title: Nuclear Factor, Erythroid 2-Like 2 Regulates Expression of Type 3 Inositol 1,4,5-Trisphosphate Receptor and Calcium Signaling in Cholangiocytes.
abstract: OBJECTIVE
  Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3.
  
  
  METHODS
  The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases.
  
  
  RESULTS
  We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders.
  
  
  CONCLUSIONS
  The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies.
authors: Liver Center, Yale University School of Medicine, New Haven, Connecticut; Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors: Jittima
authors: J
authors: Weerachayaphorn
authors: Liver Center, Yale University School of Medicine, New Haven, Connecticut.
authors: Maria Jimena
authors: MJ
authors: Amaya
authors: Liver Center, Yale University School of Medicine, New Haven, Connecticut.
authors: Carlo
authors: C
authors: Spirli
authors: Department of Anatomy, Phramongkutklao College of Medicine, Bangkok, Thailand.
authors: Piyachat
authors: P
authors: Chansela
authors: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
authors: Kisha A
authors: KA
authors: Mitchell-Richards
authors: Liver Center, Yale University School of Medicine, New Haven, Connecticut.
authors: Meenakshisundaram
authors: M
authors: Ananthanarayanan
authors: Liver Center, Yale University School of Medicine, New Haven, Connecticut. Electronic address: michael.nathanson@yale.edu.
authors: Michael H
authors: MH
authors: Nathanson
mesh_terms: D000818:Animals; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000378:metabolism*; D020013:Calcium Signaling / Q000235:genetics* / Q000502:physiology; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D055785:Gene Knockdown Techniques; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism; D018384:Oxidative Stress / Q000235:genetics; D051381:Rats; D015398:Signal Transduction / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C506929:ITPR3 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D014157:Transcription Factors
keywords: Gene Regulation; Primary Biliary Cirrhosis; Signal Transduction