The guanylate binding protein-1

The endothelium is among the largest organs in the body. Stimuli originating from the blood or from neighbouring cells, like inflammatory cytokines (IC), lead to structural and functional alterations of vascular endothelial cells (EC). These alterations are often referred to as “EC activation”. Activated EC play a key role in different physiological processes like during immune response, in menstruation and in pathological processes like inflammation, allergy, viral infections, atherosclerosis and tumour angiogenesis. The human guanylate binding protein-1 (GBP-1) is a protein of the family of large GTPases. GBP-1 is characterized by a high turnover GTPase activity. Previous work showed that GBP-1 mRNA expression is induced by IC in EC and that GBP-1 is the specific mediator of the anti-proliferative effect of IC on EC in vitro. The main goals of this work were first, to investigate whether GBP-1 may be a molecular marker of IC-activated EC at the protein level in vitro. Second, to investigate GBP-1 expression in human healthy and/or disease tissues and to determine whether GBP-1 may be a molecular marker of IC-activated EC in vivo. To this goal mono- and poly-clonal antibodies against GBP-1 were generated. In vitro studies showed that GBP-1 expression in EC is induced by IFN-, IFN-, IL-1, IL-1 or TNF- but not by other cytokines, chemokines or growth factors. Moreover, simultaneous addition of bFGF and VEGF and IC reduced the IC-induced GBP-1 expression. This indicated that GBP-1 characterizes cells that are preferentially exposed to IC. In vivo studies using immunohistochemistry and immunofluorescence showed that GBP-1 expression is highly associated with vascular EC in a broad range of human tissues. This was confirmed by the simultaneous detection of GBP-1 and the EC-associated marker CD31. Notably, GBP-1 expression was undetectable in healthy skin. In contrast, GBP-1 was highly expressed in vessels of skin diseases with a high inflammatory component including psoriasis, adverse drug reactions and Kaposi’s sarcoma. This indicated that GBP-1 characterizes IC-activated EC in vivo. Further immunohistochemical studies on Kaposi’s sarcoma demonstrated that GBP-1 expression and EC cell proliferation are inversely related. This indicated that GBP-1 may also mediate the anti-proliferative effect of IC on EC in vivo. Finally, GBP-1 was found to be secreted by EC stimulated with IFN- and IFN- in vitro. This finding was confirmed by immunoprecipitation of GBP-1 from cell culture supernatants and by a novel ELISA developed for the detection of GBP-1 in solution. Further characterization of the mechanism of secretion demonstrated that GBP-1 release is due to an 3 Summary energy-dependent mechanism and is not due to cell death. Most importantly, circulating GBP-1 could be detected in increased concentrations in the blood of patients that were subjected to IFN–-therapy or in patients with inflammatory diseases. These findings indicated that GBP-1 is a novel marker of inflammatory vessel activation. Specifically, the serological detection of GBP-1 may open new perspectives for the early detection of inflammatory activation of EC in patients with inflammatory diseases

Mir34a constrains pancreatic carcinogenesis

Several studies have shown that over 70 different microRNAs are aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC), affecting proliferation, apoptosis, metabolism, EMT and metastasis. The most important genetic alterations driving PDAC are a constitutive active mutation of the oncogene Kras and loss of function of the tumour suppressor Tp53 gene. Since the MicroRNA 34a (Mir34a) is a direct target of Tp53 it may critically contribute to the suppression of PDAC. Mir34a is epigenetically silenced in numerous cancers, including PDAC, where Mir34a down-regulation has been associated with poor patient prognosis. To determine whether Mir34a represents a suppressor of PDAC formation we generated an in vivo PDAC-mouse model harbouring pancreas-specific loss of Mir34a (Kras(G12D);Mir34a(Delta/Delta)). Histological analysis of Kras(G12D);Mir34a(Delta/Delta) mice revealed an accelerated formation of pre-neoplastic lesions and a faster PDAC development, compared to Kras(G12D) controls. Here we show that the accelerated phenotype is driven by an early up-regulation of the pro-inflammatory cytokines TNFA and IL6 in normal acinar cells and accompanied by the recruitment of immune cells. Our results imply that Mir34a restrains PDAC development by modulating the immune microenvironment of PDAC, thus defining Mir34a restauration as a potential therapeutic strategy for inhibition of PDAC development

Intracellular Trafficking of Guanylate-Binding Proteins Is Regulated by Heterodimerization in a Hierarchical Manner

Guanylate-binding proteins (GBPs) belong to the dynamin family of large GTPases and represent the major IFN-γ-induced proteins. Here we systematically investigated the mechanisms regulating the subcellular localization of GBPs. Three GBPs (GBP-1, GBP-2 and GBP-5) carry a C-terminal CaaX-prenylation signal, which is typical for small GTPases of the Ras family, and increases the membrane affinity of proteins. In this study, we demonstrated that GBP-1, GBP-2 and GBP-5 are prenylated in vivo and that prenylation is required for the membrane association of GBP-1, GBP-2 and GBP-5. Using co-immunoprecipitation, yeast-two-hybrid analysis and fluorescence complementation assays, we showed for the first time that GBPs are able to homodimerize in vivo and that the membrane association of GBPs is regulated by dimerization similarly to dynamin. Interestingly, GBPs could also heterodimerize. This resulted in hierarchical positioning effects on the intracellular localization of the proteins. Specifically, GBP-1 recruited GBP-5 and GBP-2 into its own cellular compartment and GBP-5 repositioned GBP-2. In addition, GBP-1, GBP-2 and GBP-5 were able to redirect non-prenylated GBPs to their compartment in a prenylation-dependent manner. Overall, these findings prove in vivo the ability of GBPs to dimerize, indicate that heterodimerization regulates sub-cellular localization of GBPs and underscore putative membrane-associated functions of this family of proteins

Notch signaling is required for exocrine regeneration after acute pancreatitis

BACKGROUND & AIMS: The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. METHODS: Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. RESULTS: Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. CONCLUSIONS: Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells

The guanylate binding protein-1 GTPase controls the invasive and angiogenic capability of endothelial cells through inhibition of MMP-1 expression

Expression of the large GTPase guanylate binding protein-1 (GBP-1) is induced by inflammatory cytokines (ICs) in endothelial cells (ECs), and the helical domain of the molecule mediates the repression of EC proliferation by ICs. Here we show that the expression of GBP-1 and of the matrix metalloproteinase-1 (MMP-1) are inversely related in vitro and in vivo, and that GBP-1 selectively inhibits the expression of MMP-1 in ECs, but not the expression of other proteases. The GTPase activity of GBP-1 was necessary for this effect, which inhibited invasiveness and tube-forming capability of ECs in three-dimensional collagen-I matrices. A GTPase-deficient mutant (D184N-GBP-1) operated as a transdominant inhibitor of wild-type GBP-1 and rescued MMP-1 expression in the presence of ICs. Expression of D184N-GBP-1, as well as paracrine supplementation of MMP-1, restored the tube-forming capability of ECs in the presence of wild-type GBP-1. The latter finding indicated that the inhibition of capillary formation is specifically due to the repression of MMP-1 expression by GBP-1, and is not affected by the anti-proliferative activity of the helical domain of GBP-1. These findings substantiate the role of GBP-1 as a major regulator of the anti-angiogenic response of ECs to ICs