Inhibition of the tyrosine phosphatase SHP-2 suppresses angiogenesis in vitro and in vivo

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo (p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction ( p ! 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis ( annexin V staining, p ! 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n=3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth. Copyright (C) 2007 S. Karger AG, Basel

Molecular Networks in FGF Signaling: Flotillin-1 and Cbl-Associated Protein Compete for the Binding to Fibroblast Growth Factor Receptor Substrate 2

Fibroblast growth factor receptor substrate 2 (FRS2α) is a signaling adaptor protein that regulates downstream signaling of many receptor tyrosine kinases. During signal transduction, FRS2 can be both tyrosine and threonine phosphorylated and forms signaling complexes with other adaptor proteins and tyrosine phosphatases. We have here identified flotillin-1 and the cbl-associated protein/ponsin (CAP) as novel interaction partners of FRS2. Flotillin-1 binds to the phosphotyrosine binding domain (PTB) of FRS2 and competes for the binding with the fibroblast growth factor receptor. Flotillin-1 knockdown results in increased Tyr phosphorylation of FRS2, in line with the inhibition of ERK activity in the absence of flotillin-1. CAP directly interacts with FRS2 by means of its sorbin homology (SoHo) domain, which has previously been shown to interact with flotillin-1. In addition, the third SH3 domain in CAP binds to FRS2. Due to the overlapping binding domains, CAP and flotillin-1 appear to compete for the binding to FRS2. Thus, our results reveal a novel signaling network containing FRS2, CAP and flotillin-1, whose successive interactions are most likely required to regulate receptor tyrosine kinase signaling, especially the mitogen activated protein kinase pathway

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair. © 2014 Nakajima et al

Galectin-8 in IgA Nephritis: Decreased Binding of IgA by Galectin-8 Affinity Chromatography and Associated Increased Binding in Non-IgA Serum Glycoproteins

Background Immunoglobulin A nephritis (IgAN) is the most common primary glomerulonephritis worldwide. It is caused by accumulation of IgA1-containing immune complexes in the kidney resulting in renal failure, which is thought to be due to altered glycosylation of IgA with a decrease of 2-3-sialylated galactosides (NeuAc alpha 2-3Gal). less thanbrgreater than less thanbrgreater thanPurpose The purpose of this study was to analyze whether altered glycosylation of IgA would lead to an altered binding to galectin-8, an endogenous lectin with strong affinity for 2-3-sialylated galactosides. Galectins are a family of beta-galactoside-binding proteins; by binding various glycoproteins, they play important roles in the regulation of cellular functions in inflammation and immunity. Hence, an altered binding of IgA to galectin-8 could lead to pathologic immune functions, such as glomerulonephritis. less thanbrgreater than less thanbrgreater thanMethods Affinity chromatography of serum glycoproteins on the human sialogalactoside-binding lectin galectin-8N permitted quantitation of bound and unbound fractions, including IgA. less thanbrgreater than less thanbrgreater thanResults Analysis of similar to 100 IgA nephritis sera showed that the galectin-8N unbound fraction of IgA increased compared to similar to 100 controls, consistent with the known loss of galactosylation. A subgroup of similar to 15% of the IgAN patients had a ratio of galectin-8 bound/unbound IgA andlt;0.09, not found for any of the controls. Unexpectedly, the galectin-8N-binding fraction of serum glycoproteins other than IgA increased in the sera of IgAN patients but not in controls, suggesting a previously unrecognized change in this disease. less thanbrgreater than less thanbrgreater thanConclusion This is the first study that relates a galectin, an endogenous lectin family, to IgA nephritis and thus should stimulate new avenues of research into the pathophysiology of the disease.Funding Agencies|Swedish Research Council (Vetenskapsradet)|2008-3356|Swedish Foundation for Swedish Research|FFL4|Swedish Healthcare System (ALF)||Region Skane||</p

Domain Analysis Reveals That a Deubiquitinating Enzyme USP13 Performs Non-Activating Catalysis for Lys63-Linked Polyubiquitin

Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus, the non-activating catalysis of USP13 for K63-polyUb chains implies that it may function differently from USP5 in cellular deubiquitination processes

Galectin-1 Is Part of Human Trophoblast Invasion Machinery - A Functional Study In Vitro

Interactions of glycoconjugates with endogenous galectins, have been long proposed to participate in several reproductive processes including implantation. In human placenta gal-1, gal-3, gal-8, and gal-13 proteins are known to be present. Each of them has been proposed to play multiple functions, but so far no clear picture has emerged. We hypothesized that gal-1 participates in trophoblast invasion, and conducted Matrigel invasion assay using isolated cytotrophoblast from first trimester placenta and HTR-8/SVneo cell line to test it.<0.001) by Ox-gal-1 at 1 µg/ml. Both sets of results confirmed involvement of gal-1 in trophoblast invasion. Galectin profile of isolated cytotrophoblast and HTR-8/SVneo cells was established using RT-PCR and real-time PCR and found to consist of gal-1, gal-3 and gal-8 for both cell types. Only gal-1 was located at the trophoblast cell membrane, as determined by FACS analysis, which is consistent with the results of the functional tests.These findings qualify gal-1 as a member of human trophoblast cell invasion machinery

Group subsidiaries, tax minimization and offshore financial centres: Mapping organizational structures to establish the ‘in-betweener’ advantage

International business and public policy research have examined the techniques that multinational enterprises (MNEs) use to shift revenues to subsidiaries in offshore financial centres (OFCs) in order to minimize tax liability and arbitrage for their advantage. While study of such tax arbitrage strategies has looked to geographical locations and legal dimensions to better understand these strategies, it has ignored the structural and organizational relationship between MNEs and their subsidiaries. We define two distinct types of OFC-based corporate entities based on their location among and apparent control over other MNE affiliates: ‘stand-alone’ OFCs at the end of a chain of MNE subsidiaries; and ‘in-betweener’ OFCs with equity control over further entities and hence apparent flexibility to redirect profits to other MNE subsidiaries further down the chain. We hypothesize that when MNEs have in-betweener OFCs controlling a substantial share of overall MNE profits, this indicates greater MNE interest in aggressive tax planning (ATP). We then evaluate empirical support for our claims based on an ‘equity mapping’ approach identifying stand-alone and in-betweener OFCs in 100 of the largest MNEs operating globally. This study demonstrates that a key factor determining tax arbitrage is not the amount of value registered on OFC subsidiaries’ balance sheets, but rather the portion of the group’s operating revenues and net income controlled by OFC subsidiaries. National taxing authorities could benefit from tracking in-betweener OFC locations and behaviour to counter ATP strategies, decrease sovereign arbitrage, and increase MNE tax revenue