title: PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.
abstract: OBJECTIVE
  A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM.
  
  
  METHODS
  Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements.
  
  
  RESULTS
  PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death.
  
  
  CONCLUSIONS
  The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Mellado-Gil
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Carmen María
authors.initials: CM
authors.lastname: Jiménez-Moreno
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Martin-Montalvo
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Alvarez-Mercado
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Esther
authors.initials: E
authors.lastname: Fuente-Martin
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Nadia
authors.initials: N
authors.lastname: Cobo-Vuilleumier
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Petra Isabel
authors.initials: PI
authors.lastname: Lorenzo
authors.affiliation: Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain.|Instituto de Bioingeniería, Universidad Miguel Hernandez, Elche, Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Bru-Tari
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Irene de Gracia
authors.initials: Ide G
authors.lastname: Herrera-Gómez
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain.
authors.forename: Livia
authors.initials: L
authors.lastname: López-Noriega
authors.affiliation: Medical Genome Project, Genomics & Bioinformatics Platform of Andalusia, Seville, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Pérez-Florido
authors.affiliation: Medical Genome Project, Genomics & Bioinformatics Platform of Andalusia, Seville, Spain.|Edinburgh Genomics, University of Edinburgh, Edinburgh, UK.
authors.forename: Javier
authors.initials: J
authors.lastname: Santoyo-López
authors.affiliation: Department of Internal Medicine, Ulm University Medical Centre, Ulm, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Spyrantis
authors.affiliation: Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland.
authors.forename: Paolo
authors.initials: P
authors.lastname: Meda
authors.affiliation: Department of Internal Medicine, Ulm University Medical Centre, Ulm, Germany.|Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Republic of Singapore.|Imperial College, London, UK.
authors.forename: Bernhard O
authors.initials: BO
authors.lastname: Boehm
authors.affiliation: Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Spain.|Instituto de Bioingeniería, Universidad Miguel Hernandez, Elche, Spain.
authors.forename: Ivan
authors.initials: I
authors.lastname: Quesada
authors.affiliation: Pancreatic Islet Development and Regeneration Unit, Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Avda Américo Vespucio, Parque Científico y Tecnológico Cartuja 93, 41092, Seville, Spain. benoit.gauthier@cabimer.es.
authors.forename: Benoit R
authors.initials: BR
authors.lastname: Gauthier
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D003922:Diabetes Mellitus, Type 1 / Q000378:metabolism* / Q000473:pathology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005260:Female; D018398:Homeodomain Proteins / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008817:Mice, Mutant Strains; D051761:Paired Box Transcription Factors / Q000378:metabolism*
chemical_list: D018398:Homeodomain Proteins; D051761:Paired Box Transcription Factors; C110218:Pax4 protein, mouse
keywords: Beta cell degeneration; ER homeostasis; PAX4; RIP-B7.1; Type 1 diabetes
references.citation: Nat Rev Drug Discov. 2014 Jun;13(6):465-76
references.pmid: 24854413
references.citation: Eur J Immunol. 2009 Sep;39(9):2403-11
references.pmid: 19670381
references.citation: Nat Genet. 1998 Oct;20(2):143-8
references.pmid: 9771706
references.citation: Cell Metab. 2014 Jan 7;19(1):109-21
references.pmid: 24332968
references.citation: Diabetologia. 2004 Mar;47(3):581-9
references.pmid: 14767595
references.citation: Diabetes. 2011 Jun;60(6):1705-15
references.pmid: 21521872
references.citation: Sci Rep. 2013;3:1804
references.pmid: 23652855
references.citation: Diabetes. 2005 Feb;54(2):452-61
references.pmid: 15677503
references.citation: Islets. 2014;6(4):e982376
references.pmid: 25437380
references.citation: EBioMedicine. 2015 Mar 17;2(5):378-85
references.pmid: 26137583
references.citation: Genesis. 2005 Oct;43(2):80-6
references.pmid: 16108006
references.citation: J Biol Chem. 2011 Sep 16;286(37):32300-12
references.pmid: 21784843
references.citation: Diabetes. 2011 Feb;60(2):525-36
references.pmid: 20980460
references.citation: J Ultrastruct Res. 1969 Jan;26(1):31-43
references.pmid: 4887011
references.citation: Endocrinology. 2009 Feb;150(2):607-15
references.pmid: 18818288
references.citation: Nature. 1997 Mar 27;386(6623):399-402
references.pmid: 9121556
references.citation: Diabetes. 2005 Dec;54 Suppl 2:S97-107
references.pmid: 16306347
references.citation: Mech Dev. 2007 Feb;124(2):97-107
references.pmid: 17196797
references.citation: Cell Transplant. 2013;22(11):2135-45
references.pmid: 23067523
references.citation: Int Immunol. 2000 Jan;12(1):9-17
references.pmid: 10607745
references.citation: Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10845-50
references.pmid: 11526215
references.citation: Diabetes. 2013 Aug;62(8):2870-8
references.pmid: 23835340
references.citation: Adv Immunol. 2008;100:125-49
references.pmid: 19111165
references.citation: J Biol Chem. 2004 Jul 23;279(30):31121-30
references.pmid: 15151993
references.citation: Eur J Immunol. 2007 Aug;37(8):2097-103
references.pmid: 17615584
references.citation: Nat Rev Cancer. 2006 Jan;6(1):52-62
references.pmid: 16397527
references.citation: Diabetes. 2014 Feb;63(2):619-31
references.pmid: 24353176
references.citation: Antioxid Redox Signal. 2014 Jul 20;21(3):396-413
references.pmid: 24702237
references.citation: Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):19090-5
references.pmid: 19855005
references.citation: Endocrinology. 2013 Oct;154(10):3515-24
references.pmid: 23867214
references.citation: J Mol Endocrinol. 2008 Feb;40(2):37-45
references.pmid: 18234907
references.citation: Nat Genet. 2000 Aug;25(4):406-9
references.pmid: 10932183
references.citation: Antimicrob Agents Chemother. 2014 Jun;58(6):3342-7
references.pmid: 24687497
references.citation: J Biol Chem. 2014 Apr 18;289(16):11454-64
references.pmid: 24610783
references.citation: Science. 2007 Jun 1;316(5829):1341-5
references.pmid: 17463248
references.citation: Endocrinology. 1997 Apr;138(4):1736-41
references.pmid: 9075738
references.citation: Cold Spring Harb Perspect Med. 2012 Sep;2(9):a007666
references.pmid: 22951443
references.citation: PLoS One. 2011;6(7):e22609
references.pmid: 21799917
references.citation: Cell Metab. 2009 Aug;10(2):110-8
references.pmid: 19656489
references.citation: Diabetes. 2014 Jun;63(6):1978-93
references.pmid: 24379348
references.citation: Nat Immunol. 2005 Dec;6(12):1245-52
references.pmid: 16286920
references.citation: Hum Mol Genet. 2004 Dec 15;13(24):3151-9
references.pmid: 15509590
references.citation: Diabetes. 2002 Feb;51 Suppl 1:S144-51
references.pmid: 11815474
references.citation: J Autoimmun. 2006 Feb;26(1):7-15
references.pmid: 16338119
references.citation: Int J Biochem Cell Biol. 2012 Jun;44(6):842-6
references.pmid: 22373697
references.citation: Proc Natl Acad Sci U S A. 2014 Aug 19;111(33):E3405-14
references.pmid: 25092329