Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.National Science Foundation (U.S.) (NSF EFRI grant 735997)National Institutes of Health (U.S.) (NIH Cell Migration Consortium grant GM06346)National Institutes of Health (U.S.) (NIH Cell Decision Processes Center grant GM68762)National Institutes of Health (U.S.) (NIH grant GM69668)National Institutes of Health (U.S.) (NIH grant GM81336

Expression of Odorant Receptor Family, Type 2 OR in the Aquatic Olfactory Cavity of Amphibian Frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions

Etude des interactions entre une protéine humaine de liaison aux odorants et ses partenaires, odorants et récepteurs olfactifs

Les molécules odorantes, généralement hydrophobes, activent les récepteurs olfactifs (RO) exprimés à la membrane des neurones sensitifs recouverts d une couche de mucus hydrophile. Des protéines liant les odorants, les OBP (Odorant-Binding Protein) présentes dans le mucus olfactif transporteraient les odorants à travers le mucus vers les RO. On s est intéressé dans ce travail à un variant humain d OBP, hOBP-2A qui présente une spécificité de liaison pour les aldéhydes et les acides aliphatiques. Par une approche de mutagenèse dirigée, on a pu montrer le rôle fondamental dans cette spécificité d une lysine de la cavité de hOBP-2A. L expression fonctionnelle d un RO, OR1G1, a montré son activation par des composés se liant à hOBP-2A. Une étude comparative de l activation avec et sans OBP a révélé qu elle n avait pas d impact sur l activité du récepteur OR1G1. Ce qui n exclue pas la possibilité que les OBP interviennent dans d autres processus biologiques que le transport des odorants.Odorant molecules, generally hydrophobic, activate the olfactory receptors (OR) embedded in the membrane of sensitive neurons which are covered with an aqueous layer of mucus. It was proposed that odorant-binding proteins, named OBP, transport odorants through olfactory mucus towards OR. In this work we studied a human OBP variant, hOBP-2A, which has a narrow specificity for aliphatic aldehydes and acids. Using site directed mutagenesis, we demonstrated the fundamental role, in this specificity, of a lysin located in the hydrophobic cavity of hOBP-2A. The functional expression of an olfactory receptor, OR1G1, revealed an activation of this receptor by some compounds that bind to hOBP-2A. Study of the activation of OR1G1 with and without hOBP-2A revealed that hOBP-2A did not have impact on OR1G1 activity. One cannot however exclude the possibility that the OBP is involved in other biological processes than odorants transport.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Identification of a lysyl residue defining the binding specificity of a human odorant-binding protein

International audienc

Identification of a lysyl residue defining the binding specificity of a human odorant-binding protein

International audienc

Stx transport in annexin A1 depleted cells is regulated by PKCδ and PLA2.

<p>(A) Golgi transport of Shiga toxin was evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040429#s4" target="_blank">materials and methods</a> by quantification of sulfated ShigaB in HeLa cells transfected with siRNA against annexin A1 or non targeting siRNA, pretreated with the indicated inhibitors. Data from Stx sulfation are plotted as percentages of the value obtained for HeLa cells transfected with control siRNA and treated with DMSO. The white and black bars represent ShigaB-sulf2 sulfation for control and annexin A1 knockdown cells respectively. Data presented are the average of 3 independent experiments, each performed in parallel, error bars indicating standard error of the mean. *p<0.05 indicates statistically significant change between annexin A1 knockdown cells and the corresponding control siRNA treated cells. (B) After treatment with either 5 µM ONO-RS-082 for 30 min or 30 µM MAFP for 1 h, HeLa cells were incubated for 30 min with ShigaB before fixation and staining as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040429#s4" target="_blank">materials and methods</a> section with antibodies against TGN46 and ShigaB. Panel shows representative confocal pictures, scale bars 20 μm. Graphic shows quantification of amount of ShigaB colocalized with TGN46 in one representative experiment plotted as percentage of control condition. Data presented for one representative experiment (n = 3) are the average of at least 30 cells per condition. Quantifications were obtained with Zen 2009 software from Zeiss, error bars indicating standard error of the mean.</p

Annexin A1 and A2: Roles in Retrograde Trafficking of Shiga Toxin

<div><p>Annexins constitute a family of calcium and membrane binding proteins. As annexin A1 and A2 have previously been linked to various membrane trafficking events, we initiated this study to investigate the role of these annexins in the uptake and intracellular transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Once endocytosed, both toxins are retrogradely transported from endosomes to the Golgi apparatus and the endoplasmic reticulum before being targeted to the cytosol where they inhibit protein synthesis. This study was performed to obtain new information both about toxin transport and the function of annexin A1 and annexin A2. Our data show that depletion of annexin A1 or A2 alters the retrograde transport of Stx but not ricin, without affecting toxin binding or internalization. Knockdown of annexin A1 increases Golgi transport of Stx, whereas knockdown of annexin A2 slightly decreases the same transport step. Interestingly, annexin A1 was found in proximity to cytoplasmic phospholipase A2 (cPLA₂), and the basal as well as the increased Golgi transport of Stx upon annexin A1 knockdown is dependent on cPLA₂ activity. In conclusion, annexin A1 and A2 have different roles in Stx transport to the <em>trans</em>-Golgi network. The most prominent role is played by annexin A1 which normally works as a negative regulator of retrograde transport from the endosomes to the Golgi network, most likely by complex formation and inhibition of cPLA₂.</p> </div

ShigaB transport to the Golgi is regulated by annexin A1 and A2.

<p>HeLa cells were transfected with control, annexin A1 or A2 siRNA for 72 h. For sulfation measurements, cells were starved in the presence of radioactive sulfate for 3 h. ShigaB-sulf2 or Ricin-sulf1 was then added and the incubation proceeded for an additional one or two hours, respectively. Cells were lyzed, ShigaB or ricin immunoprecipitated, separated by electrophoresis and analyzed by autoradiography. The protein knockdown level was investigated in total cell lysates by immunoblotting. (A) Cell lysates were analyzed by western blotting (upper panel) with the indicated antibodies demonstrating protein knockdown of annexin A1 and A2 by the indicated siRNA oligos. Hsp90 represents loading control. Autoradiography (lower panel) showing results from the corresponding sulfation experiment. (B) and (C) Quantative data from protein sulfation for ShigaB and ricin respectively, plotted as percentages of control values. Quantifications of sulfation are the average of 3–8 independent experiments, each performed in parallel, error bars indicating standard error of the mean; *p<0.05, **p<0.005 indicate statistically significant change.</p