title: [Effects of wild-type PTEN overexpression and its mutation on F-actin in activated hepatic stellate cells].
abstract: Objective:To investigate the effect of overexpression of wild-type phosphatase and tensin homolog (PTEN) deleted on chromosome 10 and its mutant G129E (exhibiting the activity of protein phosphatase and losing the activity of lipid phosphatase) on F-actin in activated hepatic stellate cells (HSCs) cultured in vitro.Methods:The activated hepatic stellate cell-T6 (HSC-T6) cells were cultured in vitro, and activated HSCs were transfected with adenovirus that carried wild-type PTEN gene and G129E gene using transient transfection. The HSCs were divided into the following groups: control group, which was transfected with DMEM medium instead of virus solution; Ad-GFP group, which was transfected with the empty adenovirus vector with the expression of green fluorescent protein (GFP); Ad-PTEN group, which was transfected with the recombinant adenovirus with wild-type PTEN gene and GFP expression; Ad-G129E group, which was transfected with the recombinant adenovirus with G129E gene and GFP expression. Western blot and quantitative real-time PCR were used to measure the protein and mRNA expression of PTEN in activated HSCs; under a laser scanning confocal microscope (LSCM), phalloidine labeled with the fluorescein tetramethylrhodamine isothiocyanate (TRITC) was used to observe the morphology of HSCs, distribution and fluorescence intensity of F-actin, and changes in pseudopodia and stress fibers, and a calcium fluorescence probe (Rhod-2/AM) was used to measure the changes in Ca(2+) concentration in HSCs. A one-way analysis of variance was used for comparison between multiple groups, and the least significant difference test was used for comparison between two groups.Results:Wild-type PTEN and G129E genes were highly expressed in activated HSCs. In the control group and the Ad-GFP group, HSCs had a starlike or polygonal shape, F-actin was reconfigured and formed a large number of stress fibers which stretched across the whole cell, and layered pseudopodia were seen around the cell. In the Ad-PTEN group and the Ad-G129E group, the HSCs had a fusiform shape, F-actin was mainly seen around the cell, a small number of stress fibers were seen inside the cell, and layered pseudopodia around the cell disappeared. The Ad-PTEN group and the Ad-G129E group had significant reductions in the fluorescence intensity of F-actin compared with the control group and the Ad-GFP group (357.67±13.39/377.25±14.55 vs 961.87±27.33/954.68±20.71,F= 1783.486,P< 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P> 0.05). The Ad-PTEN group and the Ad-G129E group had significant reductions in the relative concentration of Ca(2+) compared with the control group and the Ad-GFP group (251.60±90.88/352.18±146.01 vs 1953.95±132.99/1937.57±115.17,F= 834.988,P< 0.05), while there were no significant differences between the Ad-PTEN group and the Ad-G129E group, as well as between the control group and the Ad-GFP group (P> 0.05).Conclusion:The overexpressed wild-type PTEN and its mutant G129E can significantly inhibit the formation and reconfiguration of cytoskeletal protein F-actin and reduce the concentration of Ca2+ in activated HSCs in vitro. In addition, there are no significant differences in the above effects between wild-type PTEN and G129E.
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: L S
authors.initials: LS
authors.lastname: Hao
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: Y L
authors.initials: YL
authors.lastname: Liu
authors.affiliation: The Basic Medical College of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: G L
authors.initials: GL
authors.lastname: Zhang
authors.affiliation: The Life Science College of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: X J
authors.initials: XJ
authors.lastname: Song
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: Y L
authors.initials: YL
authors.lastname: Wang
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Gastroenterology, the Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei Province 063000, China.
authors.forename: L M
authors.initials: LM
authors.lastname: Jin
mesh_terms: D000199:Actins / Q000378:metabolism*; D000256:Adenoviridae; D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D005822:Genetic Vectors; D055166:Hepatic Stellate Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D051059:PTEN Phosphohydrolase / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D014162:Transfection
chemical_list: D000199:Actins; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human
keywords: Calciumion concentration; Cytoskeletal protein; F-actin; Hepatic stellate cells; PTEN