title: A GAPDH-mediated trans-nitrosylation pathway is required for feedback inhibition of bile salt synthesis in rat liver.
abstract: OBJECTIVE
  Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals.
  
  
  METHODS
  We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers.
  
  
  RESULTS
  Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions.
  
  
  CONCLUSIONS
  In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain. Electronic address: crodriguez@unav.es.
authors: Carlos M
authors: CM
authors: Rodríguez-Ortigosa
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors: Jon
authors: J
authors: Celay
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors: Israel
authors: I
authors: Olivas
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain.
authors: Nerea
authors: N
authors: Juanarena
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain.
authors: Sara
authors: S
authors: Arcelus
authors: Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain.
authors: Iker
authors: I
authors: Uriarte
authors: Laboratory of Experimental Hepatology and Drug Targeting, University of Salamanca, Salamanca, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Salamanca, Spain.
authors: José Juan G
authors: JJ
authors: Marín
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain.
authors: Matias A
authors: MA
authors: Avila
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain.
authors: Juan F
authors: JF
authors: Medina
authors: Division of Hepatology and Gene Therapy, Center for Applied Medical Research, University of Navarra, Pamplona, Spain; Centro de Investigación Biomédica en Red en el área temática de Enfermedades Hepáticas y Digestivas (CIBERehd), Pamplona, Spain; Liver Unit, University of Navarra Clinic, Pamplona, Spain. Electronic address: jprieto@unav.es.
authors: Jesus
authors: J
authors: Prieto
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000096:biosynthesis*; D002478:Cells, Cultured; D020355:Cholates / Q000008:administration & dosage; D002779:Cholestasis / Q000201:enzymology* / Q000235:genetics; D002790:Cholesterol 7-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D025461:Feedback, Physiological; D015971:Gene Expression Regulation, Enzymologic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D056464:Histone Deacetylase 2 / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D056564:Sirtuin 1 / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
chemical_list: D001647:Bile Acids and Salts; D020355:Cholates; D004791:Enzyme Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; C099713:nuclear receptor subfamily 0, group B, member 2; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; C532838:CYP7A1 protein, rat; D002790:Cholesterol 7-alpha-Hydroxylase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C488368:Sirt1 protein, rat; D056564:Sirtuin 1; C533426:Hdac2 protein, rat; D056464:Histone Deacetylase 2
keywords: Cholestasis; Cholesterol 7α-Hydroxylase; Histone Deacetylase; Nitric Oxide