©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction of Heparin-binding EGF-like Growth Factor Expression during Myogenesis
ACTIVATION OF THE GENE BY MyoD AND LOCALIZATION OF THE TRANSMEMBRANE FORM OF THE PROTEIN ON THE MYOTUBE SURFACE (*)

(Received for publication, March 24, 1995; and in revised form, May 23, 1995)

Xiaorong Chen (1) Gerhard Raab (1) Urban Deutsch (1) Jianchun Zhang (1) Robert M. Ezzell (2) Michael Klagsbrun (1)(§)

From the  (1)Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115 and the (2)Surgery Research Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

HB-EGF (^1)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-alpha(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) .

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) 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) .

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 alpha (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) .

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.


EXPERIMENTAL PROCEDURES

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(2)PB(3), 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 10^5 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 10^6 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 beta-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 10^4 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(2), 0.1 mM CaCl(2) and fixed for 20 min at room temperature in 2% paraformaldehyde/HEPES buffer(44) . Fixed cells were washed twice with PBS, 1 mM MgCl(2), 0.1 mM CaCl(2), 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(2), 1 mM CaCl(2), 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.


RESULTS

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.

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-kappaB, 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.


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.



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, B(2)proB(3)) 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).


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(2)PB(3), 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(2)PB(3) (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.



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.

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.


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(2)PB(3) (&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.


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). 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.



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.

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) .


Figure 7: Transient activation of HB-EGF promoter activity during C2C12 muscle cell differentiation. Top, light microscopy at 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).


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-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.



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 10^7 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.


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.




DISCUSSION

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 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.

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, 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. (^2)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) .

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-beta (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. (^3)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.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants CA37392 and GM47397 (to M. K.), a Deutsche Forschungsgemeinschaft award (to G. R.) and an American Chemical Society Grant CB-148 (to R. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
To whom correspondence and reprint requests should be addressed: Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: 617-355-7503; Fax: 617-355-7291.

^1
The abbreviations used are: HB-EGF, heparin-binding epidermal growth factor-like growth factor; HB-EGF, 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.

^2
G. Raab, S. K. Dey, and M. Klagsbrun, submitted for publication.

^3
X. Chen, unpublished results.


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