Abstract
NONMUSCLE MYOSIN HEAVY CHAIN IIA IS IMPORTANT
FOR HEPATIC STELLATE CELL CONTRACTILITY AND
INTRACELLULAR CALCIUM SIGNALLING
Z.A. Liu1, E. Van Rossen1, H. Reynaert1, L.A. van Grunsven1, J. Van
Daele2, J.-P. Timmermans2, A. Geerts1. 1Department of Cell Biology,
Vrije Universiteit Brussel, Jette, 2Laboratory of Cell Biology & Histology,
University of Antwerp, Antwerp, Belgium
E-mail: [email protected]
Background: Activated hepatic stellate cells develop contractile properties
that contribute to the pathogenesis of portal hypertension.
Aim: To investigate the role of nonmuscle myosin isoform IIA in mouse
hepatic stellate cells (mHSCs).
Methods: mHSCs were isolated, purified and cultured under standard
conditions. Nonmuscle myosin heavy chain IIA (NMMHC-IIA) expression
and knockdown were examined by QRT-PCR, Western blotting,
immunofluorescence, and confocal microscopy. Double staining and
co-immunoprecipitation were performed on NMMHC-IIA/a-SMA, and
NMMHC-IIA/vinculin. Contractile force generation was examined by
silicone wrinkle formation assay. Intracellular Ca2+ increase in response
to ET-1 was measured using Fluo-4 as a fluorescent calcium indicator.
Results: NMMHC-IIA expression was decreased at both mRNA (~75%)
and protein (~89%) levels in cells transfected with NMMHC-IIA siRNA.
Knockdown of NMMHC-IIA led to an over-all reduction in cell size.
Moreover, it led to decreased numbers of stress fibers and decreased steady
state levels of a-SMA protein (?53%). Confocal microscopy demonstrated
that NMMHC-IIA and a-SMA colocalized in stress fibers. The colocalization
of NMMHC-IIA and a-SMA was further demonstrated by co-
IP. In activated mHSCs, double staining of NMMHC-IIA and vinculin,
showed that the stress fibers ended in vinculin-containing focal adhesions.
Knockdown of NMMHC-IIA led to fewer and smaller focal adhesions.
Wrinkle formation experiments on a silicone substrate demonstrated that
NMMHC-IIA knock down abolished ET-1-induced cell contraction. Finally,
intracellular [Ca2+] release in response to ET-1 was blocked in the
NMHC IIA siRNA-transfected cells.
Conclusions: In mHSCs, nonmuscle myosin IIA plays pivotal roles in
formation and stabilization of stress fibers, contractile force generation
and intracellular [Ca2+] signalling.
FOR HEPATIC STELLATE CELL CONTRACTILITY AND
INTRACELLULAR CALCIUM SIGNALLING
Z.A. Liu1, E. Van Rossen1, H. Reynaert1, L.A. van Grunsven1, J. Van
Daele2, J.-P. Timmermans2, A. Geerts1. 1Department of Cell Biology,
Vrije Universiteit Brussel, Jette, 2Laboratory of Cell Biology & Histology,
University of Antwerp, Antwerp, Belgium
E-mail: [email protected]
Background: Activated hepatic stellate cells develop contractile properties
that contribute to the pathogenesis of portal hypertension.
Aim: To investigate the role of nonmuscle myosin isoform IIA in mouse
hepatic stellate cells (mHSCs).
Methods: mHSCs were isolated, purified and cultured under standard
conditions. Nonmuscle myosin heavy chain IIA (NMMHC-IIA) expression
and knockdown were examined by QRT-PCR, Western blotting,
immunofluorescence, and confocal microscopy. Double staining and
co-immunoprecipitation were performed on NMMHC-IIA/a-SMA, and
NMMHC-IIA/vinculin. Contractile force generation was examined by
silicone wrinkle formation assay. Intracellular Ca2+ increase in response
to ET-1 was measured using Fluo-4 as a fluorescent calcium indicator.
Results: NMMHC-IIA expression was decreased at both mRNA (~75%)
and protein (~89%) levels in cells transfected with NMMHC-IIA siRNA.
Knockdown of NMMHC-IIA led to an over-all reduction in cell size.
Moreover, it led to decreased numbers of stress fibers and decreased steady
state levels of a-SMA protein (?53%). Confocal microscopy demonstrated
that NMMHC-IIA and a-SMA colocalized in stress fibers. The colocalization
of NMMHC-IIA and a-SMA was further demonstrated by co-
IP. In activated mHSCs, double staining of NMMHC-IIA and vinculin,
showed that the stress fibers ended in vinculin-containing focal adhesions.
Knockdown of NMMHC-IIA led to fewer and smaller focal adhesions.
Wrinkle formation experiments on a silicone substrate demonstrated that
NMMHC-IIA knock down abolished ET-1-induced cell contraction. Finally,
intracellular [Ca2+] release in response to ET-1 was blocked in the
NMHC IIA siRNA-transfected cells.
Conclusions: In mHSCs, nonmuscle myosin IIA plays pivotal roles in
formation and stabilization of stress fibers, contractile force generation
and intracellular [Ca2+] signalling.
| Original language | English |
|---|---|
| Title of host publication | Journal of Hepatology |
| Pages | 185-185 |
| Number of pages | 1 |
| Volume | 48 |
| Publication status | Published - Apr 2008 |
| Event | Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet - Stockholm, Sweden Duration: 21 Sept 2009 → 25 Sept 2009 |
Publication series
| Name | |
|---|---|
| Number | 488 |
Conference
| Conference | Finds and Results from the Swedish Cyprus Expedition: A Gender Perspective at the Medelhavsmuseet |
|---|---|
| Country/Territory | Sweden |
| City | Stockholm |
| Period | 21/09/09 → 25/09/09 |
Keywords
- HSC contraction
- calcium signaling
- myosin IIA
- LIVER FIBROSIS