title: Pancreatic and duodenal homeobox protein 1 (Pdx-1) maintains endoplasmic reticulum calcium levels through transcriptional regulation of sarco-endoplasmic reticulum calcium ATPase 2b (SERCA2b) in the islet β cell.
abstract: Although the pancreatic duodenal homeobox 1 (Pdx-1) transcription factor is known to play an indispensable role in β cell development and secretory function, recent data also implicate Pdx-1 in the maintenance of endoplasmic reticulum (ER) health. The sarco-endoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) gradient between the cytosol and ER lumen. In models of diabetes, our data demonstrated loss of β cell Pdx-1 that occurs in parallel with altered SERCA2b expression, whereas in silico analysis of the SERCA2b promoter revealed multiple putative Pdx-1 binding sites. We hypothesized that Pdx-1 loss under inflammatory and diabetic conditions leads to decreased SERCA2b levels and activity with concomitant alterations in ER health. To test this, siRNA-mediated knockdown of Pdx-1 was performed in INS-1 cells. The results revealed reduced SERCA2b expression and decreased ER Ca(2+), which was measured using fluorescence lifetime imaging microscopy. Cotransfection of human Pdx-1 with a reporter fused to the human SERCA2 promoter increased luciferase activity 3- to 4-fold relative to an empty vector control, and direct binding of Pdx-1 to the proximal SERCA2 promoter was confirmed by chromatin immunoprecipitation. To determine whether restoration of SERCA2b could rescue ER stress induced by Pdx-1 loss, Pdx1(+/-) mice were fed a high-fat diet. Isolated islets demonstrated an increased spliced-to-total Xbp1 ratio, whereas SERCA2b overexpression reduced the Xbp1 ratio to that of wild-type controls. Together, these results identify SERCA2b as a novel transcriptional target of Pdx-1 and define a role for altered ER Ca(2+) regulation in Pdx-1-deficient states.
authors.affiliation: From the Departments of Biochemistry and Molecular Biology.
authors.forename: Justin S
authors.initials: JS
authors.lastname: Johnson
authors.affiliation: Medicine, and.
authors.forename: Tatsuyoshi
authors.initials: T
authors.lastname: Kono
authors.affiliation: Cellular and Integrative Physiology and.
authors.forename: Xin
authors.initials: X
authors.lastname: Tong
authors.affiliation: Cellular and Integrative Physiology and.
authors.forename: Wataru R
authors.initials: WR
authors.lastname: Yamamoto
authors.affiliation: the Departamento de Bioquimica, Facultad de Medicina, National Autonomous University of México, México City, 04510 México.
authors.forename: Angel
authors.initials: A
authors.lastname: Zarain-Herzberg
authors.affiliation: the Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, and Department of Biomolecular Chemistry, University of Wisconsin Madison School of Medicine and Public Health, Madison, Wisconsin 53705.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Merrins
authors.affiliation: the Department of Pharmacology and Brehm Center for Diabetes Research, University of Michigan Medical School, Ann Arbor, Michigan 48105.
authors.forename: Leslie S
authors.initials: LS
authors.lastname: Satin
authors.affiliation: the Pôle d'Endocrinologie, Diabète et Nutrition, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, 1348 Belgium, and.
authors.forename: Patrick
authors.initials: P
authors.lastname: Gilon
authors.affiliation: From the Departments of Biochemistry and Molecular Biology, Medicine, and Cellular and Integrative Physiology and the Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202, the Roudebush Veterans Affairs Medical Center, Indianapolis, Indiana 46202 cevansmo@iu.edu.
authors.forename: Carmella
authors.initials: C
authors.lastname: Evans-Molina
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D003920:Diabetes Mellitus / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence / Q000379:methods; D041681:NIH 3T3 Cells; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics* / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism
chemical_list: D018398:Homeodomain Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C506947:Atp2a2 protein, mouse; D002118:Calcium
keywords: Calcium ATPase; Diabetes; Endoplasmic Reticulum Stress (ER Stress); Pancreatic And Duodenal Homeobox 1; Pancreatic Islet; Sarco-endoplasmic Reticulum Calcium ATPase; β Cell