Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system. Oncogene (2010) 29, 752-762; doi: 10.1038/onc.2009.379; published online 9 November 2009

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.

A novel reciprocal and biphasic relationship between membrane cholesterol and beta-secretase activity in SH-SY5Y cells and in human platelets.

Research into the cause of Alzheimer's disease (AD) has identified strong connections to cholesterol. Cholesterol and cholesterol esters can modulate amyloid precursor protein (APP) processing, thus altering production of the A beta peptides that deposit in cortical amyloid plaques. Processing depends on the encounter between APP and cellular secretases, and is thus subject to the influence of cholesterol-dependent factors including protein trafficking, and distribution between membrane subdomains. We have directly investigated endogenous membrane beta-secretase activity in the presence of a range of membrane cholesterol levels in SH-SY5Y human neuroblastoma cells and human platelets. Membrane cholesterol significantly influenced membrane beta-secretase activity in a biphasic manner, with positive correlations at higher membrane cholesterol levels, and negative correlations at lower membrane cholesterol levels. Platelets from individuals with AD or mild cognitive impairment (n = 172) were significantly more likely to lie within the negative correlation zone than control platelets (n = 171). Pharmacological inhibition of SH-SY5Y beta-secretase activity resulted in increased membrane cholesterol levels. Our findings are consistent with the existence of a homeostatic feedback loop between membrane cholesterol level and membrane beta-secretase activity, and suggest that this regulatory mechanism is disrupted in platelets from individuals with cognitive impairment.

Genetic Variation in the alpha 7 Nicotinic Acetylcholine Receptor is Associated with Delusional Symptoms in Alzheimer's Disease

Psychotic symptoms are common in Alzheimer's disease (AD) and have a negative impact oil quality of life. It is suggested that psychotic symptoms may be attributed to genetic risk factors which are revealed during neurodegeneration. CHRNA7, the gene for the alpha 7 nicotinic acetylcholine receptor, has been associated with schizophrenia in linkage and association Studies. Hence we investigated single SNPs and haplotypes in CHRNA7 in relation to AD with psychosis in a large, well-characterised and previously described cohort within the Northern Ireland population. A significant association between delusions and the T allele of rs6494223 (P = 0.014, OR = 1.63, Cl 1.22-2.17) was found. This suggests that the alpha 7 receptor may be a suitable target for the treatment of AD with psychosis.

Alpha7 nicotinic acetylcholine receptor gene and reduced risk of Alzheimer's disease.

Background: Sporadic Alzheimer's disease (AD) is a common disabling disease of complex aetiology for which there are limited therapeutic options. We sought to investigate the role of the alpha 7 nicotinic acetylcholine receptor gene (CHRNA7) in influencing risk of AD in a large population. CHRNA7 is a strong candidate gene for AD for several reasons: (1) its expression is altered differentially in the AD brain; (2) it interacts directly with beta amyloid peptide (A beta(42)); and (3) agonist activation induces several neuroprotective pathways.

Keratoconus: an analysis of corneal asymmetry

Background: Keratoconus, a non-inflammatory corneal ectasia, is reported to have bilateral involvement in over 90% of patients. The purpose of this study was to quantify the extent of asymmetry of disease at presentation to a regional corneal clinic.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

BACKGROUND: Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development.

METHODOLOGY/PRINCIPAL FINDINGS: Cathepsin S expression and secretion from endothelial cells was characterised using RT-PCR and western blotting. We further show that cathepsin S promotes pericellular hydrolysis of extracellular matrix components in the tumour microenvironment and facilitates endothelial invasion. The cathepsin S inhibitory antibody, Fsn0503, blocks extracellular proteolysis, inhibiting endothelial invasion and tube formation in cell-based assays. The anti-angiogenic effects of Fsn0503 were also shown in vivo where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.

CONCLUSIONS/SIGNIFICANCE: Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis.

Vasodilator-Stimulated Phosphoprotein Regulates Inside-Out Signaling of beta(2) Integrins in Neutrophils

The monomeric GTPase Rap1 controls functional activation of beta2 integrins in leukocytes. In this article, we describe a novel mechanism by which the chemoattractant fMLP activates Rap1 and inside-out signaling of beta2 integrins. We found that fMLP-induced activation of Rap1 in human polymorphonuclear leukocytes or neutrophils and differentiated PLB-985 cells was blocked by inhibitors of the NO/guanosine-3',5'-cyclic monophosphate-dependent protein kinase (cGKI) pathway [N-(3-(aminomethyl)benzyl)acetamidine, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, DT-3 peptide, 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphothioate, Rp-isomer triethylammonium salt-guanosine-3',5'-cyclic monophosphate], indicating that the downstream signaling events in Rap1 activation involve the production of NO and guanosine-3',5'-cyclic monophosphate, as well as the activation of cGKI. Silencing the expression of vasodilator-stimulated phosphoprotein (VASP), a substrate of cGKI, in resting PLB-985 cells or mice neutrophils led to constitutive activation of Rap1. In parallel, silencing VASP in differentiated PLB-985 cells led to recruitment of C3G, a guanine nucleotide exchange factor for Rap1, to the plasma membrane. Expression of murine GFP-tagged phosphodeficient VASP Ser235Ala mutant (murine serine 235 of VASP corresponds to human serine 239) in PLB-985 cells blunted fMLP-induced translocation of C3G to the membrane and activation of Rap1. Thus, bacterial fMLP triggers cGKI-dependent phosphorylation of human VASP on serine 239 and, thereby, controls membrane recruitment of C3G, which is required for activation of Rap1 and beta2 integrin-dependent antibacterial functions of neutrophils.

Respiratory syncytial virus NS1 protein degrades STAT2 by using the elongin-cullin E3 ligase

Respiratory syncytial virus (RSV) infection causes bronchiolitis and pneumonia in infants. RSV has a linear single-stranded RNA genome encoding 11 proteins, 2 of which are nonstructural (NS1 and NS2). RSV specifically downregulates STAT2 protein expression, thus enabling the virus to evade the host type I interferon response. Degradation of STAT2 requires proteasomal activity and is dependent on the expression of RSV NS1 and NS2 (NS1/2). Here we investigate whether RSV NS proteins can assemble ubiquitin ligase (E3) enzymes to target STAT2 to the proteasome. We demonstrate that NS1 contains elongin C and cullin 2 binding consensus sequences and can interact with elongin C and cullin 2 in vitro; therefore, NS1 has the potential to act as an E3 ligase. By knocking down expression of specific endogenous E3 ligase components using small interfering RNA, NS1/2, or RSV-induced STAT2, degradation is prevented. These results indicate that E3 ligase activity is crucial for the ability of RSV to degrade STAT2. These data may provide the basis for therapeutic intervention against RSV and/or logically designed live attenuated RSV vaccines.