Department of Medicine and Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110
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Abstract |
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Previously, we have suggested that vascular
cell adhesion molecule-1 (VCAM-1) and its integrin receptor 4
1 mediate cell-cell interactions important
for skeletal myogenesis. Expression of the receptors
subsequently subsides in muscle after birth. Here, we
examine the mechanism regulating VCAM-1 gene expression in muscle. An enhancer located between the
TATA box and the transcriptional start site is responsible for VCAM-1 gene expression in muscle
this element is inactive in endothelial cells where VCAM-1 expression is dependent on nuclear factor
B sites and
inflammatory cytokines. We identify interferon regulatory factor-2 (IRF-2), a member of the interferon regulatory factor family, as the enhancer-binding transcription factor and show that expression of IRF-2 parallels that of VCAM-1 during mouse skeletal myogenesis.
IRF-2 is not dependent upon cytokines for expression
or activity, and it has been shown to act as a repressor
in other nonmuscle cell types. We show that the basic
repressor motif located near the COOH-terminal of
IRF-2 is not active in muscle cells, but instead an acidic region in the center of the molecule functions as a
transactivating domain. Although IRF-2 and VCAM-1
expression diminishes on adult muscle fiber, they are
retained on myogenic stem cells (satellite cells). These
satellite cells proliferate and fuse to regenerate muscle
fiber after injury or disease. We present evidence that VCAM-1 on satellite cells mediates their interaction
with
4
1(+) leukocytes that invade the muscle after
injury or disease. We propose that VCAM-1 on endothelium generally recruits leukocytes to muscle after injury, whereas subsequent interaction with VCAM-1 on
regenerating muscle cells focuses the invading leukocytes specifically to the sites of regeneration.
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Introduction |
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VASCULAR cell adhesion molecule-1 (VCAM-1)1 is a
member of the immunoglobulin gene superfamily
that is expressed on the surface of endothelial cells
in response to inflammation (Osborn et al., 1989; Elices
et al., 1990
; Rice et al., 1991
). Through its interaction with
the integrin receptors,
4
1 and
4
7, on T-cells, monocytes, and eosinophils, VCAM-1 targets these inflammatory cells to cytokine-stimulated endothelium (Erle et al.,
1991
; Freedman et al., 1991
; Miyake et al., 1991
; Scheeren
et al., 1991
; Schimizu et al., 1991; Ruegg et al., 1992
).
In addition to its role in inflammation, VCAM-1-deficient mice demonstrated essential roles for VCAM-1 during embryonic development (Gurtner et al., 1995; Kwee
et al., 1995
). Mice lacking VCAM-1 died at two different
developmental stages. Most VCAM-1-deficient embryos
died before embryonic day (E) 11.5 due to a failure of the
allantois to fuse with the chorion. In wild-type embryos, VCAM-1 and its receptor,
4
1, are expressed on the allantois and chorion, respectively (Kwee et al., 1995
). Embryos that survived these defects in the extraembryonic
membranes died by E12.5 due to cardiac abnormalities, including defects of the ventricular myocardium and intraventricular septum and a failure to form an epicardium. In
wild-type embryos, VCAM-1 is expressed on the outer layer of the myocardium, which physically interacts with
epicardial cells that express
4
1 (Kwee et al., 1995
; Sheppard et al., 1994
). Significantly,
4-deficient embryos exhibit similar defects in placental and cardiac development
(Yang et al., 1995
). These results suggest important roles
for VCAM-1 and
4
1 in both placental and cardiac development.
Other studies have suggested that VCAM-1 also plays a
role in the differentiation of skeletal muscle; however,
VCAM-1-deficient embryos die before the role of VCAM-1
can be examined in this process. Mammalian skeletal muscle differentiation occurs in two stages, each represented
by a distinct population of myoblasts (Ontell, 1977; Ross
et al., 1987
; Stockdale and Miller, 1987
; Ontell et al., 1988
).
Around E12 in the mouse, an early-born population of
myoblasts (primary myoblasts) fuse to form primary myotubes. Then, between E14 and E16, a second, distinct wave
of myoblasts (secondary myoblasts) appear and align
themselves along the primary myotubes, where they proliferate and appear to use primary myotubes as a template
for fusion into secondary myotubes. These secondary myotubes comprise most adult muscle fiber. VCAM-1 is present
on secondary myoblasts and portions of secondary myotubes that are apposed to primary myotubes, while
4
1 is
present on primary myotubes (Rosen et al., 1992
). This
alignment suggests that these receptors play a role in secondary myogenesis. Antibodies that block the interaction
between VCAM-1 and
4
1 inhibited myoblast fusion in
culture (Rosen et al., 1992
), and expression of antisense
4
RNA blocked myoblast fusion in vitro and prevented
fusion into muscle in vivo (unpublished observations),
providing further evidence of a role for these proteins in
myogenesis. Expression of both VCAM-1 and
4
1 is developmental-specific since neither receptor is found in normal adult muscle fibers.
In endothelial cells where VCAM-1 expression is dependent upon inflammatory cytokines, silencer elements
(identified as octamer binding sites) restrict VCAM-1 gene
promoter activity in unstimulated endothelial cells (Iademarco et al., 1992, 1993
). Inflammatory cytokines overcome the negative activity of the octamers and cause activation of the promoter through two adjacent nuclear factor
B sites located at positions
77 and
63 bp in the VCAM-1
gene promoter. In contrast to endothelial cells, where
VCAM-1 expression is dependent upon cytokines, there is
high basal expression of VCAM-1 on muscle cells. An element between the TATA box and the transcriptional start
site appears to override the activity of other promoter elements causing constitutive VCAM-1 expression; this element
is not active in endothelial cells. The nucleotide sequence
of the VCAM-1 gene promoter in this region, between positions
17 and
5 bp, matches the consensus for an interferon (IFN) regulatory factor (IRF) binding element (Neish
et al., 1995
). The IRF family of transcription factors binds
to these DNA sequences classically found in the IFN-
and IFN-
gene promoters. However, a number of other
genes contain IRF elements, and not all of the IRF family members are regulated by cytokines. The IRF family
members are characterized by a highly conserved amino-terminal DNA binding domain that contains a repeated
tryptophan motif (Veals et al., 1992
), but exhibit minimal
sequence similarity outside this region. The IRF family
contains both activators and repressors of transcription. Transfection studies using the IFN-
and other IFN-
responsive gene promoters have shown that IRF-1 is a
transcriptional activator while IRF-2 is a repressor (Harada
et al., 1989
, 1990
; Reis et al., 1992
). However, other studies
have demonstrated that IRF-2 contains a latent activation
domain (which becomes evident when the repressor domain is deleted; Yamamoto et al., 1994
) that can activate
transcription from some gene promoters (Vaughan et al., 1995
).
Here, we demonstrate that IRF-2 interacts with the VCAM-1 gene promoter in muscle cells and is responsible for the transcriptional activation of the VCAM-1 gene promoter. We show that the repressor domain is inactive in muscle cells, and using a series of IRF-2 deletion mutants, we identify the transactivating domain in IRF-2 that is responsible for transcriptional activity during myogenesis. We show that IRF-2 expression closely parallels that of VCAM-1 in differentiating skeletal muscle. Additionally, we examined VCAM-1 expression during muscle regeneration, both in response to muscle injury and in a murine model of muscular dystrophy. Muscle regeneration differs from embryonic myogenesis in several ways. Myogenesis in the embryo occurs in two stages and involves distinct populations of myoblasts while regenerating muscle is derived from muscle satellite cells, which are stem cell-like precursors that are normally quiescent and do not express muscle transcription factors. In response to injury and disease, these satellite cells proliferate and fuse into muscle fibers. Muscle regeneration is intimately associated with inflammation, and in addition to their obvious role in inflammation, invading leukocytes may provide a trigger for proliferation of satellite cells that initiates the regeneration process. We present evidence that VCAM-1 is expressed on satellite cells and newly forming myotubes, where it plays a novel role in recruitment of leukocytes to specific sites of myogenic regeneration.
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Materials and Methods |
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Cell Culture and DNA Transfection
C2C12 mouse myoblasts were maintained in DME supplemented with
13% FBS. For transient transfections, 20 µg of reporter plasmid were
transfected by the calcium phosphate technique (Iademarco et al., 1992)
followed by DMSO shocking (8 min in 12% DMSO in DME) 18 h later.
Cells were maintained as myoblasts in 13% FBS, and cell extracts were
prepared 36 h after transfection. 1 µg of TKluc, which contains the thymidine kinase promoter fused to the firefly luciferase gene, was cotransfected as an internal control and luciferase activity was used to correct for
transfection efficiency. Luciferase and chloramphenicol acetyl transferase gene (CAT) assays were done as described previously (Iademarco et al.,
1992
). NIH 3T3 cells were maintained in DME supplemented with 10%
FBS. P19 cells were maintained in
-MEM containing 10% FBS and differentiated with retinoic acid as described previously (Sheppard et al.,
1995
).
Cell Adhesion Assays
C2C12 cells were transferred to 96-well plates and allowed to adhere for
18 h. Ramos cells were washed twice with PBS and resuspended in DME
with 10% FBS alone or with 2 µg/ml anti-4 antisera (HP 2/1, a gift from
Dr. F. Sanchez-Madrid, Hospital de la Princesa, Madrid, Spain) or control
anti-myosin heavy chain antisera (a gift from Dr. R. Kopan, Washington
University). Approximately 105 Ramos cells were allowed to attach to the
C2C12 monolayers for 1 h. The plates were then washed three times with
PBS and photographed.
Plasmid Construction
VCAM-1 promoter constructs have been described previously (Iademarco et al., 1992). Mutant
32 VCAMCAT plasmids were constructed by cloning double-stranded synthetic oligonucleotides corresponding to
positions
32 to +8 bp of the VCAM-1 gene promoter into the PstI and
HindIII sites of SkCAT-Pst/Bam (Rosen et al., 1991). IRF-1 and IRF-2
expression plasmids (Lin et al., 1994
) were a gift from Dr. John Hiscott
(McGill University, Montreal, Quebec, Canada).
Gel Retardation Assays
Nuclear extracts were prepared from C2C12 cells using a modified Dignam protocol (Dignam et al., 1983). Briefly, cells were harvested and
rinsed. The cells were then lysed through a syringe in hypotonic buffer (10 mM Hepes, pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 1 mM PMSF, and 1 mM
DTT). 10 µg of nuclear extract was assayed for binding using 32P-labeled,
double stranded oligonucleotides. The probe for the VCAM-1 IRF site
spanned
32 to +8 bp of the VCAM-1 gene. For gel retardation assays,
nuclear extract was incubated with probe on ice for 30 min in binding
buffer (10 mM Tris-HCl, pH 7.9, 50 mM NaCl, 1 mM DTT, 1 mM EDTA,
5% glycerol, 1 µg nonspecific DNA, 2.5 mM MgCl2, 2.5 mM MnCl2). For
supershift analysis, nuclear extract was incubated with 2.5 µl anti-IRF-2
or control anti-c-rel antisera (Santa Cruz Biotechnology, Santa Cruz, CA)
for 45 min on ice before addition of binding buffer. The sequences of the oligonucleotides used are as follows: VCAM-1, 5'-TTTATAAAGCACAGACTTTCTATTTCACTCCGCGGTATCTGCA-3'; and HLA, 5'-ATTCCCCACTCCCCTGAGTTTCACTTCTTCTCCCAAC-3'.
Western Blot Analysis
Whole cell extracts were prepared from cultured cells in 10 mM Tris-HCl
(pH 6.8) and 0.1% SDS. Muscle extracts were prepared by homogenization of embryonic limbs or adult hindlimb muscle as previously described
(Andrew and Appel, 1973). Whole cell extracts were subjected to Western
blot analysis using anti-IRF-1 and anti-IRF-2 (COOH-terminal) antibodies as described previously (Nguyen et al., 1995
). Anti-IRF-1 and anti-
IRF-2 antisera (Nguyen et al., 1995
) were a gift from Dr. Hiscott.
Immunostaining and Muscle Injury
Immunostaining of embryonic mouse sections and sections of adult skeletal muscle was done as we have described (Rosen et al., 1992). Immunostaining for IRF-2 was confirmed with three different antisera, each to a
distinct region of IRF-2. For muscle damage, the gastrocnemius muscle in
the hindlimb of adult C3H mice was exposed surgically and damaged by
either cutting or freezing (results were similar with both damaging techniques). At various times after muscle damage, the area of damage or a
control region from the other undamaged hindlimb was dissected and frozen in O.C.T. (Miles-Yeda Inc., Elkhart, IN) as described previously
(Rosen et al., 1992
). Sections were immunostained for VCAM-1, NCAM,
and CD45 as described previously (Rosen et al., 1992
). Anti-CD45 was a
gift from Dr. M. Thomas (Washington University).
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Results |
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Expression of VCAM-1 in Skeletal Muscle Cells Is Controlled by an Element 3' of the TATA Box
Previously, we found that a 2.1-kb fragment of the
VCAM-1 gene promoter is transcriptionally active in
C2C12 myoblasts and that deletion of the promoter to
32 bp, which is immediately upstream of the TATA box,
did not inhibit promoter activity (Iademarco et al., 1993
).
Here, we demonstrate that mutation of the region 3' of the
TATA box, between positions
21 and
5 bp, in the context of the
32-,
288-, and
2.1-kb VCAMCAT reporter
constructs inhibited VCAM-1 promoter activity in C2C12
myoblasts (Fig. 1). These results suggest that the region
between
21 and
5 bp is necessary for the activity of the
VCAM-1 gene promoter in skeletal myoblasts.
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Mutations Identify an IRF Element in the VCAM-1 Gene Promoter
To more precisely identify regions critical for VCAM-1
gene promoter activity, a series of mutant constructs was
created, and their activities were examined in transfection
assays in C2C12 myoblasts (Fig. 2). This analysis identified
an essential DNA sequence in the VCAM-1 gene promoter that matches the consensus binding site for the IRF
family of transcription factors, G(A)AAANNGAAA(G/C) (T/C) (Tanaka et al., 1993). Mutations in either or both
half-sites of the IRF-like DNA binding sequence decreased
transcriptional activity. Additionally, mutations at positions
14 and
8 bp disrupted promoter activity; such
mutations in the IRF binding sequence have previously
been shown to disrupt IRF binding (Neish et al., 1995
).
However, mutations in the nonconserved spacer (NN) between the half-sites or in the sequences 5' or 3' of the IRF
element had little or no effect. This mutational analysis
suggests that the enhancer in the VCAM-1 gene promoter
is an IRF site and that this site is responsible for VCAM-1
expression in muscle cells.
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IRF-2 Is Expressed in Myoblasts
We examined expression of IRF family proteins in C2C12
myoblasts. Extracts of C2C12 myoblasts were tested for
IRF-1, IRF-2, and IRF-4 expression by Western blot analysis. Other IRF family members, ICSBP and IGSF3,
were not tested. (ICSBP is expressed only in macrophages
and lymphocytes [Driggers et al., 1990
; Nelson et al.,
1993
], and IGSF3
is activated only in response to IFN treatment [Kessler et al., 1990
].) IRF-2 was the only IRF
protein detected in the C2C12 extracts (Fig. 3 A, and data
not shown). As a control, IRF-2 expression was examined
in P19 embryonic carcinoma cells. It has been demonstrated previously that IRF-2 is not expressed in undifferentiated P19 cells, but it is expressed when P19 cells are induced to differentiate into neural cells in response to retinoic acid treatment (Harada et al., 1990
). Interestingly, we have found previously that VCAM-1 expression is also
induced upon P19 cell differentiation (Sheppard et al., 1995
).
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While IRF-1 was not normally expressed in C2C12 cells,
it is expressed in response to treatment with IFN- (Fig. 3
B). Accordingly, VCAM-1 expression has been shown
previously to be induced by IFN-
(Thornhill et al., 1991
;
Sikorski et al., 1993
; Gao and Issekutz, 1996
; Seko et al.,
1996
; Prudhomme et al., 1996
). As a control, we show that
IRF-1 is also induced by IFN-
in NIH 3T3 cells (Fig. 3 B).
These patterns of expression suggest that IRF-2 is the IRF family member that normally regulates VCAM-1 gene
transcription in muscle cells; however, other IRF proteins,
such as IRF-1, may be able to substitute for IRF-2 (e.g.,
during inflammation).
IRF-2 Binds to the VCAM-1 IRF Element
Next, the ability of IRF-2 to bind to the IRF element from
the VCAM-1 gene promoter was examined. Synthetic oligonucleotides containing the VCAM-1 gene IRF element
were used in gel shift assays with extracts from 293 cells
transfected with an IRF-2 expression vector. Two specific
complexes were observed with extract from the IRF-
2-transfected cells, and mutation of the IRF site eliminated
binding of these complexes to the probe; antisera against IRF-2 supershifted these complexes (data not shown). A
control IRF binding element from the HLA-B7 gene promoter (Johnson and Pober, 1994) formed similar complexes and efficiently competed for binding to the VCAM-1
site. These results indicate that the VCAM-1 gene IRF element can bind IRF-2.
To determine which IRF proteins bind the VCAM-1 gene IRF element in C2C12 cells, the IRF element was used in gel retardation assays with nuclear extract from C2C12 cells (Fig. 4). A single major complex was observed, along with several minor, more slowly migrating complexes. The IRF element from the HLA-B7 probe efficiently competed for binding to the VCAM-1 IRF element. Mutation of the IRF site eliminates formation of the specific complexes and, accordingly, the mutant probe did not compete for binding of nuclear proteins to the wild-type probe. Formation of all specific complexes was blocked by the IRF-2 antibody, resulting in accumulation of the nonspecific complex; an irrelevant antibody had no effect. These results demonstrate that IRF-2 is responsible for all of the binding to the IRF site in the VCAM-1 gene promoter in myoblasts, and thus IRF-2 appears to be the IRF family member responsible for controlling VCAM-1 expression in C2C12 cells.
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IRF-2 Transactivates the VCAM-1 Gene Promoter
It was surprising that IRF-2 is the family member that
binds to the VCAM-1 gene promoter in C2C12 cells since
it has been demonstrated to be a transcriptional repressor
in lymphoid cells. However, it has been demonstrated that
IRF-2 contains a latent transcriptional activation domain
that is apparent upon deletion or mutation of the transcriptional repression domain (Yamamoto et al., 1994).
Recent studies have demonstrated that IRF-2 activates transcription, suggesting that the activation domain can
function in the context of the wild-type protein (Vaughan
et al., 1995
; Luo and Skalnik, 1996). To demonstrate that
IRF-2 activates transcription from the VCAM-1 gene promoter, a vector that overexpresses the protein was cotransfected with the
32 or
2.1 VCAMCAT reporter construct into C2C12 myoblasts (Fig. 5). Overexpression
of IRF-2 significantly increased CAT activity (Fig. 5 C),
suggesting that it activates the VCAM-1 gene promoter in
myoblasts through interaction with the IRF element.
|
Although IRF-1 is not normally expressed in the C2C12
cells, it is expressed in response to IFN- treatment (see
above). Therefore, the possibility that cotransfection of an
IRF-1 expression vector would activate the VCAM-1 gene
promoter was examined (Fig. 5 C). Overexpression of
IRF-1 activated the promoter to a level similar to that observed with IRF-2, suggesting that other IRF proteins, if
present, can substitute for IRF-2 in activation of the
VCAM-1 gene promoter.
The Acidic Region of IRF-2 Is Required for Transcriptional Activation
Previous mutational studies of IRF-2 have identified the
basic, COOH-terminal region of IRF-2 (amino acids 325-
349) as a transcriptional repressor motif (Yamamoto et al.,
1994). Deletion or mutation of this inhibitory COOH-terminal region revealed a latent activation domain in the
central region of the protein; this region (amino acids 182-
218) is relatively acidic. However, it is unclear whether this
acidic activation domain is responsible for activation in the
context of the full-length protein. To localize the region of
IRF-2 required for transcriptional activation, a series of
IRF-2 deletion mutants was tested in transfection assays for ability to transactivate the VCAM-1 gene promoter
(Fig. 5, B and C). Deletion of the COOH-terminal region,
which acted as a repressor in other systems, had no effect
on transcriptional activity. However, deletions into the
acidic central domain of the protein eliminated transcriptional activation of the VCAM-1 gene promoter. Therefore, this acidic domain is necessary for transcriptional activation in myoblasts, while the COOH-terminal repressor
motif of the protein appears to be inactive in myoblasts.
IRF-2 deletion mutants that lack the acidic transcriptional activation domain, but contain the DNA binding
domain, should act as a dominant negative and displace
wild-type IRF-2 from the VCAM-1 gene promoter, thereby
blocking transcription (Fig. 5, A and B). Expression of the
DNA binding domain of IRF-2 indeed functioned as a
dominant negative that blocked transactivation by full-length IRF-2 and inhibited transcriptional activity of the
32 and
2.1 VCAMCAT reporter constructs (Fig. 5, C
and D). IRF-2 constructs were transfected into 293 cells,
and nuclear extracts were examined in gel retardation assays for binding to an IRF binding site (Fig. 5 E). The results demonstrate that expression of full-length IRF-2 or
the IRF-2 deletion mutants that contain only the DNA
binding domain results in similar DNA binding activity in
the nucleus. Taken together, the above results provide further evidence of an important role for IRF proteins in activating VCAM-1 gene expression in muscle cells.
Expression of IRF-2 Immediately Precedes VCAM-1 Expression in Differentiating Skeletal Muscle Cells in the Mouse
During skeletal myogenesis in the mouse, VCAM-1-positive cells appear relatively late, around E16 and remain
present until at least postnatal day 2 (Rosen et al., 1992;
Sheppard et al., 1994
). However, no VCAM-1 is detectable on adult muscle fibers. Therefore, we examined
whether IRF-2 expression correlates with VCAM-1 expression. Extracts from embryonic (E16 and E18) limb muscle and adult hindlimb muscle were examined for IRF-2
by Western blot analysis (Fig. 3 A). We found that IRF-2
is indeed expressed in limb muscle during embryogenesis,
but is not expressed in adult skeletal muscle.
Next, we compared the patterns of VCAM-1, IRF-2,
and the myogenic marker myosin heavy chain during mouse
development. At E11, myosin was already evident in the
differentiating somites (Fig. 6 A, a). No immunostaining
for IRF-2 was evident at E11 in the muscle or anywhere
else in the embryo (Fig. 6 A, b). We have demonstrated previously that VCAM-1 does not appear until relatively
late in muscle differentiation process (well after myosin)
(Rosen et al., 1992), and accordingly it is not yet evident at
E11 (Fig. 6 A, c). At E15, expression of IRF-2 was evident
on myogenic cells in skeletal muscle masses (Fig. 6 B, a
and c)
no immunostaining was observed in other areas of
the embryos. No VCAM-1 was evident on low power
views of the muscle (Fig. 6 B, b). At this time, IRF-2 was
only evident on a subset of the myosin-positive cells in the
muscle masses (Fig. 6 B, d-f). Higher power views of the muscle masses showed very low levels of VCAM-1 expression in the muscle masses (Fig. 6 B, g-i). By E18, IRF-2 expression was evident on all skeletal muscle masses (Fig. 6
C, a and b). In contrast to E15, where IRF-2 was only expressed on a subset of myosin-positive cells within a muscle
mass, by E18 all of the myosin-positive cells were IRF-2
positive (Fig. 6 C, c-e). Also by E18, VCAM-1 expression
had increased and the protein was evident around the IRF-2-positive cells in the muscle masses (Fig. 6 C, f-h).
These results suggest that IRF-2 expression precedes that
of VCAM-1 on differentiating muscle cells. These patterns
of IRF-2 immunostaining are consistent with the expression of IRF-2 detected by Western blot analysis described
above. The pattern of IRF-2 expression in the embryos
was surprisingly muscle specific, further suggesting an important role for the protein in the late stages of myogenesis.
|
IRF-2 and VCAM-1 Are Downregulated in Adult Skeletal Muscle Fibers, but Are Retained on Satellite Stem Cells
We have found previously that VCAM-1 expression diminishes in adult muscle fibers; however, the protein is retained on myogenic stem cells known as satellite cells
(Rosen et al., 1992). Therefore, we wondered whether
IRF-2 expression also resembled that of VCAM-1 in adult
mice. Indeed, as with VCAM-1, we found that expression of IRF-1 diminished on adult myofibers, but it persisted on
VCAM-1-positive cells that appeared to be scattered
among the muscle fibers and located under the basement
membrane surrounding the muscle fibers (Fig. 7, A-E). As
we have shown previously, these cells also express NCAM,
which is a marker for satellite cells (Rosen et al., 1992
; Fig.
7, F and G). The above results suggest that lack of IRF-2 on adult muscle fibers may be responsible for the loss of
VCAM-1 expression in adult muscle. Additionally, we
conclude that IRF-2 is expressed in satellite cells and that
this expression may be responsible for the presence of
VCAM-1 on these cells.
|
Expression of VCAM-1 during Muscle Regeneration
In the adult mouse, the myogenic program can be reactivated after muscle injury or in diseases such as muscular
dystrophy. However, myogenesis after muscle injury or
disease is distinct from embryonic myogenesis in that stem
cell-like satellite cells proliferate and fuse to form muscle
fibers. VCAM-1 is expressed on resting satellite cells in
adult muscle; however, to determine whether VCAM-1
continues to be expressed on proliferating satellite cells
and newly forming myotubes after muscle injury, the gastrocnemius muscle in the mouse hindlimb was exposed
surgically and cut. 6 d after the surgery, mice were killed
and muscle in the damaged region and control muscle
from the undamaged hindlimb were removed, and frozen
sections were prepared for immunostaining. Fig. 8, A and
B, shows immunostaining for laminin, which is a component of the basement membrane surrounding individual
muscle fibers, in normal and damaged muscle. Note the
presence of small, newly forming myotubes in the area of
muscle damage. NCAM is a marker of the proliferating
satellite cells and immature myotubes (Rosen et al., 1992).
Neither VCAM-1 nor NCAM were evident on undamaged adult muscle fibers (Fig. 8, C and D). However, the
proteins were coexpressed on what appear to be proliferating satellite cells and immature myotubes formed by the
fusion of these satellite cells (Fig. 8, E and F). Together,
these results indicate that VCAM-1 is expressed during regeneration of myogenic tissue.
|
Expression of VCAM-1 in Regenerating Muscle Tissue Is Closely Associated with Infiltrating Inflammatory Cells
Are IRF-2 and 4
1, the VCAM-1 ligand, also expressed
on proliferating satellite cells and the new myotubes
formed from the fusion of these cells? This question has
proven difficult to address because the muscle injury is accompanied by a large influx of IRF-2-positive,
4
1-positive leukocytes (data not shown), and the number of these
leukocytes makes it difficult to determine whether the regenerating muscle cells themselves also express these proteins. IRF-2 was originally identified in lymphocytes, where it functions as a repressor, and
4
1 was originally
characterized on leukocytes, where it plays an important
role in targeting the leukocytes to the endothelium, which
expresses VCAM-1 in response to inflammatory cytokines.
It is then difficult to determine whether an interaction between VCAM-1 and
4
1 has a role in muscle regeneration as we propose during embryonic development. However,
we noticed that the infiltrating lymphocytes (which were
followed by expression of the leukocyte markers CD45,
CD3, and
4
1) were selectively and very tightly associated with VCAM-1(+) cells (Fig. 8, I-L and data not
shown). None of the lymphocyte markers were evident in
undamaged muscle.
VCAM-1 Is Expressed on Adult Muscle in Dystrophin-deficient Mice, Where It Is also Associated with Infiltrating Leukocytes
Dystrophin-deficient mice are a model for human muscular dystrophy and show continuous muscle damage and regeneration. Sections of gastrocnemius muscle from dystrophin-deficient mice were immunostained for VCAM-1 and CD45 to detect infiltrating leukocytes. As in muscle damage, VCAM-1 was expressed on what appear to be proliferating satellite cells and newly forming myotubes, and this expression was closely associated with infiltrating leukocytes (Fig. 8, M and N; results not shown).
Binding of VCAM-1 on C2C12 Satellite Cells to 4
1
on Lymphocytes Mediates Cell-Cell Interactions
Our results in vivo suggest that infiltrating leukocytes are
being targeted selectively to satellite cells and newly forming myotubes arising from satellite cell fusion at sites of
muscle damage. To determine whether this interaction
might be mediated by VCAM-1 on the satellite cells and
4
1 on infiltrating leukocytes (all except neutrophils), we
asked whether antibodies to
4
1 would block interaction
of lymphocytes with C2C12 satellite cells. C2C12 cells
were grown on tissue culture dishes as monolayers, and
then the T-cell line Ramos was allowed to adhere to the
C2C12 cells. The Ramos cells bound efficiently to the
C2C12 cells, and addition of blocking antibodies
4
1 on
the lymphocytes blocked the cell-cell interactions, whereas
a control antibody to myosin heavy chain has no effect
(Fig. 9, A-E). These results demonstrate that VCAM-1 expression can mediate the adhesion of leukocytes to satellite cells through interaction with
4
1 on the leukocytes.
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Discussion |
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We show that IRF-2 regulates transcription of the VCAM-1 gene in C2C12 cells. In vivo expression of IRF-2 is remarkably muscle specific during embryogenesis and is expressed in a pattern consistent with a regulator of VCAM-1 expression in muscle. IRF-2 appears relatively late in muscle differentiation, after myosin heavy chain expression and just preceding VCAM-1 expression. As with VCAM-1, IRF-2 expression is lost in adult muscle fibers; however, IFR-2 is maintained on adult satellite cells, which we propose is responsible for the expression of VCAM-1 in these muscle stem cells. These studies not only provide a mechanism for how VCAM-1 expression is controlled during myogenesis, they also provide the first evidence of a role for IRF-2 in muscle differentiation and regeneration.
Most transcription factors associated with skeletal myogenesis (i.e., myoD, myf-5, etc.) are expressed early in myoblasts and are thought to drive expression of genes such as
myosin that are important for the differentiation process.
We present evidence here that IRF-2 is expressed after
myosin, and is thus a marker of late stages of myogenesis.
IRF-2 is then the first transcription factor that we are
aware of that specifically marks these late myogenic
stages. We propose that it regulates expression of VCAM-1 (and perhaps other late stage myogenic genes). Myogenesis in the mouse and human occur in two waves (see references in Rosen et al., 1992). Initially, a primary generation
of myoblasts appear and fuse to form primary myotubes.
These primary myotubes are
4
1 positive, but VCAM-1
negative. Then, a second generation of myoblasts appears.
These myoblasts are VCAM-1 positive (and
4
1 negative) and appear to use the interaction with
4
1 on primary myotubes to align themselves along the primary myotubes where they, in turn, fuse into myotubes. Most of the
muscle fibers are then comprised of such secondary myotubes. It is thus conceivable that IRF-2 is a marker of the
second generation myoblasts.
IRF-2 contains both transcriptional activation and repression domains. In lymphocytes, IRF-2 appears to function primarily as a transcriptional repressor (Harada et al.,
1989, 1990
). However, we demonstrate here that in muscle
cells the protein functions as a transcriptional activator. In
L929 cells, it has been shown that deletion of the COOH-terminal repressor domain converts IRF-2 into a transcriptional activator (Yamamoto et al., 1990). These results
suggest that the repressor domain may be dominant over the transactivation domain. Conceivably, a tissue-specific
corepressor molecule may determine whether the repressor domain is active, and in the absence of repressor activity, the molecule functions as a transcriptional activator by
default.
We have previously found that VCAM-1 and 4
1 appear to have a role in myogenesis in culture and in vivo
(Rosen et al., 1992
; unpublished observations). Additionally, here we present evidence of a novel role for VCAM-1
in muscle regeneration. We show that leukocytes are targeted to proliferating satellite cells and newly forming myotubes after muscle injury and disease. In culture, we demonstrate that this interaction is mediated by VCAM-1 on the satellite cells and
4
1 on leukocytes. We propose a
model where VCAM-1 appears on endothelial cells in response to muscle damage, and this is responsible at least in
part for general recruitment of leukocytes to muscle after
injury or disease. After the leukocytes invade the muscle
tissue, they are then focused to sites of regeneration by
their interaction with VCAM-1 on the regenerating muscle cells.
Hepatocyte growth factor (HGF)/scatter factor interacts
with the c-met tyrosine kinase receptor and this can signal
satellite cell proliferation (Anastasi et al., 1996). It is likely
that c-met is responsible for expansion of the myogenic
cell population during development and for stimulating
proliferation of resting satellite cells in response to muscle
injury. c-met also stimulates hepatocyte proliferation during liver regeneration (Ishiki et al., 1992
) and keratinocyte
proliferation during wound healing (Dunsmore et al.,
1996
). Although nothing is known of how the c-met pathway is triggered in response to injury and disease, one
common aspect of muscle damage, liver regeneration, and
wound healing is the influx of leukocytes into the injury/
disease site. It is possible that cytokines or proteases released from the infiltrating leukocytes play a role in triggering expression/activation of HGF/scatter factor or c-met
in the region of VCAM-1-positive satellite cells that proliferate and fuse into myotubes after muscle damage. We
suggest that cytokines released by these leukocytes may
directly activate HGF/scatter factor or cause its release,
which in turn triggers the satellite cell proliferation. One
role of VCAM-1 in such a process would be to target the
infiltrating leukocytes to the sites of muscle regeneration.
These conclusions are supported in tissue culture by the
finding that VCAM-1 on C2C12 satellite cells can efficiently mediate interaction with
4
1-positive lymphocytes. Additionally, although VCAM-1 does not appear to
have any signaling capabilities, its ligand
4
1 on leukocytes does. It has been demonstrated that binding of
4
1
to VCAM-1 can activate a signal transduction cascade in
leukocytes and this interaction, in conjunction with cross-linking of the T-cell receptor, can provide an efficient coactivator role. Therefore, interaction of leukocytes with
VCAM-1 not only provides a mechanism for targeting leukocytes to specific areas for muscle regeneration, it also
provides a trigger for leukocyte activation and the resulting release of cytokines and proteases into the tissue.
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Footnotes |
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Address correspondence to Douglas C. Dean, Campus Box 8069, Washington University School of Medicine, St. Louis, MO 63110. Tel.: (314) 362-8989. Fax: (314) 362-8987. E-mail: dean{at}im.wustl.edu
Received for publication 5 June 1997 and in revised form 3 November 1997.
We thank Dr. J. Hiscott for anti-IRF-1 and anti-IRF-2 antisera, and IRF-1
and IRF-2 expression plasmids, Dr. M. Thomas for anti-CD45 antisera,
Dr. F. Sanchez-Madrid for anti-4 antisera, and Dr. R. Kopan for antimyosin antisera.
These studies were supported by grants to D.C. Dean from the National Institutes of Health.
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Abbreviations used in this paper |
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CAT, chloramphenicol acetyl transferase gene; E, embryonic day; IFN, interferon; IRF, interferon regulatory factor; VCAM-1, vascular cell adhesion molecule-1.
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