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

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

Nuclear factor-kappaB motif and interferon-alpha-stimulated response element co-operate in the activation of guanylate-binding protein-1 expression by inflammatory cytokines in endothelial cells.

The large GTPase GBP-1 (guanylate-binding protein-1) is a major IFN-gamma (interferon-gamma)-induced protein with potent anti-angiogenic activity in endothelial cells. An ISRE (IFN-alpha-stimulated response element) is necessary and sufficient for the induction of GBP-1 expression by IFN-gamma. Recently, we have shown that in vivo GBP-1 expression is strongly endothelial-cell-associated and is, in addition to IFN-gamma, also activated by interleukin-1beta and tumour necrosis factor-alpha, both in vitro and in vivo [Lubeseder-Martellato, Guenzi, Jörg, Töpolt, Naschberger, Kremmer, Zietz, Tschachler, Hutzler, Schwemmle et al. (2002) Am. J. Pathol. 161, 1749-1759; Guenzi, Töpolt, Cornali, Lubeseder-Martellato, Jörg, Matzen, Zietz, Kremmer, Nappi, Schwemmle et al. (2001) EMBO J. 20, 5568-5577]. In the present study, we identified a NF-kappaB (nuclear factor kappaB)-binding motif that, together with ISRE, is required for the induction of GBP-1 expression by interleukin-1beta and tumour necrosis factor-alpha. Deactivation of the NF-kappaB motif reduced the additive effects of combinations of these cytokines with IFN-gamma by more than 50%. Importantly, NF-kappaB p50 rather than p65 activated the GBP-1 promoter. The NF-kappaB motif and ISRE were detected in an almost identical spatial organization, as in the GBP-1 promoter, in the promoter regions of various inflammation-associated genes. Therefore both motifs may constitute a cooperative inflammatory cytokine response module that regulates GBP-1 expression. Our findings may open new perspectives for the use of NF-kappaB inhibitors to support angiogenesis in inflammatory diseases including ischaemia

Oncogenic KRas-induced Increase in Fluid-phase Endocytosis is Dependent on N-WASP and is Required for the Formation of Pancreatic Preneoplastic Lesions

Fluid-phase endocytosis is a homeostatic process with an unknown role in tumor initiation. The driver mutation in pancreatic ductal adenocarcinoma (PDAC) is constitutively active KRasG12D, which induces neoplastic transformation of acinar cells through acinar-to-ductal metaplasia (ADM). We have previously shown that KRasG12D-induced ADM is dependent on RAC1 and EGF receptor (EGFR) by a not fully clarified mechanism. Using three-dimensional mouse and human acinar tissue cultures and genetically engineered mouse models, we provide evidence that (i) KRasG12D leads to EGFR-dependent sustained fluid-phase endocytosis (FPE) during acinar metaplasia; (ii) variations in plasma membrane tension increase FPE and lead to ADM in vitro independently of EGFR; and (iii) that RAC1 regulates ADM formation partially through actin-dependent regulation of FPE. In addition, mice with a pancreas-specific deletion of the Neural-Wiskott–Aldrich syndrome protein (N-WASP), a regulator of F-actin, have reduced FPE and impaired ADM emphasizing the in vivo relevance of our findings. This work defines a new role of FPE as a tumor initiating mechanism

The helical domain of GBP-1 mediates the inhibition of endothelial cell proliferation by inflammatory cytokines

Inflammatory cytokines (IC) activate endothelial cell adhesiveness for monocytes and inhibit endothelial cell growth. Here we report the identification of the human guanylate binding protein-1 (GBP-1) as the key and specific mediator of the anti-proliferative effect of IC on endothelial cells. GBP-1 expression was induced by IC, downregulated by angiogenic growth factors, and inversely related to cell proliferation both in vitro in microvascular and macrovascular endothelial cells and in vivo in vessel endothelial cells of Kaposi’s sarcoma. Experimental modulation of GBP-1 expression demonstrated that GBP-1 mediates selectively the anti-proliferative effect of IC, without affecting endothelial cell adhesiveness for monocytes. GBP-1 anti-proliferative activity did not affect ERK-1/2 activation, occurred in the absence of apoptosis, was found to be independent of the GTPase activity and isoprenylation of the molecule, but was specifically mediated by the C-terminal helical domain of the protein. These results define GBP-1 as an important tool for dissection of the complex activity of IC on endothelial cells, and detection and specific modulation of the IC-activated non-proliferating phenotype of endothelial cells in vascular diseases

Human Guanylate Binding Protein-1 Is a Secreted GTPase Present in Increased Concentrations in the Cerebrospinal Fluid of Patients with Bacterial Meningitis

Interferon-γ-induced GTPases are key to the protective immunity against microbial and viral pathogens. As yet, the cell interior has been regarded as the exclusive residence of these proteins. Here we show that a member of this group, human guanylate binding protein-1 (hGBP-1), is secreted from cells. Secretion occurred in the absence of a leader peptide via a nonclassical, likely ABC transporter-dependent, pathway, was independent of hGBP-1 GTPase activity and isoprenylation, and did not require additional interferon-γ-induced factors. Interestingly, hGBP-1 was only secreted from endothelial cells but not from any of the nine different cell types tested. Clinically most important was the detection of significantly (P < 0.001, Mann-Whitney U-test) increased hGBP-1 concentrations in the cerebrospinal fluid of patients with bacterial meningitis (n = 32) as compared to control patients (n = 74). In this first report of a secreted GTPase, we demonstrate that secreted hGBP-1 may be a useful surrogate marker for diagnosis of bacterial meningitis