Original Article
Copyright ©2011 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Hepatol. Jul 27, 2011; 3(7): 184-197
Published online Jul 27, 2011. doi: 10.4254/wjh.v3.i7.184
Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells
Cathy C Moore, Ashley M Lakner, Christopher M Yengo, Laura W Schrum
Cathy C Moore, Ashley M Lakner, Christopher M Yengo, Laura W Schrum, Department of Biology, University of North Carolina at Charlotte, Charlotte, NC 28223, United States
Laura W Schrum, Liver, Digestive and Metabolic Disorders Laboratory, Carolinas Medical Center, Charlotte, NC 28203, United States
Author contributions: Moore CC, Yengo CM and Schrum LW developed the experimental design; Moore CC performed the research; Moore CC, Lakner AM, Yengo CM and Schrum LW analyzed the data; Moore CC, Lakner AM and Schrum LW organized and edited the paper.
Correspondence to: Laura W Schrum, PhD, Research Group Director, Liver, Digestive and Metabolic Disorders Laboratory, Carolinas Medical Center, 1000 Blythe Blvd, Charlotte, NC 28203, United States. laura.schrum@carolinashealthcare.org
Telephone: +1-704-3559670 Fax:+1-704-3557648
Received: January 6, 2011
Revised: May 6, 2011
Accepted: May 13, 2011
Published online: July 27, 2011

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 Sprague-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 in primary 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 significant increases seen in all pair-wise 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 significant 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 studies 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 expression were downregulated with siRNA. NMM II-A and/or II-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM II-A and II-B inhibition had no significant effect on HSC contraction; however, contraction was inhibited with the myosin II inhibitor, blebbistatin (38.7% ± 1.9%).

CONCLUSION: Increased expression of NMM II-A and II-B regulates HSC migration, while other myosin IIclasses likely modulate contraction, contributing to development and severity of liver fibrosis.

Keywords: Hepatic stellate cells, Nonmuscle myosin II, Migration, Contraction, Blebbistatin, Hepatic injury