Nonmuscle myosin Ⅱ regulates migration but not contraction in rat hepatic stellate cells  

作　　者：Cathy C Moore Ashley M Lakner Christopher M Yengo Laura W Schrum 

机构地区：[1]Department of Biology,University of North Carolina at Charlotte,Charlotte,NC 28223,United States [2]Liver,Digestive and Metabolic Disorders Laboratory,Carolinas Medical Center

出　　处：《World Journal of Hepatology》2011年第7期184-197,共14页世界肝病学杂志（英文版）（电子版）

基　　金：Supported by NIH Grant AA14891 (awarded to LS)

摘　　要：AIM: To identify and characterize the function of non-mu-scle myosin Ⅱ (NMM Ⅱ) isoforms in primary rat hepatic stellate cells (HSCs).METHODS: Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM Ⅱ isoform (Ⅱ-A, Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry. Using a selective myosin Ⅱ inhibitor and siRNA-mediated knockdown of each isoform, NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS: NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs (Day 14) with sig-niflicant increases seen in all pairwise comparisons (Ⅱ-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; Ⅱ-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (Ⅱ-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; Ⅱ-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM Ⅱ-A and Ⅱ-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro stud-ies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction, NMM Ⅱ-A and Ⅱ-B expres-sion were downregulated with siRNA. NMM Ⅱ-A and/or Ⅱ-B siRNA AIM:To identify and characterize the function of nonmuscle myosin Ⅱ(NMM Ⅱ) isoforms in primary rat hepatic stellate cells(HSCs).METHODS:Primary HSCs were isolated from male Spra-gue-Dawley rats by pronase/collagenase digestion.Total RNA and protein were harvested from quiescent and culture-activated HSCs.NMM Ⅱ isoform(Ⅱ-A,Ⅱ-B and Ⅱ-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively.NMM Ⅱ protein localization was visualized in vitro using immunocytochemical analysis.For in vivo assessment,liver tissue was harvested from bile duct-ligated(BDL) rats and NMM Ⅱisoform expression determined by immunohistochemistry.Using a selective myosin Ⅱ inhibitor and si RNA-mediated knockdown of each isoform,NMM Ⅱ functionality inprimary rat HSCs was determined by contraction and migration assays.RESULTS:NMM Ⅱ-A and Ⅱ-B mRNA expression was increased in culture-activated HSCs(Day 14) with signif icant increases seen in all pairwise comparisons(Ⅱ-A:12.67 ± 0.99(quiescent) vs 17.36 ± 0.78(Day 14),P < 0.05;Ⅱ-B:4.94 ± 0.62(quiescent) vs 13.90 ± 0.85(Day 14),P < 0.001).Protein expression exhibited similar expression patterns(Ⅱ-A:1.87 ± 2.50(quiescent) vs 58.64 ± 8.76(Day 14),P < 0.05;Ⅱ-B:1.17 ± 1.93(quiescent) vs 103.71 ± 21.73(Day 14),P < 0.05).No signif icant differences were observed in NMM Ⅱ-C mRNA and protein expression between quiescent and activated HSCs.In culture-activated HSCs,NMM Ⅱ-A and Ⅱ-B merged with Factin at the cellular periphery and throughout cytoplasm respectively.In vitro studies showed increased expression of NMM Ⅱ-B in HSCs activated by BDL compared to shamoperated animals.There were no apparent increases of NMM Ⅱ-A and Ⅱ-C protein expression in HSCs during hepatic BDL injury.To determine the contribution of NMM Ⅱ-A and Ⅱ-B to migration and contraction,NMM Ⅱ-A and Ⅱ-B expression were downregulated with siRNA.NMM Ⅱ-A and/or Ⅱ-B siRNA inhibited HSC migration by approximately 25% compa

关 键 词：HEPATIC stellate cells Nonmuscle MYOSIN Ⅱ MIGRATION CONTRACTION BLEBBISTATIN HEPATIC injury 

分 类 号：Q26[生物学—细胞生物学]