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      EGFR Signaling Promotes β-Cell Proliferation and Survivin Expression during Pregnancy

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          Abstract

          Placental lactogen (PL) induced serotonergic signaling is essential for gestational β-cell mass expansion. We have previously shown that intact Epidermal growth factor –receptor (EGFR) function is a crucial component of this pathway. We now explored more specifically the link between EGFR and pregnancy-induced β-cell mass compensation. Islets were isolated from wild-type and β-cell-specific EGFR-dominant negative mice (E1-DN), stimulated with PL and analyzed for β-cell proliferation and expression of genes involved in gestational β-cell growth. β-cell mass dynamics were analyzed both with traditional morphometrical methods and three-dimensional optical projection tomography (OPT) of whole-mount insulin-stained pancreata. Insulin-positive volume analyzed with OPT increased 1.4-fold at gestational day 18.5 (GD18.5) when compared to non-pregnant mice. Number of islets peaked by GD13.5 (680 vs 1134 islets per pancreas, non-pregnant vs. GD13.5). PL stimulated beta cell proliferation in the wild-type islets, whereas the proliferative response was absent in the E1-DN mouse islets. Serotonin synthesizing enzymes were upregulated similarly in both the wild-type and E1-DN mice. However, while survivin ( Birc5) mRNA was upregulated 5.5-fold during pregnancy in the wild-type islets, no change was seen in the E1-DN pregnant islets. PL induced survivin expression also in isolated islets and this was blocked by EGFR inhibitor gefitinib, mTOR inhibitor rapamycin and MEK inhibitor PD0325901. Our 3D-volumetric analysis of β-cell mass expansion during murine pregnancy revealed that islet number increases during pregnancy. In addition, our results suggest that EGFR signaling is required for lactogen-induced survivin expression via MAPK and mTOR pathways.

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          IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs.

          Survivin is a member of the inhibitor of apoptosis protein (IAP) family. We investigated the antiapoptotic mechanism of Survivin, as well as its expression in 60 human tumor cell lines used for the National Cancer Institute's anticancer drug screening program. In cotransfection experiments, cell death induced by Bax or Fas (CD 95) was partially inhibited (mean +/- SD, 65% +/- 8%) by Survivin, whereas XIAP, another IAP family member, almost completely blocked cell death (93% +/- 4%) under the same conditions. Survivin and XIAP also protected 293 cells from apoptosis induced by overexpression of procaspase-3 and -7 and inhibited the processing of these zymogens into active caspases. In vitro binding experiments indicated that, like other IAP-family proteins, Survivin binds specifically to the terminal effector cell death proteases, caspase-3 and -7, but not to the proximal initiator protease caspase-8. Using a cell-free system in which cytosolic extracts were derived from control- or Survivin-transfected cells and where caspases were activated either by addition of cytochrome c and dATP or by adding recombinant active caspase-8, Survivin was able to substantially reduce caspase activity, as measured by cleavage of a tetrapeptide substrate, AspGluValAsp-aminofluorocoumarin. Similar results were obtained in intact cells when Survivin was overexpressed by gene transfection and caspase activation was induced by the anticancer drug etoposide. Survivin was expressed in all 60 cancer cell lines analyzed, with highest levels in breast and lung cancers and lowest levels in renal cancers. These findings indicate that Survivin, which is commonly expressed in human tumor cell lines, can bind the effector cell death proteases caspase-3 and -7 in vitro and inhibits caspase activity and cell death in cells exposed to diverse apoptotic stimuli. Although quantitative differences may exist, these observations suggest commonality in the mechanisms used by IAP-family proteins to suppress apoptosis.
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            Role of transactivation of the EGF receptor in signalling by G-protein-coupled receptors.

            Transduction of a mitogenic signal from the cell membrane to the nucleus involves the adapter proteins SHC and Grb2, which mediate activation of the Ras/mitogen-activated protein (MAP) kinase pathway. In contrast to receptor tyrosine kinases (RTKs), the signalling steps leading to Ras/MAP kinase activation by G-protein-coupled receptors (GPCRs) are still poorly characterized but appear to include beta gamma subunits of heterotrimeric G-proteins and as-yet unidentified tyrosine kinases. We report here that the epidermal growth factor receptor (EGFR) and the neu oncoprotein become rapidly tyrosine-phosphorylated upon stimulation of Rat-1 cells with the GPCR agonists endothelin-1, lysophosphatic acid and thrombin, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by either the selective tyrphostin AG1478 or a dominant-negative EGFR mutant suppressed MAP kinase activation and strongly inhibited induction of fos gene expression and DNA synthesis. Our results demonstrate a role for RTKs as downstream mediators in GPCR mitogenic signalling and suggest a ligand-independent mechanism of RTK activation through intracellular signal crosstalk.
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              Adaptive changes in pancreatic beta cell fractional area and beta cell turnover in human pregnancy

              Aims/hypothesis We sought to establish the extent and basis for adaptive changes in beta cell numbers in human pregnancy. Methods Pancreas was obtained at autopsy from women who had died while pregnant (n = 18), post-partum (n = 6) or were not pregnant at or shortly before death (controls; n = 20). Pancreases were evaluated for fractional pancreatic beta cell area, islet size and islet fraction of beta cells, beta cell replication (Ki67) and apoptosis (TUNEL), and indirect markers of beta cell neogenesis (insulin-positive cells in ducts and scattered beta cells in pancreas). Results The pancreatic fractional beta cell area was increased by ∼1.4-fold in human pregnancy, with no change in mean beta cell size. In pregnancy there were more small islets rather than an increase in islet size or beta cells per islet. No increase in beta cell replication or change in beta cell apoptosis was detected, but duct cells positive for insulin and scattered beta cells were increased with pregnancy. Conclusions/interpretation The adaptive increase in beta cell numbers in human pregnancy is not as great as in most reports in rodents. This increase in humans is achieved by increased numbers of beta cells in apparently new small islets, rather than duplication of beta cells in existing islets, which is characteristic of pregnancy in rodents. Electronic supplementary material The online version of this article (doi:10.1007/s00125-010-1809-6) contains supplementary material, which is available to authorised users.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                2 April 2014
                : 9
                : 4
                : e93651
                Affiliations
                [1 ]Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Helsinki, Finland
                [2 ]Children's Hospital, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland
                University of Bremen, Germany
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: EH JU JP PJM TO. Performed the experiments: EH JU JP. Analyzed the data: EH JU PJM TO. Contributed reagents/materials/analysis tools: JP. Wrote the paper: EH PJM TO.

                [¤]

                Current address: Department of Gene Technology,Tallinn University of Technology, Tallinn, Estonia

                Article
                PONE-D-13-40906
                10.1371/journal.pone.0093651
                3973552
                24695557
                730628e2-7948-4960-96d3-597b9ba81b8d
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 7 October 2013
                : 8 March 2014
                Page count
                Pages: 11
                Funding
                These studies were funded by the Diabetes Research Foundation ( www.diabetestutkimus.fi), the Helsinki University Central Hospital, the Foundation for Pediatric Research ( www.lastentautientutkimussaatio.fi), the Sigrid Jusélius Foundation ( www.sigridjuselius.fi), the Helsinki Biomedical Graduate Program ( www.hbgs.helsinki.fi), the Academy of Finland (Grant no 257157) ( www.aka.fi), the Orion-Farmos Research Foundation and the Biomedicum Helsinki Foundation ( www.biomedicum.fi). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Anatomy
                Endocrine System
                Pancreas
                Cell Biology
                Cell Processes
                Cell Cycle and Cell Division
                Mitogens
                Cell Growth
                Molecular Cell Biology
                Medicine and Health Sciences
                Diagnostic Medicine
                Diagnostic Radiology
                Tomography
                Computed Axial Tomography
                Endocrinology
                Diabetic Endocrinology
                Metabolic Disorders
                Diabetes Mellitus
                Gestational Diabetes
                Type 1 Diabetes
                Radiology and Imaging
                Women's Health
                Maternal Health
                Pregnancy
                Obstetrics and Gynecology
                Research and Analysis Methods
                Model Organisms
                Animal Models
                Mouse Models

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                Uncategorized

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