|
|
|
|
|
|
|
||
© The Rockefeller University Press,
0021-9525/1997//1477 $5.00
The Journal of Cell Biology, Volume 139, Number 6,
, 1997 1477-1484
Article |
Sarcomeric Gene Expression and Contractility in Myofibroblasts
Myofibroblasts are unusual cells that share morphological and functional features of muscle and nonmuscle cells. Such cells are thought to control liver blood flow and kidney glomerular filtration rate by having unique contractile properties. To determine how these cells achieve their contractile properties and their resemblance to muscle cells, we have characterized two myofibroblast cell lines. Here, we demonstrate that myofibroblast cell lines from kidney mesangial cells (BHK) and liver stellate cells activate extensive programs of muscle gene expression including a wide variety of muscle structural proteins. In BHK cells, six different striated myosin heavy chain isoforms and many thin filament proteins, including troponin T and tropomyosin are expressed. Liver stellate cells express a limited subset of the muscle thick filament proteins expressed in BHK cells. Although these cells are mitotically active and do not morphologically differentiate into myotubes, we show that MyoD and myogenin are expressed and functional in both cell types. Finally, these cells contract in response to endothelin-1 (ET-1); and we show that ET-1 treatment increases the expression of sarcomeric myosin.
Abbreviations used in this paper: ET-1, endothelin 1; MLC, myosin light chain; MRF, myogenic regulatory factors; MyHC, myosin heavy chain isoforms; NGS, normal goat serum; TnT, troponin T.
MYOFIBROBLASTS are cells of mesenchymal origin that are characterized by a fibroblastic appearance with some ultrastructural features of muscle cells. First described in wound retraction, they are found in almost all organ systems including the external theca of the ovarian follicle, the pulmonary alveolar septa, and the stromal cells of the rat intestinal villi (Kaye et al., 1968; O'Shea, 1970; Gabbiani et al., 1971; Kapanci et al., 1974; Skalli et al., 1986; Skalli and Gabbiani, 1988; Czernobilisky et al., 1989; Sappino et al., 1990). They are also implicated in pathological situations, such as liver cirrhosis and kidney glomerular fibrosis (for review see Skalli and Gabbiani, 1988). It has been hypothesized that myofibroblasts have specialized contractile properties in vivo since they can contract in response to endothelin 1 (ET-1),1 a vasoconstrictive peptide that is activated during injury (Kon et al., 1989; Moore et al., 1992).
The myofibroblasts of liver are called stellate cells and are located in the space of Disse, which is the region between the hepatocytes and the sinusoids. They are involved in retinoid metabolism and are the main producers of extracellular matrix proteins (Wake, 1971; De Leuw et al., 1984). During liver injury, the stellate cells undergo an activation process characterized by a decrease in vitamin A content and an increase in desmin, an intermediate filament protein normally found in muscle. Their close proximity to the endothelial lining of the sinusoids and their contractility in response to ET-1 have led to the hypothesis that they might function as liver pericytes, which are cells that can be found wrapped around capillaries (Diaz-Flores et al., 1991; Ogata et al., 1993; Housset et al., 1995). In the kidney, the mesangial cells located in the glomeruli are myofibroblasts (Johnson et al., 1992). These myofibroblasts are involved in the generation of mediators of inflammation, synthesis of cytokines, production and breakdown of basement membrane, as well as uptake of macromolecules (Elema et al., 1976; McCausland et al., 1977; Martin et al., 1989; Ohyama et al., 1990; Abboud et al., 1991). In addition, they contract and relax in response to vasoactive agents, including ET-1 (Schlondorff et al., 1984; Schlondorff, 1987; Badr et al., 1989). Their contractile properties are thought to control the rate of glomerular filtration by changing capillary surface area and to mediate wound healing after glomerular injury.
The expression of sarcomeric proteins has traditionally been thought to be restricted to striated muscle where their assembly into the sarcomere is highly regulated and ordered. Nonmuscle cells express many homologues of sarcomeric proteins, but there is significant sequence divergence between contractile proteins of muscle and nonmuscle cells. For example, nonmuscle myosin and striated muscle myosin are only 50% identical, and can have up to a 10-fold difference in their ATPase activities. In contrast, all eight striated muscle myosins share >80% amino acid sequence identity (for review see Weiss and Leinwand, 1996). Myofibroblasts have been broadly characterized by their expression of two proteins found in muscle, smooth muscle 
-actin and desmin (for review see Sappino et al., 1990). There have been reports of additional muscle proteins expressed in myofibroblasts. An adult skeletal fast IId MyHC gene was found to be expressed in a liver stellate cell line (Ogata et al., 1993). In addition, one study has shown that BHK cells, which we propose are kidney myofibroblasts, express several muscle proteins, including titin (Schaart et al., 1991).
Very little is known about the mechanisms whereby myofibroblasts achieve their contractile properties nor about the components that define their "muscle-like" phenotype. To define and determine how these cells achieve their intermediate phenotype and to define the molecules that confer contractility to these cells, we have characterized two cell lines corresponding to liver and kidney myofibroblasts. The liver stellate cell line was derived from a cirrhotic rat liver, and we present data that BHK cells are a model of glomerular mesangial cells. We found extensive expression of sarcomeric proteins in BHK cells and more limited expression in liver stellate cells. Two myogenic regulators, myogenin and MyoD, appear to be responsible for activating the muscle program of gene expression in these two myofibroblast cell types. We also demonstrate that ET-1 is a potent stimulus of contraction and that this contraction is correlated with a stimulation of striated muscle protein gene expression.
 |
Materials and Methods |
|---|
 TopAbstract Materials and Methods ResultsDiscussionReferences |
|---|
Immunochemistry
Mouse liver was fixed overnight at 4°C in fresh 4% formaldehyde in PBS (171 mM NaCl, 6 mM Na2HPO4, 6 mM KH2PO4, and 3 mM KCl). Tissue was embedded in paraffin following standard procedures (Watkins, 1989). 6-µm sections were deparaffinized, blocked overnight at 4°C in 5% normal goat serum (NGS), 2.5% BSA in PBS. HRP-labeled secondary antibody was visualized with 3-amino-9-ethylcarbazole (Vector Labs, Inc., Burlingame, CA). Antibody incubations were performed overnight at 4°C. Sections were counterstained with Gill's hematoxylin to visualize nuclei (Vector Labs, Inc.). Coverslips were fixed in 3.7% formaldehyde in PBS for 5 min, permeabilized in ice-cold acetone for 2 min, and then blocked in 10% NGS for 1 h at room temperature. Subsequent antibody incubations were performed for 1 h at room temperature.
Immunoelectron Microscopy
All incubations were performed at 4°C unless otherwise indicated. Cells were grown on coverslips that were washed with cold PBS three times for 5 min each, fixed with 4% paraformaldehyde in PBS for 30 min, and then washed with PBS as above. Cells were permeabilized with 1% Triton X-100 for 1 min, rinsed with PBS, and then free aldehydes were quenched with 0.05% sodium borohydride in PBS for 1 h at 4°C. Cells were again rinsed in PBS, and then blocked overnight at 4°C in 2% NGS in PBS. Cells were washed three times for 5 min each in 1% BSA, 0.05% Tween-20 in PBS (BSA/Tween/PBS). Primary antibody was diluted in 1% BSA, 0.05% Tween-20, and then incubated overnight. Cells were washed three times for 5 min each with BSA/Tween/PBS and incubated for 12–16 h with secondary antibody, 5 nm gold-conjugated, goat anti–mouse IgG (Ted Pella, Redding, CA). Cells were then washed with 0.05% Tween-20 in PBS, five times for 10 min at room temperature. Cells were then washed in 0.1 M sodium cacodylate, pH 7.4, and postfixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate. Coverslips were removed from the culture dishes for embedding, staining, and sectioning as previously described (Luft, 1961; Sabatini et al., 1963). Serial sections were cut parallel to the plane of the substratum, and examined in an electron microscope (100C; JEOL USA Inc., Peabody, MA) at an accelerating voltage of 80 kV.
Immunoblot Analysis
Protein expression of liver stellate cells and BHK cells was examined by immunoblotting. 100-mm dishes were washed twice in PBS, scraped in cytoskeletal buffer (50 mM KCl, 10 mM KPO4, 2 mM MgCl, 0.5 mM EDTA, 2 mM DTT), and then lysed by freeze thawing. Lysate was diluted in Laemmli sample buffer (Laemmli, 1970). Protein concentration was determined by Bradford assay, and 10 µg of each protein sample were electrophoresed on a 7.5% SDS-PAGE, transferred to 0.2 µm nitrocellulose membrane (Schleicher and Schuell, Keene, NH), and then membrane blocked with 5% nonfat milk in PBS for 2 h at room temperature. Electrophoresis and electroblotting followed standard protocols (Laemmli, 1970; Towbin et al., 1979). Primary antibody incubations were performed at 4°C overnight. Secondary antibody incubation was performed at room temperature for 2 h. Immunoreactive proteins were visualized using chemiluminescence (Amersham Corp., Arlington Heights, IL).
Proliferation Assay
Cells were plated at density of 7.44 x 105 in 100-mm dishes and maintained in serum-free medium for 24 h and treated with 10–10 M ET-1 for 24 h. Cells were trypsinized with 1 ml of trypsin/EDTA and counted with an hemocytometer 24 h after addition of ET-1.
Collagen Contraction Assay
Collagen solution, vitrogen 100 (Celltrix Laboratories, Palo Alto, CA), containing liver stellate cells (2 x 105 cells/ml) or BHK (7.5 x 105 cells/ml) were prepared as previously described (Moon and Tranquillo, 1993), with the exception that 0.5 ml of partially gelled collagen was pipetted into wells containing warm silicon oil instead of 0.1 ml of collagen.
CAT Assays on Cultured Cell Lysates
Transient transfections were performed using lipofectamine as described by the manufacturer (GIBCO BRL). 48 h after transfections, dishes were washed three times in PBS. Cell lysate was prepared by freeze thawing. The amount of protein in the supernatant was measured by the Bradford assay using BSA as the standard (Bio-Rad Laboratories, Hercules, CA). chloramphenicol acetyl transferase (CAT) assays were performed on 10 µg of total protein. CAT assays were done by TLC with an incubation for 2 h at 37°C (Gorman et al., 1982).
Antibodies
Primary monoclonal antibodies were F59 (Miller et al., 1989), antiembryonic myosin heavy chain, F.1652 (a gift from Dr. H. Blau, Stanford University School of Medicine, Palo Alto, CA), antiperinatal myosin heavy chain, N3.36 (a gift from Dr. H. Blau), anti-MyoD and antimyogenin (Novocastra, Burlingame, CA), antisarcomeric actin, HUC1-1 (a gift from Dr. J. Lessard, University of Cincinnati, Cincinnati, OH), anti-myosin light chain (MLC) 1,3 (a gift from Dr. F. Stockdale, Stanford University School of Medicine). Monoclonal antibodies against tropomyosin, troponin T (TnT), desmin, and titin were purchased from Sigma Chemical Co. Secondary antibodies conjugated to fluorescein and HRP were obtained from Jackson Immunoresearch Laboratories (West Grove, PA). Actin was visualized with rhodamine phalloidin (Molecular Probes, Eugene, OR).
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-actin and desmin, they also express many skeletal muscle structural proteins (Table I). Both the number and category are different between the two myofibroblast cell types. As shown in Table I, the liver stellate cells express three skeletal muscle myosin heavy chain isoforms (MyHCs) including one developmental isoform and two adult isoforms. To confirm that sarcomeric proteins are expressed in vivo in liver myofibroblasts, serial sections from a mouse liver were stained with a sarcomeric MyHC antibody. As shown in Fig. 1, the staining is localized to the liver stellate cells.
|
|
-cardiac MyHC, which suggests that only the skeletal muscle program is activated in these two myofibroblast cell types. Unlike the liver stellate cell line, the BHK cell line also expresses sarcomeric thin filament proteins, including and β tropomyosin and TnT. To determine whether the expression of sarcomeric proteins was homogeneous across the cell population, cells were stained with an antibody that recognizes all sarcomeric MyHCs, F59 (Miller et al., 1989). Approximately 10% of liver stellate cells expressed sarcomeric MyHC in culture, whereas 30% of BHK cells expressed sarcomeric MyHC under standard growth conditions (Fig. 2). In those cells expressing sarcomeric MyHC, the level of expression corresponds to 0.01% of total protein in liver stellate cells and 0.1% of total protein in BHK cells. This compares to 0.2–0.5% of total protein for nonmuscle MyHC (data not shown).
|
|
|
|
|
-actin in vivo (Sappino et al., 1990; Gabbiani, 1994). ET-1 has been shown to induce contractility of cultured stellate cells and mesangial cells (Badr et al., 1989; Rockey et al., 1993). To determine if ET-1 can induce the contraction of BHK and the liver stellate cell line characterized here, we performed a collagen contraction assay in the presence and absence of ET-1, using mouse L cells as a negative control. In the absence of exogenously added ET-1, myofibroblasts exhibited limited contractility, while L cells did not. As shown in Fig. 7, there was a dramatic contractile response to ET-1 that was blocked by an endothelin antagonist in both liver stellate cells and BHK cells.
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-actin (Tsutsumi et al., 1987; Ramadori et al., 1990; Rockey et al., 1992). In addition, the cells contract in response to treatment with ET-1. The discovery of extensive sarcomeric protein gene expression is consistent with the idea that these cells may be uniquely equipped for the contractile activities required for wound healing. A key characteristic of these cells is their expression of sarcomeric myosins. Myosins are the molecular motors responsible for movement in all eukaryotic cells. It seems logical to attribute the acquisition of unique contractile capacity of myofibroblasts to the expression of sarcomeric proteins. Sarcomeric myosin was previously thought to perform a single function, that of muscle contraction in the context of the sarcomere, whereas the myosin expressed in nonmuscle cells has been shown to be involved in diverse cellular functions, ranging from cytokinesis to morphogenesis (DeLozanne and Spudich, 1987; Fukui et al., 1990). Given that many nonmuscle cells actively divide, one requirement for the nonmuscle myosin filaments is that they be able to undergo rounds of assembly and disassembly. The muscle sarcomere is a much more stable structure and most changes in its composition are thought to occur by exchange, in and out, of existing structures (Wenderoth and Eisenberg, 1987). Clearly, the sarcomeric proteins expressed in myofibroblasts are able to function in an actively dividing cell. Sarcomeric MyHC in liver stellate cells and BHK cells is organized differently than the nonmuscle MyHC, but despite the presence of most of the components of the sarcomere in BHK cells, sarcomeres do not form. BHK cells are quite remarkable in that they express every skeletal muscle structural protein we have tested (except titin), including ones that are normally developmentally regulated. As mentioned earlier, there is a report that under certain growth conditions, BHK cells can even express titin (Schaart et al., 1991). It seems logical that the sarcomeric motor molecules provide unique contractile properties to myofibroblasts during processes such as wound healing.
The activation of the myogenic program in muscle precursor cells involves a number of regulatory factors that regulate the expression of sarcomeric proteins (Olson, 1990; Tapscott and Weintraub, 1991; Buckingham, 1992). MyoD, Myf-5, MRF4, and myogenin act as transcriptional activators of many skeletal muscle genes (Davis et al., 1987; Braun et al., 1989; Rhodes et al., 1989; Wright et al., 1989; Braun et al., 1990; Miner and Wold, 1990; Edmonson and Olson, 1989). The expression of MyoD and myogenin in cultured liver stellate cells and BHK cells suggests that the muscle program in these specialized nonmuscle cells is activated by some of the same factors that are used during myogenesis. This is further supported by the transient transfection assays, which indicate that MRF-binding sites in the MLC enhancer are targets for the action of these factors in myofibroblasts. It is very intriguing that these cells express both myogenic regulators and muscle structural proteins, and yet they are not terminally differentiated, nor are they morphologically differentiated. It is also interesting that these two cell types express distinct programs of muscle gene expression, with the liver stellate cells expressing a more limited program. What limits muscle gene expression in liver stellate cells to the thick filament?
The wound healing response is a complex process that involves interplay of extracellular matrix components, cytokines, and biologically active peptides, such as members of the endothelin family (Slavin, 1996; Mutsaers et al., 1997). ET-1 is a powerful vasoconstrictive agent synthesized by vascular endothelial cells (Yanigasawa et al., 1988). Significant amounts of ET-1 are produced by nonendothelial cells such as mesangial cells and liver stellate cells. In the liver, the stellate cells have abundant ET-1 receptors (Housset et al., 1993; Rockey et al., 1995). In the kidney, a number of glomerular cells, including the mesangial cells synthesize ET-1 and also express ET receptors (Kohzuki et al., 1989; Kohan et al., 1992). There is an increased level of ET-1 during liver and kidney injury (Ohta et al., 1991; Moore et al., 1992; Asbert et al., 1993). Cultured liver stellate cells and mesangial cells display increased contractility in response to ET-1 (Badr et al., 1989; Kon et al., 1989; Katoh et al., 1990; Rockey et al., 1993). Furthermore, intravenous injection of ET-1 directly into rat kidneys led to a decrease in glomerular filtration rate and renal blood flow with a concomitant contraction of mesangial cells cultured from the injected rats. These effects were reversed by injection of an anti–ET-1 antibody (Kon et al., 1989). Likewise, liver stellate cells treated with ET antagonists showed a decrease in smooth muscle -actin expression (Rockey and Chung, 1996). In this study, we confirm that an established liver stellate cell line and BHK cells can contract in response to ET-1. In addition, we demonstrate that there is a significant increase in the number of cells expressing sarcomeric MyHC. This is additional evidence supporting a functional role for the sarcomeric proteins expressed in these two myofibroblast cell types. This contractile response is not accompanied by an increase in cell proliferation (Mayer, D.H.G., and L.A. Leinwand, unpublished observation). We also demonstrate that the contractility of liver stellate cells and mesangial cells is blocked by the presence of an ET-1 antagonist.
There are numerous questions about these intriguing cells that will form the basis for future investigation. Future experiments will be directed towards identifying pathways that lead to myofibroblast cell types in vivo as well as determining the effects of disrupting sarcomeric protein expression on liver stellate cell and BHK cell contractility. Additionally, it will be interesting to determine the block to terminal differentiation that prevents the full differentiation potential of the myogenic regulators.
Submitted: 18 August 1997
Revised: 25 September 1997
Address all correspondence to Leslie A. Leinwand, Department of Molecular, Cellular and Developmental Biology, University of Colorado, Campus Box 347, Boulder, CO 80309-0347. Tel.: (303) 492-7606. Fax: (303) 492-8907. E-mail: leinwand{at}stripe.colorado.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abboud HE. Resident glomerular cells in glomerular injury: mesangial cells, Semin Nephrol, 1991, 11, 304–310.[Medline]
Asbert M, Ginhs A & Ginhs P. Circulating level of endothelin in cirrhosis, Gastroenterology, 1993, 104, 1485–1491.[Medline]
Badr KF, Murray JJ, Breyer MD, Takahashi K, Inagami T & Harris RC. Mesangial cell, glomerular and renal vascular responses to endothelin in the rat kidney, J Clin Invest, 1989, 83, 336–342.[Medline]
Benzonana G, Skalli O & Gabbiani G. Correlation between the distribution of smooth muscle and non muscle myosins and -smooth muscle actin in normal and pathological soft tissues, Cell Motil Cytoskeleton, 1988, 11, 260–274.[Medline]
Braun T, Buschhausen-Denker G, Bober E, Tannich E & Arnold HH. A novel human muscle factor related but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts, EMBO (Eur Mol Biol Organ) J, 1989, 8, 701–709.[Medline]
Braun T, Bober E, Winter B, Rosenthal N & Arnold HH. Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12, EMBO (Eur Mol Biol Organ) J, 1990, 9, 821–831.[Medline]
Buckingham M. Making muscle in mammals, Trends Genet, 1992, 8, 144–148.[Medline]
Buskin JN & Hauschka SD. Identification of a myocyte-specific nuclear factor which binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene, Mol Cell Biol, 1989, 9, 2627–2640.
Citi S, Smith RC & Kendrick-Jones J. Effects of light chain phosphorylation and skeletal myosin on the stability of nonmuscle myosin filaments, J Mol Biol, 1987, 198, 253–262.[Medline]
Czernobilisky BE, Shezen E, Lifschitz-Mercer B, Fogel M, Luzon A, Jacob N, Skalli O & Gabbiani G. Alpha-smooth muscle actin (-SM actin) in normal human ovaries, in ovarian stromal hyperplasia and in ovarian neoplasms, Virchows Arch, 1989, 57, 55–65.
Davis RL, Weintraub H & Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts, Cell, 1987, 51, 987–1000.[Medline]
De Leuw AM, McCarthy SB, Geerts A & Knook DL. Purified rat liver fat-storing cells in culture divide and contain collagen, Hepatology, 1984, 4, 392–403.[Medline]
DeLozanne A & Spudich JA. Disruption of the Dictyostelium discoideummyosin heavy chain gene by homologous recombination, Science, 1987, 236, 1086–1091.
Diaz-Flores L, Gutierrez R, Verela H, Rancel N & Valladares F. Microvascular pericytes: a review of their morphological and functional characteristics, Histol Histopathol, 1991, 6, 269–286.[Medline]
Edmonson DG & Olson EN. A gene with homology to the mycsimilarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program, Genes Dev, 1989, 3, 628–640.
Elema, J.D., Hoyer J.R., and R.L. Vernier. 1976. The glomerular mesangium: uptake and transport of intravenously injected colloidal carbon in rats. Kidney Int. 9:395–406.
Fukui Y, DeLozanne A & Spudich JA. Structure and function of the cytoskeleton of a Dictyostelium discoideummyosin-defective mutant, J Cell Biol, 1990, 110, 367–378.
Gabbiani G. Modulation of fibroblastic cytoskeletal features during wound healing and fibrosis, Pathol Res Pract, 1994, 190, 851–853.[Medline]
Gabbiani G, Ryan GB & Majno G. Presence of modified fibroblasts in granulation tissue and their possible role in wound contraction, Experientia (Basel), 1971, 27, 549–550.
Gorman CA, Moffat LF & Howard BH. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells, Mol Cell Biol, 1982, 2, 1044–1051.
Housset CN, Rockey DC & Bissel DM. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1, Proc Natl Acad Sci USA, 1993, 90, 9266–9270.
Housset CN, Rockey DC, Friedman SL & Bissel DM. Hepatic lipocytes: a major target for endothelin-1, J Hepatol, 1995, 22, 55–60.[Medline]
Johnson RJ, Floege J, Yoshimura A, Lida H, Couser WG & Alpers CE. The activated mesangial cell: a glomerular "myofibroblast"? , J Am Soc Nephrol, 1992, 2, 190–197.
Kapanci Y, Assimacopoulos A, Irle C, Zwahlen A & Gabbiani G. "Contractile interstitial cells" in pulmonary alveolar septa: A possible regulator of ventilation/perfusion ratio? , J Cell Biol, 1974, 60, 375–392.
Katoh T, Chang H, Uchida S, Okuda T & Kurokawa K. Direct effects of endothelin in the rat kidney, Am J Physiol, 1990, 258, 397–402.
Kaye GI, Lane N & Pascal PR. Colonic pericryptal fibroblast sheath: replication, migration, and cytodifferentiation of a mesenchymal cell-system in adult tissue. Fine structural aspects of rabbit and human colon, Gastroenterology, 1968, 60, 515–520.[Medline]
Kohan DE, Hughes AK & Perkins SL. Characterization of endothelin receptors in the inner medullary collecting duct of the rat, J Biol Chem, 1992, 267, 12336–12340.
Kohzuki M, Johnson CI, Chai SY, Casley DJ & Mendelsohn FAO. Localization of endothelin receptors in rat kidney, Eur J Pharmacol, 1989, 160, 193–194.[Medline]
Kon V, Yoshioka T, Fogo A & Ichikawa I. Glomerular actions of endothelin in vivo. , J Clin Invest, 1989, 83, 1762–1767.[Medline]
Korn ED & Hammer JA III. Myosins of nonmuscle cells, Annu Rev Biophys Biophys Chem, 1988, 17, 23–45.[Medline]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 1970, 237, 680–685.
Luft JH. Improvements in epoxy resin embedding methods, J Biophys Biochem Cytol, 1961, 9, 409–414.
Mak KM, Leo MA & Lieber CS. Alcoholic liver injury in baboons: transformation of lipocytes to transitional cells, Gastroenterology, 1984, 87, 188–200.[Medline]
Martin J, Davies M, Thomas G & Lovett DH. Human mesangial cells secrete a GBM-degrading neutral proteinase and a specific inhibitor, Kidney Int, 1989, 36, 790–801.[Medline]
McCausland IP, Gavin JB & Herdson PB. The distribution of dextran macromolecules in the glomeruli of normal and nephrotoxic rats, Pathology, 1977, 9, 123–128.[Medline]
Miller JB, Teal SB & Stockdale FE. Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression, J Biol Chem, 1989, 264, 13122–13130.
Miner JH & Wold B. Herculin, a fourth member of the MyoD family of myogenic regulatory genes, Proc Natl Acad Sci USA, 1990, 87, 1089–1093.
Molkentin JD & Olson EN. Defining the regulatory networks for muscle development, Curr Opin Gen Dev, 1996, 6, 445–453.[Medline]
Moncman CL, Rindt H, Robbins J & Winkelman DA. Segregated assembly of muscle myosin expressed in nonmuscle cells, Mol Biol Cell, 1993, 4, 1051–1067.[Abstract]
Moon AG & Tranquillo R. Fibroblast-populated collagen microsphere assay of cell traction force: part 1. Continuum model, AIChE (American Institute of Chemical Engineers) J, 1993, 39, 163–177.
Moore K, Wendon J, Frazer M, Karani J, Williams R & Badr K. Plasma endothelin immunoreactivity in liver disease and the hepatorenal syndrome, N Engl J Med, 1992, 327, 1774–1778.[Abstract]
Mutsaers SE, Bishop JE, McGrouther G & Laurent GJ. Mechanisms of tissue repair: from wound healing to fibrosis, Int J Biochem Cell Biol, 1997, 29, 5–17.[Medline]
Ogata I, Saez CG, Greenwel P, Ponce ML, Geerts A, Leinwand L & Rojkind M. Rat liver fat-storing cell lines express sarcomeric myosin heavy chain mRNA and protein, Cell Motil Cytoskeleton, 1993, 26, 125–132.[Medline]
Ohta K, Hirata Y, Shichiri M, Kanno K, Emori T, Tomika K & Marumo F. Urinary excretion of endothelin 1 in normal subjects and patients with renal diseases, Kidney Int, 1991, 39, 307–311.[Medline]
Ohyama K, Seyer JM, Raghow R & Kang AH. Extracellular matrix phenotype of rat mesangial cells in culture. Biosynthesis of collagen types I, III, IV, and a low molecular weight collagenous component and their regulation by dexamethasone, J Lab Clin Med, 1990, 116, 219–227.[Medline]
Olson EN. MyoD family: a paradigm for development? , Genes Dev, 1990, 4, 1454–1461.
O'Shea JD. An ultrastructural study of smooth-muscle like cells in the theca of the ovarian follicle of the rat, Anat Record, 1970, 167, 137–140.
Ramadori G, Veit T, Schwogler S, Dienes HP, Knittel T, Reider H & Meyer zum Buschenfelde K-H. Expression of the gene of the -smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells, Virchows Archiv B Cell Pathol, 1990, 59, 349–357.
Rhodes SJ & Konienczny SF. Identification of MRF4: a new member of the muscle regulatory factor gene family, Genes Dev, 1989, 3, 2050–2061.
Rockey DC. Characterization of endothelin receptors mediating rat hepatic stellate cell contraction, Biochem Biophys Res Commun, 1995, 207, 725–731.[Medline]
Rockey DC & Chung JJ. Endothelin antagonism in experimental hepatic fibrosis, J Clin Invest, 1996, 98, 1381–1388.[Medline]
Rockey DC, Boyles JK, Gabbiani G & Friedman SL. Hepatic lipocytes express smooth-muscle actin upon activation in vivo and in culture, J Submicrosc Cytol Pathol, 1992, 24, 193–203.[Medline]
Rockey DC, Housset CN & Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo, J Clin Invest, 1993, 92, 1795–1804.[Medline]
Rosenthal N, Kornhauser JM, Donoghue M, Rosen KM & Merlie JP. Myosin light chain enhancer activates muscle-specific, developmentally regulated expression in transgenic mice, Proc Natl Acad Sci USA, 1989, 86, 7780–7784.
Sabatini DD, Bensch K & Barnett RJ. Cytochemistry and electron microscopy: The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation, J Cell Biol, 1963, 17, 19–58.
Sappino AP, Schurch W & Gabbiani G. Biology of disease: differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic regulation, Lab Invest, 1990, 63, 144–161.[Medline]
Schaart G, Pieper FR, Kuijpers HJH, Bloemendal H & Ramaekers FC S. Baby hamster kidney (BHK-21/C13) cells can express striated muscle type proteins, Differentiation, 1991, 46, 105–115.[Medline]
Schlondorff D. The glomerular mesangial cell: an expanding role for a specialized pericyte, FASEB (Fed Am Soc Exp Biol) J, 1987, 1, 272–281.[Abstract]
Schlondorff D, Satriano JA, Hagege J, Perez J & Baud L. Effect of platelet activating factor and serum treated zymosan on PGE2 synthesis, arachidonic acid release and contraction of cultured rat mesangial cells, J Clin Invest, 1984, 73, 1227–1231.[Medline]
Skalli, O., and G. Gabbiani. 1988. The biology of myofibroblasts: relationships to wound contraction and fibrocontractive diseases. In The Molecular and Cellular Biology of Wound Repair. R.M.F. Clarck, and P.M. Henson, editors. Plenum Publishing Corp., New York. 373–402.
Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gilessen D & Gabbiani G. A monoclonal antibody for -smooth muscle actin, a new probe for smooth muscle differentiation, J Cell Biol, 1986, 103, 2787–2797.
Slavin J. The role of cytokines in wound healing, J Pathol, 1996, 178, 5–10.[Medline]
Tapscott SJ & Weintraub H. MyoD and the regulation of myogenesis by basic helix-loop-helix proteins, J Clin Invest, 1991, 87, 1133–1138.[Medline]
Tsutsumi M, Takada A & Takase S. Characterization of desmin-positive rat liver sinusoidal cells, Hepatology, 1987, 7, 277–284.[Medline]
Towbin H, Staehelin T & Gordon J. Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedures and some applications, Proc Natl Acad Sci USA, 1979, 76, 4350–4354.
Verkhovsky AB, Svitkina TM & Borisy GG. Myosin II filament assembly in the active lamella of fibroblasts: Their morphogenesis and role in the formation of actin filament bundles, J Cell Biol, 1995, 131, 989–1002.
Wake K. "Sternzellen": perisinusoidal cells with reference to storage of vitamin A, Am J Anat, 1971, 132, 429–462.[Medline]
Watkins, S. 1989. In situ hybridization and immunohistochemistry. In Current Protocols in Molecular Biology. S.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, editors. John Wiley and Sons. New York.
Weiss A & Leinwand LA. The mammalian myosin heavy chain gene family, Annu Rev Cell Dev Biol, 1996, 12, 417–439.[Medline]
Wenderoth MP & Eisenberg BR. Incorporation of nascent myosin heavy chains into thick filaments of cardiac myocytes in thyroid-treated rabbits, J Cell Biol, 1987, 105, 2771–2780.
Wentworth BM, Donoghue M, Engert JC, Berglund EB & Rosenthal N. Paired MyoD binding sites regulate myosin light chain gene expression, Proc Natl Acad Sci USA, 1991, 88, 1242–1246.
Wright WE, Binder M & Lin VK. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD1, Cell, 1989, 56, 607–617.[Medline]
Yanigasawa M, Kurihara H, Kimura S, Tomobe Y, Koboyashi M, Mitsui Y, Goto K & Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature, 1988, 332, 411–415.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
) after addition of 10–10 M ET-1 (•) and 5 x 10–7 M ET-1 antagonist (
). Each contraction assay was performed in triplicate. Data are presented as mean values ± standard error.