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Autosomal dominant mutations in sarcomere proteins such as the cardiac troponin T ( TNNT2) are the main genetic causes of human hypertrophic cardiomyopathy and dilated cardiomyopathy. Allele-specific silencing by RNA interference (ASP-RNAi) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. Here, we propose ASP-RNAi as a possible strategy to specifically knockdown mutant alleles coding for R92Q and R173W mutant TNNT2 proteins, identified in hypertrophic and dilated cardiomyopathy, respectively. Different siRNAs were designed and validated by luciferase reporter assay and following analysis in HEK293T cells expressing either the wild-type or mutant TNNT2 alleles. This study is the first exploration of ASP-RNAi on TNNT2-R173W and TNNT2-R92Q mutations in vitro and gives a base for further application of allele silencing as a therapeutic treatment for TNNT2-mutation-associated cardiomyopathies.

Blocking the signaling activated by the plasma membrane receptor CD93 has recently been demonstrated a useful tool in antiangiogenic treatment and oncotherapy. In the proliferating endothelium, CD93 regulates cell adhesion, migration, and vascular maturation, yet it is unclear how CD93 interacts with the extracellular matrix activating signaling pathways involved in the vascular remodeling. Here for the first time we show that in endothelial cells CD93 is structured as a dimer and that this oligomeric form is physiologically instrumental for the binding of CD93 to its ligand Multimerin-2. Crystallographic X-ray analysis of recombinant CD93 reveals the crucial role played by the C-type lectin-like and sushi-like domains in arranging as an antiparallel dimer to achieve a functional binding state, providing key information for the future design of new drugs able to hamper CD93 function in neovascular pathologies.

The glycogen synthase kinase 3β (GSK-3β) is a ubiquitous enzyme that is a validated target for the development of potential therapeutics useful in several diseases including retinal degeneration. Aiming at developing an innovative class of allosteric inhibitors of GSK-3β potentially useful for retinal degeneration, we explored the class of squaramides. The developed compounds (6 a-l) were obtained through a nontoxic one-pot synthetic protocol, which employs low-cost goods and avoids any purification step. Ethanol was used as the reaction solvent, simultaneously allowing the pure reaction products' recovery (by precipitation). Out of this set of squaramides, 6 j stood out, from computational and enzymatic converging data, as an ATP non-competitive inhibitor of GSK-3β of micromolar potency. When engaged in cellular studies using retinal pigment epithelial cells (ARPE-19) transfected with a luciferase reporter gene under the control of T-cell factor/lymphoid enhancer factor (TCF/LEF) binding sites, 6 j was able to dose-dependently induce β-catenin nuclear accumulation, as shown by the increased luciferase activity at a concentration of 2.5 μM.

Endothelial cell migration is essential to angiogenesis, enabling the outgrowth of new blood vessels both in physiological and pathological contexts. Migration requires the activation of several signaling pathways, the elucidation of which expands the opportunity to develop new drugs to be used in antiangiogenic therapy. In the proliferating endothelium, the interaction between the transmembrane glycoprotein CD93 and the extracellular matrix activates signaling pathways that regulate cell adhesion, migration, and vascular maturation. Here we identify a pathway, comprising CD93, the adaptor proteins Cbl and Crk, and the small GTPases Rac1, Cdc42, and RhoA, which we propose acts as a regulator of cytoskeletal movements responsible for endothelial cell migration. In this framework, phosphorylation of Cbl on tyrosine 774 leads to the interaction with Crk, which acts as a downstream integrator in the CD93-mediated signaling regulating cell polarity and migration. Moreover, confocal microscopy analyses of GTPase biosensors show that CD93 drives coordinated activation of Rho-proteins at the cell edge of migratory endothelial cells. In conclusion, together with the demonstration of the key contribution of CD93 to the migratory process in living cells, these findings suggest that the signaling triggered by CD93 converges to the activation and modulation of the Rho GTPase signaling pathways regulating cell dynamics.

During skeletal myogenesis, the zinc-finger transcription factors SNAI1 and SNAI2, are expressed in proliferating myoblasts and regulate the transition to terminally differentiated myotubes while repressing pro-differentiation genes. Here, we demonstrate that SNAI1 is upregulated in vivo during the early phase of muscle regeneration induced by bupivacaine injury. Using shRNA-mediated gene silencing in C2C12 myoblasts and whole-transcriptome microarray analysis, we identified a collection of genes belonging to the endoplasmic reticulum (ER) stress pathway whose expression, induced by myogenic differentiation, was upregulated in absence of SNAI1. Among these, key ER stress genes, such as Atf3, Ddit3/Chop, Hspa5/Bip, and Fgf21, a myokine involved in muscle differentiation, were strongly upregulated. Furthermore, by promoter mutant analysis and Chromatin immune precipitation assay, we demonstrated that SNAI1 represses Fgf21 and Atf3 in proliferating myoblasts by directly binding to multiple E boxes in their respective promoter regions. Together, these data describe a new regulatory mechanism of myogenic differentiation involving the direct repressive action of SNAI1 on ER stress and Fgf21 expression, ultimately contributing to maintaining the proliferative and undifferentiated state of myoblasts.

The use of light-responsive proteins to control both living or synthetic cells, is at the core of the expanding fields of optogenetics and synthetic biology. It is thus apparent that a richer reaction toolbox for the preparation of such systems is of fundamental importance. Here, we provide a proof-of-principle demonstration that Morita-Baylis-Hillman adducts can be employed to perform a facile site-specific, irreversible and diastereoselective click-functionalization of a lysine residue buried into a lipophilic binding pocket and yielding an unnatural chromophore with an extended π-system. In doing so we effectively open the path to the in vitro preparation of a library of synthetic proteins structurally reminiscent of xanthopsin eubacterial photoreceptors. We argue that such a library, made of variable unnatural chromophores inserted in an easy-to-mutate and crystallize retinoic acid transporter, significantly expand the scope of the recently introduced rhodopsin mimics as both optogenetic and "lab-on-a-molecule" tools.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Background In the endothelium, the single-pass membrane protein CD93, through its interaction with the extracellular matrix protein Multimerin-2, activates signaling pathways that are critical for vascular development and angiogenesis. Trafficking of adhesion molecules through endosomal compartments modulates their signaling output. However, the mechanistic basis coordinating CD93 recycling and its implications for endothelial cell (EC) function remain elusive. Methods Human umbilical vein ECs (HUVECs) and human dermal blood ECs (HDBEC) were used in this study. Fluorescence confocal microscopy was employed to follow CD93 retrieval, recycling, and protein colocalization in spreading cells. To better define CD93 trafficking, drug treatments and transfected chimeric wild type and mutant CD93 proteins were used. The scratch assay was used to evaluate cell migration. Gene silencing strategies, flow citometry, and quantification of migratory capability were used to determine the role of Rab5c during CD93 recycling to the cell surface. Results Here, we identify the recycling pathway of CD93 following EC adhesion and migration. We show that the cytoplasmic domain of CD93, by its interaction with Moesin and F-actin, is instrumental for CD93 retrieval in adhering and migrating cells and that aberrant endosomal trafficking of CD93 prevents its localization at the leading edge of migration. Moreover, the small GTPase Rab5c turns out to be a key component of the molecular machinery that is able to drive CD93 recycling to the EC surface. Finally, in the Rab5c endosomal compartment CD93 forms a complex with Multimerin-2 and active β1 integrin, which is recycled back to the basolaterally-polarized cell surface by clathrin-independent endocytosis. Conclusions Our findings, focusing on the pro-angiogenic receptor CD93, unveil the mechanisms of its polarized trafficking during EC adhesion and migration, opening novel therapeutic opportunities for angiogenic diseases. Electronic supplementary material The online version of this article (10.1186/s12964-019-0375-x) contains supplementary material, which is available to authorized users.

During angiogenesis, cell adhesion molecules expressed on the endothelial cell surface promote the growth and survival of newly forming vessels. Hence, elucidation of the signaling pathways activated by cell-to-matrix adhesion may assist in the discovery of new targets to be used in antiangiogenic therapy. In proliferating endothelial cells, the single-pass transmembrane glycoprotein CD93 has recently emerged as an important endothelial cell adhesion molecule regulating vascular maturation. In this study, we unveil a signaling pathway triggered by CD93 that regulates actin cytoskeletal dynamics responsible of endothelial cell adhesion. We show that the Src-dependent phosphorylation of CD93 and the adaptor protein Cbl leads to the recruitment of Crk, which works as a downstream integrator in the CD93-mediated signaling. Moreover, confocal microscopy analysis of FRET-based biosensors shows that CD93 drives the coordinated activation of Rac1 and RhoA at the cell edge of spreading cells, thus promoting the establishment of cell polarity and adhesion required for cell motility.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats&rsquo, eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Aims. Anethole dithiolethione (ADT) is a marketed drug to treat xerostomia. Its mechanism of action is still unknown, but several preclinical studies indicate that it is able to increase intracellular glutathione (GSH) and protect against oxidative stress. Here, we investigated the molecular mechanisms behind these effects. Results. Oral treatment of rats confirmed the GSH enhancing properties of ADT; among the different organs examined in this study, only the kidney showed a significant GSH increase that was already observed at low-dose treatments. The increase in GSH correlated with a decrease in γ-glutamyltranspeptidase (γ-GT) activity of the different tissues. In vitro and ex vivo experiments with tubular renal cells and isolated perfused rat kidney showed that the cellular uptake of intact GSH was correlated with the extracellular concentrations of GSH. Conclusions. The prominent in vivopharmacological effect of ADT was a marked increase of GSH concentration in the kidney and a decrease of some systemic and renal biomarkers of oxidative stress. In particular, by inhibition of γ-GT activity, it decreased the production cysteinylglycine, a thiol that has prooxidant effects as the consequence of its autooxidation. The activity of ADT as GSH enhancer in both the circulation and the kidney was long-lasting. All these characteristics make ADT a promising drug to protect the kidney, and in particular proximal tubule cells, from xenobiotic-induced damage.

PURPOSE. The purpose of this study was to investigate the involvement of CD93 and Multimerin-2 in three choroidal neovascularization (CNV) models and to evaluate their contribution in the neovascular progression of age-related macular degeneration (AMD). METHODS. Choroidal neovascular membranes collected during surgery from AMD patients were analyzed by microscopy methods. Laser-induced CNV mouse models and choroid sprouting assays (CSAs) were carried out using the CD93 knockout mouse model. An original ex vivo CSA of vascular angiogenesis, employing choroid tissues isolated from human donors, was developed. RESULTS. In contrast to healthy choroid endothelium, hyperproliferative choroidal endothelial cells (ECs) of AMD patients expressed high levels of CD93, and Multimerin-2 was abundantly deposited along the choroidal neovasculature. CD93 knockout mice showed a significant reduced neovascularization after laser photocoagulation, and their choroidal ECs displayed a decreased ability to produce sprouts in ex vivo angiogenesis assays. Moreover, the presence of an antibody able to hamper the CD93/Multimerin-2 interaction reduced vascular sprouting in the human CSA. CONCLUSIONS. Our results demonstrate that CD93 and its interaction with Multimerin-2 play an important role in pathological vascularization of the choroid, disclosing new possibilities for therapeutic intervention to neovascular AMD.

Several drugs are currently in use as glutathione (GSH) enhancers in clinical, pre-clinical and experimental research. Here we compare the ability of N-acetylcysteine (NAC), 2-oxothiazolidine-4-carboxylic acid (OTC), glutathione ethyl ester (GSH-EE) and N-acetylcysteine ethyl ester (NACET) to increase the intracellular concentration of GSH using primary human umbilical vein endothelial cells (HUVEC) as in vitro model. Our experiments highlighted that NACET is largely the most efficient molecule in increasing the intracellular levels of GSH, cysteine, and γ-glutamylcysteine. This is because NACET is lipophilic and can freely cross plasma membrane but, inside the cell, it is de-esterified to the more hydrophilic NAC, which, in turn, is trapped into the cell and slowly transformed into cysteine. The higher availability of cysteine is matched by an increase in GSH synthesis, cysteine availability being the rate limiting step for this reaction. Surprisingly, the increase in GSH concentration was not linear but peaked at 0.5 mM NACET and gradually decreased when cells were treated with higher concentrations of NACET. We demonstrated that this puzzling ceiling effect was due to the fact that NAC released from NACET turned out to be a competitive inhibitor of the enzyme glutamate-cysteine ligase, with a Ki value of 3.2 mM. By using a cell culture medium lacking of cysteine and methionine, we could demonstrate that the slight increase in intracellular levels of cysteine and GSH induced by NAC in HUVEC grown in standard medium was due to the reduction of the cystine present in the medium itself there rather than to the action of NAC as Cys pro-drug. This fact may explain why NAC works well as GSH enhancer at very high concentrations in pre-clinical and in vitro studies, whereas it failed in most clinical trials.

In patients with age-related macular degeneration (AMD), choroidal neovascularization is the major cause of severe visual loss. In these patients, the persistence of neovascular growth despite vascular endothelial growth factor-A blockage needs the discovery of new endothelial cell targets. The glycoprotein CD93, highly expressed in activated endothelial cells, has been recently involved in the regulation of the angiogenic process both as transmembrane and soluble protein. Choroidal neovascular membranes from patients affected by AMD were examined by immunofluorescence using anti-CD93 and anti-von Willebrand factor antibodies. Blood vessels within intraocular and extraocular neoplasias were used as controls for CD93 expression. All choroidal neovascular membranes displayed strong CD93 staining in the von Willebrand factor-positive endothelial cells, consistently with the analyses showing a high colocalization coefficient in the blood vessels. Intraocular and extraocular tumor vessels showed similar results, whereas the normal choroid displayed blood vessels with only faint CD93 staining. Additionally, the concentration of soluble CD93 was determined in the aqueous humor of patients affected by naive neovascular AMD by enzyme-linked immunosorbent assays. Age-matched cataract patients served as controls. Soluble CD93 was significantly increased in the aqueous humor of naive neovascular AMD patients and tended to decrease after treatment with an antiangiogenic drug. In conclusion, both transmembrane and soluble CD93 are overexpressed in patients with neovascular AMD, indicating that CD93 may represent a potential new antiangiogenic target in the treatment of choroidal neovascularization. J. Cell. Physiol. 232: 1767-1773, 2017. © 2016 Wiley Periodicals, Inc.

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of AKU treatment is palliative and little is known about its physiopathology. Neovascularization is involved in the pathogenesis of systemic inflammatory rheumatic diseases, a family of related disorders that includes AKU. Here, we investigated the presence of neoangiogenesis in AKU synovium and healthy controls. Synovium from AKU patients, who had undergone total joint replacement or arthroscopy, or from healthy patients without any history of rheumatic diseases, who underwent surgical operation following sport trauma was subjected to hematoxylin and eosin staining. Histologic grades were assigned for clinical disease activity and synovitis based on cellular content of the synovium. By immunofluorescence microscopy, using different endothelial cell markers, we observed large vascularization in AKU but not in healthy synovium. Moreover, Western blotting and quantification analyses confirmed strong expression of endothelial cell markers in AKU synovial tissues. Importantly, AKU synovium vascular endothelium expressed high levels of β-dystroglycan, a protein previously involved in the regulation of angiogenesis in osteoarthritic synovium. This is the first report providing experimental evidences that new blood vessels are formed in AKU synovial tissues, opening new perspectives for AKU therapy.

Purpose This work was aimed to further characterize cells of idiopathic epiretinal membranes (iERMs). We wanted to determine the contribution of 90-kDa heat shock protein (HSP90) to sustain the transforming growth factor-β (TGF-β)-mediated signal transduction pathway in iERM. Methods Immunofluorescence and confocal microscopy were carried out on deplasticized sections from 36 epiretinal membranes processed for electron microscopy and on frozen sections from five additional samples with antibodies against α-smooth muscle actin (αSMA), vimentin, glial fibrillary acidic protein (GFAP), SMAD2, HSP90α, type-II TGF-β1 receptor (TβRII), type-I collagen, and type-IV collagen. In addition, Muller MIO-M1 cells were transfected with HSP90 and challenged with TGF-β1. Results Double and triple labeling experiments showed that a variable number of TβRII+ cells were present in 94.1% of tested iERMs and they were mostly GFAP-/αSMA+/vimentin+/HSP90α+. In almost half of the cases these cells contained type-I collagen, suggesting their involvement in matrix deposition. HSP90 overexpressing MIO-M1 cells challenged with TGF-β1 showed increased levels of TβRII, SMAD2, SMAD3, and phosphor-SMAD2. Nuclear SMAD2 staining could be observed in HSP90α+ cells on frozen sections of iERMs. Conclusions Cells in iERMs that express TβRII are also HSP90α+ and show the antigenic profile of myofibroblast-like cells as they are GFAP-/αSMA+/vimentin+. HSP90α-overexpressing MIO-M1 cells challenged with TGF-β1 showed an increased activation of the SMAD pathway implying that HSP90α might play a role in sustaining the TGF-β1-induced fibrotic response of iERM cells.

Controversy still exists regarding the role of the TGF-β in neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Here, we measured the concentrations of active TGF-β1, TGF-β2, and TGF-β3 by ELISA in the aqueous humor of 20 patients affected by nAMD, who received 3 consecutive monthly intravitreal injections of anti-VEGF-A antibody. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). The same samples were used in a luciferase-based reporter assay to test the TGF-β pathway activation. Active TGF-β1 concentrations in the aqueous humor were below the minimum detectable dose. Active TGF-β2 concentrations were significantly lower at baseline and at month 1, compared to controls. No significant differences in active TGF-β3 concentration were found among the sample groups. Moreover, TGF-β pathway activation was significantly lower at baseline compared to controls. Our data corroborate an anti-angiogenic role for TGF-β2 in nAMD. This should be considered from the perspective of a therapy using TGF-β inhibitors.

The multifunctional transforming growth factors-beta (TGF-βs) have been extensively studied regarding their role in the pathogenesis of neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Despite this, their effect remains somewhat controversial. Indeed, both pro- and antiangiogenic activities have been suggested for TGF-β signaling in the development and progression of nAMD, and opposite therapies have been proposed targeting the inhibition or activation of the TGF-β pathway. The present article summarizes the current literature linking TGF-β and nAMD, and reviews experimental data supporting both pro- and antiangiogenic hypotheses, taking into account the limitations of the experimental approaches.

We have introduced protein thiolation index (PTI), i.e. the molar ratio of the sum of all low molecular mass thiols bound to plasma proteins to protein free cysteinyl residues, as a sensitive biomarker of oxidative stress. According to the original procedure its determination requires a rapid separation of plasma and a specific treatment of samples to stabilize thiols. Here we demonstrate that samples can be collected without use of any anticoagulant to prevent blood clotting and without any stabilization of thiols too. This simplification of the determination of PTI makes its analysis more feasible also in routine clinical laboratories.

Purpose The purpose of this study was to evaluate the expression of high-temperature requirement A serine peptidase 1 (HTRA1), TGF-β1, bone morphogenetic protein 4 (BMP4), growth differentiation factor 6 (GDF6), and VEGFA proteins in the aqueous humor of patients with naive choroidal neovascularization (nCNV) secondary to AMD. Methods We measured by ELISA the concentrations of HTRA1, TGF-β1, BMP4, GDF6, and VEGFA in the aqueous humor of 23 patients affected by nCNV who received three consecutive monthly intravitreal injections of 0.5 mg ranibizumab. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). Twenty-three age-matched cataract patients served as controls. Results Bone morphogenetic protein 4 and GDF6 were not detectable in any samples. Baseline HTRA1 was higher than controls (P < 0.0001) and higher than both the month 1 (P < 0.0001) and the month 2 (P < 0.0001) values. Baseline VEGFA was higher than controls (P < 0.0001), not different from month 1 value (P = 0.0821), but higher than month 2 value (P < 0.0001). Baseline TGF-β1 was higher than controls (P = 0.0015) and not different from month 1 (P = 0.129) and month 2 values (P = 0.5529). No correlation was found in naive patients between concentrations of HTRA1 and TGF-β1, HTRA 1 and VEGFA, or TGF-β1 and VEGFA. Conclusions In nCNV patients, HTRA1 and TGF-β1 were significantly higher compared to controls. After treatment, TGF-β1 was persistently elevated, while HTRA1 returned to control levels, suggesting the involvement of TGF-β1 and HTRA1 in neovascular AMD and a VEGFA-independent role for TGF-β1.

Age-related macular degeneration (AMD) is a progressive chronic disease that currently represents the leading cause of irreversible vision loss in the western world. Experimental and clinical evidence has demonstrated that vascular endothelial growth factor A (VEGF-A) plays an important role in promoting the choroidal neovascularization that characterizes the wet form of AMD. Intravitreal injection of anti- VEGF-A agents is the current treatment of choice for neovascular AMD (nAMD). These agents have brought about dramatic changes in the treatment of nAMD, but most patients require frequently repeated injections and regular long-term follow-up, with a significant percentage of them showing resistance to anti-VEGF-A drugs. Thus, the identification of additional therapies that could improve the treatment protocols is needed. There are numerous areas of investigation into new treatments, with increasing efforts being made to study drugs that address various targets along the angiogenic signaling cascade, or other pathways related to the onset of nAMD. The aim of the present review is to summarize and discuss promising new therapies and targets that have the potential to improve outcomes and to lengthen treatment durability, especially in patients with recalcitrant or recurrent forms of nAMD.

Sustained signalling at the immune synapse (IS) requires the synaptic delivery of recycling endosome-associated T cell antigen receptors (TCRs). IFT20, a component of the intraflagellar transport system, controls TCR recycling to the IS as a complex with IFT57 and IFT88. Here, we used quantitative mass spectrometry to identify additional interaction partners of IFT20 in Jurkat T cells. In addition to IFT57 and IFT88, the analysis revealed new binding partners, including IFT54 (also known as TRAF3IP1), GMAP-210 (also known as TRIP11), Arp2/3 complex subunit-3 (ARPC3), COP9 signalosome subunit-1 (CSN1, also known as GPS1) and ERGIC-53 (also known as LMAN1). A direct interaction between IFT20 and both IFT54 and GMAP-210 was confirmed in pulldown assays. Confocal imaging of antigen-specific conjugates using T cells depleted of these proteins by RNA interference showed that TCR accumulation and phosphotyrosine signalling at the IS were impaired in the absence of IFT54, ARPC3 or ERGIC-53. Similar to in IFT20-deficient T cells, this defect resulted from a reduced ability of endosomal TCRs to polarize to the IS despite a correct translocation of the centrosome towards the antigen-presenting cell contact. Our data underscore the traffic-related role of an IFT20 complex that includes components of the intracellular trafficking machinery in IS assembly.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

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Endogenous thiols undergo rapid and reversible oxidation to disulfides when exposed to oxidants and are, therefore, suitable biomarkers of oxidative stress. However, accurate analysis of thiols in blood is frequently compromised by their artifactual oxidation during sample manipulation, which spuriously elevates the disulfide levels. Here, we describe a validated pre-analytical procedure that prevents both artifactual oxidation of thiols during sample manipulation and their oxidative decay for months in biosamples that are stored at -80°C. Addition of N-ethylmaleimide to blood samples from healthy donors was used to stabilize whole blood, red blood cells, platelets and plasma disulfides, whereas addition of citrate buffer followed by dilution of plasma with H2O was used to stabilize plasma thiols. The concentrations of thiols and disulfides were stable in all biosamples for at least 6 months when analyzed by UV/Vis HPLC at regular intervals. Only 3 ml of blood were needed to perform the analyses of thiols and disulfides in the different blood fractions. This pre-analytical procedure is reliable for use in both animal and human prospective studies. Its ease of implementation makes the method suitable for application to multicenter studies where blood samples are collected by different sites and personnel and are shipped to specific specialized laboratories.

CD93 is a transmembrane glycoprotein predominantly expressed in endothelial cells. Although CD93 displays proangiogenic activity, its molecular function in angiogenesis still needs to be clarified. To get molecular insight into the biological role of CD93 in the endothelium, we performed proteomic analyses to examine changes in the protein profile of endothelial cells after CD93 silencing. Among differentially expressed proteins, we identified dystroglycan, a laminin-binding protein involved in angiogenesis, whose expression is increased in vascular endothelial cells within malignant tumors. Using immunofluorescence, FRET, and proximity ligation analyses, we observed a close interaction between CD93 and β-dystroglycan. Moreover, silencing experiments showed that CD93 and dystroglycan promoted endothelial cell migration and organization into capillary-like structures. CD93 proved to be phosphorylated on tyrosine 628 and 644 following cell adhesion on laminin through dystroglycan. This phosphorylation was shown to be necessary for a proper endothelial migratory phenotype. Moreover, we showed that during cell spreading phosphorylated CD93 recruited the signaling protein Cbl, which in turn was phosphorylated on tyrosine 774. Altogether, our results identify a new signaling pathway which is activated by the cooperation between CD93 and dystroglycan and involved in the control of endothelial cell function.

One of the most important questions in cell biology concerns how cells reorganize after sensing polarity cues. In the present study, we describe the formation of an actin-rich domain on the apical surface of human primary endothelial cells adhering to the substrate and investigate its role in cell polarity. We used confocal immunofluorescence procedures to follow the redistribution of proteins required for endothelial cell polarity during spreading initiation. Activated Moesin, vascular endothelial cadherin and partitioning defective 3 were found to be localized in the apical domain, whereas podocalyxin and caveolin-1 were distributed along the microtubule cytoskeleton axis, oriented from the centrosome to the cortical actin-rich domain. Moreover, activated signaling molecules were localized in the core of the apical domain in tight association with filamentous actin. During cell attachment, loss of the apical domain by Moesin silencing or drug disruption of the actin cytoskeleton caused irregular cell spreading and mislocalization of polarity markers. In conclusion, our results suggest that the apical domain that forms during the spreading process is a structural organizer of cell polarity by regulating trafficking and activation of signaling proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00418-012-0965-9) contains supplementary material, which is available to authorized users.

Vascular Endothelial Growth Factor-A (VEGF-A) is a key molecule in normal and tumor angiogenesis. This study addresses the role of c-ABL as a novel downstream target of VEGF-A in primary Human Umbilical Vein Endothelial Cells (HUVEC). On the basis of immunoprecipitation experiments, in vitro kinase assay and RNA interference, we demonstrate that VEGF-A induces the c-ABL kinase activity through the VEGF Receptor-2/Phosphatidylinositol-3-Kinase pathway. By treating HUVEC with the specific tyrosine kinase inhibitor STI571 and over-expressing a dominant negative c-ABL mutant, we show that the VEGF-A-activated c-ABL reduces the amplitude of Mitogen-Activated Protein Kinases (ERK1/2, JNKs and p38) activation in a dose-dependent manner by a negative feedback mechanism. By analysis of the adaptor proteins NCK1 and GRB2 mutants we further show that the negative loop on p38 is mediated by c-ABL phosphorylation at tyrosine 105 of the adaptor protein NCK1, while the phosphorylation at tyrosine 209 of GRB2 down-modulates ERK1/2 and JNKs signaling. These findings suggest that c-ABL function is to establish a correct and tightly controlled response of endothelial cells to VEGF-A during the angiogenic process.

Rationale : Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown. Objective : To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion. Methods and Results : We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK. Conclusions : These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.

The analysis of the global thiol-disulfide redox status in tissues and cells is a challenging task since thiols and disulfides can undergo artificial oxido-reductions during sample manipulation. Because of this, the measured values, in particular for disulfides, can have a significant bias. Whereas this methodological problem has already been addressed in samples of red blood cells and solid tissues, a reliable method to measure thiols and disulfides in cell cultures has not been previously reported. Here, we demonstrate that the major artifact occurring during thiol and disulfide analysis in cultured cells is represented by glutathione disulfide (GSSG) and S-glutathionylated proteins (PSSG) overestimation, due to artificial oxidation of glutathione (GSH) during sample manipulation, and that this methodological problem can be solved by the addition of N-ethylmaleimide (NEM) immediately after culture medium removal. Basal levels of GSSG and PSSG in different lines of cultured cells were 3-5 and 10-20 folds higher, respectively, when the cells were processed without NEM. NEM pre-treatment also prevented the artificial reduction of disulfides that occurs during the pre-analytical phase when cells are exposed to an oxidant stimulus. In fact, in the absence of NEM, after medium removal, GSH, GSSG and PSSG levels restored their initial values within 15-30 min, due to the activity of reductases and the lack of the oxidant. The newly developed protocol was used to measure the thiol-disulfide redox status in 16 different line cells routinely used for biomedical research both under basal conditions and after treatment with disulfiram, a thiol-specific oxidant (0-200 μM concentration range). Our data indicate that, in most cell lines, treatment with disulfiram affected the levels of GSH and GSSG only at the highest concentration. On the other hand, PSSG levels increased significantly also at the lower concentrations of the drug, and the rise was remarkable (from 100 to 1000 folds at 200 μM concentration) and dose-dependent for almost all the cell lines. These data support the suitability of the analysis of PSSG in cultured cells as a biomarker of oxidative stress.

Ten-eleven translocation (Tet) genes encode for a family of hydroxymethylase enzymes involved in regulating DNA methylation dynamics. Tet1 is highly expressed in mouse embryonic stem cells (ESCs) where it plays a critical role the pluripotency maintenance. Tet1 is also involved in cell reprogramming events and in cancer progression. Although the functional role of Tet1 has been largely studied, its regulation is poorly understood. Here we show that Tet1 gene is regulated, both in mouse and human ESCs, by the stemness specific factors Oct3/4, Nanog and by Myc. Thus Tet1 is integrated in the pluripotency transcriptional network of ESCs. We found that Tet1 is switched off by cell proliferation in adult cells and tissues with a consequent genome-wide reduction of 5hmC, which is more evident in hypermethylated regions and promoters. Tet1 downmodulation is mediated by the Polycomb repressive complex 2 (PRC2) through H3K27me3 histone mark deposition. This study expands the knowledge about Tet1 involvement in stemness circuits in ESCs and provides evidence for a transcriptional relationship between Tet1 and PRC2 in adult proliferating cells improving our understanding of the crosstalk between the epigenetic events mediated by these factors.

Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial-mesenchymal transition, is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs, Snai1 does not respond to TGFβ or BMP4 signaling but it is induced by retinoic acid treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα, the dissociation of the Polycomb repressor complex 2 which results in the decrease of H3K27me3, and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c-Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes. Stem Cells 2015;33:742–750

Objective:Embryonic stem cells (ESC) pluripotency is maintained through a complex interplay of different signaling pathways and a network of transcription factors. Although much is known about this pluripotency selfrenewal circuitry, the molecular events that lead ESC to exit from pluripotency and begin differentiation are currently less known. All-trans retinoic acid (atRA), plays important and pleiotropic roles in embryonic development and ESC differentiation. The objective of the present study is to check the autocrine synthesis of atRA by ESC, test its role in spontaneous differentiation of ESC as embryoid bodies (EBs), and analyze the expression of the enzymes and proteins involved in atRA synthesis pathway. Methods: ESC, undifferentiated or differentiating as EBs, were grown in absence or presence of atRA, retinol, or the RAR antagonist CD2665, and Brachyury expression was analyzed as marker of ESC differentiation state. ESC or EBs-conditioned medium was produced in absence or presence of retinol and tested on RARE-luciferase reporter cells. RT-qPCR analysis of atRA biosynthetic pathway components was performed on undifferentiated or differentiating ESC. Finally, microarray gene expression profile was used to identify direct atRA target genes in ESC. Results: Here, we demonstrate that atRA promotes early steps of ESC differentiation, and that ESC increase their capacity to synthesize atRA during spontaneous differentiation as EBs, up-regulating RDH1, RDH10, ADH3, RALDH2, and CRABP2. Among 35 transcription factors (TFs) regulated by atRA in ESC, 3 TFs up-regulated (Snai1, Gata6, Cdx1) are known to be involved in ESC pluripotency exit and 3 TFs down-regulated (Otx2, Id2 and Arid1a) are involved in ESC pluripotency maintaining. Conclusion: Cultivation and controlled differentiation of ESC has opened new frontiers both in regenerative medicine and biology of development. Here, we demonstrated that RA is synthesized by ESC during spontaneous differentiation as EBs and takes an active role to promote their own differentiation process.

// Maurizio Orlandini 1 , Federico Galvagni 1 , Monia Bardelli 1,5 , Marina Rocchigiani 1 , Claudia Lentucci 1 , Francesca Anselmi 1 , Alessio Zippo 1,6 , Luca Bini 2 , Salvatore Oliviero 3,4 1 Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro, 2 – 53100 Siena, Italy. 2 Department of Life Sciences, University of Siena, Via A. Moro, 2 – 53100 Siena, Italy. 3 HuGeF, Via Nizza, 52 – 10126 Torino, Italy. 4 Department of Life Sciences and Systems Biology, University of Torino, Via Accademia Albertina, 13 – 10123 Torino, Italy. 5 Novartis Vaccines, Via Fiorentina, 1 – 53100 Siena, Italy. 6 INGM, Via F. Sforza, 35 – 20122 Milano, Italy. Correspondence: Maurizio Orlandini, email: // Keywords : monoclonal antibody, endothelial cell, angiogenesis, C1qRp, AA4. Received : March 12, 2014 Accepted : April 07, 2014 Published : April 07, 2014 Abstract The inhibition of tumor angiogenesis is one of the main challenges in cancer therapy. With the aim of developing monoclonal antibodies able to inhibit angiogenesis, we immunized mice with proliferating human umbilical vein endothelial cells. We generated a library of monoclonal antibodies able to recognize antigens expressed on endothelial cells and screened the antibodies for their ability to inhibit endothelial cell proliferation, migration, and sprouting in vitro . Here, we show that the antibody, designated as 4E1, is able to neutralize the formation of new vessels both in vitro and in vivo without affecting endothelial cell survival. By mass spectrometry we identified CD93 as the antigen bound by 4E1 and mapped the recognized epitope. CD93 is a transmembrane protein heavily glycosylated preferentially expressed in the vascular endothelium. CD93 silencing by lentiviral-mediated small hairpin RNA expression impairs human endothelial cell proliferation, migration, and sprouting. Altogether these findings reveal 4E1 as a novel antiangiogenic antibody and identify CD93 as a new target suitable for antiangiogenic therapy.

Embryonic stem cell (ESC) self-renewal and pluripotency is maintained by an external signaling pathways and intrinsic regulatory networks involving ESC-specific transcriptional complexes (mainly formed by OCT3/4, Sox2 and Nanog proteins), the Polycomb repressive complex 2 (PRC2) and DNA methylation [1], [2], [3], [4], [5], [6], [7], [8]. Among these, Nanog represents the more ESC specific factor and its repression correlates with the loss of pluripotency and ESC differentiation [9], [10], [11]. During ESC early differentiation, many development-associated genes become upregulated and although, in general, much is known about the pluripotency self-renewal circuitry, the molecular events that lead ESCs to exit from pluripotency and begin differentiation are largely unknown. Snai1 is one the most early induced genes during ESC differentiation in vitro and in vivo [12], [13]. Here we show that Snai1 is able to directly repress several stemness-associated genes including Nanog. We use a ESC stable-line expressing a inducible Snai1 protein. We here show microarray analysis of embryonic stem cells (ESC) expressing Snail-ER at various time points of induction with 4-OH. Data were deposited in Gene Expression Omnibus (GEO) datasets under reference GSE57854 and here: http://epigenetics.hugef-research.org/data.php.

The dystrophin gene transcription is up-regulated during muscle cell differentiation. Its expression in muscle cells is induced by the binding of the positive regulators serum response factor and dystrophin promoter bending factor (DPBF) on a regulatory CArG element present on the promoter. Here we show that the dystrophin CArG box is also recognized by the zinc finger nuclear factor YY1. Transient transfection experiments show that YY1 negatively regulates dystrophin transcription in C2C12 muscle cells. On the dystrophin CArG element YY1 competes with the structural factor DPBF. We further show that YY1 and DPBF binding to the CArG element induce opposite DNA bends suggesting that their binding induces alternative promoter structures. Along with C2C12 myotube formation YY1 is reduced and we observed that YY1, but not DPBF, is a substrate of m-calpain, a protease that is up-regulated in muscle cell differentiation. Thus, high levels of YY1 in non-differentiated muscle cells down-regulate the dystrophin promoter, at least in part, by interfering with the spatial organization of the promoter.

The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T)6GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoter-bending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.

Vasculogenesis and angiogenesis, the fundamental processes by which new blood vessels are formed, involve the proliferation, migration, and remodeling of endothelial cells. Dynamic adhesion of endothelial cells to extracellular matrix plays a fundamental role in all these events. Key regulators of endothelial cells adhesion and migration are the αvβ3 and uPA-uPAR complexes. The αvβ3 integrin heterodimer is the receptor for extracellular matrix components such as vitronectin and is overexpressed on the cell surface of angiogenic endothelial cells, but not quiescent cells lining normal vessels. The uPA-uPAR complex contributes to extracellular matrix remodeling by mediating proteolytic activity at the leading edge of migrating cells. We recently reported that the FOSL1 transcription factor of the AP-1 family plays a pivotal role in the regulation of the level of the αvβ3 and uPA-uPAR complexes on the surface of endothelial cells. In this commentary, we review the current knowledge of αv and β3 transcriptional regulation in endothelial cells and discuss the role of FOSL1 in angiogenesis.

SummaryThe de novo DNA methyltransferase 3-like (Dnmt3L) is a catalytically inactive DNA methyltransferase that cooperates with Dnmt3a and Dnmt3b to methylate DNA. Dnmt3L is highly expressed in mouse embryonic stem cells (ESCs), but its function in these cells is unknown. Through genome-wide analysis of Dnmt3L knockdown in ESCs, we found that Dnmt3L is a positive regulator of methylation at the gene bodies of housekeeping genes and, more surprisingly, is also a negative regulator of methylation at promoters of bivalent genes. Dnmt3L is required for the differentiation of ESCs into primordial germ cells (PGCs) through the activation of the homeotic gene Rhox5. We demonstrate that Dnmt3L interacts with the Polycomb PRC2 complex in competition with the DNA methyltransferases Dnmt3a and Dnmt3b to maintain low methylation levels at the H3K27me3 regions. Thus, in ESCs, Dnmt3L counteracts the activity of de novo DNA methylases to maintain hypomethylation at promoters of bivalent developmental genes.

Growth factor stimulation induces c-Jun-dependent survival of primary endothelial cells. However, the mechanism of c-Jun anti-apoptotic activity has not been identified. We here demonstrate that in response to growth factor treatment, primary human endothelial cells as well as mouse fibroblasts respond with an increased expression of c-Jun that forms a complex with ATF2. This complex activates the expression of the anti-apoptotic protein Bcl-X(L). By site-directed mutagenesis experiments, we identified two AP-1-binding sites located within the proximal promoter of the Bcl-X gene. Site-directed mutagenesis demonstrated that these AP-1 sites are required for the transcriptional activation of the promoter. Chromatin immunoprecipitation experiments show that in response to growth factor treatment, the heterodimer c-Jun.ATF2 binds to these functional AP-1 sites. Silencing of either c-Jun or ATF2 demonstrated that both nuclear factors are required for the activation of the proximal Bcl-X promoter. Taken together, our experiments provide evidence that growth factor-independent signaling pathways converge in the formation of an active c-Jun.AFT2 dimer, which induces the expression of the anti-apoptotic factor Bcl-X(L) that mediates a pro-survival response.

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin- 1. Caveolin- 1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.

Vascular endothelial growth factor receptor-3 (VEGFR-3) plays a key role for the remodeling of the primary capillary plexus in the embryo and contributes to angiogenesis and lymphangiogenesis in the adult. However, VEGFR-3 signal transduction pathways remain to be elucidated. Here we investigated VEGFR-3 signaling in primary human umbilical vein endothelial cells (HUVECs) by the systematic mutation of the tyrosine residues potentially involved in VEGFR-3 signaling and identified the tyrosines critical for its function. Y1068 was shown to be essential for the kinase activity of the receptor. Y1063 signals the receptor-mediated survival by recruiting CRKI/II to the activated receptor, inducing a signaling cascade that, via mitogen-activated protein kinase kinase-4 (MKK4), activates c-Jun N-terminal kinase-1/2 (JNK1/2). Inhibition of JNK1/2 function either by specific peptide inhibitor JNKI1 or by RNA interference (RNAi) demonstrated that activation of JNK1/2 is required for a VEGFR-3–dependent prosurvival signaling. Y1230/Y1231 contributes, together with Y1337, to proliferation, migration, and survival of endothelial cells. Phospho-Y1230/Y1231 directly recruits growth factor receptor–bonus protein (GRB2) to the receptor, inducing the activation of both AKT and extracellular signal–related kinase 1/2 (ERK1/2) signaling. Finally, we observed that Y1063 and Y1230/Y1231 signaling converge to induce c-JUN expression, and RNAi experiments demonstrated that c-JUN is required for growth factor–induced prosurvival signaling in primary endothelial cells.

The tyrosine kinase receptor fetal liver kinase 1 (Flk-1) plays a crucial role in vasculogenesis and angiogenesis, but its target genes remain elusive. Comparing Flk-1+/+ with Flk-1-/- embryonic stem (ES) cells, we identified transcripts regulated by the vascular endothelial growth factor A (VEGF-A)/Flk-1 pathway at an early stage of their differentiation to endothelial and mural precursors. Further analysis of a number of these genes (Nm23-M1, Nm23-M2, Slug, Set, pp32, Cbp, Ship-1, Btk, and Pim-1) showed that their products were transiently up-regulated in vivo in endothelial cells (ECs) during angiogenesis of the ovary, and their mRNA was rapidly induced in vitro by VEGF-A in human umbilical cord vein endothelial cells (HUVECs). Functional analysis by RNA interference (RNAi) in ES cells induced to differentiate demonstrated that Pim-1 is required for their differentiation into ECs and smooth muscle cells (SMCs). In HUVECs, RNAi showed that Pim-1 is required in ECs for VEGF-A-dependent proliferation and migration. The identification of Flk-1 target genes should help in elucidating the molecular pathways that govern the vasculogenesis and angiogenesis processes. (Blood. 2004;103:4536-4544)

Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a downregulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Repression of SPARC contributes to the oncogenic process by facilitating tumor development in vivo. A proximal promoter fragment, designated � 124/ þ 16, is responsible for high constitutive activity of the SPARC gene and is the target of repression by v-Jun. In this paper, using electrophoretic mobility shift and pull-down assays in vitro, and transient transfections and chromatin immunoprecipitation assays in Sp1/3-deficient Drosophila SL2 cells and in chick embryo fibroblasts, we show that (i) Sp1 and/or Sp3 is required for constitutive activation of SPARC transcription, by binding directly to the GGA-rich � 92/� 57 fragment; and (ii) v-Jun does not bind � 124/ þ 16 directly, but binds to the GGA-rich fragment indirectly, most likely through a physical interaction with Sp1/3. Moreover, a transactivation-proficient v-Jun derivative, designated v-Jun/cebp/glz, which cannot bind Jun DNA motifs anymore and cannot heterodimerize, is still capable of downregulating SPARC efficiently. Taken together, these data strongly suggest that v-Jun downregulates SPARC through the formation of a DNA–Sp1/3–v-Jun, chromatin-associated complex. Oncogene (2003) 22, 4047–4061. doi:10.1038/sj.onc.1206713