distinct roles for non-muscle myosin ii isoforms in mouse hepatic stellate cells

Research Article

Distinct roles for non-muscle myosin II isoforms in mousehepatic stellate cells

Zhenan Liu1, Elke Van Rossen1, Jean-Pierre Timmermans2, Albert Geerts1,�, Leo A. van Grunsven1,��,Hendrik Reynaert1,⇑,��

1Liver Cell Biology Lab, Vrije Universiteit Brussel (V.U.B.), Belgium; 2Laboratory of Cell Biology and Histology, Department of Veterinary Sciences,University of Antwerp (UA), Belgium

Background & Aims: Upon liver injury, hepatic stellate cells � 2010 European Association for the Study of the Liver. Published
(HSCs) undergo dramatic morphological and functional changesincluding migration and contraction. In the present study, weinvestigated the role of myosin II isoforms in the developmentof the contractile phenotype of mouse HSCs, which are consid-ered therapeutic targets to decrease portal hypertension andfibrosis.Methods: We characterized the expression of myosin IIA and IIBin primary mouse HSCs and addressed their function by geneknock-down using isoform-specific siRNAs.Results: We found that myosin IIA and IIB are differentiallyexpressed and localized and have clearly different functions inHSCs. Myosin IIA is mainly located in the subcortical area of qui-escent HSCs and at a-SMA-containing stress fibres after activa-tion, while myosin IIB is located in the cytoplasm and at theedge of protrusions of quiescent HSCs, at stress fibres of activatedcells, and at the leading edge of lamellipodia. Knock-down ofmyosin IIA in HSCs influences cell size and shape, results in thedisruption of stress fibres and in a decrease of focal adhesions,and inhibits contractility and intra-cellular Ca2+ release butincreases cell migration. Myosin IIB contributes to the extensionof lamellipodia and cell spreading but has no direct role in stressfibres and focal adhesion formation, contraction, or intra-cellularCa2+ signalling.Conclusions: In mouse HSCs, myosin IIA and IIB clearly fulfil dis-tinct roles. Our results provide an insight into the contractilemachinery of HSCs, that could be important in the search fornew molecules to treat portal hypertension.
Journal of Hepatology 20

Keywords: Hepatic stellate cells; Myosin II isoforms; Contraction; Migration;Intra-cellular calcium.Received 28 January 2010; received in revised form 2 June 2010; accepted 10 June2010; available online 26 August 2010⇑ Corresponding author. Address: Liver Cell Biology Lab, Faculty of Medicine andPharmacy, Vrije Universiteit Brussel (V.U.B.), Laarbeeklaan 103, 1090 Brussels,Belgium. Tel.: +32 2 477 6811; fax: +32 2 477 6810.E-mail address: [email protected] (H. Reynaert).

� Prof. Geerts passed away during the completion of the study.�� Contributed equally to the paper.

Abbreviations: HSCs, hepatic stellate cells; Myosin IIA (B), myosin II isoform, non-muscle myosin heavy chain IIA (B), Myh9 (10); a-SMA, a-smooth muscle actin;ET-1, endothelin-1; PBS, phosphate buffered saline; [Ca2+]i, intra-cellular Ca2+; FA,focal adhesion.

by Elsevier B.V. All rights reserved.

Introduction

Upon liver injury of any aetiology, quiescent hepatic stellate cells(HSCs) transform into myofibroblast-like cells, which are theeffector cells of fibroproliferative disorders characterized by thedestruction of the normal tissue architecture and the develop-ment of fibrosis [1]. HSCs are located in the perisinusoidal spaceof Disse, with long cytoplasmic processes extending betweenhepatocytes and around sinusoids in normal liver. In normal liver,HSCs play a pivotal role in vitamin A metabolism, synthesis anddegradation of extracellular matrix, and regulation of sinusoidalblood flow [2,3]. Following liver injury, HSCs transform intoa-SMA-expressing activated HSCs, which are characterized byincreased proliferation, contractility, fibrogenesis, cytokine secre-tion, and matrix degradation [1]. Several ligands bind to theirplasma membrane receptors, activate the Rho/Rho kinase path-way, elevate cytoplasmic Ca2+ concentration ([Ca2+]i), and inducecontraction of HSCs [4,5]. Activated HSCs play an important rolein the pathophysiology of fibrosis and portal hypertension,thereby providing an important framework for defining thera-peutic targets [2,6].

Myosins are a superfamily of actin-based molecular motorproteins divided into at least 20 classes. The three-non-musclemyosins (hereafter referred to as myosin IIA, IIB, and IIC) consisteach of 4 myosin light chains associated with 2 heavy chains,either Non-Muscle Heavy Chain IIA, B or C, which are encodedby Myh9, Myh10, and Myh14 genes, respectively [7]. HSC contrac-tion can be reduced using inhibitors of the Rho-kinase (Y-27632),myosin light chain kinase (ML-7) or non-muscle myosin II ATPase(Blebbistatin), indicating that the generation of the contractileforce by HSCs requires myosin [4,8,9]. In the present study, weexamined the role of myosin II isoforms in the formation of theHSC cytoskeleton and their influence on mouse HSC (mHSC) con-tractility and migration. By siRNA-mediated knock-down studieswe showed that myosin IIA is necessary for stress fibre and focaladhesion formation, contraction, and Ca2+ flux in HSCs. MyosinIIB does not seem to contribute to these processes, apart fromplaying a role in the formation of lamellipodia.

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mailto:[email protected]

JOURNAL OF HEPATOLOGY
Materials and methods
Isolation and culture of mHSCs

Animals were treated according to the institution’s guidelines for the care and useof laboratory animals in research, approved by the local ethical committee. HSCsused in this study were isolated from adult Balb/C mice by a modified collage-nase–pronase digestion. HSCs in 1-day cultures were >96% pure as determinedby lipid droplet content and morphology.

siRNA-mediated gene silencing

For siRNA-mediated gene silencing, pools of siRNAs specific for mouse myosinIIA or IIB ON-TARGETplus SMARTpool L-040013-00-0005 (myh9) andL-062322-00-0005 (myh10)(Dharmacon Research Inc., Chicago, USA) were used(see Fig. 2A and B). Cells were plated in 24-well plates on uncoated coverslips or6-well plates at the indicated days depending on experimental requirementsand siRNAs were transfected using HiPerFect (QIAGEN, Venlo, The Netherlands).The negative control consisted of fluorescently labelled siRNAs (siGlo� Green,Dharmacon).

Myosin IIB

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Fig. 1. Differential expression and localization of myosin IIA and IIB during mHSC acat different time-points in culture. (B–F). Subcellular distribution of myosin IIA and IIB wshows the co-localization of myosin IIA and a-SMA at the level of stress fibres and the co-F were visualized using confocal microscopy. The scale bar represents: 50 lm in B-E, an


Statistical analysis

All experiments were repeated with mHSCs from at least three isolations. Datawere expressed as means ± SEM. All statistical tests were performed using Graph-Pad Prism version 4.0 (GraphPad Software, San Diego, CA, USA). One-way ANOVA(Dunnett’s multiple comparison test) was used for wrinkle and intra-cellular Ca2+

release assays and two-way ANOVA (Post tests) for wound-healing assays.

Results

Differential expression and localization of myosin II isoforms duringmHSC activation

Freshly isolated mHSCs trans differentiate into myofibroblast-like cells when put in culture using normal tissue culture dishes,reminding the in vivo HSC activation process induced by chronicliver damage. In culture-activated mHSCs (day 2, 5, 8, 11, and 14),Myosin IIA (Myh9) RNA was readily detected at every time point,

Myo

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tivation. (A) Western blot images of myosin II isoforms present in primary mHSCsith a-SMA and b-actin at different time points of HSC activation. Inserted picturelocalization of myosin IIB and b-actin at the leading edge of lamellipodia. B, D, andd 100 lm in F. Representative images of 6 independent HSC isolations.

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while myosin IIB (Myh10) was up-regulated during HSC activa-tion (Table 4). In addition to the non-muscle isoforms, smoothmuscle myosin (SM2, Myh11) RNA was induced upon activationbut we were unable to detect SM2 at the protein level (Table 4).All other class II myosin isoforms were expressed at relativelylow levels when compared to the control tissue. We thereforefocused our investigation on the group of non-muscle myosins,i.e., myosin IIA, IIB, and IIC. At the protein level, myosin IIB wasnearly undetectable in early cultures and was strongly up-regu-lated during HSC activation, while expression of myosin IIA wasconstant over time, and myosin IIC undetectable (Fig. 1A). At
Myosin IIB

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Fig. 2. Morphological analysis of HSCs after knock-down of myosin IIA/IIB. (A) RT-PCRof myosin IIA or IIB. (C–E) Knock-down of myosin IIA/IIB does not influence cell prolifeexpressed as mean ± SEM of three independent experiments. (F) Morphological analysis. Cspecific siRNAs (IIA siRNA/IIB siRNA), or treated with blebbistatin (BLEB) 50 lM for 2 h.experiments were carried out at least three times.

134 Journal of Hepatology 201

day 11, when HSCs are fully activated, myosin IIA and IIB showeddistinct subcellular localizations; myosin IIA was mainlyexpressed in stress fibres, while myosin IIB was expressed instress fibres and lamellipodia (Fig. 1B–F). Co-staining of thesemyosin isoforms with a-SMA and b-actin in activated HSCsshowed that myosin IIA and a-SMA co-localized at stress fibres(Fig. 1C and D) and myosin IIB and b-actin co-localized at theleading edge of lamellipodia (Fig. 1E and F). Quiescent HSCs(day 2 in culture) showed myosin IIA expression mainly in thesubcortical area while myosin IIB was localized in the cytoplasmand at the edge of cell protrusions.

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and (B) Western blots confirm that the siRNAs specifically inhibit the expressionration nor a-SMA and proCOL1a1 mRNA expression. The graphs show the resultsells were transfected at D01 (or D07) with irrelevant siRNAs (Cont), myosin IIA/-IIB

B and F are representative images of at least three independent HSC isolations, all

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Myosin IIA and IIB play different role in HSCs morphology
The differential expression and localization of myosin IIA and IIBin HSCs suggest a different role of these myosins during HSC acti-vation. To test this hypothesis, we performed functional assaysfollowing myosin IIA or IIB-specific siRNA-mediated gene silenc-ing. The specificity of myosin II isoform siRNAs was confirmed bythe analysis of both RNA and protein expression of myosins, 4days after transfection (Fig. 2A and B). Knock-down of myosinIIA or IIB did not seem to influence the culture-induced prolifer-ation of HSCs nor the expression of a-SMA and procollagen1a1,both well-accepted markers for HSC activation (Fig. 2C–E). Dur-ing the in vitro activation, HSCs form stress fibres to enable cellgrowth and they form lamellipodia for cell spreading. Whenmyosin IIA siRNAs were transfected, we observed that HSCsremained smaller (day 5) or became smaller (day 11) in compar-ison to control cells. In both instances, no obvious cell death wasobserved and cells developed and retained lamellipodia (Fig. 2F).The size of cell bodies did not show large differences in cellstransfected with myosin IIB siRNA, both at day 1 and 7 but asmaller number of lamellipodia was observed (Fig. 2F). We con-firmed those observations by treating cells with blebbistatin, amyosin II ATPase inhibitor, inhibiting both myosin IIA and IIB[8]. In blebbistatin-treated cells, both the size of cell bodies andthe number of lamellipodia were reduced (Fig. 2F). These resultssuggest that during HSC activation, myosin IIA plays a role in theregulation of cell size and shape, while myosin IIB is implicated inthe formation of lamellipodia.

Myosin IIA is required for the stabilization of stress fibres

To further explore how myosin II isoforms influence cell mor-phology, we performed double-staining of the two myosin II

IIA s

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Fig. 3. Dual immunoflourescence staining of myosin IIA/ IIB with a-SMA/b-actin. Dtransfected HSCs. (B) Dual immunofluorescence staining of myosin IIB with a-SMA/b-aimages of 6 independent HSC isolations.


isoforms (IIA/IIB) with different actin isoforms (a-SMA/b-actin),following siRNA-mediated gene silencing. HSCs transfected atday 7 and imaged at day 11 showed a strong reduction ina-SMA-containing stress fibres when myosin IIA was silenced,and close to normal a-SMA-containing stress fibres when myo-sin IIB was silenced, although, myosin IIB also localized to stressfibres. (Fig. 3A and B). Surprisingly, knock-down of myosin IIAalso decreased the formation of b-actin and myosin IIB filamentsin the cytoplasm but not at the leading edge of lamellipodia,suggesting that myosin IIA plays a key role in the overall fila-ment formation in the cytoplasm. As expected, the knock-downof myosin IIB in HSCs reduced the filaments of b-actin at theleading edge of lamellipodia and partially in the cytoplasm. Incontrast, knock-down of myosin IIA did not seem to influencethe b-actin expression at the leading edge of lamellipodia(Fig. 3A and B).

Myosin IIA is necessary for mHSC contraction

Considering the observation that myosin IIA and IIB show a dif-ferent subcellular expression and have different functions withrespect to cytoskeleton and lamellipodia formation, we postu-lated that their function in cell contraction would differ. To testthis hypothesis, we performed silicone wrinkle assays to deter-mine which myosin II isoform is implicated in this process. Con-traction of cultured HSCs on a silicone layer caused wrinkling ofthe substrate, which increased following HSC activation. Weobserved that HSCs cultured on silicone at day 11 showedstrong wrinkle formation, which disappeared after a 2 h blebb-istatin treatment (Fig. S4A). HSCs transfected with irrelevantsiRNAs at day 1, generated small wrinkles at day 5 whichincreased in number and intensity over time (Fig. S4B). In cellstransfected with myosin IIB siRNAs, wrinkle formation was only

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ual immunoflourescence staining of myosin IIA with a-SMA/b-actin in siRNA-ctin in siRNA-transfected HSCs. The scale bar represents 50 lm. Representative

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slightly affected when compared with control cells. Interest-ingly, in cells transfected with myosin IIA specific siRNAs, wrin-kle formation was strongly reduced or even disappearedcompletely (Fig. S4B and C). Furthermore, cells formed wrinklesat the level of the cell body, that is where the stress fibres arelocated, and not at the leading edge of lamellipodia. Similarresults were obtained for Endothelin-1(ET-1) -induced cell con-traction, no ET-1-induced dynamic changes were observed inmyosin IIA siRNA-treated HSCs. Control and myosin IIB knock-down HSCs showed either an increase in wrinkle number percell or stronger wrinkle formation when ET-1 was added(Fig. 4D).
before Bleb Bleb

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Fig. 4. Silicone substrate wrinkle assay. (A) Blebbistatin treatment results in reduced wtreated with blebbistatin (50 lM) for 2 h. (B) Representative images of HSCs were on siliisoform-specific siRNAs (IIA siRNA, IIB siRNA) at day 1 or day 7 and imaged at day 5wrinkles (day 11). Each bar represents mean ± SEM (n = 3 experiments with HSCs from thof wrinkles/total number of cells. (D) ET-1-induced dynamic changes in the wrinkles forsiRNAs. Images show the same cell before and after ET-1 (4 � 10�8 M) stimulation (10 m


Myosin IIA knock-down impairs increase in ET-1-induced [Ca2+]i

release

Since we could show that myosin IIA is involved in contraction,which in many cell types involves Ca2+ transients, we carried out[Ca2+]i measurements using Fluo-4 as an indicator of free Ca2+ inthe cell. Control cells exposed to ET-1 showed a clear increase in[Ca2+]i, and 74% of cells showed a clear increase in fluorescence lessthan 4 s after ET-1 addition (Fig. 5A and B). When myosin IIA siRNA-transfected cells were exposed to ET-1, the majority of cells (84%)remained unresponsive (Fig. 5B), and no increase in [Ca2+]i wasvisualized in these cells (Fig. 5A). When cells were transfected with

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rinkle formation on a silicone substrate. Cells cultured on silicone at day 11 werecone substrates. Cells were transfected with irrelevant siRNAs (Cont) or myosin IIand at day 11, respectively. (C) Quantification of HSC-induced silicone substrateree independent isolations, *p <0.05). The average wrinkles per cell = total numbermed on a silicone substrate with cells transfected by control and myosin IIA or IIB

in).

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Fig. 5. Myosin IIA knock-down impairs [Ca2+]i increase in HSCs induced by ET-1. (A) Representative images of time-course [Ca2+]i measurements following addition ofET-1 (4 � 10�8 M) in HSCs. Cells were transfected at day 7 with control or myosin IIA/IIB siRNAs and measurements were carried out at day 11 following a 24 h starvation(images shown: before, at addition of ET-1 and 3.96, 11.82 s after addition of ET-1). (B) The graph shows the percentage of cells responsive to ET-1. Each bar representsmean values ± SEM (n = 3, triplicate experiment with cells from three independent isolations, *p <0.05). Cells are considered responsive when [Ca2+]i increased >100% ascompared to the basal level.


myosin IIB siRNAs, 57% of the cells were responsive to the additionof ET-1. This decrease in cell response, in comparison with that ofcontrol cells, was not statistically significant (Fig. 5B). These resultsimply that in HSCs mainly myosin IIA is necessary for [Ca2+]i

increase in response to ET-1.

Myosin IIA is necessary for proper distribution of the endoplasmicreticulum

To obtain a mechanistic insight into the surprising finding that theknock-down of myosin IIA suppressed ET-1-induced [Ca2+]i

increase in HSCs, we examined the effects of myosin IIA siRNA-mediated knock-down on several components of the ET-1 signal-ling cascade. There was no significant change in ETB receptorexpression, neither at the mRNA level nor at the protein level incells transfected with myosin IIA siRNAs (ETA receptor was nearlyundetectable; Fig. 6A–C). Furthermore, the knock-down of myosinIIA led to a slight, but statistically insignificant increase in ET-1-induced IP1 formation, a stable breakdown product of IP3. Anotherpossibility is a spatial disruption between the site of IP3 produc-tion and IP3 receptors on the endoplasmic reticulum (ER) [10,11]in the myosin IIA knock-down cells. To investigate this option,we used Protein Disulphide Isomerase (PDI) as a marker of ER.We found that the knock-down of myosin IIA resulted in a changeof ER distribution; in myosin IIA siRNA-transfected cells the ERseemed collapsed around the nucleus, while in control cells itwas spread out from the nucleus into the cytoplasm (Fig. 6E).

Myosin IIA knock-down enhances migration of HSCs

Migration of HSCs is initiated during hepatic injury and is essen-tial for wound healing and fibrogenesis. We used ‘wound healing


assays’ to investigate the role of individual myosin II isoforms incell migration. Knock-down of myosin IIA led to an enhancedmigration of HSCs when compared to the migration of controlcells, while knock-down of myosin IIB resulted in slightlydecreased migration (Fig. 7A). Although, we repeatedly observedthis, the latter effect was statistically insignificant (Fig. 7A). Sim-ilar results were obtained in cell chemotaxis studies using PDGF-induced transwell assays. Knock-down of myosin IIA resulted inan increase in transwell migration both in control conditionsand in PDGF-induced cell chemotaxis while knock-down of myo-sin IIB did not influence the migration significantly (Fig. 7B).

Myosin IIA knock-down reduces the number and size of vinculin-containing focal adhesions

We hypothesized that the enhanced migration in myosin IIA siR-NA-transfected HSCs was due to a reduction in focal adhesions[12]. Hence, we investigated whether focal adhesions were affectedin myosin IIA/IIB siRNA-transfected HSCs. The staining of vinculin, amajor component of focal adhesions, showed that the number andsize of vinculin-containing focal adhesions were strongly reducedin myosin IIA knock-down cells. In contrast, the knock-down ofmyosin IIB had no effect on the formation of vinculin-containingfocal adhesions (Fig. 8). To explore this further, we performed celladhesion assays on collagen after myosin II knock-down. We couldnot observe significant differences in cell adhesion between cellstransfected with control, myosin IIA, or myosin IIB siRNAs.

Discussion

In the present study we have demonstrated that myosin IIA ishighly expressed and plays a number of vital roles in HSCs. One

1 vol. 54 j 132–141 137

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Fig. 6. Effects of myosin IIA siRNA-mediated knock-down on components of the ET-1 signalling cascade. (A) The expression of endothelin receptor A (ETA) and B (ETB)protein in mHSCs at different time-points in culture (ETA was nearly undetectable after day 8). (B and C) Knock-down of myosin IIA does not influence the expression ofendothelin receptor B (ETB) both at the protein level and at the mRNA level. (D) IP-One ELISA assay showing no blockade of IP-One accumulation in myosin IIA siRNAtreated cells. Untransfected HSCs were used as control (normal and normal + ET-1). (A–D) The experiments were repeated three times with HSCs from three independentisolations. (E) Double staining of PDI and myosin IIA. Cells were transfected with control or myosin IIA siRNAs at day 7 in culture. Following 3-h-serum-free starvation atday 11, cells were incubated with ET-1 (4 � 10�8 M) for 10 min and fixed for staining. The scale bar represents 50 lm.

Research Article

of the most striking changes occurring during activation of HSCsis their gain in size. Myosin IIA appears to play a very importantrole in this obvious change since the knock-down of this protein


in 1-day-old HSCs keeps the cells small, while myosin IIA knock-down in 7-day-cultured cells seems to cause a disintegration ofthe already formed stress fibres and a reduction in the cell size.

1 vol. 54 j 132–141

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Fig. 7. Myosin IIA knock-down enhances migration of HSCs. (A) Representative images of cells taken at 0, 24, and 48 h (h) after wounding (left), and quantification of HSCmigration (right). The graph shows cell migrated distance (lm) after 24 h and 48 h. Each bar represents mean values ± SEM (n = 3, **p <0.01. ns: not significant). Scale bar:50 lm. (B) PDGF-BB-induced transwell migration. siRNA-transfected HSCs were plated at day 10 in the transwell and migration was determined after 18 h. The graphshows the results expressed as mean ± SEM of three independent experiments in duplicate, *p <0.05, **p <0.01.


10X

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Fig. 8. Knock-down of myosin IIA decreases the number and size of vinculin-containing focal adhesions. Cells were transfected at day 7 and fixed at day 11 for vinculinstaining. Representative images were taken by fluorescence microscopy with original objective 10� (Scale bar: 100 lm) and 40� (Scale bar: 50 lm). Inserted pictures areblow-ups of the dashed region in the original images.


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These changes are not observed in myosin IIB knock-down cells(Figs. 2–4). This finding is in line with studies conducted in othercell types in which cell size or shape changes were observed fol-lowing the inhibition or knock-down of myosin IIA [13,14].Besides their increased size, activated HSCs also develop lamelli-podia, cellular protrusions that interact and attach to their envi-ronment via different adhesion molecules. Knock-down ofmyosin IIA does not affect the formation of lamellipodia inmHSCs; however, when cells are transfected with myosin IIB siR-NAs, a clear reduction in lamellipodia can be observed, suggestingthat myosin IIB is implicated in the formation of lamellipodia. Arole for myosin IIA in lamellipodia protrusion has been reportedin studies on gastric parietal cells [15]. Nevertheless, in other celltypes, such as fibroblasts and neurons, myosin IIB is responsiblefor the protrusion of lamellipodia [13,16]. From our data, we con-clude that in HSCs, lamellipodia formation largely depends onmyosin IIB.
Knock-down of myosin IIA in quiescent HSCs leads to theinability to form a-SMA-containing stress fibres and to the disap-pearance of already formed stress fibres present in activated HSCs(Figs. 2E, 3). In these myosin IIA-low cells, the cytoplasmic a-SMAdistribution remains diffuse, suggesting that it is unable to inte-grate into microfilaments. We have further demonstrated thatmyosin IIA is indispensable for stellate cell contraction. Indeed,in myosin IIA-depleted HSCs, no ET-1-induced [Ca2+]i. increasewas observed and HSCs were not able to contract. In contrast,no obvious changes in Ca2+ transients and contraction wereobserved in cells transfected with myosin IIB siRNAs. Theseobservations are in agreement with findings in human foreskinfibroblasts [13]. Recently, there has been a considerable progressin the understanding of the signalling mechanisms regulating thecontraction in HSCs. The major pathways, i.e., the Rho pathway[4,17] and Ca2+ signalling [18], tend to become important whenHSCs become activated. It has been reported that actin filamentsin smooth muscle cells may be an integral component of Ca2+ reg-ulation and/or signal transduction in receptor-coupled mecha-nisms and that there is a relationship between myosinphosphorylation and intra-cellular [Ca2+]i [19]. We have alsofound that myosin IIA and the ET-1-induced [Ca2+]i increase arelinked, but from our experimental data we cannot conclude thatthe disruption of stress fibres by down-regulation of myosin IIAcauses the observed inhibition of [Ca2+]i release. It has been pos-tulated that disruption of the cytoskeleton results in the rear-rangement of the ER, which alters the spatial relationshipbetween the sites of IP3 production and the IP3 receptors on theER [10,11]. Since there was no significant change in either theET receptor expression or the production of IP3 (Fig. 6), webelieve that this disruption in cytoskeleton architecture couldbe the reason for the absence of the [Ca2+]i increase. In fact, weobserved that the knock-down of myosin IIA resulted in a majorrearrangement of the ER. In this scenario, it is conceivable that IP3

cannot interact with its receptors on the ER. Another possibility isthat the knock-down of myosin IIA changes cell size and shape,leading to spatial conformational changes in signalling networks[20,21], which may lead to IP3 not being able to reach its recep-tors. Until now, the mechanism that involves microfilaments incalcium release is very speculative. Our results are in accordancewith reported data in NIH 3T3 cells [11] and support the spatialconformational-coupling model for calcium signalling [20,21].

We have observed that wound-induced migration of HSCswas accelerated by the knock-down of myosin IIA (Fig. 7). One


might think that the loss of contraction automatically results inthe loss of migration, but as shown in lung carcinoma cells andprimary mouse embryonic fibroblasts [13,22], the loss of contrac-tion due to myosin IIA siRNA/blebbistatin still enables migration.In addition, an earlier study showed that cell spreading wasaccompanied by a loss of cortical myosin II and actinomyosin-based contractility [23]. Our results in HSCs corroborate theseobservations and are in line with earlier studies suggesting that,in the absence of myosin IIA, migration strategies might switchand rather depend on microtubule-dependent dendritic exten-sions [24,25]. It has been reported that stress fibres associatewith focal adhesions, and a reduction in the number of focaladhesions can lead to a less adherent phenotype which facilitatesthe movement of the leading edge and the rear end [26]. Webelieve that the explanation for this enhanced wound-inducedmigration in knock-down myosin IIA cells lies in the fact thatthe knock-down of myosin IIA results in the loss of stress fibresand focal adhesions (Figs. 3,4,8) as well as in the changes in cellmorphology suitable for cell migration. We did not find signifi-cant differences in cell-substrates attachment assays. The latteris not entirely surprising since cell attachment to substrates ismainly mediated by binding of cell surface integrins. However,integrins can switch between relaxed and tensioned states inresponse to myosin II-generated forces [27]. Nevertheless, theknock-down of myosin II did not influence cell-substrates attach-ment. Blebbistatin treatment of mHSCs in cell suspension didinfluence attachment to collagen [8]. We cannot explain this dis-crepancy, but it has been reported that other myosins areinvolved in the regulation of integrin integrity or function, suchas myosin X [28], whose activity might also be affected byblebbistatin.

In summary, we propose that myosin IIA and IIB fulfil differentfunctions in HSCs. Myosin IIA is necessary for the development ofa normal actinomyosin cytoskeleton and for determining cellshape. Moreover, myosin IIA regulates cell adhesion and cell con-tractility, allows intra-cellular Ca2+ signalling, and suppresses cellmigration. In contrast, myosin IIB does not seem to influence theseprocesses in HSCs but rather plays a role in the formation and pro-trusion of lamellipodia. The knowledge of the different rolesplayed by these myosin II isoforms in stellate cell contractionand migration provides further insight into the contractilemachinery of HSCs which could be important in the search ofnew molecules to treat portal hypertension.

Funding

This study was funded by FWO-V (Fonds voor WetenschappelijkOnderzoek – Vlaanderen), grants G.0652.06; OZR (ResearchCouncil of Vrije Universiteit Brussel) grants GOA48, OZR1226,OZR1924; Roche and Schering-Plough unconditional grant.

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Acknowledgements

We remember Professor Albert Geerts, who passed away duringthe finalization of this study. We are grateful to him for all his

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enthusiasm and support which made the realization of this pro-ject possible. We express our warmest thanks to Jan Van Daelefor assistance with confocal microscopy and Ca2+-imaging andall the members of the Liver Cell Biology Lab for their help andtechnical assistance.
References

[1] Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmaticcells of the liver. Physiol Rev 2008;88 (1):125–172.

[2] Reynaert H, Thompson MG, Thomas T, Geerts A. Hepatic stellate cells: role inmicrocirculation and pathophysiology of portal hypertension. Gut 2002;50(4):571–581.

[3] Soon Jr RK, Yee Jr HF. Stellate cell contraction: role, regulation, and potentialtherapeutic target. Clin Liver Dis 2008;12 (4):791–803, viii.

[4] Melton AC, Datta A, Yee Jr HF. [Ca2+]i-independent contractile forcegeneration by rat hepatic stellate cells in response to endothelin-1. Am JPhysiol Gastrointest Liver Physiol 2006;290 (1):G7–13.

[5] Pinzani M, Failli P, Ruocco C, Casini A, Milani S, Baldi E, et al. Fat-storing cellsas liver-specific pericytes. Spatial dynamics of agonist-stimulated intracel-lular calcium transients. J Clin Invest 1992;90 (2):642–646.

[6] Rockey DC. Hepatic blood flow regulation by stellate cells in normal andinjured liver. Semin Liver Dis 2001;21 (3):337–349.

[7] Golomb E, Ma X, Jana SS, Preston YA, Kawamoto S, Shoham NG, et al.Identification and characterization of nonmuscle myosin II-C, a new memberof the myosin II family. J Biol Chem 2004;279 (4):2800–2808.

[8] Liu Z, Grunsven LAv, Rossen EV, Timmermans J-P, Geerts A, et al. Blebbistatininhibits contraction and accelerates migration in mouse hepatic stellatecells. Br J Pharmacol 2010;159 (2):304–315.

[9] Saab S, Tam SP, Tran BN, Melton AC, Tangkijvanich P, Wong H, et al. Myosinmediates contractile force generation by hepatic stellate cells in response toendothelin-1. J Biomed Sci 2002;9 (6 Pt 2):607–612.

[10] Lee HS, Park CS, Lee YM, Suk HY, Clemons TC, Choi OH. Antigen-inducedCa2+ mobilization in RBL-2H3 cells: role of I(1, 4, 5)P3 and S1P and necessityof I(1, 4, 5)P3 production. Cell Calcium 2005;38 (6):581–592.

[11] Ribeiro CM, Reece J, Putney Jr JW. Role of the cytoskeleton in calciumsignaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry. J Biol Chem1997;272 (42):26555–26561.

[12] Hinz B, Dugina V, Ballestrem C, Wehrle-Haller B, Chaponnier C. Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibro-blasts. Mol Biol Cell 2003;14 (6):2508–2519.

[13] Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS, Yamada KM.Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk.Nat Cell Biol 2007;9 (3):299–309.


[14] Niggli V, Schmid M, Nievergelt A. Differential roles of Rho-kinase andmyosin light chain kinase in regulating shape, adhesion, and migration ofHT1080 fibrosarcoma cells. Biochem Biophys Res Commun 2006;343(2):602–608.

[15] Zhou R, Watson C, Fu C, Yao X, Forte JG. Myosin II is present in gastricparietal cells and required for lamellipodial dynamics associated with cellactivation. Am J Physiol Cell Physiol 2003;285 (3):C662–C673.

[16] Lo CM, Buxton DB, Chua GC, Dembo M, Adelstein RS, Wang YL. Nonmusclemyosin IIb is involved in the guidance of fibroblast migration. Mol Biol Cell2004;15 (3):982–989.

[17] Yee Jr HF, Melton AC, Tran BN. RhoA/rho-associated kinase mediatesfibroblast contractile force generation. Biochem Biophys Res Commun2001;280 (5):1340–1345.

[18] Bataller R, Gasull X, Gines P, Hellemans K, Gorbig MN, Nicolas JM, et al. Invitro and in vivo activation of rat hepatic stellate cells results in de novoexpression of L-type voltage-operated calcium channels. Hepatology2001;33 (4):956–962.

[19] Tseng S, Kim R, Kim T, Morgan KG, Hai CM. F-actin disruption attenuatesagonist-induced [Ca2+], myosin phosphorylation, and force in smoothmuscle. Am J Physiol 1997;272 (6 Pt 1):C1960–C1967.

[20] Neves SR, Tsokas P, Sarkar A, Grace EA, Rangamani P, Taubenfeld SM, et al.Cell shape and negative links in regulatory motifs together control spatialinformation flow in signaling networks. Cell 2008;133 (4):666–680.

[21] Pomorski P, Targos B, Baranska J. Rearrangement of the endoplasmicreticulum and calcium transient formation: the computational approach.Biochem Biophys Res Commun 2005;328 (4):1126–1132.

[22] Sandquist JC, Swenson KI, Demali KA, Burridge K, Means AR. Rho kinasedifferentially regulates phosphorylation of nonmuscle myosin II isoforms Aand B during cell rounding and migration. J Biol Chem 2006;281(47):35873–35883.

[23] van Leeuwen FN, van Delft S, Kain HE, van der Kammen RA, Collard JG. Racregulates phosphorylation of the myosin-II heavy chain, actinomyosindisassembly and cell spreading. Nat Cell Biol 1999;1 (4):242–248.

[24] Rhee S, Jiang H, Ho CH, Grinnell F. Microtubule function in fibroblastspreading is modulated according to the tension state of cell-matrixinteractions. Proc Natl Acad Sci USA 2007;104 (13):5425–5430.

[25] Shutova MS, Alexandrova AY, Vasiliev JM. Regulation of polarity in cellsdevoid of actin bundle system after treatment with inhibitors of myosin IIactivity. Cell Motil Cytoskeleton 2008;65 (9):734–746.

[26] Dulyaninova NG, House RP, Betapudi V, Bresnick AR. Myosin-IIA heavy-chainphosphorylation regulates the motility of MDA-MB-231 carcinoma cells.Mol Biol Cell 2007;18 (8):3144–3155.

[27] Friedland JC, Lee MH, Boettiger D. Mechanically activated integrin switchcontrols alpha5beta1 function. Science 2009;323 (5914):642–644.

[28] Toyoshima F, Nishida E. Integrin-mediated adhesion orients the spindleparallel to the substratum in an EB1- and myosin X-dependent manner.EMBO J 2007;26 (6):1487–1498.

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distinct roles for non-muscle myosin ii isoforms in mouse hepatic stellate cells

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