (1.54±0.09; n = 3) kidneys. The DDSO number was 5 in WT and Fzd4-/-, and only 4 in Fzd6-/-. Scaling characteristics such as diameter and length of vessel segments were altered in mutants, whereas bifurcation angles were not different from WT. Fzd4 and Fzd6 deletion increased vessel resistance, calculated using the Hagen-Poiseuille equation, for each DDSO, and decreased the density and the homogeneity of the distal vessel segments. Our results show that our methodology is suitable for 3D quantitative characterization of vascular networks, and that Fzd4 and Fzd6 genes have a deep patterning effect on arterial vessel morphogenesis that may determine its functional efficiency

A PDZ-binding Motif as a Critical Determinant of Rho Guanine Exchange Factor Function and Cell Phenotype

We identified a Rho guanine exchange factor (GEF) expressed as two splice variants, which differ only in either having or lacking a Postsynaptic density 95, Disk large, Zona occludens-1 (PDZ) motif. The PDZ adaptor protein synectin bound the longer splice variant, Syx1, which was targeted to the plasma membrane in a synectin-dependent manner. The shorter variant, Syx2, was diffusely distributed in the cytoplasm. Fluorescence resonance energy transfer (FRET) imaging revealed similar differences between the spatial patterns of active RhoA in Syx1 versus Syx2-expressing cells. Expression of Syx1 augmented endothelial cell (EC) migration and tube formation, whereas Syx2 expression did not. It appears, therefore, that synectin-dependent targeting of Syx is critical to its contribution to these EC functions. Although agonist-stimulated global RhoA activity was similar in Syx1- and Syx2-expressing cells, basal RhoA activity was surprisingly higher in the latter. Out of 23 cell types, we found a significant level of endogenous Syx2 expression only in brain tumor cells, which also exhibited high basal RhoA activity. We found that the activity level of JNK, which mediates transcriptional regulation downstream of RhoA, is elevated in a Syx2-dependent manner in these cells, possibly contributing to their tumorigenicity