- Email: [email protected]
Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells
Journal ojHepatology 1999; 31: 91-99 Printed in Denmark . All rights reserved Munksgaard Copenhagen
Copyright 0 European Association jar the Study of the Liver 1999 Journal of Hepatology ISSN 0168-8278
Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells Masaki Kate’,
Hiroaki
‘Third Department
Iwamoto’, Nobuhiko Higashi’, Rie Sugimoto2, Koutaro Uchimura’, Hironori Sakai’, Makoto Nakamuta’ and Hajime Nawata’
of Internal Medicine
and ‘Department of Clinical Chemistry and Laboratory Kyushu University. Fukuoka, Japan
Medicine.
Seiya Tada’,
Faculty of Medicine,
Background/Aims: In the fibrotic response to liver injury, hepatic stellate cells are activated, leading to the myofibroblastic cell shape, with actin cytoskeletal reorganization and increased extracellular matrix production. The reorganization of actin cytoskeleton suggests that the small GTP binding protein Rho might modulate the process of this myofibroblastic change. The aim of this study was to investigate the role of Rho in the phenotypic changes of hepatic stellate cells. Methods: The phenotypic changes were investigated by the overexpression of Rho regulator, Rho GDI or dominant negative mutant of Rho in mouse hepatic stellate cell line, GRX cells. In activated rat hepatic stellate cells, the effects of microinjection of Botulinus toxin C3, which is the specific inhibitor for Rho, were analyzed. Furthermore, the effect of C3 on the type I collagen accumulation in hepatic stellate cells was investigated.
Results: Overexpression of Rho GDI or the dominant negative mutant of Rho caused the shrinkage cell shape and suppressed stress fiber formation. Microinjection of toxin C3 caused a markedly distorted cell shape and the disappearance of stress fibers in rat stellate cells. In addition, C3 strongly suppressed collagen accumulation in activated stellate cells. Conclusions: These results suggest that Rho regulates the actin cytoskeletal reorganization, and may be implicated in the collagen accumulation in activated stellate cells. These findings provide evidence for the role of Rho in the myofibroblastic phenotype in hepatic stellate cells.
H
vated cells develop a flat and spread-out cell shape, with prominent microfilaments, including stress fiber formation. In addition, these morphological changes are accompanied by the production of various extracellular matrices (ECM), including fibronectin and type I collagen, finally causing liver fibrosis (8). In studies of cultured HSCs (9,10), experimental fibrosis (8), and human liver disease (1 l), HSCs have been found to be the major cellular source of ECM (12-14). It is well understood that the Rho small GTP binding protein regulates various cell functions, such as cell morphology, smooth-muscle contraction, platelet aggregation, cell motility and cytokinesis, through the reorganization of actin filaments (15-19). The Rho receives upstream signals through its regulators and is converted from the GDP-bound inactive form to the GTP-bound active form, which then interacts with its individual downstream target molecules and transduces signals to
stellate cells (HSCs, also referred to as Ito cells, fat-storing cells, and lipocytes) are nonparenchymal liver cells with a characteristic stellate morphology residing in the perisinusoidal space of Disse (l3). They contain variable amounts of lipid droplets, mainly consisting of vitamin A. In the inflammatory condition, HSCs undergo an activation process in which dramatic changes occur in morphology and cellular metabolism. The activated HSCs show characteristic morphological changes to myofibroblast-like cells with the expression of a-smooth muscle actin (4-7). The actiEPATIC
Received I1 August; revised 17 December 1998; accepted II January 1999
Correspondence: Makoto Nakamuta, Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: 8192 642 5282. Fax: 8192 642 5287. e-mail: [email protected]
Key words: Actin cytoskeleton; Botulinus toxin C3; Collagen; Hepatic stellate cell; Liver fibrosis; Rho; Rho GDI; Stress fiber.
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each downstream pathway (15,l 6). The biological functions of Rho have been clarified by the use of dominant negative or dominant active mutants of Rho, Rho GDP dissociation inhibitor (Rho GDI), and Botulinus toxin C3 (15-l 7). Rho GDI, a negative regulator of Rho family members, selectively interacts with the GDP-bound form of Rho to inhibit the conversion from the GDPbound inactive form to the GTP-bound active form (16,20). C3 is an exoenzyme which selectively ADP-ribosylates an Asn41, located in the effector domain of Rho, and inhibits the function of Rho (21,22). To assess the mechanism of phenotypic changes of HSCs, we analyzed the regulation of actin cytoskeletal reorganization via the Rho-dependent signaling pathway using rat HSC primary cell cultures and mouse hepatic stellate cell line, GRX cells. The roles of Rho in the regulation of actin cytoskeleton were evaluated by analyses of cell-shape changes and stress fiber formation in Rho CD1 and Rho dominant negative mutant overexpressed GRX cells, and C3 microinjected rat HSCs. The effect of C3 on the accumulation of type I collagen in activated HSCs was also analyzed. Our results indicate that the Rho-dependent signaling pathway is involved in the process of myofibroblastic changes via the reorganization of actin cytoskeleton in HSCs.
(Lab-Tek, Nunc, IL. USA). Twenty-four hours later, cells were washed with PBS and incubated with transfection mixture containing 250 ng of plasmid and 1.25 ul of SuperFect transfection reagent (Qiagen, Hilden, Germany) in DMEM. After 3 h, the cells were washed and incubated for 2 days in DMEM supplemented with 10% FCS. Microinjection Recombinant Botulinus toxin C3 (BIOMOL Research Laboratories. Plymouth Meeting, PA, USA) was microinjected into living HSCs cultured in grid culture dish (Nunc) as described previously (26,27). Glass capillaries (Femtotips. Eppendorf, Germany) were used to microinject. Injection pressure was controlled using Eppendorf Microinjector model 5246. C3 dissolved in buffer A (20 mM TrislHCl (pH 7.4) 20 mM NaCl, 2 mM MgClz, 100 uM ATP 0.1 mM EDTA. and 1 mM 2-mercaptoethanol) was microinjected at 50 &ml. Microinjection was confirmed by co-microinjection with 5 mg/ml of rabbit IgG. About 30 cells were usually microinjected within 10 min, followed by incubation for 60 min. The Trypan blue exclusion test showed more than 90% of the cells survived the microinjection procedure. Fluorescence microscopy For immunofluorescence microscopy. cells were fixed in 3.7% formaldehyde for 10 min, rinsed in PBS, and permeabilized for 5 min in 0.2% TritonX-100, followed by incubation with 10% FCS in PBS for 1 h. Then the cells were incubated with primary antibody (QElO, anti-Myc antibody (Genosys Biotechnologies, UK)) or anti-Rho antibody for 1 h, followed by staining with fluorescein-phalloidin (Molecular Probes, Eugene. OR) mixed with rhodamine-conjugated goat anti-mouse IgG (Leinco Technologies. Inc., St. Louis, MO, USA) or rhodamine-phalloidin (Molecular Probes) mixed with fluoresceinconjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. respectively. The slides were washed in PBS. and observed using a Zeiss-Axiophot microscope.
Materials and Methods Cell isolation and culture HSCs were isolated from normal male Wistar rats by sequential in situ perfusion with collagenase and digestion with pronase, following centrifugation in a double-layered (17%/l 1.5%) Metrizamide solution (Sigma Chemical, St. Louis, MO, USA), as described previously (23). HSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% fetal calf serum (FCS), 50 U/ml penicillin, and 50 mg/ml streptomycin. Experiments described in this study were performed on cells between the second and fourth serial passages and in three rat HSC primary cell cultures. Mouse hepatic stellate cell line, GRX, was kindly supplied by Dr. R. Borojevic (Rio de Janeiro, Brazil), and cultured in DMEM supplemented with 10% FCS (24). All animal experiments were conducted in accordance with the institutional ethical guidelines of Kyushu University. Immunoblotting For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), culture dishes were washed with phosphate-buffered saline (PBS) and dissolved by solubilizing buffer containing 25 mM Tris / HCl (pH 7.4), 1 mM DTT, 1 mM EDTA, and 1% TritonX-100. Samples containing 30 ug protein were electrophoresed on 13% SDSpolyacrylamide gel, and transferred to nitrocellulose filter (BA85, Schleicher & Schuell, Germany). After blocking, the filter was probed with 1: 100 dilution of polyclonal anti-Rho antibody (Santa Cruz Biotechnology, CA, USA). Biotin-conjugated anti-rabbit IgG (Zymed, CA, USA) was used as a second antibody. The antigen-antibody complex was visualized using alkaline phosphatase-conjugated avidin-biotin complex. Transfection A plasmid expressing Rho GDI expressing dominant negative (Tl9N)] were kindly supplied Osaka, Japan) (25). GRX cells
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[pEF-myc-Rho GDI], and a plasmid mutant of RhoA LpEF-myc-RhoA by Dr. Y. Takai (Osaka University, were seeded in X-chamber glass slides
Type I collagen as.say Eight day-cultured HSCs were incubated in serum-free medium in the presence or absence of 20 &ml of C3 for 48 h. Type I collagen was then determined in culture media by an enzyme-linked immunosorbent assay (ELISA) method as described (28). Anti-rat type I collagen antibody (LSL. Japan) was used as first antibody and peroxidaseconjugated goat antibody against rabbit IgG (Organon Teknika Corporation, Durham, NC, USA) was used as second antibody. Ral tail tendon collagen type 1 (Advance Biofactures Corporation, Lymbrook, NY, USA) was used as standard. Results were expressed as micrograms of collagen per microgram of cellular DNA, which was determined by a fluorometric assay according to the method of Brunk et al. (29). Northern blot analysis Total cellular RNA was extracted from 5X IO6 cells using RNeasy kit (Qiagen). Six micrograms of total RNA was size-fractionated on a l.O’% agarose gel in MOPS buffer (20 mM), pH 7.0, transferred to a Hybond-N nylon membrane (Amersham Life Science, Buckinghamshire, UK). This membrane was probed by an alkaline phosphatase enzyme-labeled rat al(I) collagen or rat glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) using Gene ImagesT” Alkphos Direct=” labeling and detection system (Amersham) according to the manufacturer’s instructions. Briefly, the filter was hybridized at 50°C for 16 h and then washed sequentially with primary wash buffer (2 M urea. 0.1% SDS, 50 mM sodium phosphate, 150 mM NaCl, 10 mM MgCl,, pH 7.0) at 57°C for 20 min twice. and with secondary wash buffer (50 mM Tris, 100 mM NaCl, 2 mM MgClz) for 10 min at room temperature. The detection was performed using the CDPStarTM chemiluminescent detection system (Amersham). The filter was exposed to x-ray films at room temperature for 1.5 h. The intensity of the hybridized bands was measured by a CCD image sensor (Densitograph AE6900F, Atto, Tokyo. Japan). Rat al(I) collagen and rat GAPDH cDNA probes were obtained
Role of Rho in hepatic stellate cells by reverse transcription-polymerase chain reaction (RTPCR) using the SuperScript Preamplification system (Life Technologies, Inc., Rockville, MD, USA). The PCR primers were based on published nucleotide sequences for rat al(I) collagen (30) (5’-CCT GCT GGA CCC CGA GGA AAC-3’ (sense); 5’- CGG TTC ACC AGG GTC GCC ATT-3’ (antisense)), rat GAPDH (GenBank accession number, U75401) (5’-AGG CCA GCC TCG TCT CAT AG-3’ (sense); 5’GTT AGC GGA TCT CGC TCC TG-3’ (antisense)). The PCR products were subcloned into pGEM T-easy vector (Promega Corp., Madison, WI, USA) and sequenced with the dideoxy chain termination technique using Sequenase version 2.0 (US Biochemical Corp., Cleveland, OH, USA). Statistical analysis Data were expressed as means&SD. Comparisons were made using the unpaired Student’s r-test. Differences were considered significant at a pcO.05.
phalloidin staining. The isolated HSCs showed small cell shape, and actin fibers were only detected at the cell surface and filopodia in the day 1 cultures (Fig. 1A). In the cultures on day 2, about 15% of HSCs showed a mildly spread cell shape and stress fiber formation (Fig. 1B). The ratio of activated HSCs was increased in a time-dependent manner (52% in day 4 cultures, Fig. 1C). Finally, almost all cells showed the activated phenotype in day-8 cultures, with well-spread cell shape and extended stress fiber formation (Fig. 1D). These phenotypic changes occurred under these conditions in a time-dependent manner, indicating that cytoskeletal reorganization was implicated in the activation of HSCs.
Results Morphological activation
and cytoskeletal
changes during HSCs
To assess the time-dependent changes of cell shape and actin cytoskeleton during activation of HSCs, freshly isolated HSCs from rat liver were cultured on plastic dishes for 1, 2, 4 and 8 days, followed by rhodamine-
Expression and localization GRX cells
of Rho in rat HSCs and
We next determined the expression of Rho by immunoblotting and immunocytochemical analyses. Rho was detected at molecular weight 21 kDa by immuno-
Fig. 1. Time-dependent activation of HSCs in culture. HSCs isolated from rat liver were plated on plastic dishes, followed by rhodamine-phalloidin staining at the indicatedperiods (original magnification 200X). A, day I cultures; B, day 2 cultures; C, day 4 cultures; D, day 8 cultures.
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Fig. 2. Expression and localization of Rho in rat HSCs (day 8 cultures) and GRX cells. (A) Expression of RhoA in rat HSCs and GRX cells. The cells plated on plastic dishes were solubilized, and the samples were electrophoresed on 13% SDSPAGE, followed by immunoblotting with anti-RhoA antibody. The protein size markers: ovalbumin, Mr=48,000; carbonic anhydrase, Mr=34,200; soybean trypsin inhibitor, Mr=28,400; lysozyme, Mr=20,500. The arrow indicates RhoA. (B) Localization of RhoA in rat HSCs. The activated HSCs were incubated with anti-RhoA antibody, followed by$uoresceinconjugated goat anti-rabbit IgG (original magntjication 400X). The results shown are representative of three independent experiments.
blotting in both rat HSCs and mouse stellate cell line, GRX cells (Fig. 2A). We also analyzed the expression of Rho in quiescent HSCs just isolated from rat liver, but it was found to be at almost the same level as that of activated HSCs (data not shown). In immunocytochemistry, Rho was diffusely detected in the cytoplasm of activated HSCs (Fig. 2B), which was similar in Rat2 cells or MDCK cells (31,32). Effect of Rho GDI or dominant negative mutant of RhoA on the cell shape and actin cytoskeleton in GRX cells
To analyze the role of Rho in the cytoskeletal reorganization in hepatic stellate cells, a plasmid expressing Rho GDI [pEF-myc-Rho GDI] or a plasmid expressing dominant negative mutant of RhoA [pEFmyc-RhoA (T19N)] was transfected into stellate cell line, GRX cells. Transfected cells were detected by staining with anti-Myc antibody, and the actin cytoskeleton of these cells was analyzed with phalloidin staining. The transfection efficiency of each plasmid was about 3%. We first transfected these plasmids into rat subcultured HSCs, but successfully transfected cells were not detected in these ceils. GRX cells showed well-spread cell shape and extended stress fiber formation (Fig. 3A). In the case of pEF-mycRho GDI or pEF-myc-RhoA (T19N) transfection, both the Myc-tagged proteins were diffusely stained throughout the cytoplasm in the transfected cells 94
(Fig. 3B and D). When Rho GDI was overexpressed in GRX cells, the cell became round and retractile and stress fibers were intensively suppressed 48 h after transfection (Fig. 3C). Overexpression of the dominant negative mutant of RhoA also caused similar results (Fig. 3E). These results suggest that Rho-dependent signaling pathway plays an important role in maintaining the myofibroblastic cell shape via the cytoskeletal reorganization in HSCs.
Effect of Botulinus toxin C3 on cell shape and actin cytoskeleton in rat HSCs
To assess the role of Rho in HSCs thoroughly, exoenzyme C3 was microinjected into the rat HSCs. Rabbit IgG was co-microinjected to detect the microinjected cells. The successfully microinjected cells were stained with fluorescein-conjugated anti-rabbit IgG (Fig. 4A and C), and the actin cytoskeleton of these cells was analyzed with rhodamine-phalloidin staining (Fig. 4B and D). Control cells microinjected with rabbit IgG only demonstrated the typical stellate morphology with a distended cell shape containing well-developed stress fibers (Fig. 4A and B). The C3 microinjected HSC was still distended but showed a highly arborized shape, concomitant with the disappearance of stress fibers (Fig. 4C and D). It is intriguing that the C3 microinjected cells left beaded dendritic processes attached to the dish, suggesting the focal adhesion of
Role of Rho in hepatic stellate cells
Fig. 3. Overexpression of Rho GDI or dominant negative mutant of RhoA in GRX cells. A plasmid expressing Rho GDI [pEF-myc-Rho GDI], or a plasmid expressing dominant negative mutant of RhoA [pEF-myc-RhoA (T19N) / was transfected into GRX cells. After 2 days, the immunocytochemical analyses were performed (original magn$cation 640X). A, normal control; B and C, pEF-myc-Rho GDI transfected cell; D and E, pEF-myc-RhoA (T19N) transfected cell. B and D, stained with anti-A4yc antibody; A, C, and E, stained with Jluorescein-phalloidin. The results shown are representative of three independent experiments.
HSCs was not destroyed by C3-mediated of endogenous Rho.
inactivation
Effect of Botulinus toxin C3 on the collagen accumulation in rat HSCs
Isolated rat HSCs cultured in DMEM supplemented with 20% FCS changed shape as described above (Fig. lD), and HSCs in this condition produce an increased amount of type I collagen (data not shown). To assess the functional role of Rho in the activated HSCs, type I collagen in culture media was analyzed after 2 days
incubation in the presence or absence of C3. The Trypan blue exclusion test showed more than 90% of the cells survived these treatments. Type I collagen in culture media was dramatically decreased in the presence of C3 (Fig. 5A). We also analyzed al(I) collagen mRNA level of HSCs in each sample by Northern blot analysis, using the level of GAPDH mRNA as standard. In contrast to the effect of C3 on collagen accumulation, there was no significant difference in al(I) collagen mRNA in the presence or absence of C3 (Fig. 5B). 95
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Fig. 4. Microinjection of exoenzyme C3 into primary culture HSCs. Recombinant Botulinus toxin C3 was microinjected into living HSCs. Microinjection was confirmed by co-microinjection with rabbit IgG. After 60 min incubation, the cells were fixed and double-stained with Jluorescein-conjugated goat anti-rabbit IgG (A and C) or rhodamine-phalloidin (B and D) (original magnification 400X). A and B, microinjected with IgG only; C and D, microinjected with Q and IgG. The arrows indicate microinjected cells. The results shown are representative of three independent experiments.
Discussion HSCs play a central role in the development of liver fibrosis (l-3). HSCs are activated in the inflammatory condition, and it has been demonstrated that multiple cytokines including TGFB and PDGF, and ECM regulate HSC activation (33-36). Once HSCs were activated, they expressed the smooth muscle cytoskeletal markers including a smooth muscle actin and desmin, and showed the appearance of extensive actin microfilaments which are typical of smooth muscle cells but absent from conventional fibroblasts (4-7). These characteristic changes lead HSCs to the contractile phenotype in culture and in vivo. Especially the activated HSCs contracted in response to treatment with substance P or endothelin-1, and it is suggested that HSCs are involved in the regulation of hepatic sinusoidal microcirculation related to the portal hypertension in liver cirrhosis (37,38). It is, therefore, important
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to understand the mechanism by which the actin cytoskeletal reorganization is regulated in HSCs. We demonstrated in this study that Rho is expressed in HSCs and plays an important role in the regulation of cell shape and actin-cytoskeletal reorganization in the activated HSCs. Rho is a member of the Rho subfamily which belongs to the small GTP binding protein superfamily (15-19). The mammalian Rho family consists of at least Rho subfamily, Rat subfamily, CdcQHs, and TClO. The Rho subfamily consists of five members, RhoA, B, C, D, and E, and only this Rho subfamily is ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3, and analyzed extensively by this feature (21,22). C3 ADP-ribosylates Asn-41 of Rho, which is located in the putative effector domain, and the ADP-ribosylation impairs the function of Rho. The Rho subfamily is involved primarily in the regulation of actin cytoskeleton, such as cell
Role of Rho in hepatic stellate cells
morphology, smooth muscle contraction, platelet aggregation, cell motility, cytokinesis, and lymphocyte aggregation. Recently it has been revealed that Rho plays crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development (19). The HSCs changed shape from small rounded cells to well-spread (flattened) cells, and showed a time-dependent formation of stress fibers, suggesting the activation of a regulatory mechanism of actin cytoskeletal reorganization including Rho-dependent signaling pathway. Rho was expressed in both activated HSCs and GRX cells. In the quiescent HSCs freshly isolated from rat liver, Rho was detected at the same amount in the activated HSCs (data not shown). Therefore, the expression of this protein itself is not altered by the activation process. And it seems likely that the GTPbound active form of Rho might be increased by HSC activation, because overexpression of both Rho GDI and the dominant negative mutant of RhoA caused the shrinkage of cell shape and decreased the stress fiber formation in GRX cells. We have shown that the
microinjection of C3 dramatically distorted the cell shape and also decreased stress fiber formation in the activated HSCs. These results suggest that Rho regulates the actin cytoskeletal reorganization along with the phenotypic changes of HSCs. We also transfected Rho-dominant active plasmid, pef-myc-RhoA(V14) (25), since the use of dominant active mutant of Rho would further enhance the effect of Rho. The number of cells transfected with this mutant plasmid, however, was always much less than that of the cells transfected with Rho GDI or Rho dominant negative plasmid. It is possible that overexpression of Rho may lead to cytotoxicity or prevent cell adhesion in GRX cells where Rho is already activated; however, more study is needed on this aspect. We demonstrated that C3 decreased the amount of type I collagen in the culture media in activated HSCs. The level of collagen al(I) mRNA, however, was not affected by the C3 treatment, suggesting that inactivation of Rho did not alter the transcription level of the collagen gene. Because the production or destruction mechanism of collagen in HSCs involves very
,; c3 . rpO.0002
1
T
+
II
+
I
Fig. 5’. Effect of C3 on the collagen accumulation in activated HSCs. (A) Type I collagen level in culture media in the presence or absence of Q. C3 was added to the rat HSCs in the cultures of day 8, and type I collagen in culture media was analyzed as described in Materials and Methods. Results were expressed as micrograms of collagen per micrograms of cellular DNA, and the mean&SD (n=3) are presented. * p
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complicated processes, it is difficult to explain the exact reason for this phenomenon in our assay system. It has been reported that the vesicular transports along the biosynthetic and secretory pathways involve the Rho-mediated mechanism (19), and therefore, the effect of C3 on the decrease of collagen accumulation might be mediated by this mechanism. Recently, Carloni et al. reported that the expression of integrin in HSCs and the adhesion of HSCs to ECM components elicited the increase in tyrosine phosphorylation of focal adhesion kinase (FAK) (39,40), suggesting that the integrin and FAK-dependent signaling pathway plays an important role in the function of HSCs. It is supposed that Rho mediates integrindependent multiple cellular function, and Rho is involved in the regulation of FAK phosphorylation (4143). Therefore, Rho may regulate the cytoskeletal reorganization of HSCs via the integrin and FAK-mediated signaling pathway. We have demonstrated in this study that Rho is implicated in the phenotypic changes of HSCs, mainly in the regulation of actin cytoskeletal reorganization. Furthermore, our results suggest that Rho might regulate other signaling pathways, including collagen accumulation in activated HSCs. Further investigations are necessary to clarify the roles of Rho in the functions of HSCs.
Acknowledgements This work was supported by a Grant-in-aid (10670485) for scientific research from the Ministry of Education, Science and Culture, Japan. We are grateful to Dr. Y. Takai (Department of Molecular Biology and Biochemistry, Osaka University ) for supplying pEF-mycRho GDI and pEF-myc-RhoA (T19N), and for many helpful suggestions. We also thank K. Tsuru for expert secretarial assistance.
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38. Rockey DC. Characterization of endothelin receptors mediating rat hepatic stellate cell contraction. Biochem Biophys Res Commun 1995; 207: 725-31. 39. Carloni V, Romanelli RG, Pinzani M, Laffi G, Gentilini I? Expression and function of integrin receptors for collagen and laminin in cultured human hepatic stellate cells. Gastroenterology 1996; 110: 1127-36. 40. Carloni \! Romanelli RG, Pinzani M, Laffi G, Gentilini F! Focal adhesion kinase and phospholipaseCy involvement in adhesion and migration of human hepatic stellate cells. Gastroenterology 1997; 112: 522-31. 41. Kumagai N, Morii N, Fujisawa K, Yoshimasa T, Nakao K, Narumiya S. Lysophosphatidic acid induces tyrosin phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells. FEBS Lett 1993; 329: 273-6. 42. Rankin S, Morii N, Narumiya S, Rozengurt E. Botulinus C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin. FEBS Lett 1994; 354: 315-9. 43. Seckl MJ, Morii N, Narumiya S, Rozengurt E. Guanosine 5’3-0-(thio) triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells. Role of p21 rho. J Biol Chem 1995; 270: 6984-90.
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Copyright 0 European Association jar the Study of the Liver 1999 Journal of Hepatology ISSN 0168-8278
Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells Masaki Kate’,
Hiroaki
‘Third Department
Iwamoto’, Nobuhiko Higashi’, Rie Sugimoto2, Koutaro Uchimura’, Hironori Sakai’, Makoto Nakamuta’ and Hajime Nawata’
of Internal Medicine
and ‘Department of Clinical Chemistry and Laboratory Kyushu University. Fukuoka, Japan
Medicine.
Seiya Tada’,
Faculty of Medicine,
Background/Aims: In the fibrotic response to liver injury, hepatic stellate cells are activated, leading to the myofibroblastic cell shape, with actin cytoskeletal reorganization and increased extracellular matrix production. The reorganization of actin cytoskeleton suggests that the small GTP binding protein Rho might modulate the process of this myofibroblastic change. The aim of this study was to investigate the role of Rho in the phenotypic changes of hepatic stellate cells. Methods: The phenotypic changes were investigated by the overexpression of Rho regulator, Rho GDI or dominant negative mutant of Rho in mouse hepatic stellate cell line, GRX cells. In activated rat hepatic stellate cells, the effects of microinjection of Botulinus toxin C3, which is the specific inhibitor for Rho, were analyzed. Furthermore, the effect of C3 on the type I collagen accumulation in hepatic stellate cells was investigated.
Results: Overexpression of Rho GDI or the dominant negative mutant of Rho caused the shrinkage cell shape and suppressed stress fiber formation. Microinjection of toxin C3 caused a markedly distorted cell shape and the disappearance of stress fibers in rat stellate cells. In addition, C3 strongly suppressed collagen accumulation in activated stellate cells. Conclusions: These results suggest that Rho regulates the actin cytoskeletal reorganization, and may be implicated in the collagen accumulation in activated stellate cells. These findings provide evidence for the role of Rho in the myofibroblastic phenotype in hepatic stellate cells.
H
vated cells develop a flat and spread-out cell shape, with prominent microfilaments, including stress fiber formation. In addition, these morphological changes are accompanied by the production of various extracellular matrices (ECM), including fibronectin and type I collagen, finally causing liver fibrosis (8). In studies of cultured HSCs (9,10), experimental fibrosis (8), and human liver disease (1 l), HSCs have been found to be the major cellular source of ECM (12-14). It is well understood that the Rho small GTP binding protein regulates various cell functions, such as cell morphology, smooth-muscle contraction, platelet aggregation, cell motility and cytokinesis, through the reorganization of actin filaments (15-19). The Rho receives upstream signals through its regulators and is converted from the GDP-bound inactive form to the GTP-bound active form, which then interacts with its individual downstream target molecules and transduces signals to
stellate cells (HSCs, also referred to as Ito cells, fat-storing cells, and lipocytes) are nonparenchymal liver cells with a characteristic stellate morphology residing in the perisinusoidal space of Disse (l3). They contain variable amounts of lipid droplets, mainly consisting of vitamin A. In the inflammatory condition, HSCs undergo an activation process in which dramatic changes occur in morphology and cellular metabolism. The activated HSCs show characteristic morphological changes to myofibroblast-like cells with the expression of a-smooth muscle actin (4-7). The actiEPATIC
Received I1 August; revised 17 December 1998; accepted II January 1999
Correspondence: Makoto Nakamuta, Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Tel: 8192 642 5282. Fax: 8192 642 5287. e-mail: [email protected]
Key words: Actin cytoskeleton; Botulinus toxin C3; Collagen; Hepatic stellate cell; Liver fibrosis; Rho; Rho GDI; Stress fiber.
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each downstream pathway (15,l 6). The biological functions of Rho have been clarified by the use of dominant negative or dominant active mutants of Rho, Rho GDP dissociation inhibitor (Rho GDI), and Botulinus toxin C3 (15-l 7). Rho GDI, a negative regulator of Rho family members, selectively interacts with the GDP-bound form of Rho to inhibit the conversion from the GDPbound inactive form to the GTP-bound active form (16,20). C3 is an exoenzyme which selectively ADP-ribosylates an Asn41, located in the effector domain of Rho, and inhibits the function of Rho (21,22). To assess the mechanism of phenotypic changes of HSCs, we analyzed the regulation of actin cytoskeletal reorganization via the Rho-dependent signaling pathway using rat HSC primary cell cultures and mouse hepatic stellate cell line, GRX cells. The roles of Rho in the regulation of actin cytoskeleton were evaluated by analyses of cell-shape changes and stress fiber formation in Rho CD1 and Rho dominant negative mutant overexpressed GRX cells, and C3 microinjected rat HSCs. The effect of C3 on the accumulation of type I collagen in activated HSCs was also analyzed. Our results indicate that the Rho-dependent signaling pathway is involved in the process of myofibroblastic changes via the reorganization of actin cytoskeleton in HSCs.
(Lab-Tek, Nunc, IL. USA). Twenty-four hours later, cells were washed with PBS and incubated with transfection mixture containing 250 ng of plasmid and 1.25 ul of SuperFect transfection reagent (Qiagen, Hilden, Germany) in DMEM. After 3 h, the cells were washed and incubated for 2 days in DMEM supplemented with 10% FCS. Microinjection Recombinant Botulinus toxin C3 (BIOMOL Research Laboratories. Plymouth Meeting, PA, USA) was microinjected into living HSCs cultured in grid culture dish (Nunc) as described previously (26,27). Glass capillaries (Femtotips. Eppendorf, Germany) were used to microinject. Injection pressure was controlled using Eppendorf Microinjector model 5246. C3 dissolved in buffer A (20 mM TrislHCl (pH 7.4) 20 mM NaCl, 2 mM MgClz, 100 uM ATP 0.1 mM EDTA. and 1 mM 2-mercaptoethanol) was microinjected at 50 &ml. Microinjection was confirmed by co-microinjection with 5 mg/ml of rabbit IgG. About 30 cells were usually microinjected within 10 min, followed by incubation for 60 min. The Trypan blue exclusion test showed more than 90% of the cells survived the microinjection procedure. Fluorescence microscopy For immunofluorescence microscopy. cells were fixed in 3.7% formaldehyde for 10 min, rinsed in PBS, and permeabilized for 5 min in 0.2% TritonX-100, followed by incubation with 10% FCS in PBS for 1 h. Then the cells were incubated with primary antibody (QElO, anti-Myc antibody (Genosys Biotechnologies, UK)) or anti-Rho antibody for 1 h, followed by staining with fluorescein-phalloidin (Molecular Probes, Eugene. OR) mixed with rhodamine-conjugated goat anti-mouse IgG (Leinco Technologies. Inc., St. Louis, MO, USA) or rhodamine-phalloidin (Molecular Probes) mixed with fluoresceinconjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. respectively. The slides were washed in PBS. and observed using a Zeiss-Axiophot microscope.
Materials and Methods Cell isolation and culture HSCs were isolated from normal male Wistar rats by sequential in situ perfusion with collagenase and digestion with pronase, following centrifugation in a double-layered (17%/l 1.5%) Metrizamide solution (Sigma Chemical, St. Louis, MO, USA), as described previously (23). HSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 20% fetal calf serum (FCS), 50 U/ml penicillin, and 50 mg/ml streptomycin. Experiments described in this study were performed on cells between the second and fourth serial passages and in three rat HSC primary cell cultures. Mouse hepatic stellate cell line, GRX, was kindly supplied by Dr. R. Borojevic (Rio de Janeiro, Brazil), and cultured in DMEM supplemented with 10% FCS (24). All animal experiments were conducted in accordance with the institutional ethical guidelines of Kyushu University. Immunoblotting For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), culture dishes were washed with phosphate-buffered saline (PBS) and dissolved by solubilizing buffer containing 25 mM Tris / HCl (pH 7.4), 1 mM DTT, 1 mM EDTA, and 1% TritonX-100. Samples containing 30 ug protein were electrophoresed on 13% SDSpolyacrylamide gel, and transferred to nitrocellulose filter (BA85, Schleicher & Schuell, Germany). After blocking, the filter was probed with 1: 100 dilution of polyclonal anti-Rho antibody (Santa Cruz Biotechnology, CA, USA). Biotin-conjugated anti-rabbit IgG (Zymed, CA, USA) was used as a second antibody. The antigen-antibody complex was visualized using alkaline phosphatase-conjugated avidin-biotin complex. Transfection A plasmid expressing Rho GDI expressing dominant negative (Tl9N)] were kindly supplied Osaka, Japan) (25). GRX cells
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[pEF-myc-Rho GDI], and a plasmid mutant of RhoA LpEF-myc-RhoA by Dr. Y. Takai (Osaka University, were seeded in X-chamber glass slides
Type I collagen as.say Eight day-cultured HSCs were incubated in serum-free medium in the presence or absence of 20 &ml of C3 for 48 h. Type I collagen was then determined in culture media by an enzyme-linked immunosorbent assay (ELISA) method as described (28). Anti-rat type I collagen antibody (LSL. Japan) was used as first antibody and peroxidaseconjugated goat antibody against rabbit IgG (Organon Teknika Corporation, Durham, NC, USA) was used as second antibody. Ral tail tendon collagen type 1 (Advance Biofactures Corporation, Lymbrook, NY, USA) was used as standard. Results were expressed as micrograms of collagen per microgram of cellular DNA, which was determined by a fluorometric assay according to the method of Brunk et al. (29). Northern blot analysis Total cellular RNA was extracted from 5X IO6 cells using RNeasy kit (Qiagen). Six micrograms of total RNA was size-fractionated on a l.O’% agarose gel in MOPS buffer (20 mM), pH 7.0, transferred to a Hybond-N nylon membrane (Amersham Life Science, Buckinghamshire, UK). This membrane was probed by an alkaline phosphatase enzyme-labeled rat al(I) collagen or rat glyceraldehyde 3-phosphatase dehydrogenase (GAPDH) using Gene ImagesT” Alkphos Direct=” labeling and detection system (Amersham) according to the manufacturer’s instructions. Briefly, the filter was hybridized at 50°C for 16 h and then washed sequentially with primary wash buffer (2 M urea. 0.1% SDS, 50 mM sodium phosphate, 150 mM NaCl, 10 mM MgCl,, pH 7.0) at 57°C for 20 min twice. and with secondary wash buffer (50 mM Tris, 100 mM NaCl, 2 mM MgClz) for 10 min at room temperature. The detection was performed using the CDPStarTM chemiluminescent detection system (Amersham). The filter was exposed to x-ray films at room temperature for 1.5 h. The intensity of the hybridized bands was measured by a CCD image sensor (Densitograph AE6900F, Atto, Tokyo. Japan). Rat al(I) collagen and rat GAPDH cDNA probes were obtained
Role of Rho in hepatic stellate cells by reverse transcription-polymerase chain reaction (RTPCR) using the SuperScript Preamplification system (Life Technologies, Inc., Rockville, MD, USA). The PCR primers were based on published nucleotide sequences for rat al(I) collagen (30) (5’-CCT GCT GGA CCC CGA GGA AAC-3’ (sense); 5’- CGG TTC ACC AGG GTC GCC ATT-3’ (antisense)), rat GAPDH (GenBank accession number, U75401) (5’-AGG CCA GCC TCG TCT CAT AG-3’ (sense); 5’GTT AGC GGA TCT CGC TCC TG-3’ (antisense)). The PCR products were subcloned into pGEM T-easy vector (Promega Corp., Madison, WI, USA) and sequenced with the dideoxy chain termination technique using Sequenase version 2.0 (US Biochemical Corp., Cleveland, OH, USA). Statistical analysis Data were expressed as means&SD. Comparisons were made using the unpaired Student’s r-test. Differences were considered significant at a pcO.05.
phalloidin staining. The isolated HSCs showed small cell shape, and actin fibers were only detected at the cell surface and filopodia in the day 1 cultures (Fig. 1A). In the cultures on day 2, about 15% of HSCs showed a mildly spread cell shape and stress fiber formation (Fig. 1B). The ratio of activated HSCs was increased in a time-dependent manner (52% in day 4 cultures, Fig. 1C). Finally, almost all cells showed the activated phenotype in day-8 cultures, with well-spread cell shape and extended stress fiber formation (Fig. 1D). These phenotypic changes occurred under these conditions in a time-dependent manner, indicating that cytoskeletal reorganization was implicated in the activation of HSCs.
Results Morphological activation
and cytoskeletal
changes during HSCs
To assess the time-dependent changes of cell shape and actin cytoskeleton during activation of HSCs, freshly isolated HSCs from rat liver were cultured on plastic dishes for 1, 2, 4 and 8 days, followed by rhodamine-
Expression and localization GRX cells
of Rho in rat HSCs and
We next determined the expression of Rho by immunoblotting and immunocytochemical analyses. Rho was detected at molecular weight 21 kDa by immuno-
Fig. 1. Time-dependent activation of HSCs in culture. HSCs isolated from rat liver were plated on plastic dishes, followed by rhodamine-phalloidin staining at the indicatedperiods (original magnification 200X). A, day I cultures; B, day 2 cultures; C, day 4 cultures; D, day 8 cultures.
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Fig. 2. Expression and localization of Rho in rat HSCs (day 8 cultures) and GRX cells. (A) Expression of RhoA in rat HSCs and GRX cells. The cells plated on plastic dishes were solubilized, and the samples were electrophoresed on 13% SDSPAGE, followed by immunoblotting with anti-RhoA antibody. The protein size markers: ovalbumin, Mr=48,000; carbonic anhydrase, Mr=34,200; soybean trypsin inhibitor, Mr=28,400; lysozyme, Mr=20,500. The arrow indicates RhoA. (B) Localization of RhoA in rat HSCs. The activated HSCs were incubated with anti-RhoA antibody, followed by$uoresceinconjugated goat anti-rabbit IgG (original magntjication 400X). The results shown are representative of three independent experiments.
blotting in both rat HSCs and mouse stellate cell line, GRX cells (Fig. 2A). We also analyzed the expression of Rho in quiescent HSCs just isolated from rat liver, but it was found to be at almost the same level as that of activated HSCs (data not shown). In immunocytochemistry, Rho was diffusely detected in the cytoplasm of activated HSCs (Fig. 2B), which was similar in Rat2 cells or MDCK cells (31,32). Effect of Rho GDI or dominant negative mutant of RhoA on the cell shape and actin cytoskeleton in GRX cells
To analyze the role of Rho in the cytoskeletal reorganization in hepatic stellate cells, a plasmid expressing Rho GDI [pEF-myc-Rho GDI] or a plasmid expressing dominant negative mutant of RhoA [pEFmyc-RhoA (T19N)] was transfected into stellate cell line, GRX cells. Transfected cells were detected by staining with anti-Myc antibody, and the actin cytoskeleton of these cells was analyzed with phalloidin staining. The transfection efficiency of each plasmid was about 3%. We first transfected these plasmids into rat subcultured HSCs, but successfully transfected cells were not detected in these ceils. GRX cells showed well-spread cell shape and extended stress fiber formation (Fig. 3A). In the case of pEF-mycRho GDI or pEF-myc-RhoA (T19N) transfection, both the Myc-tagged proteins were diffusely stained throughout the cytoplasm in the transfected cells 94
(Fig. 3B and D). When Rho GDI was overexpressed in GRX cells, the cell became round and retractile and stress fibers were intensively suppressed 48 h after transfection (Fig. 3C). Overexpression of the dominant negative mutant of RhoA also caused similar results (Fig. 3E). These results suggest that Rho-dependent signaling pathway plays an important role in maintaining the myofibroblastic cell shape via the cytoskeletal reorganization in HSCs.
Effect of Botulinus toxin C3 on cell shape and actin cytoskeleton in rat HSCs
To assess the role of Rho in HSCs thoroughly, exoenzyme C3 was microinjected into the rat HSCs. Rabbit IgG was co-microinjected to detect the microinjected cells. The successfully microinjected cells were stained with fluorescein-conjugated anti-rabbit IgG (Fig. 4A and C), and the actin cytoskeleton of these cells was analyzed with rhodamine-phalloidin staining (Fig. 4B and D). Control cells microinjected with rabbit IgG only demonstrated the typical stellate morphology with a distended cell shape containing well-developed stress fibers (Fig. 4A and B). The C3 microinjected HSC was still distended but showed a highly arborized shape, concomitant with the disappearance of stress fibers (Fig. 4C and D). It is intriguing that the C3 microinjected cells left beaded dendritic processes attached to the dish, suggesting the focal adhesion of
Role of Rho in hepatic stellate cells
Fig. 3. Overexpression of Rho GDI or dominant negative mutant of RhoA in GRX cells. A plasmid expressing Rho GDI [pEF-myc-Rho GDI], or a plasmid expressing dominant negative mutant of RhoA [pEF-myc-RhoA (T19N) / was transfected into GRX cells. After 2 days, the immunocytochemical analyses were performed (original magn$cation 640X). A, normal control; B and C, pEF-myc-Rho GDI transfected cell; D and E, pEF-myc-RhoA (T19N) transfected cell. B and D, stained with anti-A4yc antibody; A, C, and E, stained with Jluorescein-phalloidin. The results shown are representative of three independent experiments.
HSCs was not destroyed by C3-mediated of endogenous Rho.
inactivation
Effect of Botulinus toxin C3 on the collagen accumulation in rat HSCs
Isolated rat HSCs cultured in DMEM supplemented with 20% FCS changed shape as described above (Fig. lD), and HSCs in this condition produce an increased amount of type I collagen (data not shown). To assess the functional role of Rho in the activated HSCs, type I collagen in culture media was analyzed after 2 days
incubation in the presence or absence of C3. The Trypan blue exclusion test showed more than 90% of the cells survived these treatments. Type I collagen in culture media was dramatically decreased in the presence of C3 (Fig. 5A). We also analyzed al(I) collagen mRNA level of HSCs in each sample by Northern blot analysis, using the level of GAPDH mRNA as standard. In contrast to the effect of C3 on collagen accumulation, there was no significant difference in al(I) collagen mRNA in the presence or absence of C3 (Fig. 5B). 95
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Fig. 4. Microinjection of exoenzyme C3 into primary culture HSCs. Recombinant Botulinus toxin C3 was microinjected into living HSCs. Microinjection was confirmed by co-microinjection with rabbit IgG. After 60 min incubation, the cells were fixed and double-stained with Jluorescein-conjugated goat anti-rabbit IgG (A and C) or rhodamine-phalloidin (B and D) (original magnification 400X). A and B, microinjected with IgG only; C and D, microinjected with Q and IgG. The arrows indicate microinjected cells. The results shown are representative of three independent experiments.
Discussion HSCs play a central role in the development of liver fibrosis (l-3). HSCs are activated in the inflammatory condition, and it has been demonstrated that multiple cytokines including TGFB and PDGF, and ECM regulate HSC activation (33-36). Once HSCs were activated, they expressed the smooth muscle cytoskeletal markers including a smooth muscle actin and desmin, and showed the appearance of extensive actin microfilaments which are typical of smooth muscle cells but absent from conventional fibroblasts (4-7). These characteristic changes lead HSCs to the contractile phenotype in culture and in vivo. Especially the activated HSCs contracted in response to treatment with substance P or endothelin-1, and it is suggested that HSCs are involved in the regulation of hepatic sinusoidal microcirculation related to the portal hypertension in liver cirrhosis (37,38). It is, therefore, important
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to understand the mechanism by which the actin cytoskeletal reorganization is regulated in HSCs. We demonstrated in this study that Rho is expressed in HSCs and plays an important role in the regulation of cell shape and actin-cytoskeletal reorganization in the activated HSCs. Rho is a member of the Rho subfamily which belongs to the small GTP binding protein superfamily (15-19). The mammalian Rho family consists of at least Rho subfamily, Rat subfamily, CdcQHs, and TClO. The Rho subfamily consists of five members, RhoA, B, C, D, and E, and only this Rho subfamily is ADP-ribosylated by Clostridium botulinum ADP-ribosyltransferase C3, and analyzed extensively by this feature (21,22). C3 ADP-ribosylates Asn-41 of Rho, which is located in the putative effector domain, and the ADP-ribosylation impairs the function of Rho. The Rho subfamily is involved primarily in the regulation of actin cytoskeleton, such as cell
Role of Rho in hepatic stellate cells
morphology, smooth muscle contraction, platelet aggregation, cell motility, cytokinesis, and lymphocyte aggregation. Recently it has been revealed that Rho plays crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development (19). The HSCs changed shape from small rounded cells to well-spread (flattened) cells, and showed a time-dependent formation of stress fibers, suggesting the activation of a regulatory mechanism of actin cytoskeletal reorganization including Rho-dependent signaling pathway. Rho was expressed in both activated HSCs and GRX cells. In the quiescent HSCs freshly isolated from rat liver, Rho was detected at the same amount in the activated HSCs (data not shown). Therefore, the expression of this protein itself is not altered by the activation process. And it seems likely that the GTPbound active form of Rho might be increased by HSC activation, because overexpression of both Rho GDI and the dominant negative mutant of RhoA caused the shrinkage of cell shape and decreased the stress fiber formation in GRX cells. We have shown that the
microinjection of C3 dramatically distorted the cell shape and also decreased stress fiber formation in the activated HSCs. These results suggest that Rho regulates the actin cytoskeletal reorganization along with the phenotypic changes of HSCs. We also transfected Rho-dominant active plasmid, pef-myc-RhoA(V14) (25), since the use of dominant active mutant of Rho would further enhance the effect of Rho. The number of cells transfected with this mutant plasmid, however, was always much less than that of the cells transfected with Rho GDI or Rho dominant negative plasmid. It is possible that overexpression of Rho may lead to cytotoxicity or prevent cell adhesion in GRX cells where Rho is already activated; however, more study is needed on this aspect. We demonstrated that C3 decreased the amount of type I collagen in the culture media in activated HSCs. The level of collagen al(I) mRNA, however, was not affected by the C3 treatment, suggesting that inactivation of Rho did not alter the transcription level of the collagen gene. Because the production or destruction mechanism of collagen in HSCs involves very
,; c3 . rpO.0002
1
T
+
II
+
I
Fig. 5’. Effect of C3 on the collagen accumulation in activated HSCs. (A) Type I collagen level in culture media in the presence or absence of Q. C3 was added to the rat HSCs in the cultures of day 8, and type I collagen in culture media was analyzed as described in Materials and Methods. Results were expressed as micrograms of collagen per micrograms of cellular DNA, and the mean&SD (n=3) are presented. * p
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complicated processes, it is difficult to explain the exact reason for this phenomenon in our assay system. It has been reported that the vesicular transports along the biosynthetic and secretory pathways involve the Rho-mediated mechanism (19), and therefore, the effect of C3 on the decrease of collagen accumulation might be mediated by this mechanism. Recently, Carloni et al. reported that the expression of integrin in HSCs and the adhesion of HSCs to ECM components elicited the increase in tyrosine phosphorylation of focal adhesion kinase (FAK) (39,40), suggesting that the integrin and FAK-dependent signaling pathway plays an important role in the function of HSCs. It is supposed that Rho mediates integrindependent multiple cellular function, and Rho is involved in the regulation of FAK phosphorylation (4143). Therefore, Rho may regulate the cytoskeletal reorganization of HSCs via the integrin and FAK-mediated signaling pathway. We have demonstrated in this study that Rho is implicated in the phenotypic changes of HSCs, mainly in the regulation of actin cytoskeletal reorganization. Furthermore, our results suggest that Rho might regulate other signaling pathways, including collagen accumulation in activated HSCs. Further investigations are necessary to clarify the roles of Rho in the functions of HSCs.
Acknowledgements This work was supported by a Grant-in-aid (10670485) for scientific research from the Ministry of Education, Science and Culture, Japan. We are grateful to Dr. Y. Takai (Department of Molecular Biology and Biochemistry, Osaka University ) for supplying pEF-mycRho GDI and pEF-myc-RhoA (T19N), and for many helpful suggestions. We also thank K. Tsuru for expert secretarial assistance.
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