(Received for publication, March 24, 1995; and in revised form, May 23, 1995)
From the
Heparin-binding epidermal growth factor-like growth factor
(HB-EGF) gene expression and protein localization were analyzed during
the process of myogenic differentiation. The mouse HB-EGF gene was
isolated, and a 1.8-kilobase genomic fragment flanking the 5` end of
the cDNA was cloned. This fragment contains two sequences which match
the consensus CANNTG sequence for E-boxes, binding sites for the MyoD
family of DNA-binding transcription factors that regulate myogenesis.
Accordingly, HB-EGF synthesis was analyzed in 10T1/2 cells and C2C12
cells which are used commonly for the study of myogenesis. HB-EGF gene
expression was up-regulated in both cell types during myogenesis. In
10T1/2 cells, direct activation of HB-EGF gene expression by MyoD was
shown in that: i) transient transfection of these cells with a plasmid
expressing MyoD resulted in a 10-20-fold increase in endogenous
HB-EGF mRNA levels; ii) co-transfection of MyoD and an HB-EGF
promoter-reporter plasmid resulted in a 5-10-fold increase in
reporter activity, an increase that was abrogated by deletion of a
putative HB-EGF proximal E-box sequence; and iii) incubation of MyoD
protein with a 25-base pair double-stranded oligonucleotide
corresponding to the HB-EGF proximal E-box sequence resulted in
retarded electrophoretic mobility of the oligonucleotide. In C2C12
cells, differentiation of myoblasts into myotubes resulted in a
40-50-fold increase in HB-EGF promoter activity. In addition,
immunostaining and laser confocal microscopy detected HB-EGF protein in
C2C12 myotubes but not in myoblasts. The HB-EGF produced was in its
transmembrane form and localized to the myotube surface. Taken
together, it was concluded that during skeletal muscle cell
differentiation, MyoD plays a direct role in activating HB-EGF gene
expression and that HB-EGF protein is expressed preferentially in
myotubes and in its membrane-anchored form.
HB-EGF ( HB-EGF exists also as a
biologically active membrane-spanning protein. Transmembrane forms of
HB-EGF with molecular masses of 21.5-24 kDa, compared to
14-17-kDa forms of mature HB-EGF have been purified from insect
cells infected with a recombinant baculovirus construct encoding the
entire open reading frame (ORF) of HB-EGF(9) . Transmembrane
HB-EGF (HB-EGF The HB-EGF gene is expressed in a
wide variety of cells and tissues. Northern blot analysis of tissues
indicates that the highest levels of HB-EGF mRNA are found in lung,
skeletal muscle, and heart(15) . HB-EGF gene expression in
cultured cells can be induced, particularly in vascular and
inflammatory cells, by a variety of agents. For example, in cultured
SMC, HB-EGF mRNA levels are increased by phorbol ester, thrombin,
angiotensin II, and growth
factors(16, 17, 18) ; in endothelial cells,
by tumor necrosis factor The inducibility of HB-EGF gene expression in vitro and in vivo prompted us to isolate and characterize the HB-EGF
promoter in order to study the mechanisms of HB-EGF gene regulation.
Recently, the structure of the human HB-EGF gene was reported and shown
to encode the 6 exon/5 intron structure characteristic of members of
the EGF family(24) . In that study, a promoter-reporter
construct composed of a 2.0-kb genomic fragment of human HB-EGF
5`-flanking sequence linked to the bacterial chloramphenicol
acetyltransferase gene (CAT) was active in endothelial cells.
Concurrently, our laboratory has been analyzing the mouse HB-EGF gene
and its promoter. Upon isolating and sequencing a 1.8-kb mouse HB-EGF
genomic fragment that flanks the 5` end of the cDNA, we noticed the
presence of a CAGGTG and a CACCTG sequence located upstream from the
HB-EGF transcription start site. These sequences correspond to the
CANNTG consensus sequence that identifies the
E-box(25, 26, 27) , the DNA binding site for
the MyoD family of transcription factors which currently include
MyoD(28, 29, 30) , myogenin(31) ,
Myf-5(32) , and MRF4, also known as Myf-6 or Herculin (33, 34, 35) . MyoD family members all have a
characteristic basic helix-loop-helix (bHLH) structure and are
expressed exclusively in skeletal muscle. Each of these transcription
factors forms heterodimers with other bHLH proteins known as E proteins
and bind to the E-box consensus sequence, resulting in the
transcription of muscle-specific
genes(25, 26, 27) . The presence of
E-boxes in the HB-EGF promoter and our finding that human, rat, and
mouse skeletal muscle tissues are among the most abundant tissue
sources of HB-EGF mRNA (15) suggested that HB-EGF gene
expression might be associated with myogenesis. In this report, we
demonstrate that, during myogenesis, HB-EGF gene expression is
up-regulated, that MyoD acts directly to transactivate the HB-EGF gene,
that HB-EGF protein can be detected only in myotubes, and that it is
the membrane-anchored form of HB-EGF, localized to the myotube surface,
that is produced preferentially.
The 5` transcriptional start site for HB-EGF mRNA was determined by
primer extension analysis (Fig. 1). Northern blot analysis of
total RNA confirmed a previous report (15) that lung expressed
high levels of HB-EGF mRNA, brain expressed a moderate amount, and
liver did not express any detectable levels (Fig. 1A),
These RNA samples were used as templates for a reverse transcription
reaction (Fig. 1B). Two oligonucleotides of about 40
nucleotides in length corresponding to 5` sequences of mouse HB-EGF
cDNA were used as primers. The primer HB1 (-219 to -179;
see Fig. 2) gave rise to extension products using lung and brain
RNA but not liver RNA (Fig. 1B), and the amounts of
extended product correlated directly to the levels of mRNA expression
as detected by Northern blotting. An adenosine at position -261
relative to the ATG initiation codon and 42 bp upstream of the 5` end
of the cDNA clone, was identified as the first base of the transcript
by comparison to a sequencing reaction using the same primer (Fig. 1B). A similar conclusion was obtained using
primer HB2, which is complementary to sequences downstream of the ATG
initiation codon (+24 to +63, data not shown). By comparison,
the human HB-EGF gene has a major transcription start site at position
-275 relative to the ATG initiation codon, 14 bp upstream of the
5` end of the cDNA sequence(24) .
Figure 1:
Primer extension analysis of the
HB-EGF 5` end transcription start site. A, Northern blot
analysis of total RNA (20 µg/lane) prepared from mouse liver,
brain, and lung tissues, respectively, probed with mouse HB-EGF cDNA.
The position of the 2.5-kb HB-EGF transcript is marked with an arrow. B, primer extension of endogenous HB-EGF transcripts. Lanes 4-6, a primer, HB1, corresponding to the HB-EGF 5`
sequences -219 to -179, was hybridized to 50 µg of
total RNA derived from liver, brain, and lung, respectively, and
extended with avian myeloblastosis virus reverse transcriptase as
described under ``Experimental Procedures.'' The position of
the transcription start site was determined by comparison of the primer
extension product (indicated by an arrow) with the HB1-primed
dideoxy sequencing reaction of the HB-EGF genomic plasmid analyzed in
parallel on the same polyacrylamide gel (right).
Figure 2:
Nucleotide sequence of the mouse HB-EGF
5`-flanking sequence region. A 1.837-kb MboI-NotI
5`-HB-EGF genomic fragment was subcloned and sequenced. The HB-EGF
nucleotide sequence is numbered relative to the first nucleotide of the
ATG initiation codon (+1) for HB-EGF and the corresponding amino
acids are shown in single-letter code. The major transcription
start site is marked with a bent arrow. The sequence of the
oligonucleotide (HB1) used to determine this site by primer extension
analysis is indicated by a bracket. An atypical TATA motif
upstream of the major transcription start site is indicated by a double underline. Potential transcription factor binding sites
are underlined in boldface and labeled. The
nucleotide sequence of the upstream portion of intron 1 is designated
by lowercase letters.
The sequence of the
1,837-bp 5`-flanking region containing the putative HB-EGF promoter was
determined (Fig. 2). Consistent with a single start site, an
atypical TATA motif (TTATT) was located 29 bp upstream of the
transcription start site, although no consensus CCAAT element was
present. The upstream sequences were further analyzed for the presence
of potential binding sites for transcription factors by searching for
motifs that are listed in the SITES table of the transcription factor
data base (45) using the ``pattern'' program of the
GCG program package. Several potential binding sites for the
transcription factor Sp1 were found. In addition, potential MyoD
(E-box), Pit-1, NF-
Figure 3:
Transient expression of HB-EGF promoter
activity in SMC. Top, a schematic depiction of the HB-EGF
promoter-luciferase construct. Top line, the 5`-flanking
sequence region starting from the MboI restriction site at
-1837, together with exon 1 (black box) containing a NotI restriction site and the HB-EGF ATG translation
initiation codon. Bottom line, the HB-EGF promoter-luciferase
construct (pHB-EGF-Luc) in which the luciferase gene is substituted at
the NotI site in HB-EGF exon 1 for the rest of this exon and
its downstream regions. Bottom, induction of pHB-EGF promoter
activity in SMC. FHVSMC were transfected with pHB-EGF-Luc DNA and
plated in the absence(-) or presence of 200 nM phorbol
ester, 2 units/ml human thrombin, or 50 µM lyso-PC. After
40 h, cells were harvested and assayed for luciferase activity. A
promoterless luciferase construct was used as a negative
control.
Figure 4:
MyoD induction of HB-EGF promoter activity
and mRNA expression in 10T1/2 cells. A, HB-EGF promoter
activity. Mouse 10 T1/2 fibroblasts were transfected with pHB-EGF-Luc
plasmid DNA alone(-), or together with either pEMSV-MyoD (MyoD), or pEMSV-MyoD, B
In addition, HB-EGF mRNA levels were increased in 10T1/2
cells by about 10-20-fold upon transfection of the MyoD
expression plasmid, but not the MyoD mutant, as determined by Northern
blot analysis (Fig. 4B). Thus, MyoD activates exogenous
HB-EGF promoter activity and stimulates endogenous mRNA expression as
well. Taken together, these results demonstrate that MyoD activates
HB-EGF gene transcription and that this activation is mediated by the
interaction of MyoD with the HB-EGF promoter.
Figure 5:
E-box-dependent activation of the HB-EGF
promoter. Two HB-EGF promoter-luciferase constructs were prepared,
using either the wild type HB-EGF promoter (panel 1) or an
HB-EGF promoter with the proximal E-box deleted (panel 2). For
comparison, a 2.0-kb bFGF promoter-luciferase plasmid was used (panel 3). The 10T1/2 fibroblasts were transfected with the
appropriate promoter-luciferase plasmid, either alone (▪) or
together with pEMSV-MyoD (&cjs2108;), or with the non-DNA binding MyoD
mutant, pEMSV-MyoD, B
Figure 6:
Electrophoretic mobility retardation of
HB-EGF E-box oligonucleotide by MyoD + E12 proteins. A,
retardation of putative HB-EGF E-box oligonucleotide. Complementary
strands of oligonucleotides were synthesized corresponding to a 24-bp
region containing an E-box binding site in the muscle creatine kinase
promoter (MCK, lanes 1 and 4), a 25-bp
region in the putative HB-EGF proximal E-box (-518 to -493) (HB-EGF, lanes 2 and 5), and the same HB-EGF
E-box sequence but containing 4 substitution mutations in the core
sequence (Mutated, lane 3).
The
specificity of HB-EGF proximal E-box oligonucleotide interactions with
MyoD + E12 was demonstrated further by the competition of binding
that occurred using an excess of unlabeled oligonucleotides (Fig. 6B). The retardation of the radiolabeled muscle
creatine kinase oligonucleotide by MyoD + E12 (Fig. 6B, lane 1) was inhibited competitively
by a 100-fold molar excess of unlabeled HB- EGF proximal E-box
oligonucleotide (Fig. 6B, lane 4). Similarly,
the retardation of the radiolabeled HB-EGF proximal E-box
oligonucleotide (Fig. 6B, lane 2) was
inhibited competitively by a 100-fold molar excess of unlabeled muscle
creatine kinase E-box (Fig. 6B, lane 3). As a
control, no electrophoretic mobility gel shift occurred in the absence
of the MyoD + E12 in vitro translation product (Fig. 6B, lane 5). Taken together, these
results suggest that MyoD + E12 protein is capable of binding
directly to the HB-EGF proximal E-box DNA.
Figure 7:
Transient activation of HB-EGF promoter
activity during C2C12 muscle cell differentiation. Top, light
microscopy at
Figure 8:
Schematic representation of the mouse
HB-EGF gene showing putative HB-EGF protein domains in the cDNA ORF.
HB-EGF is synthesized as a 208-amino-acid precursor consisting of the
following putative domains: signal peptide, propeptide, mature HB-EGF
(the 86 amino acids shown in black with a C-terminal EGF-like
domain containing the 6 cysteine residues characteristic of the EGF
family, mature HB-EGF is released from the precursor after processing),
transmembrane domain, and cytoplasmic domain. Polyclonal rabbit
antibody 3100 is directed against a synthetic peptide corresponding to
the C-terminal 16 amino acids (in brackets).
Figure 9:
Synthesis of HB-EGF-AP protein in stable
C2C12 transfectants. Stable transfectants of C2C12 myoblasts expressing
HB-EGF
Next, the synthesis of endogenous C2C12 HB-EGF protein was examined.
Heparin affinity column analysis of conditioned medium did not detect
any release of HB-EGF by 2.5
Figure 10:
Synthesis of endogenous HB-EGF in C2C12
cells. C2C12 myotubes (a and b) and myoblasts (c and d) were immunostained with anti-HB-EGF antibody 3100
directed against the cytoplasmic tail as depicted in Fig. 8. The
cells were examined by confocal immunofluorescence microscopy (a and c) and by phase contrast microscopy (b and d). Magnification bars are 20 µm. Arrows indicate myotubes that are
immunostained.
In this study, we have demonstrated that HB-EGF gene
expression is induced during myogenesis and that MyoD plays a key role
in modulating HB-EGF transcription during this process. Furthermore,
synthesis of HB-EGF protein is induced and it is the transmembrane form
of HB-EGF, which is produced preferentially, appearing on the myotube
surface. The impetus for analyzing HB-EGF expression in myogenesis was
our isolation, identification, and characterization of the mouse HB-EGF
promoter. It was noted that the mouse HB-EGF promoter has two sequences
located at nucleotide positions -510 (proximal) and -1606
(distal) relative to the first nucleotide of the ATG initiation codon,
respectively, which match the CANNTG consensus sequence for E-boxes
that are found in many muscle-specific gene promoters and enhancers.
E-boxes are binding sites for the class of myogenic bHLH proteins which
are muscle-specific transcription factors that include MyoD, myogenin,
Myf-5, and
MRF4(25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) . The regulation of HB-EGF gene expression by MyoD was analyzed in
10T1/2 cells. These cells are fibroblasts that do not express
functional MyoD but, when transfected with MyoD cDNA, form stable
myogenic clones with the potential to undergo myogenesis(30) .
Using 10T1/2 cells, we have accumulated the following evidence to
demonstrate a role for MyoD in regulating HB-EGF gene expression. (i)
HB-EGF promoter activity and endogenous HB-EGF mRNA levels are not
readily detectable in 10T1/2 cells. However, both are increased
10-20-fold upon transfection of 10T1/2 cells with a MyoD
expression plasmid, but not upon transfection of cells with a MyoD
mutant incapable of binding to DNA. By comparison, myoD gene
expression has no effect on bFGF promoter activity. (ii) The ability of
MyoD to transactivate HB-EGF gene expression is abolished when a mutant
HB-EGF promoter is used in which the putative proximal E-box has been
deleted. Deleting the putative distal E-box lowers MyoD transactivating
activity substantially but not as efficiently as deleting the proximal
E-box. (iii) MyoD + E12 proteins synthesized in vitro interact with a C2C12 cells are a model system for studying myogenesis in
vitro(47) . C2C12 myoblasts can be differentiated into
myotubes in culture and are therefore ideal for analyzing changes that
occur in HB-EGF gene expression, protein synthesis, and localization
during skeletal muscle differentiation. When C2C12 myoblasts are
differentiated into myotubes, there is a 40-50-fold increase in
HB-EGF promoter activity, consistent with the increase in HB-EGF gene
expression that occurs concomitant with expression of MyoD in 10T1/2
cells. Analysis of HB-EGF protein synthesis is made more complicated
because this growth factor, like other members of the EGF
family(4) , is synthesized as a transmembrane protein,
HB-EGF The role of
HB-EGF in myogenesis appears to differ from those of other growth
factors such as FGF (49, 50, 51) and
transforming growth factor-
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
L36024[GenBank],
L36025[GenBank],
L36026[GenBank], and
L36027[GenBank].
)is a member of the EGF family that was
identified initially in the conditioned medium of macrophages and
macrophage-like U-937 cells and subsequently purified and cloned (1, 2, 3) . This growth factor, like other
members of the EGF family(4) , is synthesized as a
membrane-anchored precursor that can be processed to release the
soluble mature form. Mature HB-EGF induces autophosphorylation of the
EGF receptor (2) and is a potent mitogen for fibroblasts,
smooth muscle cells (SMC), and keratinocytes, but not for endothelial
cells(1, 2, 3, 5) . The growth
factor was named HB-EGF because it binds tightly to immobilized
heparin, unlike EGF and transforming growth factor-
(2) . A
heparin binding domain of 21 amino acids has been identified in HB-EGF
that mediates interaction with cell surface heparan sulfate
proteoglycans(6, 7) . These interactions modulate
HB-EGF bioactivity, for example, by greatly enhancing its chemotactic
activity for SMC (6) and in enhancing cell toxicity when fused
to a Pseudomonas toxin(8) .
) is cell-associated (10, 11) and biologically active while tethered to the
cell surface. For example, HB-EGF
stimulates
phosphorylation of the EGF receptor and proliferation of adjacent cells (11) and may mediate adhesion of mouse
blastocysts(12) . In addition, HB-EGF
is the
receptor for diphtheria toxin(13) , and cells expressing the
HB-EGF
/diphtheria toxin receptor are sensitive to the
toxic effects of diphtheria toxin(10, 13) . When
HB-EGF
is processed to release soluble mature HB-EGF by
phorbol esters, cells become diphtheria toxin-resistant, evidence for
loss of the membrane-anchored form of HB-EGF(10) . Recently,
the diphtheria toxin binding domain of HB-EGF has been localized to the
EGF-like region (14) .
(19) and lysophosphatidylcholine
(lyso-PC)(20) ; in monocytes/macrophages by phorbol esters and
lyso-PC (3, 21) ; and in CD4
T
lymphocytes by serum(22) . HB-EGF gene expression is regulated in vivo as well. For example, HB-EGF mRNA and/or protein
levels are increased in response to injury (5) and in response
to hyperoxia which leads to pulmonary hypertension (23) .
Materials
Minimum essential medium (MEM), Dulbecco's modified
Eagle's medium (DMEM), horse serum, RPMI 1640, and G418-sulfate
(geneticin) were obtained from Life Technologies, Inc. Fetal calf serum
(FCS) was obtained from Intergen (Purchase, NY). Iron-fortified bovine
calf serum was obtained from JRH Bioscience (Lenexa, KS). GPS
(100 = 29.2 mg/ml glutamine, 10,000 units/ml penicillin,
10 mg/ml streptomycin) was obtained from Irvine Scientific. Human
recombinant thrombin and phorbol ester were purchased from Sigma.
Lyso-PC (palmitoyl, C16:0) was purchased from Avanti Polar Lipids.
Mouse liver, brain, and lung tissue were isolated from C57BL/6 mice
purchased from The Charles River Laboratories. Anti-alkaline
phosphatase antibody 1801 was obtained from Biomedix Biotech Inc.
(Foster City, CA). FITC antibody was purchased from Sigma. The cloning
and characterization of mouse HB-EGF cDNA has been described
previously(15) . MyoD (pEMSV-MyoD) and MyoD mutant (pEMSV-MyoD,
B
PB
, a single point amino acid mutation in the
DNA binding region) expression plasmids were gifts from Dr. Andrew
Lassar (Harvard Medical School)(28, 29, 36) .
The E12 expression plasmid was a gift of Dr. Jian Wang
(Children's Hospital, Boston, MA). The bFGF promoter construct,
P2.0 CAT(37) , was a gift of Dr. Robert Florkiewicz (Whittier
Institute, La Jolla, CA). The luciferase vector (pGL2-basic) was
purchased from Promega.
Cell Culture
Fetal human vascular smooth muscle cells (FHVSMC) were
maintained in DMEM/10% FCS/PS as described previously(16) .
C3H10T1/2 (10T1/2) fibroblasts were obtained from Dr. Jian Wang
(Children's Hospital, Boston, MA) and were maintained in DMEM/15%
FCS/PS. After transfection with the MyoD expression plasmids, 10T1/2
cells were transferred to differentiation medium (DMEM/2% horse
serum/PS) for analysis of HB-EGF promoter activity and mRNA expression.
C2C12 myoblasts were obtained from Dr. Jian Wang (Children's
Hospital, Boston MA) and were maintained in DMEM supplemented with 20%
FCS for growth. To differentiate C2C12 myoblasts into myotubes, cells
were grown to 100% confluence in growth medium, washed with
phosphate-buffered saline (PBS), and switched to differentiation medium
(DMEM/2% horse serum/PS). Differentiation occurred within 48 to 72 h.Cloning of Genomic DNA
A mouse adult DBA/2J liver genomic library cloned into
EMBL3 (Catalog No. ML 1009d) was purchased from Clontech. The
library was screened with a
P-labeled (nick translation)
1.6-kb EcoRI-KpnI fragment derived from the 3` end of
mouse HB-EGF cDNA or with a 470-bp
P-labeled probe
generated by polymerase chain reaction that corresponded to the 5` end
of mouse HB-EGF cDNA using methods previously described(38) .
Hybridization was carried out as described previously(16) .
Washes were performed with 2
SSC plus 0.1% SDS at 65 °C.
Positive clones were digested with restriction enzymes SalI
and EcoRI and subcloned into a Bluescript KS II(+) vector
(Stratagene). Clones containing HB-EGF exons were identified by
Southern blotting using the mouse HB-EGF cDNA as a probe. Insert
fragments were subsequently sequenced from double-stranded templates by
the dideoxy method (39) using specific oligonucleotides as
primers. The genomic nucleotide sequence has been submitted to GenBank
(accession numbers L36024, L36025, L36026, and L36027).
Primer Extension Analysis
Primer extension analysis was carried out as described
previously(40) . Briefly, two specific oligonucleotides
complementary to the HB-EGF cDNA sequences, HB1 (5`-
CAGAGTCGGTCCGCGCGTCCACTCCAGCCCTTGAAGGTCTG-3`, corresponding to
sequences -219 to -179) and HB2
(5`-CACCAACGCGGACAACACTGCGGCCAGAAAGAGCTTCAGC-3`, corresponding to
sequences +24 to +63) were first end-labeled with T4
polynucleotide kinase and [gamma-P]ATP (DuPont
NEN). Labeled primer (1
10
cpm) was hybridized to
50 µg of total RNA derived from mouse lung, heart, and liver
overnight at 42 °C in 50% formamide, 40 mM PIPES, pH 6.4,
400 mM NaCl, 1 mM EDTA, pH 8.0. The annealed primer
was then extended with avian myeloblastosis virus reverse transcriptase
(U. S. Biochemical Corp.) as described previously (40) . The
resulting products were treated with RNase A (1 mg/ml) for 30 min at 37
°C, extracted with phenol-chloroform, precipitated with ethanol,
and separated on a 6% polyacrylamide-8 M urea gel. For precise
mapping of the initiation site, a sequencing reaction of the promoter
genomic clone using the same HB1 primer was run next to the primer
extension on the same polyacrylamide gel.
Northern Blot Analysis
Total RNA was isolated from tissues or cells by
homogenization in RNAzol ``B'' solution (Tel-Test
``B'', Inc., Friendswood, TX) followed by a ``single
step'' isolation method(41) . Northern blot analysis was
carried out as described previously with minor
modifications(16) .HB-EGF Promoter-Luciferase Activity
HB-EGF promoter-reporter constructs were made using the
vector pGL2-basic (Promega), a plasmid which contains the firefly
luciferase gene but no promoter. The HB-EGF promoter-luciferase
construct, pHB-EGF-Luc, was made by ligating a 1.7-kb MboI-NotI fragment derived from the 5` end of the
HB-EGF genomic clone (corresponding to sequences -1837 to
-155) into the KpnI/BglII site of the
polylinker situated in front of the luciferase gene. PGK-lacZ,
a LacZ reporter, plasmid was used to correct for transfection
efficiency. For transfection with HB-EGF-luciferase constructs as well
as promoterless PGL-2-basic and pCMV-luciferase controls, cells (SMC,
C2C12, 10T1/2) at 90% confluence (1.0 10
cells/transfection) were harvested by trypsinization, resuspended
in RPMI 1640/PS without FCS, and electroporated with 20 µg of
luciferase plasmids and 2 µg of PGK-lacZ DNA using a gene
pulser at settings of 960 µF and 200 V (Bio-Rad). Transfected cells
were plated in DMEM/10 or 20% FCS/PS, and, 20 h later, lysates were
prepared and equal amounts of protein were assayed for luciferase
activity using a commercially available kit (Luciferase Assay System,
Promega). Luciferase activity was measured with a luminometer (BioOrbit
1251; Pharmacia Biotech) for 30 s at 25 °C immediately following
the addition of luciferase substrate (luciferin). lacZ activity was assayed using a
-Galactosidase Enzyme Assay
System kit (Promega). To measure the induction of HB-EGF promoter
activity in FHVSMC in response to a variety of agents, cells were
electroporated with HB-EGF promoter-luciferase constructs and were
plated in the absence or presence of either 200 nM phorbol
ester, 2 units/ml human thrombin, or 50 µM lyso-PC for 48
h before being harvested for measurement of luciferase activity. To
measure HB-EGF promoter activity in C2C12 cells during differentiation,
cells were transfected first with HB-EGF promoter-reporter plasmids and
then plated into 6 well plates in growth medium (DMEM/20% FCS/PS). To
promote differentiation, wells were switched to differentiation medium
(DMEM/2% horse serum/PS) the next day. Differentiation occurred in
about 3 days at which point luciferase activity was measured. To
measure HB-EGF promoter activity in response to MyoD expression, 10T1/2
fibroblasts were co-transfected with 10 µg of HB-EGF
promoter-reporter plasmid and 20 µg of MyoD expression plasmids.
Transfected cells were plated immediately into differentiation medium
(DMEM/2% horse serum/PS), and, 24 h later, cell lysates were prepared
and assayed for luciferase activity. A bFGF-promoter-luciferase
construct was made by ligating a 2.0-kb SalI-XhoI
fragment of pF2.0 CAT into the XhoI site of pGL2 basic, and
the correct orientation of clones was screened by restriction analysis.
HB-EGF Promoter E-box Deletion Mutants
To prepare an HB-EGF promoter-luciferase construct in which
the E-box most proximal to the transcription initiation site was
deleted, a genomic subclone that contains a 580-bp XbaI/NotI promoter fragment (containing sequences
-646 to -155) cloned in pBluescript was digested with the
restriction enzymes XcmI and PpuMI to delete the
sequences between -605 and -428. The DNA was then
blunt-ended with Klenow, and the largest fragment was isolated and
self-ligated. The insert of the resulting clone was released by
restriction digestion with XbaI/SacII and ligated
into the corresponding XbaI-SacII site of the wild
type HB-EGF promoter-Luc construct.Electrophoretic Mobility Shift Assays
Synthesis of MyoD + E12 Proteins in
Vitro
To prepare MyoD + E12 protein for electrophoretic
mobility shift analysis, transcription and translation reactions were
carried out in vitro using a commercially available kit,
TNT T3 Coupled Reticulocyte Lysate System
(Promega) according to the manufacturer's instructions. One
µg each of MyoD and E12 cDNA plasmids were incubated with
TNT rabbit reticulocyte lysate in the presence of T3 RNA
polymerase and a mixture of amino acids lacking leucine and methionine
in a 50-µl total volume at 30 °C for 120 min. To monitor MyoD
+ E12 protein production,
S-labeled proteins were
synthesized in parallel using L-[
S]methionine (>800 Ci/mmol;
DuPont NEN) and analyzed on a 10% SDS-polyacrylamide gel
electrophoresis polyacrylamide gel.
Oligonucleotides
Complementary strands of
oligonucleotides were synthesized corresponding to: (i) an HB-EGF
promoter sequence encompassing -518 to -493 that contains
the putative proximal E-box binding site
(5`-CGGTCTTCGCCACCTGCTGCTGGTCATTC-3`, (ii) a similar sequence but
mutated by making substitutions in the E-box core
(5`CGGTCTTCTCTAGATGCTGGTCATTC-3`), and (iii) a bona fide MyoD E-box
binding site obtained from the muscle creatine kinase enhancer
(5`-GATCCCCCAACACCTGCTGCCTGA-3`)(29) . The oligonucleotides
were first labeled with [-
P]ATP (DuPont
NEN) using T4 polynucleotide kinase (New England Biolabs) and then
annealed to form double-stranded oligonucleotides.
Gel Shift Assays
Assays were performed as
described previously(28) . Briefly, 5 µl of MyoD + E12
proteins synthesized in vitro were mixed with an additional 5
µl of fresh reticulocyte lysates and incubated for 20 min at 37
°C. Ten µl of 2 concentrated DNA binding mixture were
then added such that the final concentrations of each component were 20
mM HEPES, pH 7.6, 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 2 µg of
poly(dI-dC) as a nonspecific competitor (Pharmacia), and 1
10
cpm (0.1-0.3 ng) of
P-labeled
double-stranded probe. Following incubation for 15 min at room
temperature, the reaction mixtures were separated on a 5%
polyacrylamide gel in 1
TBE buffer (50 mM Tris, 50
mM boric acid, 1 mM EDTA). The polyacrylamide gel was
subsequently dried and exposed to Kodak x-ray AR film at -70
°C with an intensifying screen. For competition analysis, 100-fold
molar excess of unlabeled double-stranded oligonucleotides were
preincubated with the MyoD + E12 proteins on ice for 10 min before
the addition of the radiolabeled probe.
Expression of HB-EGF-Alkaline Phosphate Fusion
Proteins in C2C12 Cells
A construct encoding an HB-EGF-AP fusion protein
was initially prepared under the control of a cytomegalovirus promoter
for studies not reported here and then converted to a similar construct
but under control of the HB-EGF promoter. Briefly, full-length human
HB-EGF cDNA encoding the entire 208-amino-acid ORF (2) was
amplified by polymerase chain reaction using synthetic DNA
oligonucleotide primers (P20: GCT CTA GAC CAT GAA GCT GCT GCC GTC G and
P21: GCT CTA GAT CAG TGG GAA TTA GTC AT) and ligated into the XbaI site of the plasmid pRc/CMV (Invitrogen). In order to
obtain HB-EGF
epitope-tagged in the ectodomain, a 1.5-kb BglII-XbaI fragment of plasmid APtag-I (42) was blunt-ended with Klenow polymerase and then ligated
into the MscI site of the 208-amino-acid human HB-EGF ORF to
construct pHB-EGF
-AP. This manipulation created an
HB-EGF
-AP fusion protein which has the AP sequence
inserted in-frame between Leu
and Thr
of the
HB-EGF ORF, a position N-terminal to the heparin-binding region of
mature HB-EGF(2, 6, 7) . To express the
AP-tagged HB-EGF under the control of the mouse HB-EGF promoter, the
cytomegalovirus promoter of pHB-EGF
-AP was replaced by the
HB-EGF promoter. pHB-EGF
-AP was digested with restriction
enzymes BglII and HindIII to release the
cytomegalovirus promoter. The promoterless fragment was blunt-ended
with Klenow and ligated to a BglII linker (New England
Biolabs). The 1.8-kb MboI-NotI HB-EGF promoter
fragment was blunt-ended similarly with Klenow polymerase and ligated
to the BglII linkers (New England Biolabs). These two
fragments were subsequently digested with BglII, gel-purified,
and ligated to each other. The orientation of the clones was screened
by restriction enzyme analysis. The plasmid carrying the fusion gene
under control of the HB-EGF promoter was transfected into C2C12
myoblasts by electroporation. After 48 h, C2C12 myoblasts were
transferred to selective medium (600 µg/ml G418 sulfate).
G418-resistant clones were isolated, and each was cultured in
duplicate. One of the duplicate myoblast cultures was differentiated
into myotubes, and these were screened for the expression of HB-EGF-AP
using a monoclonal antibody directed against AP as described below. For
clones positive in expressing HB-EGF
-AP, the replicate
myoblast cultures were expanded and maintained in culture.
Immunostaining and Confocal Microscopy
Immunostaining of Myosin
To monitor the
differentiation of C2C12 myoblasts into myotubes in culture, cells were
stained with a monoclonal antibody directed against skeletal muscle
myosin heavy chain (My-32, Sigma) and analyzed with a VECTASTAIN ABC
Elite Kit (Vector Laboratories) as described previously(43) .Immunostaining of HB-EGF
C2C12 myoblasts were
grown and differentiated on coverslips. Both myoblasts and myotubes
were washed twice with ice-cold PBS, 1 mM MgCl,
0.1 mM CaCl
and fixed for 20 min at room
temperature in 2% paraformaldehyde/HEPES buffer(44) . Fixed
cells were washed twice with PBS, 1 mM MgCl
, 0.1
mM CaCl
, 100 mM Tris-HCl, pH 7.4, and
incubated for 30 min at 37 °C in normal goat serum to block
nonspecific antibody binding. For immunostaining of HB-EGF-AP, cells
expressing this fusion protein were incubated with monoclonal anti-AP
antibody 1801 (28 ng/µl) in PBS, 1 mM MgCl
, 1
mM CaCl
, 1% bovine serum albumin, 1% normal goat
serum (staining buffer) for 30 min at 37 °C, washed several times
with staining buffer at room temperature, incubated with the
FITC-conjugated secondary antibody in the same buffer for another 30
min at 37 °C, washed with staining buffer, and washed once with
PBS. Coverslips were mounted onto microscope slides using gel mount
(Biomeda, Foster City, CA), 2.5%
1,4-diazobicyclo-[2.2.2]-octane, and the slides were sealed
with nail polish. For immunostaining of endogenous mouse
HB-EGF
, an antibody(3100) directed against the cytoplasmic
domain of the human HB-EGF precursor was used(2, 15) .
This antibody had been prepared by immunizing rabbits (Lampire
Biological Laboratories, Pipersville, PA) with a synthetic peptide
representing the 16 C-terminal amino acids of the human HB-EGF
precursor (ORF amino acids 193-208, DVENEEKVKLGMTNSH) and had
been shown previously to detect the transmembrane form but not the
secreted mature form of HB-EGF(9) . To prepare an IgG fraction,
3 ml of serum were mixed 1:1 with 20 mM sodium phosphate, pH
7.0, and applied to a protein G-Sepharose column (Pharmacia). The
column was washed with the same buffer and eluted with 3 ml of 0.1 M glycine-HCl pH 2.7. Fractions were neutralized immediately,
pooled, and stored at -80 °C. C2C12 cells were grown,
differentiated, and fixed as described above. They were permeabilized
with PBS, 0.1% Triton X-100 for 5 min at room temperature and washed
several times with PBS. To reduce nonspecific binding, the primary
antibody and the secondary FITC-conjugated antibody were preincubated
on paraformaldehyde-fixed C2C12 myoblasts at 37 °C for 2 h and 4 h,
respectively. Cells were blocked as described above and then incubated
with IgG-purified polyclonal antibody 3100 (3.6 mg/ml) in staining
buffer for 1 h at 37 °C. Cells were washed for 30 min at room
temperature with staining buffer and incubated with the secondary
antibody conjugated to FITC diluted in staining buffer for 1 h at 37
°C. Washing and mounting of coverslips was as described above.
Laser Confocal Microscopy
Samples were examined
using a confocal imaging system (Bio-Rad MRC 600 attached to a Zeiss
Axiovert inverted microscope (Carl Zeiss Inc., Thornwood, NY) with a
100X Zeiss Plan-Neofluar objective). The fluorescence of the
FITC-conjugated secondary antibody was excited using the 488 nm line of
the argon-krypton mixed gas laser in the confocal microscope. The
confocal parameters of scan rate, aperture, gain, black level, and
frames accumulated were the same for all samples.
Characterization of the Mouse HB-EGF Promoter
In
order to analyze the mechanisms that regulate mouse HB-EGF gene
expression, the mouse HB-EGF promoter was isolated and characterized.
This was accomplished by screening a phage library using probes
derived from various portions of the cDNA fragment. One phage with an
insert of 16-18 kb in size was isolated using a 470-bp polymerase
chain reaction-generated probe corresponding to the 5` end of the cDNA.
This phage contained approximately 1.8 kb of upstream sequences 5` to
exon 1 in addition to all six exons of the HB-EGF gene. The phage
insert was excised by restriction digest, subcloned, and sequenced.
B, and AP-1 binding sites were also located
within the upstream 1,837-bp region.
Transcriptional Activity of an HB-EGF-Luciferase
Promoter-Reporter Construct
To determine whether the genomic
fragment flanking the 5` end of mouse HB-EGF cDNA had promoter
activity, a construct was made (pHB-EGF-Luc) that placed the firefly
luciferase gene downstream from a HB-EGF genomic fragment beginning at
a MboI site(-1837) and terminating at a 3`-NotI
site(-155) as shown schematically in Fig. 3. HB-EGF
promoter activity was assayed by measuring luciferase activity in cell
extracts prepared 40 h after transient transfection of the
promoter-reporter constructs into cells. HB-EGF promoter activity could
be demonstrated in FHVSMC and was inducible by phorbol ester, thrombin,
and lyso-PC. Compared to the nontreated control, phorbol ester,
thrombin, and lyso-PC increased promoter activity 6-, 3-, and 5-fold,
respectively (Fig. 3). These inducers have been demonstrated
previously to increase HB-EGF mRNA levels in SMC to the about same
degree (16, 17, 18) suggesting that the
HB-EGF promoter-reporter transfection assay used in our studies
measures transcriptional activity accurately.
MyoD Transactivates the HB-EGF Promoter and Induces Gene
Transcription
Since the HB-EGF promoter contains 2 E-boxes,
putative binding sites for the MyoD family of transcription factors, we
were interested in determining if MyoD regulated HB-EGF gene
expression. Mouse 10T1/2 fibroblasts were used for these experiments
because their endogenous myoD gene is methylated and is
therefore nonfunctional. Upon expression of functional exogenous MyoD,
these cells are converted into skeletal muscle-like cells(30) .
A relatively low amount of HB-EGF promoter activity was detectable in
10T1/2 cells (Fig. 4A). However, co-transfection of
10T1/2 cells with a MyoD expression plasmid, pEMSV-MyoD, together with
pHB-EGF-Luc DNA, resulted in an 8-10-fold increase in HB-EGF
promoter activity. As a control, co-transfection with a non-DNA binding
mutant of MyoD (pEMSV-MyoD, BproB
) which has a
single point amino acid mutation in the DNA binding
region(29) , did not increase HB-EGF promoter activity. For
comparison, co-expression of MyoD together with bFGF promoter-Luc DNA
had no effect on bFGF promoter activity in these cells (data not
shown).
PB
, a mutant
MyoD expression plasmid incapable of binding to DNA (MyoD
Mutant). The transfected cells were plated into differentiation
medium, and, 24 h later, cell extracts were assayed for promoter
reporter activity. B, Northern blot analysis. 10T1/2
fibroblasts were transfected with pEMSV-MyoD, B
PB
(MyoD Mutant) (lane 1) or pEMSV-MyoD plasmid (MyoD) (lane 2) and plated into differentiation
medium. After 72 h, total RNA was prepared and analyzed by Northern
blot using an HB-EGF cDNA probe. The arrow indicates the
position of the 2.5-kb HB-EGF mRNA transcript. Each lane contains equal
amounts of total RNA as ascertained by ethidium bromide
staining.
E-box Deletions Result in Lowered Activation of the
HB-EGF Promoter by MyoD
The HB-EGF promoter has a CACCTG
sequence starting at -510 (proximal) and a CAGGTG sequence
starting at -1606 (distal) (Fig. 2), which correspond to
the CANNTG consensus E-box sequence and are thus potential binding
sites for MyoD. A deletion mutant lacking the proximal E-box was
prepared by removing the sequence -605 to -428 of the
HB-EGF promoter using convenient restriction sites. The 10T1/2 cells
were co-transfected with the MyoD expression plasmid, pEMSV-MyoD,
together with either wild type HB-EGF promoter or the proximal E-box
deletion mutant (Fig. 5). Co-expression of MyoD resulted in a
5-6-fold increase in wild type HB-EGF promoter activity compared
to controls in which the myoD gene was not co-expressed, or in
which a non-DNA binding mutant of MyoD was co-expressed (Fig. 5, panel 1). The transactivating effect of MyoD expression was
abolished when cells were co-transfected with the HB-EGF proximal E-box
deletion mutant (Fig. 5, panel 2). Deletion of the
distal E-box diminished HB-EGF promoter activity also, but not as
effectively with about a 3-fold decrease occurring (data not shown).
The bFGF promoter, consistent with not having any E-boxes, was not
transactivated by MyoD at all (Fig. 5, panel 3). It was
concluded that MyoD activates HB-EGF transcription by interaction
primarily with the proximal E-box located in the HB-EGF promoter.
PB
(&cjs2113;). The
transfected cells were plated into differentiating medium and, after 24
h, cell extracts were prepared and assayed for promoter reporter
activity.
The Proximal HB-EGF E-box Binds MyoD + E12 Proteins
in Vitro
In order to test the ability of the HB-EGF proximal
E-box sequence to bind MyoD directly, the interaction of MyoD protein
and a P-labeled 25-bp double-stranded oligonucleotide
representing the proximal E-box and the surrounding regions of the
HB-EGF promoter was analyzed for an electrophoretic mobility shift on a
polyacrylamide gel (Fig. 6). Incubation of the radiolabeled
HB-EGF proximal E-box oligonucleotide probe with MyoD + E12
proteins synthesized in vitro resulted in the formation of a
complex (arrow) that was retarded in its electrophoretic mobility (Fig. 6A, lane 2). This complex had the same
mobility as a complex formed by incubation of MyoD + E12 proteins
with a radiolabeled oligonucleotide probe corresponding to a bona fide
muscle creatine kinase E-box (Fig. 6A, lane
1). When four base substitutions were made in the HB-EGF promoter
proximal E-box, no retardation of the oligonucleotide was observed (Fig. 6A, lane 3). However, we noted that
lower molecular mass complexes were formed with HB-EGF and muscle
creatine kinase E-box oligonucleotides even in the absence of MyoD
+ E12 (Fig. 6A, lanes 4 and 5).
Supershifting of the complex by binding MyoD + E12 was less
efficient with the HB-EGF oligonucleotide (Fig. 6A, lane 2) than with the muscle creatine kinase oligonucleotide (Fig. 6A, lane 1) suggesting that the HB-EGF
E-box sequence has a slightly lowered affinity for MyoD + E12
protein compared to the muscle creatine kinase E-box sequence.
P-Labeled
oligonucleotides were incubated with MyoD + E12 protein
synthesized in vitro (lanes 1-3) or, as a
control, in the presence of products of an in vitro transcription/translation reaction lacking MyoD + E12 RNA (lanes 4 and 5). The reaction products were separated
on a 5% polyacrylamide gel and analyzed by autoradiography. The
position of the retarded oligonucleotide/MyoD + E12 complex is
marked by an arrow. B, competition of retardation with excess
unlabeled oligonucleotides.
P-Labeled muscle creatine
kinase E-box (lanes 1 and 4) and HB-EGF E-box
oligonucleotides (lanes 2 and 3) were incubated with
MyoD + E12 protein synthesized in vitro in the absence of (lanes 1 and 2) or presence of 100-fold molar excess
unlabeled muscle creatine kinase E- box (lane 3) or HB-EGF
E-box (lane 4) oligonucleotides. A control in which
transcription/translation in vitro was carried in the absence
of MyoD + E12 RNA is shown in lane
5.
Regulation of HB-EGF Expression during Skeletal Muscle
Cell Differentiation
The induction by MyoD of HB-EGF promoter
activity and mRNA levels in 10T1/2 cells suggests that the expression
of the HB-EGF gene is induced during skeletal muscle differentiation.
To test this possibility, HB-EGF gene expression was analyzed in C2C12
cells. C2C12 myoblasts differentiate into myotubes when starved of
serum. This process is characterized by the fusion of myoblasts into
multinucleated cells which are immunostained strongly by antibodies
directed against myosin heavy chain (Fig. 7, top).
HB-EGF promoter activity was measured after transient transfection of
pHB-EGF-Luc DNA in myoblasts and subsequent differentiation into
myotubes (Fig. 7, bottom). No promoter activity was
detected in myoblasts. However, differentiation of the myoblasts into
myotubes was accompanied by a 40-50-fold increase in HB-EGF
promoter activity. By comparison, there was little induction of bFGF
promoter activity during the differentiation process (Fig. 7, bottom) consistent with previous findings that endogenous
levels of FGF decrease during myogenesis(46) .
500 magnification of a culture of C2C12 myoblasts (left) and a culture of C2C12 myoblasts differentiated into
myotubes by transfer from growth medium to low serum medium (right). After 4 days, C2C12 myoblast and myotube cultures
were stained for skeletal muscle myosin heavy chain using a monoclonal
antibody and avidin-biotin-peroxidase reagents that produce a
reddish-brown precipitate. Bottom, C2C12 myoblasts were
transfected with either a promoterless luciferase gene, pHB-EGF-Luc
DNA, or pbFGF-Luc DNA and plated into growth medium (left) or
differentiation medium (right). After 4 days, C2C12 cell
myoblast (left) and myotube cultures (right) were
harvested and assayed for promoter reporter activity as in Fig. 3.
Differentiated C2C12 Skeletal Muscle Cells Synthesize the
Transmembrane Form of HB-EGF
Given the large increase in HB-EGF
promoter activity associated with C2C12 cell differentiation, the next
objective was to determine whether HB-EGF protein, and if so which
form, was expressed during myogenesis. HB-EGF is synthesized as a
cell-associated membrane-spanning precursor containing a mature HB-EGF
domain and a transmembrane domain (Fig. 8). The transmembrane
form of HB-EGF can be processed to release soluble mature HB-EGF, and
cells can produce both forms. For example, some epithelial cell lines
possess mostly the transmembrane form which is associated with the cell
surface(10, 11) , while other cell types such as
monocytes/macrophages and T lymphocytes mostly release the mature form
of HB-EGF(2, 22) . The induction of HB-EGF protein
expression and its localization during C2C12 cell differentiation was
examined by immunostaining. Two approaches were used to localize HB-EGF
in C2C12 myoblasts and myotubes. In the first, C2C12 cells expressing
epitope-tagged HB-EGF-AP were prepared to take advantage of the highly
specific monoclonal anti-AP antibodies that are available commercially.
To do this, pHB-EGF-AP under control of the HB-EGF
promoter and with the the AP being positioned in the ectodomain of
transmembrane HB-EGF was transfected into C2C12 myoblasts, and stable
G418-resistant myoblast clones were selected. Cultures of myoblast
transfectants were differentiated into myotubes by exposure to low
serum. Both myoblasts and myotubes were incubated with the anti-AP
antibody and examined by confocal fluorescence microscopy (Fig. 9). The myotubes were stained by anti-AP antibody, with
most of the staining occurring on the membrane surface (Fig. 9a). On the other hand, myoblasts were not
stained (results not shown). In neither cell type was immunoreactive
HB-EGF-AP detected in the extracellular matrix (results not shown).
-AP under control of the HB-EGF promoter were
differentiated into myotubes in low serum media. Myotubes were
immunostained with anti-AP antibodies and examined by confocal
immunofluorescence microscopy (a) and by phase contrast
microscopy (b). Magnification bars are 20 µm. Arrows indicate myotubes that are
immunostained.
10
myoblasts or
myotubes (data not shown) suggesting that any endogenous HB-EGF
expressed by C2C12 cells would have to be either transmembrane or
associated with the extracellular matrix. Accordingly, C2C12 myoblasts
and myotubes were incubated with an antibody, 3100, directed against
the C-terminal 16 amino acids of the HB-EGF ORF which are in the
cytoplasmic tail as shown in the schematic in Fig. 8. This
antibody has been shown previously by Western blot to recognize only
the transmembrane and not the mature form of HB-EGF(9) . As
demonstrated by laser confocal microscopy, antibody 3100 stained the
surface of myotubes (Fig. 10a) but did not stain
myoblasts at all (Fig. 10c). In some myotubes,
transmembrane HB-EGF was localized in patches (Fig. 10a, arrows). Antibody 3100 did not
stain the extracellular matrix (data not shown). The combination of
heparin affinity column analysis and immunostaining suggest that
myoblasts do not synthesize HB-EGF protein and that, upon
differentiation, C2C12 myotubes are induced to express HB-EGF, which
appears as HB-EGF
along the myotube surface.
P-labeled oligonucleotide
representing the HB-EGF proximal E-box to form a complex that is
retarded in its electrophoretic mobility on a polyacrylamide gel. This
retarded complex co-migrates with one formed by interaction of MyoD
+ E12 proteins with a bona fide muscle creatine kinase gene
enhancer E-box sequence. Formation of either retarded complex is
inhibited competitively by excess unlabeled E-box oligonucleotides,
corresponding to either HB-EGF or muscle creatine kinase E-boxes. Taken
together, it appears that MyoD up-regulates HB-EGF transcriptional
activity in 10T1/2 cells by direct interactions with E-box sequences
found in the HB-EGF promoter. Although MyoD is a well-characterized
regulator of muscle-specific genes, activation of a growth factor gene
by MyoD has not yet been reported. It should be noted that MyoD might
not be the only regulator of HB-EGF transcription. Other bHLH proteins
such as myogenin, Myf-5, and MRF4 might also regulate HB-EGF
expression, but we have not analyzed these transcription factors yet.
, that can be processed to release the soluble
secreted mature form of HB-EGF (10, 11) . Thus, in
analyzing HB-EGF protein in C2C12 cells, it is of interest to know
whether it is the transmembrane form or the secreted mature form (or
both) which is produced. Initial analysis using heparin affinity
chromatography did not detect HB-EGF mitogenic activity in the
conditioned medium of either myoblasts or myotubes suggesting that if
HB-EGF protein were being produced by C2C12 cells, it would be
preferentially HB-EGF
. Two approaches were used to probe
for the presence of HB-EGF
. One was to express
HB-EGF
-AP in C2C12 cells under control of the HB-EGF
promoter. The epitope tag was located in the HB-EGF
ectodomain and was readily detected on the cell surface by highly
specific anti-AP antibodies. The other approach was to immunostain
C2C12 cells for endogenous mouse HB-EGF
with an antibody
directed against the C-terminal cytoplasmic tail of HB-EGF, which
recognizes HB-EGF
but cannot possibly recognize released
mature HB-EGF(9) . Thus, the analysis of HB-EGF immunostaining
was conducted with antibodies directed against both the ectodomain and
cytoplasmic domains of HB-EGF, ensuring greater accuracy. Laser
confocal microscopy of immunostained C2C12 cells revealed that both
HB-EGF
-AP and endogenous HB-EGF
were
expressed in myotubes but not in myoblasts. Furthermore, use of laser
confocal microscopy made it possible to localize both
HB-EGF
-AP, which has an immunoreactive ectodomain, and
HB-EGF
, which has an immunoreactive cytoplasmic domain, to
the myotube surface. Very little, if any, HB-EGF protein was detectable
in the cytosol or in the extracellular matrix. Taken together, these
results suggest that the synthesis of HB-EGF protein is induced during
the differentiation of C2C12 myoblasts to myotubes and that it is the
transmembrane form that is preferentially found. This pattern is
consistent with the induction of HB-EGF promoter activity during C2C12
myogenesis and with the MyoD-dependent induction of HB-EGF gene
expression accompanying 10T1/2 cell myogenesis. These results are also
consistent with our initial analysis of HB-EGF tissue distribution that
indicated that skeletal muscle is a prime source of HB-EGF mRNA
transcripts in human, rat, and mouse species(15) . The
significance of expressing the transmembrane form of HB-EGF on the
C2C12 myotube surface is not yet understood. Previous studies have
indicated that HB-EGF
expressed on the cell surface
stimulates juxtacrine EGF receptor phosphorylation (10) and
cell division (11) of adjacent cells. Recent studies indicate
that HB-EGF
is an adhesion factor for mouse blastocysts. (
)Thus, skeletal muscle cell HB-EGF
could
interact with EGF receptor and heparan sulfate proteoglycans on
adjacent muscle cells to promote cell-cell contact, cell-cell
signaling, or cell-cell adhesion facilitating myotube or myofibril
formation. Another possibility is that skeletal muscle cell
HB-EGF
could act as an adhesion and signaling factor for
other cell types, for example, neuronal cells, as does the skeletal
muscle neural adhesion molecule, N-CAM(48) .
(52) , both of which are
inhibitors of skeletal muscle cell differentiation. FGF stimulates
myoblast proliferation and inhibits myogenesis by inactivation of
myogenin via phosphorylation of a conserved site in its DNA binding
domain which abolishes the ability of myogenin to bind to
DNA(53) . Inhibition of endogenous FGF stimulates myogenin
expression and levels of endogenous FGF decrease during
myogenesis(46) . HB-EGF levels, on the other hand, increase
during myogenesis. While FGF stimulates myoblast proliferation and
inhibits differentiation, preliminary results indicate that HB-EGF does
neither. (
)Although the significance of induced HB-EGF
expression in myogenesis is not yet understood, its association with
the differentiated skeletal muscle cell state suggests strongly that it
has a different and possibly opposing role from FGF in myogenesis.
Future studies analyzing skeletal muscle HB-EGF in vivo will
be required to fully understand the role of HB-EGF in skeletal muscle
differentiation.
, transmembrane HB-EGF;
SMC, smooth muscle cells; FHVSMC, fetal human vascular smooth muscle
cells; PC, phosphatidylcholine; PS, phosphatidylserine; kb,
kilobase(s); bp, base pair(s); CAT, chloramphenicol acetyltransferase;
bHLH, basic helix-loop-helix; MEM, minimum essential medium; DMEM,
Dulbecco's modified Eagle's medium; FCS, fetal calf serum;
FITC, fluorescein isothiocyanate; FGF, fibroblast growth factor; bFGF,
basic FGF; PBS, phosphate-buffered saline; PIPES,
1,4-piperazinediethanesulfonic acid; AP, alkaline phosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.