Arai, Masahiro

Fujiwara, Kenji

Ikeda, Hitoshi

Inoue, Yukiko

Kimura, Satoshi

Matsui, Atsushi

Nagashima, Kayo

Nishikawa, Takako

Noiri, Eisei

Ogata, Itsuro

Rojkind, Marcos

Tejima, Kazuaki

Tomiya, Tomoaki

Yanase, Mikio

English

PubMed

Mikio Yanase

Hitoshi Ikeda

Atsushi Matsui

Eisei Noiri

Tomoaki Tomiya

Masahiro Arai

Yukiko Inoue

Kazuaki Tejima

Kayo Nagashima

Takako Nishikawa

Satoshi Kimura

Kenji Fujiwara

Marcos Rojkind

Itsuro Ogata

Springer - Publisher Connector

BioMed CentralComparative Hepatology
ssOpen AcceProceedings
HMG-COA reductase inhibitor modulates collagen 
GEL-contraction by hepatic myofibroblast-like stellate cell line: 
involvement of geranylgeranylated proteins
Mikio Yanase*1, Hitoshi Ikeda1, Atsushi Matsui2, Eisei Noiri3, 
Tomoaki Tomiya1, Masahiro Arai1, Yukiko Inoue1, Kazuaki Tejima1, 
Kayo Nagashima1, Takako Nishikawa1, Satoshi Kimura4, Kenji Fujiwara2, 
Marcos Rojkind5 and Itsuro Ogata1,6
Address: 1Department of Gastroenterology, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan, 2Third Department of Internal 
Medicine, Saitama Medical School, Saitama, Japan, 3Department of Nephrology, Faculty of Medicine, University of Tokyo, Tokyo, Japan, 
4Department of Infection Control and Prevention, Faculty of Medicine, University of Tokyo, Tokyo, Japan, 5Experimental Pathology Section, 
Department of Clinical Investigation, Walter Reed Army Medical Center, and Department of Biochemistry, Uniformed Services University of the 
Health Sciences, Washington, DC, USA and 6Department of Internal Medicine, Kawakita General Hospital, Tokyo, Japan
Email: Mikio Yanase* - yanase-1IM@h.u-tokyo.ac.jp; Hitoshi Ikeda - ikeda-1IM@h.u-tokyo.ac.jp; Atsushi Matsui - atsushim@saitama-
med.ac.jp; Eisei Noiri - noiri-1IM@h.u-tokyo.ac.jp; Tomoaki Tomiya - tomiya-1IM@h.u-tokyo.ac.jp; Masahiro Arai - arai-1IM@h.u-tokyo.ac.jp; 
Yukiko Inoue - yanase-1IM@h.u-tokyo.ac.jp; Kazuaki Tejima - yanase-1IM@h.u-tokyo.ac.jp; Kayo Nagashima - yanase-1IM@h.u-tokyo.ac.jp; 
Takako Nishikawa - yanase-1IM@h.u-tokyo.ac.jp; Satoshi Kimura - yanase-1IM@h.u-tokyo.ac.jp; Kenji Fujiwara - yanase-1IM@h.u-tokyo.ac.jp; 
Marcos Rojkind - marcos.rojkind@na.amedd.army.mil; Itsuro Ogata - itsuro@viola.ocn.ne.jp
* Corresponding author    
Introduction
Recently, cholesterol-independent effects of HMG-CoA
reductase inhibitors (statins) have been clarified, such as
modulation of cell morphology and/or cell-substrate
attachment [1]. Some of these effects are suggested to be
mediated by intermediate compounds of the cholesterol
synthesis, i.e., the isoprenoids [2].
Here, we investigated the effect of HMG-CoA reductase
inhibitor and/or the related compounds of the cholesterol
synthesis pathway on collagen gel-contractility, cell mor-
phology, and/or cell-substrate attachment of hepatic stel-
late cells (HSCs), using myofibroblast-like stellate cell line
derived from a CCl4-induced cirrhotic rat liver.
Methods
Materials
Simvastatin [3] was kindly provided by Merck & Co., Inc.
(Rahway, NJ) and was converted to open acid form before
use. Farnesylpyrophosphate (FPP) and geranylgeranyl-
pyrophosphate (GGPP) were obtained from Sigma
Chemical Co. (St. Louis, MO). Prenyltransferase inhibi-
tors; FTI-277, and GGTI-286, were from Calbiochem (La
Jolla, CA). A monoclonal antibody against phosphor-
ylated serine 19 of myosin regulatory light chain (anti-
MLC-pS19) was previously described [4]. A monoclonal
antibody against RhoA was from Santa Cruz Biotechnol-
ogy (Santa Cruz, CA). Other materials were purchased
commercially.
Cell culture
The hepatic myofibroblast-like stellate cell line, CFSC-8B
(CFSC), derived from a CCl4-induced cirrhotic rat liver
[5], was maintained in DMEM and was supplemented
with 10% fatal calf serum (FCS).
Collagen gel-contraction assay
Contractility of CFSCs was evaluated using collagen gel
lattices on 24-well culture plates as described previously
[4].
from 11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells
Tucson, Arizona, USA, 25–29 August, 2002
Published: 14 January 2004
Comparative Hepatology 2004, 3(Suppl 1):S21
<supplement> <title> <p>11th International Symposium on the Cells of the Hepatic Sinusoid and their Relation to Other Cells</p> </title> </supplement>
This article is available from: http://www.comparative-hepatology.com/content/3/S1/S21Page 1 of 3
(page number not for citation purposes)
Comparative Hepatology 2004, 3 http://www.comparative-hepatology.com/content/3/S1/S21F-actin staining
For visualization of F-actin, the cells were stained over-
night with TRITC-labeled phalloidin (Sigma), and then
they were observed by fluorescence microscopy.
Cell adhesion assays
Cell adhesion was measured using the electric cell-sub-
strate impedance sensor system (ECIS; Applied BioPhys-
ics, Inc., Troy, NY) as described previously [4].
Immunoblotting
Cellular proteins were separated by SDS-PAGE, and
immunoblotted using anti-MLC-pS19 or anti-RhoA.
Immunoreactive proteins were visualized using a chemi-
luminescence kit (Amersham).
Statistical analysis
Data were given as the mean value with the standard error
of it, and were analyzed by the paired Student's t test.
Results and Discussion
HMG-CoA reductase inhibitors are widely used in
patients with liver disease, such as fatty liver and/or pri-
mary liver cirrhosis, however, the effects and the mecha-
nisms of these inhibitors on the liver remain uncertain as
well in HSCs [6,7]. They block the conversion of HMG-
CoA to mevalonate, the rate-limiting step in the synthesis
of cholesterol, moreover, several recent studies have been
focused on their cholesterol-independent effects. By mod-
ulating the initial part of the cholesterol synthesis path-
way, they decrease the level of numerous important
intermediate compounds including isoprenoids includ-
ing FPP and GGPP (Figure 1). Isoprenoids are lipid attach-
ments involved in post-translational modification of
some proteins such as gamma-subunit of the heterot-
rimeric G proteins, the small G proteins as Ras, Rho, Rap,
Rab, or Ral [2]. Thus, they can modulate various biologi-
cal or physiological mechanisms.
In the present study we found that the addition of 10-5M
of simvastatin attenuated the contractile activity of colla-
gen-gel by CFSCs, which was recovered by co-addition of
10-3M of mevalonate, the direct metabolite of HMG-CoA.
The inhibitory effect of simvastatin was also cancelled by
co-addition of 10-5M of GGPP, but not by 10-5M of FPP or
squalene, the late step product of the cholesterol synthe-
sis. Moreover, the inhibitory effect was partially repro-
duced by addition of 10-5M of GGTI, not by 10-5M of FTI.
Next, we found that cell morphology and/or the
formation of stress fibers of CFSCs by F-actin staining
were abrogated by simvastatin, which were maintained in
the presence of mevalonate, and GGPP, but not of FPP.
They were also attenuated in the presence of GGTI. We
revealed further that the adhesive area of CFSCs to extra-
cellular substrate by ECIS were reduced by simvastatin
and GGTI, which were maintained in the presence of
mevalonate, and GGPP. The above observations may sug-
gest that HMG-CoA inhibitor modulates the morphologi-
cal and cytoskeletal changes through the dynamic
reorganization of actin filaments, and the cell-extracellu-
lar matrix interaction, resulting in the attenuation of the
contraction of collagen gel lattices, and that protein gera-
nylgeranylation is involved in this mechanism. Protein
prenylation of RhoA is required to functional activities of
RhoA [2]. Western blotting analyses showed that phos-
phorylated myosin regulatory light chain and prenylated
RhoA were maintained in the presence of mevalonate,
and GGPP, which were attenuated in the presence of sim-
vastatin and/or GGTI. It may be suggested that prenylated
RhoA might be associated with collagen-gel contractility,
cell morphology, and/or cell-substrate attachment of
CFSCs exerted by simvastatin and/or the isoprenoids.
In conclusion, HMG-CoA reductase inhibitor may modu-
late CFSC morphology, its attachment to surrounding
extracellular matrix and its contraction by a mechanism
involving protein geranylgeranylation.
Acknowledgements
This work was supported in part by Grant RO1AA10541 from the National 
Institute of Alcohol Abuse and Alcoholism (MR). The opinions or assertions 
contained herein are the private views of the Authors and are not to be 
construed as official or as reflecting the views of the Department of the 
Army or the Department of Defense.
References
1. Liu L, Moesner P, Kovach NL, Bailey R, Hamilton AD, Sebti SM, Har-
lan JM: Integrin-dependent leukocyte adhesion involves gera-
nylgeranylated protein(s). J Biol Chem 1999, 274:33334-33340.
2. Casey PJ, Seabra MC: Protein prenyltransferases. J Biol Chem
1996, 271:5289-5292.
3. Parker RA, Clark RW, Sit SY, Lainer TL, Grosso RA, Wright JJK:
Selective inhibition of cholesterol synthesis in liver versus
extrahepatic tissues by HMG-CoA reductase inhibitors. J Lipid
Res 1990, 31:1271-1282.
4. Yanase M, Ikeda H, Matsui A, Maekawa H, Noiri E, Tomiya T, Arai M,
Yano T, Shibata M, Ikebe M, Fujiwara K, Rojkind M, Ogata I: Lyso-
phosphatidic acid enhances collagen-gel contraction by
The cholesterol synthetic pathwayFigure 1
The cholesterol synthetic pathway.Page 2 of 3
(page number not for citation purposes)
Comparative Hepatology 2004, 3 http://www.comparative-hepatology.com/content/3/S1/S21Publish with BioMed Central   and  every 
scientist can read your work free of charge
"BioMed Central will be the most significant development for 
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central 
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
hepatic stellate cells: Association with Rho-kinase. Biochem
Biophys Res Commun 2000, 277:72-78.
5. Greenwel P, Rubin J, Schwarz M, Hertzberg EL, Rojkind M: Liver fat-
storing cell clones obtained from a CCl4-cirrhotic rat are
heterologous with regard to proliferation, expression of
extracellular matrix components, interleukin-6 and
connexin-43. Lab Invest 1993, 69:210-216.
6. Mallat A, Préaux AM, Blazejewski S, Dhumeaux D, Rosenbaum J,
Mavier P: Effect of simvastatin, an inhibitor of hydroxy-meth-
ylglutaryl coenzyme A reductase, on the growth of human
Ito cells. Hepatology 1994, 19:1095-1099.
7. Carloni V, Pinzani M, Giusti S, Romanelli RG, Parola M, Bellomo G,
Failli P, Hamilton AD, Sebti SM, Laffi G, Gentilini P: Tyrosine phos-
phorylation of focal adhesion kinase by PDGF is dependent
on Ras in human hepatic stellate cells. Hepatology 2000,
31:131-140.Page 3 of 3
(page number not for citation purposes)


Gentilini P: Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on Ras in human hepatic stellate cells. Hepatology

JJK: Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors.

JM: Integrin-dependent leukocyte adhesion involves geranylgeranylated protein(s).

Liver fatstoring cell clones obtained from a CCl4-cirrhotic rat are heterologous with regard to proliferation, expression of extracellular matrix components, interleukin-6 and connexin-43. Lab Invest

Mavier P: Effect of simvastatin, an inhibitor of hydroxy-methylglutaryl coenzyme A reductase, on the growth of human Ito cells. Hepatology

Protein prenyltransferases.

file:///data/core-remote/dit/data/Springer-OA/pdf/fc8/aHR0cDovL2xpbmsuc3ByaW5nZXIuY29tLzEwLjExODYvMTQ3Ni01OTI2LTItUzEtUzIxLnBkZg==.pdf

HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins

HMG-COA reductase inhibitor modulates collagen GEL-contraction by hepatic myofibroblast-like stellate cell line: involvement of geranylgeranylated proteins

Abstract

Similar works

Full text

Available Versions

Springer - Publisher Connector