title: Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro.
abstract: Previous work in our laboratory demonstrated that over-expression of human insulin-like growth factor-11 (hIGF-1) in the placenta corrects fetal weight deficits in mouse, rat, and rabbit models of intrauterine growth restriction without changes in placental weight. The underlying mechanisms of this effect have not been elucidated. To investigate the effect of intra-placental IGF-1 over-expression on placental function we examined amino acid transporter expression and localization in both a mouse model of placental Insufficiency (PI) and a model of human trophoblast, the BeWo Choriocarcinoma cell line. For in vitro human studies, BeWo Choriocarcinoma cells were maintained in F12 complete medium + 10%FBS. Cells were incubated in serum-free control media ± Ad-IGF-1 or Ad-LacZ for 48 h. MOIs of 10:1 and 100:1 were utilized. In BeWo, transfection efficiency was 100% at an MOI of 100:1 and Ad-IGF-1 significantly increased IGF-1 secretion, proliferation and invasion but reduced apoptosis compared to controls. In vitro, amino acid uptake was increased following Ad-IGF-1 treatment and associated with significantly increased RNA expression of SNAT1, 2, LAT1 and 4F2hc. Only SNAT2 protein expression was increased but LAT1 showed relocalization from a perinuclear location to the cytoplasm and cell membrane. For in vivo studies, timed-pregnant animals were divided into four groups on day 18; sham-operated controls, uterine artery branch ligation (UABL), UABL + Ad-hIGF-1 (10(8) PFU), UABL + Ad-LacZ (10(8) PFU). At gestational day 20, pups and placentas were harvested by C-section. Only LAT1 mRNA expression changed, showing that a reduced expression of the transporter levels in the PI model could be partially rectified with Ad-hIGF1 treatment. At the protein level, System L was reduced in PI but remained at control levels following Ad-hIGF1. The System A isoforms were differentially regulated with SNAT2 expression diminished but SNAT1 increased in PI and Ad-hIGF1 groups. Enhanced amino acid isoform transporter expression and relocalization to the membrane may be an important mechanism contributing to Ad-hIGF-1 mediated correction of placental insufficiency.
authors.affiliation: The Center for Cellular and Molecular Fetal Therapy, Division of Pediatric General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA. Electronic address: helen.jones@cchmc.org.
authors.forename: H
authors.initials: H
authors.lastname: Jones
authors.affiliation: Colorado Fetal Care Center, Children's Hospital Colorado and The University of Colorado School of Medicine, Aurora, CO, USA.
authors.forename: T
authors.initials: T
authors.lastname: Crombleholme
authors.affiliation: The Center for Cellular and Molecular Fetal Therapy, Division of Pediatric General, Thoracic and Fetal Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
authors.forename: M
authors.initials: M
authors.lastname: Habli
mesh_terms: D026905:Amino Acid Transport Systems / Q000096:biosynthesis*; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002822:Choriocarcinoma; D004195:Disease Models, Animal; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis* / Q000627:therapeutic use; D051379:Mice; D010920:Placenta / Q000378:metabolism*; D010927:Placental Insufficiency / Q000628:therapy*; D011247:Pregnancy; D014162:Transfection; D014327:Trophoblasts / Q000378:metabolism
chemical_list: D026905:Amino Acid Transport Systems; D000596:Amino Acids; D007334:Insulin-Like Growth Factor I
keywords: Amino acid; Gene therapy; Proliferation; Transport; Trophoblast