Gene expression profiling and functional assays of activated hepatic stellate cells suggest that myocardin has a role in activation.
BACKGROUND
Myofibroblast-like cells derived from transdifferentiated hepatic stellate cells (HSC) play a central role in scar formation that leads to liver fibrosis. The molecular mechanisms underlying this process are not fully understood.


OBJECTIVE
Our aim was to identify genes that are differentially regulated by HSC activation and to explore their function.


METHODS
Using oligonucleotide microarrays, we performed transcriptional analysis of the human HSC cell line, LI90, cultured on Matrigel. Microarray data were validated by quantitative real-time polymerase chain reaction and Western blotting. The function of myocardin was assessed by myocardin RNAi and overexpression.


RESULTS
Examination of Matrigel-induced deactivation of LI90 cells revealed marked downregulation of myocardin, an important transcriptional regulator in smooth and cardiac muscle development. Small interfering RNA-mediated suppression of myocardin expression in both activated LI90 and rat activated HSC resulted in loss of the phenotypic characteristics of myofibroblasts and significantly impaired the production of activated HSC markers, such as alpha-smooth muscle actin and extracellular matrix proteins like type I collagen. Overexpression of myocardin led to the upregulation of these marker genes. Myocardin was upregulated in rat primary HSC during in vitro activation and in the fibrotic liver of a dimethylnitrosamine-induced fibrosis rat model.


CONCLUSIONS
This study demonstrates that myocardin is involved in the activation of HSC; myocardin may serve as a novel therapeutic target in the treatment of liver fibrosis.
Shimada
Hideaki
H
Discovery Biology Research, Pfizer Global Research and Development Nagoya Laboratories, Pfizer Japan Inc., Aichi, Japan. hideaki.shimada@pfizer.com
Ochi
Takashi
T
Imasato
Akira
A
Morizane
Yoshihito
Y
Hori
Masatoshi
M
Ozaki
Hiroshi
H
Shinjo
Katsuhiro
K
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002469:Cell Separation; D054338:Cell Transdifferentiation / Q000235:genetics*; D003094:Collagen / Q000494:pharmacology; D015536:Down-Regulation; D004338:Drug Combinations; D015870:Gene Expression; D020869:Gene Expression Profiling; D020868:Gene Silencing; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D008106:Liver Cirrhosis, Experimental / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011509:Proteoglycans / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D000099251:Myocardin
D003094:Collagen; D004338:Drug Combinations; D007797:Laminin; D009687:Nuclear Proteins; D011509:Proteoglycans; D034741:RNA, Small Interfering; D015534:Trans-Activators; D000099251:Myocardin; C059485:matrigel