title: AKT isoforms 1 and 3 regulate basal and epidermal growth factor-stimulated SGHPL-5 trophoblast cell migration in humans.
abstract: Protein kinase B/AKT is critically involved in murine placental development and migration of human placental trophoblasts into maternal uterine tissue. However, localization of the three AKT isoforms within human placenta and their roles in extravillous trophoblasts have not been elucidated. Therefore, we analyzed the expression pattern and function of AKT1, AKT2, and AKT3 in migratory human trophoblasts using SGHPL-5 cell pools stably expressing small-hairpin microRNA (shRNAmir) against AKT1, AKT2, or AKT3 as a model. Western blot analyses using isoform-specific antibodies revealed ubiquitous expression of AKT1, AKT2, and AKT3 in primary villous and extravillous trophoblasts and the trophoblastic cell lines JEG-3, HTR-8/SVneo, and SGHPL-5. Immunofluorescence of first-trimester placentae localized AKT2 and AKT3 to the cytoplasm and nucleus, respectively, in all subtypes of cytotrophoblasts, whereas AKT1 was detected in both cellular compartments. A similar distribution of AKT isoforms was detectable in SGHPL-5 cells. Gene silencing using shRNAmir decreased protein expression of AKT1, AKT2, and AKT3 to 16%, 8%, and 11%, respectively, in SGHPL-5 cells. Compared with shRNAmir controls, proliferation and camptothecin-induced apoptosis were not affected in the different AKT knockdown cells. However, basal and epidermal growth factor (EGF)-induced trophoblast migration was significantly reduced in AKT1 and AKT3 gene-silenced cells, whereas downregulation of AKT2 was not effective. Accordingly, a decrease in EGF-stimulated phosphorylation of AKT (Ser473 and Thr308) and its downstream target mTORC1 (Ser2448) was noticed in AKT1 and AKT3 shRNAmir cell pools. In summary, the results suggest that the AKT isoforms 1 and 3 promote basal as well as EGF-induced trophoblast migration.
authors.affiliation: Department of Obstetrics and Fetal-Maternal Medicine, Reproductive Biology Unit, Medical University of Vienna, Vienna, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Haslinger
authors.forename: Sandra
authors.initials: S
authors.lastname: Haider
authors.forename: Stefan
authors.initials: S
authors.lastname: Sonderegger
authors.forename: Jan Velten
authors.initials: JV
authors.lastname: Otten
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pollheimer
authors.forename: Guy
authors.initials: G
authors.lastname: Whitley
authors.forename: Martin
authors.initials: M
authors.lastname: Knöfler
mesh_terms: D017209:Apoptosis; D002166:Camptothecin; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D015536:Down-Regulation; D004815:Epidermal Growth Factor / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D000076222:Mechanistic Target of Rapamycin Complex 1; D035683:MicroRNAs; D046912:Multiprotein Complexes; D010766:Phosphorylation; D011247:Pregnancy; D020033:Protein Isoforms / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D059004:Topoisomerase I Inhibitors; D014327:Trophoblasts / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D046912:Multiprotein Complexes; D020033:Protein Isoforms; D059004:Topoisomerase I Inhibitors; D004815:Epidermal Growth Factor; C494918:AKT1 protein, human; C494921:AKT2 protein, human; C491754:AKT3 protein, human; D000076222:Mechanistic Target of Rapamycin Complex 1; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D002166:Camptothecin