title: Sodium-coupled neutral amino acid transporter 4 functions as a regulator of protein synthesis during liver development.
abstract: OBJECTIVE
  The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes.
  
  
  METHODS
  HNF4α was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4α was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression.
  
  
  RESULTS
  The expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4α. Transactivation of AP2 was upregulated by HNF4α in HeLa cells without endogenous HNF4α. EMSA has demonstrated that HNF4α directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts.
  
  
  CONCLUSIONS
  SNAT4 functions downstream of HNF4α and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.
authors.affiliation: Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan; Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kondou
authors.forename: Masanobu
authors.initials: M
authors.lastname: Kawai
authors.forename: Kanako
authors.initials: K
authors.lastname: Tachikawa
authors.forename: Akihito
authors.initials: A
authors.lastname: Kimoto
authors.forename: Masayo
authors.initials: M
authors.lastname: Yamagata
authors.forename: Tomoko
authors.initials: T
authors.lastname: Koinuma
authors.forename: Miwa
authors.initials: M
authors.lastname: Yamazaki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nakayama
authors.forename: Sotaro
authors.initials: S
authors.lastname: Mushiake
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ozono
authors.forename: Toshimi
authors.initials: T
authors.lastname: Michigami
keywords: fetal liver organ culture; hepatocyte nuclear factor 4α; perinatal liver; primary hepatoblast culture; system A amino acid transporter