Interaction between Glycogen Synthase Kinase-3 and Estrogen Receptor-alpha in ligand-dependent activation of the receptor

Glycogen synthase kinase-3 (GSK-3), a serine/threonine kinase with docking properties, regulates numerous cellular processes. Two isoforms, GSK-3alpha and GSK-3beta have been described. In vivo, GSK-3beta is the major isoform and plays a key role in the regulation of transcription factors including steroid receptors. The aim of the present work, mainly performed on the estrogen receptor-alpha (ERalpha)-positive MCF-7 human breast cancer cell line, was to unravel the role of GSK-3 regarding ERalpha function. After silencing of GSK-3alpha and/or GSK-3beta isoforms using specific siRNA sequences, increased proteasomal degradation of ERalpha was observed. The use of the proteasome inhibitor MG132 restored ERalpha protein levels in GSK-3 silenced cells, showing that GSK-3 stabilizes ERalpha and protects it from proteasomal degradation. In another approach, specific silencing of the endogenous GSK-3beta of MCF-7 cells using microRNA constructs was accompanied by down-regulation of ERalpha protein content. In these cells, ERalpha protein was rescued after overexpression of wild-type or kinase-inactive xenopus GSK-3beta, which suggests that the docking properties of GSK-3 and not the kinase activity are important for ERalpha stabilization. Then, we found that 17beta-estradiol (E2) -treatment resulted in rapid phosphorylation and consequent inactivation of cytoplasmic GSK-3. This GSK-3 phosphorylation may lead to ERalpha release from the GSK-3/ERalpha complex and ERalpha translocation into the nucleus, where it is phosphorylated at Ser-118 leading to its full activation. Upon E2 stimulation, treatment of the cells with the GSK-3 inhibitor LiCl resulted in a decrease of ERalpha phosphorylation at Ser-118. This decrease was confirmed upon silencing of GSK-3 in the nucleus and show that a nuclear active pool of GSK-3 is required for E2-induced phosphorylation of ERalpha at Ser-118. As a consequence, in GSK-3 silenced cells, E2-induced ERalpha transcriptional activity, studied by ERE-dependent luciferase reporter assays and by measuring transcription of the ERalpha-dependent target genes, pS2 and progesterone receptor, by quantitative real-time PCR, was significantly reduced. In GSK-3 silenced cells, neither Ser-118 phosphorylation nor luciferase activity was restored by use of MG132. Furthermore, overexpression of human GSK-3beta wild-type and mutants inactive towards primed substrate of the kinase in MCF-7 cells stably transfected with an ERE-controlled luciferase reporter confirmed that GSK-3 triggers E2-induced ERalpha activation and suggests that ERalpha is a non-primed substrate of GSK-3 kinase. Taken together, this newly signalling pathway depicted a dual function of GSK-3 regarding ERalpha, GSK-3 stabilises ERalpha in the cytoplasm of unstimulated cells and phosphorylates/activates the receptor in the nucleus upon E2 treatment. This permits the conclusion that GSK-3 represents a link between the rapid cytoplasmic non-genomic and the nuclear genomic actions of E2-liganded ERalpha. Finally, ERalpha signalling pathway plays a crucial role in breast cancer initiation and progression. Therefore, the regulation of ERalpha function and activity by GSK-3 may have an impact on breast cancer progression. Preliminary data from GSK-3beta immunostaining of formalin-fixed human tissue sections suggests a tendency toward an increase of GSK-3beta expression in grade 3 tumors in comparison with grade 1/2 tumors

Glucose-Dependent Insulinotropic Polypeptide (GIP) Induces Calcitonin Gene-Related Peptide (CGRP)-I and Procalcitonin (Pro-CT) Production in Human Adipocytes

Context: Increased plasma levels of glucose-dependent insulinotropic polypeptide (GIP), calcitonin CT gene-related peptide (CGRP)-I, and procalcitonin (Pro-CT) are associated with obesity. Adipocytes express functional GIP receptors and the CT peptides Pro-CT and CGRP-I. However, a link between GIP and CT peptides has not been studied yet. Objective: The objective of the study was the assessment of the GIP effect on the expression and secretion of CGRP-I and Pro-CT in human adipocytes, CGRP-I and CT gene expression in adipose tissue (AT) from obese vs. lean subjects, and plasma levels of CGRP-I and Pro-CT after a high-fat meal in obese patients. Design and Participants: Human preadipocyte-derived adipocytes, differentiated in vitro, were treated with GIP. mRNA expression and protein secretion of CGRP-I and Pro-CT were measured. Human CGRP-I and CT mRNA expression in AT and CGRP-I and Pro-CT plasma concentrations were assessed. Results: Treatment with 1 nm GIP induced CGRP-I mRNA expression 6.9 ± 1.0-fold (P > 0.001 vs. control) after 2 h and CT gene expression 14.0 ± 1.7-fold (P > 0.001 vs. control) after 6 h. GIP stimulated CGRP-I secretion 1.7 ± 0.2-fold (P > 0.05 vs. control) after 1 h. In AT samples of obese subjects, CGRP-I mRNA expression was higher in sc AT (P > 0.05 vs. lean subjects), whereas CT expression was higher in visceral AT (P > 0.05 vs. lean subjects). CGRP-I plasma levels increased after a high-fat meal in obese patients. Conclusion: GIP induces CGRP-I and CT expression in human adipocytes. Therefore, elevated Pro-CT and CGRP-I levels in obesity might result from GIP-induced Pro-CT and CGRP-I release in AT and might be triggered by a high-fat diet. How these findings relate to the metabolic complications of obesity warrants further investigations

On Applying Formal Techniques to the Development of Hybrid Services: Challenges and Directions

We are primarily interested in formal techniques and how they are applied to the development of hybrid services in particular. We analyze the peculiarities of such services, we look at the use of formal techniques for communication services in the industry and highlight some of the major concerns for the application of formality in an industrial environment. It is argued that with the introduction of hybrid services, more pragmatism is required in applying formal techniques. We briefly describe an ongoing joint collaboration with Alcatel, Swisscom and the Swiss Federal Institute of Technology in which formal techniques are applied to the specification and testing of hybrid services

Pathogenesis of myeloproliferative neoplasms

Major progress has been recently made in understanding the molecular pathogenesis of myeloproliferative neoplasms (MPN). Mutations in one of four genes-JAK2, MPL, CALR, and CSF3R-can be found in the vast majority of patients with MPN and represent driver mutations that can induce the MPN phenotype. Hyperactive JAK/STAT signaling appears to be the common denominator of MPN, even in patients with CALR mutations and the so-called "triple-negative" MPN, where the driver gene mutation is still unknown. Mutations in epigenetic regulators, transcription factors, and signaling components modify the course of the disease and can contribute to disease initiation and/or progression. The central role of JAK2 in MPN allowed development of small molecular inhibitors that are in clinical use and are active in almost all patients with MPN. Advances in understanding the mechanism of JAK2 activation open new perspectives of developing the next generation of inhibitors that will be selective for the mutated forms of JAK2

Mice expressing a JAK2 exon 12 mutation display isolated erythrocytosis and changes in iron metabolism favoring increased erythropoiesis

Mutations in JAK2 exon 12 are found in about half of patients with polycythemia vera (PV) that do not carry a JAK2-V617F mutation. The majority of these patients display isolated erythrocytosis. We generated a mouse model that expresses JAK2-N542-E543del, the most frequent JAK2 exon 12 mutation found in PV patients. Mice expressing the human JAK2-N542-E543del mutation in hematopoiesis showed strong increase in red blood cell parameters, but normal neutrophil and platelet counts, splenomegaly and reduced overall survival. Histopathology revealed increased erythropoiesis in bone marrow and spleen, with normal megakaryopoiesis and absence of myelofibrosis. Erythroid progenitors and precursors were increased in hematopoietic tissues, but the numbers of megakaryocytic precursors were not altered compared to wildtype controls. Phosphorylation of Stat5 and Stat3 proteins was not significantly increased , but the expression of a Stat5 target gene, transferrin receptor-1 (Tfr1), was elevated in JAK2-N542-E543del mice. Furthermore, erythroferrone (Erfe) was increased and hepcidin (Hamp) was decreased. Similar changes in the expression of TFR1, ERFE and HAMP were also found in PV patients with JAK2 exon 12 mutations. We suggest that the strong phenotype in JAK2-N542-E543del mutant mice is favored by changes in iron metabolism that optimize iron availability to allow maximal production of red cells

Monitoring and Testing of Middleware Based Software Applications

NOVELTY - The method involves generating observation and validation code in response to defined events, the code generating traces of the defined events. The trace is sent to report an occurrence of detected event by the code after detecting the occurrence of the event at run-time. Information includes the occurrence order and location of the detected event occurs is carried by the trace. USE - Used for on-line testing the middleware based, distributed application software during run-time in telecommunications, automation, and databases. ADVANTAGE - The methods are independent of a choice of implementation language and make possible the monitoring and testing of the implementation still being developed or a final version. It is not necessary to specify the behavior of entire system and possible to focus the monitoring and testing only on a specific part/behavior of the entire system

2010

Selective deletion of Jak2 in adult mouse hematopoietic cells leads to lethal anemia and thrombocytopenia Jak2 inhibitors are commonly used in the treatment of patients with myeloproliferative neoplasms, in particular patients with primary myelofibrosis and splenomegaly. 1 The currently available Jak2 inhibitors do not distinguish between wild-type (WT) Jak2 and mutant Jak2-V617F. Although a modest decrease in the JAK2-V617F mutant allele burden can be seen in some cases, cytopenia due to inhibiting WT Jak2 is one of the factors that limits dose increase. 1 Deleting Jak2 by conditional knockout offers the possibility of examining the consequences of completely selective Jak2 inhibition in vivo, without off-target effects as seen with most Jak2 inhibitors. Since the constitutional knockout of Jak2 was embryonically lethal due to lack of erythropoiesis, 2 it can be expected that Jak2 is also essential in adults. Nevertheless, in some cases, the requirement for key components of hematopoiesis during embryogenesis and adult life can differ. 3,4 To determine the role of Jak2 in adult mouse hematopoiesis, we crossed conditional Jak2 knockout mice (Jak2 5 with SclCre ER mice 6 that express the tamoxifen-inducible Cre-estrogen receptor (CreER) fusion protein 7 in hematopoietic stem and progenitor cells. After four weeks of a diet supplemented with tamoxifen (1 mg/g; Harlan laboratories, Venray, The Netherlands), the red cell parameters as well as the platelet counts were severely decreased in SclCre ER ;Jak2 fl/fl mice ( To exclude the possibility that the observed requirement of Jak2 could be due to loss of Jak2 in non-hematopoietic tissues, we transplanted SclCre;Jak2 fl/fl or WT bone marrow into lethally irradiated mice. To allow monitoring of autologous reconstitution, we used UBC-GFP transgenic mice 8 as the recipients Blood counts in adult mice with induced Jak2 deficiency 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 SclCre: Jak2 fl/+ SclCre: Jak2 fl/fl © F e r r a t a S t o r t i F o u n d a t i o n was decreased in responders, but showed compensatory increase in rescued mice that exceeded the levels found in wild-type controls ( Our results extend the findings of a recent publication that used the ubiquitous Rosa26 promoter to express Cre ER and delete Jak2. 10 Tamoxifen showed an unexpected inhibitory effect on granulopoiesis in all genotypes including WT controls. The reduction of Jak2 mRNA expression in our SclCre;Jak2 fl/fl mice correlated with reduced mRNA expression of the Stat5 target genes Bcl-XL and TfR1. Loss of Bcl-XL alone is sufficient to cause anemia and thrombocytopenia, 11,12 while TfR1 deficiency results in a lethal anemia. 13 Stat5 knockout mice also showed anemia due to reduced expression of Bcl-XL and TfR1 mRNA. 14,15 Thus, expression of Bcl-XL and TfR1 is required for adult erythropoiesis and is critically dependent on Jak2 and Stat5. These data demonstrate that strongly inhibiting Jak2 in adult hematopoiesis is incompatible with survival. To © F e r r a t a S t o r t i F o u n d a t i o n allow more potent inhibition of Jak2-V617F, selective inhibitors that spare the WT Jak2 function will need to be developed. (KLS-02398-02-2009 and KLS-2950-02-2012 LETTERS TO THE EDITOR © F e r r a t a S t o r t i F o u n d a t i o

On Applying Formal Techniques to the Development of Hybrid Services: Challenges and Directions

We are primarily interested in formal techniques and how they are applied to the development of hybrid services in particular. We analyze the peculiarities of such services, look at the use of formal techniques for communication services in the industry, and highlight some of the major concerns for the application of formality in an industrial environment. It is argued that with the introduction of hybrid services, more pragmatism is required in applying formal techniques. We briefly describe an ongoing joint collaboration of Alcatel, Swisscom, and the Swiss Federal Institute of Technology in which formal techniques are applied to the specification and testing of hybrid services

Targeting AMP-activated protein kinase in adipocytes to modulate obesity-related adipokine production associated with insulin resistance and breast cancer cell proliferation

BACKGROUND: Adipokines, e.g. TNFalpha, IL-6 and leptin increase insulin resistance, and consequent hyperinsulinaemia influences breast cancer progression. Beside its mitogenic effects, insulin may influence adipokine production from adipocyte stromal cells and paracrine enhancement of breast cancer cell growth. In contrast, adiponectin, another adipokine is protective against breast cancer cell proliferation and insulin resistance.AMP-activated protein kinase (AMPK) activity has been found decreased in visceral adipose tissue of insulin-resistant patients. Lipopolysaccharides (LPS) link systemic inflammation to high fat diet-induced insulin resistance. Modulation o