Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309
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.
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 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 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 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.
Cell Culture
The liver stellate cell line, 2G, a gift from Dr. M. Rojkind (Albert Einstein
College of Medicine, New York), was maintained in MEM supplemented
with 10% FBS (HyClone, Logan, UT), penicillin G at 50 U/ml, and streptomycin at 50 µg/ml, 1% MEM nonessential amino acids, 2 mM L-glutamine. BHK cells were obtained from Dr. R. Goldman (Northwestern University, Chicago, IL) and were grown in DME with 10% tryptose phosphate
broth (Sigma Chemical Co., St. Louis, MO), 10% BCS (HyClone), penicillin G at 50 U/ml, and streptomycin at 50 µg/ml. Cells were grown at
37°C in a 5% humidified CO2 incubator. Cells were treated for 24 h with
ET-1 (Sigma Chemical Co.) at a final concentration of 10 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 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 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 Proliferation Assay
Cells were plated at density of 7.44 × 105 in 100-mm dishes and maintained in serum-free medium for 24 h and treated with 10 Collagen Contraction Assay
Collagen solution, vitrogen 100 (Celltrix Laboratories, Palo Alto, CA),
containing liver stellate cells (2 × 105 cells/ml) or BHK (7.5 × 105 cells/ml)
were prepared as previously described (Moon and Tranquillo, 1993 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 Expression of Sarcomeric Proteins in BHK and Liver
Stellate Cells
Previous reports of the expression of muscle proteins in
myofibroblasts (Table I) led us to determine the extent of
sarcomeric protein gene expression in both BHK and liver
stellate cells by immunocytochemistry and Western blotting using isoform-specific antibodies. In addition to expressing smooth muscle Table I.
Muscle Protein Expression in Myofibroblasts*
BHK cells express the entire program of developmental
and adult skeletal MyHC isoforms tested thus far. Neither
cell line expresses
To determine whether the sarcomeric MyHC expressed
in BHK and liver stellate cells interacts with the nonmuscle cytoskeleton, sarcomeric and nonmuscle MyHC were
visualized by double-label immunofluorescence. There was
no colocalization of sarcomeric and nonsarcomeric MyHC
in liver stellate cells (Fig. 2, a and c), whereas there was
some colocalization of the two MyHCs in BHK cells but
no appearance of the sarcomeric MyHC in the stress fibers
(Fig. 2, b and d). In skeletal muscle, tropomyosin and TnT
interact with the actin filaments. To determine the localization of sarcomeric tropomyosin and TnT in BHK cells,
double-label immunofluorescence was performed. As shown
in Fig. 3, there is colocalization of tropomyosin and TnT
with the actin filament network of the cell.
Organization of Sarcomeric MyHC in BHK and Liver
Stellate Cells
If the expressed sarcomeric MyHC is to play a unique contractile function in these two myofibroblast cell types, it
might be expected to be organized into filamentous structures that are distinct from the nonmuscle MyHC. It is well
established that muscle and nonmuscle MyHC filaments
are very different in size and organization (Citi et al., 1987
Myogenic Regulators Control Sarcomeric Gene
Expression in Liver Stellate Cells and BHK Cells in an
E-Box-Dependent Manner
Myogenic regulatory factors (MRFs) are responsible for
controlling the expression of muscle-specific genes during
development (for review see Molkentin and Olson, 1996
MRFs control muscle gene expression by binding to
DNA consensus sequences, called E-boxes, that are found
in the control regions of many muscle genes (Buskin and
Hauschka, 1989
ET-1-induced Contraction of BHK and Liver
Stellate Cells Is Accompanied by an Increase in
Sarcomeric MyHC Expression
Tissue repair includes the activation of resident mesenchymal cells to become myofibroblasts and the induction of
ET-1 (Mak et al., 1984
To determine if there was a change in sarcomeric MyHC
expression in response to ET-1, cells were grown for 24 h
in the presence of ET-1 and scored for the proportion of cells
expressing MyHC and the amount of MyHC expressed.
To confirm that the proportional increase in MyHC, which
was visible by immunofluorescence, was accompanied by
an increase in the amount of MyHC protein, Western analysis was also performed on the cells in the absence and
presence of ET-1. As shown in Fig. 8, there was a ninefold
increase in the proportion of MyHC-positive stellate cells
and a threefold increase in the proportion of sarcomeric,
MyHC-positive BHK cells. These increases were accompanied by a significant increase in the amount of sarcomeric MyHC (Fig. 8).
We believe that the liver stellate cell line and BHK cells
are good culture models for the myofibroblasts of the liver
and the kidney, respectively. They satisfy the established
criteria for myofibroblasts. They actively produce extracellular matrix proteins and express desmin and smooth
muscle 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 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 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 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.
; 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
).
; 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.
). 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
).
Materials and Methods
10 M and ET-A
and ET-B antagonist, PD145065 (Sigma Chemical Co.), at a final concentration of 5 × 10
7 M in serum-free medium. Transient transfections were
performed using lipofectamine as described by the manufacturer (GIBCO
BRL, Gaithersburg, MD). Myosin light chain reporter constructs were a
gift from Dr. N. Rosenthal (Harvard Medical School, Cambridge, MA).
). 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.
;
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.
). 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).
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.
), 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.
).
), 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
-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.
Fig. 1.
Sarcomeric MyHC is expressed in vivo in liver stellate
cells. Immunoperoxidase labeling of sections of liver tissue was performed to visualize liver cell types expressing sarcomeric MyHC
by using F59. Only perisinusoidal cells are positive for sarcomeric
MyHC (arrow). Bar, 10 µm.
[View Larger Version of this Image (145K GIF file)]
-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
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).
Fig. 2.
Localization of nonmuscle and sarcomeric MyHC
expressed in liver stellate
cells and BHK cells. Indirect
immunofluorescence was
performed to visualize MyHC
in liver stellate cells (a and c)
and BHK cells (b and d).
Sarcomeric MyHC (a and b)
forms punctate structures
around the plasma membrane of liver stellate cells whereas it is more uniformly
distributed in the cytoplasm
of BHK cells. Sarcomeric (a
and b) and nonmuscle MyHC
(c and d) show partial colocalization in BHK cells, but
not in liver stellate cells. Bars,
(a and c) 10 µm, and (b and
d) 20 µm.
[View Larger Version of this Image (103K GIF file)]
Fig. 3.
Localization of sarcomeric thin filament proteins in
BHK cells. Indirect immunofluorescence was performed to localize sarcomeric and
tropomyosin (a), sarcomeric TnT (c), and
the actin filaments (b and d) using anti-TnT, antisarcomeric tropomyosin, and rhodamine phalloidin. Sarcomeric TnT and sarcomeric
and
tropomyosin colocalize with the actin filaments.
Bar, 10 µm.
[View Larger Version of this Image (168K GIF file)]
;
Moncman et al., 1993
; Verkhovsky et al., 1995
). Sarcomeric thick filaments are 1.5 µm in length and composed
of 100-200 MyHC molecules, whereas nonmuscle MyHC is organized into 0.25-0.35-µm minifilaments composed of
16-28 MyHC molecules (for review see Korn and Hammer, 1988
). To examine the organization of sarcomeric
MyHC in BHK and liver stellate cells, immunogold electron microscopy was carried out. Sarcomeric MyHC expressed in BHK and liver stellate cells formed bundles of
filaments as determined by immunogold labeling (Fig. 4, a
and b) whereas nonmuscle MyHC did not (Fig. 4, c and d).
From this analysis, it is clear that there are no sarcomeres
in either of these cell lines and there is no apparent periodicity to the structures formed by the muscle MyHC.
Fig. 4.
Ultrastructural
analysis of sarcomeric MyHC
in liver stellate cells and
BHK cells. Immunoelectron
microscopy was performed to determine the organization of sarcomeric MyHC in
liver stellate cells and BHK
cells. Monoclonal antibody,
F59, was used to detect sarcomeric MyHC in liver stellate (a) and BHK cells (b);
anti--platelet myosin antibody was used to detect nonmuscle MyHC in liver stellate cells (c) and BHK cells
(d). Secondary antibodies
were 5 nm gold-conjugated IgG. Arrows denote areas in
inset for a and b. Bar, 1 µm.
[View Larger Version of this Image (142K GIF file)]
).
Since a wide array of "muscle" genes are expressed in myofibroblasts, we hypothesized that myogenic regulators are
expressed in BHK and liver stellate cells and are responsible for activation of muscle structural proteins. As determined by immunocytochemistry, MyoD (Fig. 5) and myogenin (data not shown) were detected in 100% of BHK
and liver stellate cells. Myf-5 was not detected in either
cell line (data not shown).
Fig. 5.
Localization of MyoD in liver stellate cells and BHK
cells. Indirect immunofluorescence was performed to localize
MyoD in liver stellate cells (a) and BHK cells (b) using a monoclonal anti-MyoD antibody. MyoD expressed in liver stellate cells
and BHK cells has a normal nuclear localization. Bar, 10 µm.
[View Larger Version of this Image (68K GIF file)]
). To determine whether the same programs that regulate gene expression in muscle cells also
activate the muscle genes in BHK and liver stellate cells,
skeletal myosin light chain (MLC) 1,3 promoter elements linked to the CAT reporter gene (MLC-CAT) were transfected into BHK and liver stellate cells. The wild-type construct, which has three E-boxes, is sufficient to confer muscle-specific gene expression in cultured cells and in transgenic
mice, and its activity is dependent on the presence of MRFs
(Rosenthal et al., 1989
; Wentworth et al., 1991
). The second construct is mutated in all three E-boxes and is not expressed in muscle cells (Wentworth et al., 1991
). Included
as a positive cell control were C2C12 myotubes and as negative controls, C2C12 myoblasts, and L cells. As shown
in Fig. 6, the wild-type MLC construct is as active in BHK
and stellate cells as in C2C12 differentiated muscle cells. In
addition, the activity of the MLC construct in BHK and
liver stellate cells is abolished when the E-boxes are mutated, indicating that the expression of this sarcomeric
gene in BHK and liver stellate cells is dependent on the
presence of the MRFs.
Fig. 6.
E-box dependent expression of muscle reporter constructs in myofibroblasts. Previously described MLC reporter
constructs were transfected into cell types as indicated (Wentworth et al., 1991). CAT activity is corrected for transfection efficiency by using a luciferase construct as a cotransfection reference plasmid. Transfections were carried out in triplicate. CAT
activity is presented as mean value ± standard error.
[View Larger Version of this Image (20K GIF file)]
; Sappino et al., 1990
). The activation
process involves the development of a contractile phenotype with increased expression of smooth muscle
-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.
Fig. 7.
ET-1 dependent
contractility of myofibroblasts. Contraction assays
were performed as described
in Materials and Methods.
The change in diameter of
the collagen gel was measured after 24 and 48 h in the
absence of ET-1 () after
addition of 10
10 M ET-1 (
)
and 5 × 10
7 M ET-1 antagonist (
). Each contraction assay was performed in triplicate. Data are presented as
mean values ± standard error.
[View Larger Version of this Image (16K GIF file)]
Fig. 8.
ET-1 increases
expression of sarcomeric
MyHC in myofibroblasts.
(A) Indirect immunofluorescence was performed to determine the number of sarcomeric MyHC-positive liver
stellate cells and BHK cells
before () and after (+)
treatment with 10
10 M ET-1
for 24 h. (B) Immunoblotting
was performed to determine the increase of sarcomeric
MyHC in liver stellate cells
and BHK cells in the absence
of serum (lane 1), in the presence of 10% serum (lane 2),
in the presence of 5 × 10
7 M
ET-1 antagonist (lane 3), and
in the presence of 10
10 M
ET-1 for 24 h (lane 4).
[View Larger Version of this Image (29K GIF file)]
Discussion
-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.
; 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.
;
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?
; 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.
Received for publication 18 August 1997 and in revised form 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.eduET-1, endothelin 1; MLC, myosin light chain; MRF, myogenic regulatory factors; MyHC, myosin heavy chain isoforms; NGS, normal goat serum; TnT, troponin T.
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