From the Institute of Molecular Pathology, University
of Copenhagen, Copenhagen, 2100 Denmark, the
§ Faculté de Médicine de la Timone,
Marseille, 13385 France, and the ¶ Burnham Institute, La Jolla,
California 92037
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ABSTRACT |
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The ADAM (A Disintegrin
And Metalloprotease) family of cell-surface
proteins may have an important role in cellular interactions and in
modulating cellular responses. In this report we describe a novel,
secreted form of human ADAM 12 (meltrin ), designated ADAM 12-S (S
for short), and a larger, membrane-bound form designated ADAM 12-L (L
for long form). These two forms arise by alternative splicing of a
single gene located on chromosome 10q26. Northern blotting demonstrated
that mRNAs of both forms are abundant in human term placenta and
are also present in some tumor cell lines. The ADAM 12-L transcript can
also be detected in normal human adult skeletal, cardiac, and smooth
muscle. Human A204 embryonal rhabdomyosarcoma cells that do not
differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding
the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these
transfected cells contained ectopic muscle cells of apparent mouse
origin as shown by species-specific markers. These results may have
potential applications in the development of muscle-directed gene and
cell therapies.
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INTRODUCTION |
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ADAMs1 are a recently
discovered family of membrane-anchored cell-surface proteins. They are
about 800 amino acids long and have a unique domain organization,
containing pro-, metalloprotease, disintegrin, cysteine-rich,
transmembrane, and cytoplasmic domains (1-4). Because these domains
are homologous to domains in proteins with established functions, ADAMs
have been proposed as candidates for modulating proteolysis, cell
adhesion, cell fusion, and signaling. The ADAMs have structural
similarity and ~30% sequence identity to snake venom
metalloproteases (SVMPs), which cause hemorrhage in snake bite victims
(5, 6). ADAMs and SVMPs are both members of the reprolysin subfamily of
metalloproteases (6, 7). Full-length SVMPs are processed to generate a
metalloprotease, which is able to degrade proteins of the basement
membrane such as type IV collagen and laminin (5), and a disintegrin
domain, which can inhibit the function of platelets by interacting with platelet integrin GPIIb-IIIa (8). Within the past few years the number
of identified ADAMs has expanded rapidly, and to date 18 different
members have been reported in the literature (3, 4, 9-14). For
example, ADAM 10 (Kuz) plays a critical role in neurogenesis in
Drosophila (9, 10, 15, 16); ADAM 11 is a candidate tumor
suppressor gene (17, 18); and one of the most recently identified
ADAMs, called TACE, is a tumor necrosis factor -converting enzyme
(11, 13). The most studied ADAMs are fertilin
and
(ADAM 1 and
2) (19-21) which interact with
6
1 integrin during sperm-egg
fusion in fertilization (22). In humans, however, ADAM 1 is
a non-functional pseudogene (23), indicating that different species may
utilize different sets of ADAM-integrin links.
The process of differentiation leading to cell fusion occurs in several
different tissue types including placenta, bone, and muscle. During
myoblast differentiation, the cells align and adhere to each other
before their plasma membranes merge allowing the formation of
multinucleated myotubes (24). On the assumption that myoblast fusion
may be similar to sperm-egg fusion, Yagami-Hiromasa et al.
(25) searched for homologs of ADAMs 1 and 2 in a mouse myogenic cell
line and identified ADAM 12 (meltrin ). ADAM 12 showed strong
expression in neonatal skeletal muscle and bone. In mouse C2 myoblast
cultures, the expression of ADAM 12 became apparent upon muscle cell
differentiation. Evidence for a role in muscle cell fusion was provided
by studies showing that transfection of mouse C2 cells with a minigene
of adam 12 lacking the pro- and metalloprotease domains
accelerated cell fusion, whereas antisense constructs blocked myoblast
fusion.
Here we describe the cloning of soluble and transmembrane forms of human ADAM 12 that arise by alternative splicing. We show the effects of the novel secreted form, designated ADAM 12-S, on recruitment and differentiation of ectopic muscle cells in a human tumor nude mouse model. These results may have potential applications in the development of a number of future muscle-directed gene and cell therapies.
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EXPERIMENTAL PROCEDURES |
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Isolation and Sequencing of Human ADAM 12 cDNA Clones--
A
positive prey clone (S1) was isolated from a human yeast two-hybrid
placental cDNA library (CLONTECH catalog number
HL4025AH) using a cDNA fragment of the laminin 2 chain as
bait.2 This clone was
sequenced and found to be similar to mouse ADAM 12. A probe
corresponding to the disintegrin domain (nt 1540-1963) was used to
screen a human placenta 5
-stretch plus
gt11 cDNA library
(CLONTECH catalog number HL50146). Twenty-six
positive phage were plaque-purified, and the inserts from seven of the phage were subcloned into pBluescript SK(+) (Stratagene). Sequencing was performed using either the Sequenase enzyme and reagents (Amersham Corp.) or the Vistra DNA Sequencer 725 (Amersham Corp.). DNA sequence analysis was performed using the software programs of the Wisconsin Package, version 9.1, of the Genetics Computer Group. Searches of
expressed sequences data base (dBEST) were performed using Blast server
(26).3
Chromosomal Mapping-- The chromosomal localization of the ADAM 12 gene was performed by fluorescence in situ hybridization. Metaphase spreads were prepared from phytohemagglutinin-stimulated human lymphocytes. The 5.1-kb cDNA insert of the ADAM 12-L phage (L1) in pBluescript was labeled with biotin-16-dUTP by nick translation and hybridized to the chromosome spreads as described previously (27), and the probe was detected by means of fluorescence isothiocyanate-conjugated avidin (Vector laboratories). Chromosomes were counterstained and R-banded with propidium iodide as described (28).
Analysis of Alternatively Spliced Exons--
To test the
hypothesis that the two forms of ADAM 12 were splice variants, primers
were designed to amplify the genomic DNA around the point of
divergence. Human genomic DNA from HT1080 fibrosarcoma cells was
amplified with the following primers: primer 322, a sense primer at nt
2378-2400 common to both forms of ADAM 12 (5-dGTTTGGCTTTGGAGGAAGCACAG); primer 323, an antisense primer at nt
2460-2440 of ADAM 12-S (5
-dGCTCCCTGTTGGACTCTGCAG); primer 325, a
sense primer at nt 3252-3274 of ADAM 12-S
(5
-dCAATGTAAGAGCCTAACTCCATC); and primer 324, an antisense primer at
nt 2498-2476 of ADAM 12-L (5
-dGAGATAAACCACAAATCCGGCAG). The
conditions for amplification were as follows: 1 cycle of 94 °C for 2 min, 30 cycles of 94 °C for 40 s, 55 °C for 40 s, and
72 °C for 3 min. The products were gel-purified, subcloned into the
vector pCR 2.1 (Invitrogen), and sequenced.
Analysis of ADAM 12 mRNA Expression--
Human multiple
tissue Northern blots containing ~2 µg per lane of poly(A) RNA were
purchased (CLONTECH catalog numbers 7760-1 and
7765-1). Poly(A)-enriched RNA was extracted from cultured cell lines
using the Trizol reagent (Life Technologies, Inc.), ~15 µg per lane
was fractionated by formaldehyde-agarose gel electrophoresis and
blotted onto nylon membranes using standard protocols (29). Blots were
hybridized with [32P]dCTP random primer labeled probes at
68 °C for 1 h in 10 ml per blot of QuikHyb solution
(Stratagene). Blots were washed three times at 50 °C for 15 min in
2 × SSC, 0.05% SDS, and twice for 20 min in 0.1 × SSC and
0.1% SDS. Nucleotide probes used in this work were hybridized to the
ADAM 12 pro-domain (nt 664-1007), disintegrin domain (nt 1540-1963),
or were specific for ADAM 12-S (nt 2409-3333), or specific for the
coding region (nt 2476-2717) or 3-untranslated region (nt 4227-5067)
of ADAM 12-L. A 467-bp probe specific for ADAM 12-L 8.6-kb band was
also produced via PCR using primers (5
-dACCAGGGTGTTTTGTGGTTG and
5
-dTGCTGCTTTTGTGGTTTCTG) designed after examination of EST data bases
(see below). Blots were were exposed to Kodak X-Omat AR film at
80 °C with intensifying screens.
Cell Lines and Cell Culture-- The cell lines used in this study included COS-7 (ATCC CRL 1651), HT1080 (ATCC CCL 121), A204 human embryonal rhabdomyosarcoma (ATCC HTB 82), RD human embryonal rhabdomyosarcoma (ATCC CCL 136), and HU-1 human lung adenocarcinoma (30). The cells were grown in DMEM with Glutamax I and 4500 mg/ml glucose, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C in 5% CO2. Myogenic differentiation of RD cells was induced at confluence by replacing the growth medium with DMEM containing 1% fetal bovine serum as described (31). Under the same culture conditions no myogenic differentiation of A204 cells was observed, by morphological criteria and by lack of induction of expression of myogenin mRNA by RT-PCR (not shown).
Purification of Recombinant ADAM 12 and Production of Poly- and Monoclonal Antibodies-- A plasmid for the production of recombinant ADAM 12 in Escherichia coli was constructed using the pQE32 His-tag expression vector (Qiagen). A 450-bp BamHI/XhoI fragment coding for nt 2000-2433 of ADAM 12-S was inserted at the BamHI/SalI sites of pQE32 and transformed into E. coli strain M15[pREP4]. This plasmid (p1053) codes for a 17-kDa recombinant protein containing the cysteine-rich domain and the first four amino acids (EARQ) of the unique carboxyl terminus of ADAM 12-S. Recombinant protein was purified as follows: 200 ml of E. coli culture was sedimented, lysed in 0.020 M Tris-HCl, pH 7.9, 0.5 M NaCl, 6 M guanidine HCl, centrifuged, and the supernatant applied to a column of TALON immobilized metal affinity resin (CLONTECH). After washing the resin with a urea buffer (0.020 M Tris-HCl, pH 7.9, 0.5 M NaCl, 6 M urea, 0.01 M imidazole), elution of bound material was performed with 0.05 M EDTA in 0.020 M Tris-HCl, pH 7.9, and 0.15 M NaCl. The yield from a typical preparation was ~2.5 mg.
Lewis female rats (Møllegaarden, Denmark) and female rabbits (Statens Seruminstitut, Copenhagen, Denmark) were immunized and boosted at monthly intervals with total E. coli fusion protein extract or with purified recombinant ADAM 12 derived from expression construct (p1053) emulsified in complete and incomplete Freund's adjuvant. Antisera (rb 104) were collected 10-11 days after the second and all subsequent injections. To prepare monoclonal antibodies, rats immunized and boosted eight times were given a final boost intraperitoneally, and 5 days later hybridomas were prepared by fusing spleen cells from the rat with the nonsecreting mouse myeloma P3 × 63Ag8.653 (ATCC TIB 18) as recently described (32). Supernatants of the resulting hybridomas were screened and characterized for their immunostaining of COS-7 cells transiently transfected with construct number 1095 (see below). The isotype of the rat 14E3 hybridoma was IgG2b as determined by Ouchterlony immunodiffusion using a series of anti-rat immunoglobulins purchased from Serotec and by the IsoStrip kit from Boehringer Mannheim. Hybridomas were grown in DMEM with Glutamax I and 4500 mg/ml glucose, 1 mM sodium pyruvate, 10 mM HEPES, OPI media supplement (0.15 µg/ml oxaloacetate, 0.05 µg/ml pyruvate, 0.0082 µg/ml bovine insulin (Sigma)), 50 units/ml penicillin, and 50 µg/ml streptomycin and 20% myoclone super plus fetal bovine serum (Life Technologies, Inc.) at 37 °C in 10% CO2.Transfection Assays, Immunostaining, and Immunoblotting--
A
plasmid for expression of an ADAM 12-S minigene was
constructed using the pSecTagB vector (Invitrogen). A DNA fragment
coding for the disintegrin domain, cysteine-rich domain, and the unique carboxyl terminus of ADAM 12-S was prepared by PCR amplification using
the ADAM 12-S cDNA plasmid as a template and the following primers:
5-dCCAAAGCTTGAAGTCAGGGAGTCTTTC and 5
-dCCATCTAGATCAGATGAGTGTCAGTGA. The 987-bp PCR product contained nt 1560-2528 of ADAM 12-S, with HindIII and XbaI cloning sites. This fragment was
inserted at the HindIII/XbaI sites of pSecTagB,
yielding plasmid p1095, consisting of an ADAM 12 minigene
driven by a cytomegalovirus promoter, fused to an Ig
-chain leader
sequence to allow secretion of the protein.
Heterotransplantation into Nude Mice, Morphological Examination,
Immunohistochemistry, and RT-PCR--
A204 parental cells or
transfectants were harvested by trypsin/EDTA, equilibrated in complete
medium for 1 h in suspension at 37 °C, rinsed in PBS, and
finally resuspended at a concentration of 108 cells/ml.
Tumor cells (107 per inoculum) were injected subcutaneously
via a 26-gauge needle into the back of female (6-8 week-old)
nu/nu NMRI mice (Bomholtgaard, Denmark). Mice were killed by
cervical dislocation after 6-8 weeks. Tissue specimens were fixed in
buffered formalin at room temperature or in cold 96% ethanol/glacial
acetic acid (99:1 v/v) and processed for histological examination of
hematoxylin and eosin-stained paraffin sections using standard
techniques. For electron microscopy 2- to 3-mm3 tissue
specimens were promptly fixed at room temperature in Karnovsky's fixative for 2 h. The samples were dehydrated in graded ethanols, postfixed in 2% osmium tetroxide, and embedded in Epon. One-micrometer sections were cut on an LKB ultramicrotome and stained with toluidine blue. Ultrathin sections from selected areas were collected on copper
grids, stained with uranyl acetate and lead citrate, and examined with
a Phillips 201 electron microscope. Tissue specimens were also frozen
in liquid nitrogen and stored at 70 °C for later use in
immunohistochemistry or RNA purification.
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RESULTS |
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Cloning and Sequencing of Human ADAM 12 cDNAs--
We screened
a yeast two-hybrid cDNA library with laminin 2 cDNA as
"bait," and one of the positive "prey" clones was homologous to
mouse ADAM 12 (meltrin
) but had a divergent carboxyl terminus. To
determine whether this represented an alternatively spliced form of
ADAM 12, we set out to isolate full-length cDNA for human ADAM 12. We isolated cDNA clones that cover the full-length of the human
homologue of mouse ADAM 12, designated ADAM 12-L (L for long), and
partial clones of the smaller ADAM 12-S (S for short). A map of the
clones is shown in Fig. 1A and
the nucleotide and deduced amino acid sequence in Fig.
1B.
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Chromosomal Localization--
By using fluorescent in
situ hybridization, we mapped the chromosomal localization of the
human ADAM 12 gene (ADAM12). 90% of the 30 metaphase cells
analyzed showed specific fluorescent spots on the q26 band of the long
arm of human chromosome 10 (Fig. 2).
Subsequently we searched the data base of mapped STSs on the human
genome (41)5 and identified
an STS (WI-17472) that is identical in sequence to part of the
3-untranslated region of ADAM 12-L (nt 4044-4145). WI-17472 was
placed on the distal region of chromosome 10 by radiation hybrid
mapping, in the interval between the Genethon markers D10S216 and
D10S575 (158-162 centimorgan), consistent with our cytogenetic localization of the gene. Thus the ADAM12 gene is located at
10q26.3.
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ADAM 12-L and ADAM 12-S Arise by Differential Splicing--
The
finding of two cDNA forms of ADAM 12 sharing identical 5-regions,
but diverse 3
-ends, suggested that they were alternatively spliced
versions of a single gene. This hypothesis was strengthened by the
single chromosomal localization and the observation that a probe for
the pro-domain common to both forms of ADAM 12 hybridized to a single
band in human high molecular weight genomic DNA digested with seven
different restriction enzymes (data not shown). We designed PCR primers
to amplify the genomic DNA around the point of divergence in the ADAM
12 clones. Primers 322 and 324 amplified a 4-kb, 325 and 324 a
2-kb, and 322 and 323 a 1-kb DNA fragment (Fig.
3). Sequencing of these revealed that the
ADAM 12 gene contains an intron at the point of divergence
between the clones, followed by an exon encoding the ADAM 12-S-specific
sequence, which does not appear to have any introns within it. At the
end of the ADAM 12-S sequence, about 2 kb of intron DNA are present
before the ADAM 12-L coding sequence. A consensus 5
-donor site was
found at the point of divergence between the ADAM 12-L and -S
sequences, and 3
-acceptor sites were present at the start of both the
ADAM 12-L- and ADAM 12-S-specific sequences.
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Human ADAM 12 mRNA Expression--
We examined the expression
of ADAM 12 mRNA using probes common to both forms of ADAM 12 and
probes specific for each form. Northern blot analysis with a probe for
the disintegrin domain present in both forms of human ADAM 12 revealed
three bands of 3.5, 5.4, and 8.6 kb in human full-term placenta RNA,
expressed at relative levels of 2:1:1 (Fig.
4A, lane 1). Probes specific for the ADAM 12-S clone hybridized only to the smallest 3.5-kb band
(Fig. 4A, lane 3), whereas probes specific for human ADAM 12-L hybridized to the two top bands only (Fig. 4A, lane 2).
The 3.5- and 5.4-kb bands correspond to the sizes of the full-length ADAM 12-S and -L cDNAs, although we did not isolate a full-length cDNA clone equivalent in size to the 8.6-kb band observed on
Northern blots. As the 8.6-kb band hybridized to the same probes as the 5.4-kb ADAM 12-L transcript, this suggests that the 8.6-kb transcript contained the same ADAM 12-L sequence as the 5.4-kb band but had an
extended 3-region. To clarify this further, we searched the dbEST data
base with 500 bp of the untranslated region at the 3
of the ADAM 12-L
cDNA clone and were able to assemble a partial contig covering an
additional kilobase of 3
-untranslated ADAM 12-L DNA (not shown).
Primers specific for this EST-derived region were designed and used to
amplify a 467-bp product from placenta cDNA. This probe hybridized
only to the 8.6-kb band on a placenta RNA Northern blot (data not
shown). Thus the 8.6-kb band observed on Northern blots appears to
encode the identical sequence to the ADAM 12-L cDNA but has a
longer 3
-untranslated region.
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Biological Function of ADAM 12-- To begin analyzing the distribution and function of the ADAM 12 protein, we generated poly- and monoclonal antibodies to the 17-kDa cysteine-rich domain of ADAM 12 produced in E. coli (Fig. 5A). These antibodies immunostained and reacted in Western blotting with COS-7 cells transiently transfected with an ADAM 12-S expression plasmid but not with cells transfected with a control plasmid lacking an ADAM 12 insert (Fig. 5, B and C). We then made an expression construct carrying an ADAM 12-S minigene coding for the disintegrin domain, the cysteine-rich domain, and the carboxyl terminus of ADAM 12-S. The rationale for using a minigene was based on previous studies showing that a mouse ADAM 12 minigene lacking the pro- and metalloprotease domains was biologically active, whereas the full-length form was not (25). This plasmid containing the minigene (p1095) or the vector lacking a cDNA insert as a negative control was transfected into the human rhabdomyosarcoma cells A204 that do not express detectable amounts of ADAM 12 mRNA or protein. Three stably transfected clones were obtained that expressed ADAM 12-S minigene mRNA and secreted a 42-kDa ADAM 12-S polypeptide into the medium (Fig. 5D). Like the parental A204 cells, these three cell lines showed no apparent capacity to fuse in vitro (data not shown).
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DISCUSSION |
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We have cloned the human ADAM 12 full-length cDNAs and discovered an alternatively spliced form, designated ADAM 12-S. This form of ADAM 12 has no transmembrane and cytoplasmic domains but has instead a short 34-aa carboxyl terminus. The resulting polypeptide becomes secreted, and transfection experiments indicated that this form provokes myogenesis.
Mouse ADAM 12 was first identified by Yagami-Hiromasa et al.
(25) and was called meltrin . In the same study they also isolated,
via RT-PCR, two partial sequences that they designated meltrin
and
meltrin
. Sequence comparison with other ADAM genes isolated since indicates that meltrin
is more likely to be the murine equivalent of Xenopus adam 13 (Refs. 14 and 43; not shown), whereas meltrin
shows 100% identity to nt 1289-1738 of
mouse adam 9 (40). Therefore, we prefer to use the ADAM
nomenclature and refer to meltrin
as ADAM 12.
In the present study we demonstrated that human ADAM 12 has two
alternatively spliced forms designated ADAM 12-L and ADAM 12-S.
Multiple transcripts of a single ADAM have been observed in monkey,
mouse, and human (18, 44, 45). Mouse ADAM 1I and ADAM 1
II are
identical from nucleotides 702-2492 and contain the same domain
structure but have distinct although related 5
- and 3
-ends (44).
Monkey ADAM 6 has two isoforms, although these probably reflect two
different genes (45). The only previously confirmed alternative
splicing is of the human ADAM 11 gene (18). This candidate
tumor suppressor gene for breast cancer is alternatively spliced to
generate two different transcripts, MDC-769 and MDC-524. These
transcripts differ at both the 5
- and 3
-ends. MDC-769 has full-length
cysteine-rich, transmembrane, and cytoplasmic domains, whereas MDC-524
is a truncated protein that terminates in the cysteine-rich domain.
Human ADAM 12 appears to be encoded by a single copy gene that we mapped to chromosome 10q26 by in situ hybridization. Data base searches revealed that an EST that matches the ADAM 12-L cDNA sequence was localized to the same region of chromosome 10 by radiation hybrid mapping. Within this region genes for acyl-CoA dehydrogenase, fibroblast growth factor receptor, uroporphyrinogen-III synthase, and ornithine aminotransferase have been identified. With the exception of the ornithine aminotransferase (deficiency of this enzyme causes the eye disease gyrate atrophy of the choroid and retina), no disease loci have been mapped to this region. The ADAM 8 gene also maps to 10q26.3 (46), raising the possibility that these two genes may be clustered. The other ADAMs (ADAMs 1, -2, -4, -5, and -11) that have been assigned locations on the human genome are dispersed throughout different chromosomes (47-49).
By Northern blotting we observed three human ADAM 12 mRNAs of 3.5, 5.4, and 8.6 kb, whereas in the mouse only one transcript was reported
(25). The 3.5-kb band was specific for ADAM 12-S, and the 5.4- and
8.6-kb bands were specific for ADAM 12-L. The 3.5-kb transcript arises
from alternative use of an exon that encodes an ADAM 12-S-specific
carboxyl terminus, 3-untranslated region, and polyadenylation site.
The 5.4- and 8.6-kb transcripts appear to be derived by alternative use
of polyadenylation sites in the ADAM 12-L-specific 3
-untranslated
region. We have isolated the full-length cDNA representing the
5.4-kb transcript, but the 8.6-kb transcript that appears to contain a
longer 3
-untranslated region has not been entirely isolated. The
different 3
-untranslated regions of ADAM 12-L could affect the rates
of translation or mRNA stability (50).
Analysis of the distribution patterns of the two ADAM 12 forms in normal human tissues revealed that the ADAM 12-S transcript was detected so far only in placenta, whereas the ADAM 12-L mRNAs were found in placenta and skeletal, cardiac, and smooth muscle. Splicing of the ADAM 12-S exon may be regulated by cell type-specific factors. The human ADAM 12-L transcript appears to have a more widespread expression than the mouse mRNA (25). Interestingly, both forms were detected in some tumor cell lines, indicating a possible association between ADAM 12-S and neoplasia.
Until now only two ADAMs that lack a transmembrane domain, and thus are assumed to be secreted, have been reported. These are ADAM 11/MDC-524 (18) and ADAMTS-1 (ADAM with thromobospondin motifs (12)). To this list we now add ADAM 12-S, and we have shown that it becomes secreted. The candidate tumor suppressor MDC-524 splice form was isolated from human cerebellar cDNA library and is expressed at very low levels compared with the cell membrane anchored form MDC-769 (18). ADAMTS-1, which is associated with cancer cachexia and inflammatory processes, lacks the cysteine-rich, transmembrane, and cytoplasmic domains, having instead a thrombospondin homologous domain and type I thrombospondin motifs (12). Like ADAM 12-S, these cancer-related, secreted ADAMs appear to have very restricted, low levels of expression in normal tissue. The best characterized soluble ADAM-like proteins are the snake venom metalloproteases (SVMPs) (7). The soluble ADAMs lacking the regulatory control of a transmembrane domain may be extremely potent like their SVMP counterparts. The highly restricted and low level of normal expression of these soluble ADAMs may reflect this potency, and continued comparison of ADAMs with SVMPs is warranted.
Mouse ADAM 12 has been implicated in cell fusion during C2C12 differentiation in vitro (25). In the present study we found that cells transfected with the shorter, secreted form of ADAM 12-S appear to be very potent in provoking myogenesis in vivo. We stably transfected the human embryonal rhabdomyosarcoma cell line A204 with the ADAM 12-S minigene composed of the disintegrin and cysteine-rich domains and the unique carboxyl terminus. Although a potential muscle precursor, the A204 rhabdomyosarcoma does not differentiate in vitro either spontaneously or after transfection with the ADAM 12-S minigene. However, nude mice tumors generated from these ADAM 12-S minigene transfected cells contained a striking pattern of ectopic muscle cell formation as compared with control tumors. A mixture of cells representing different stages of normal myogenesis was observed, including myoblasts and elongated multinucleated myotubes with cross-striation. These developing muscle cells were located in a disorganized pattern, as opposed to the normal adult skin muscle. Electron microscopy and immunostaining confirmed that these cells were in fact of the muscle cell lineage. Furthermore, based on combined immunostaining using mAbs specific for mouse and human antigens and RT-PCR using species-specific primers for myf-5, we conclude that these ectopic muscle cells are of an apparent murine origin rather than derived directly from the A204 human tumor cells.
What is the cell of origin for this myogenesis? There are at least two possibilities; one is the satellite cells, which are normally located in intimate relationship with existing myofibers beneath the basement membrane. Satellite cells are ubiquitous in normal adult muscle and represent the muscle progenitor cells during muscle regeneration (24). Another possible source is undifferentiated mesenchymal progenitor cells present in connective tissue. The mechanism by which ADAM 12-S may be involved in the recruitment and differentiation of muscle progenitor cells is not clear, and how it acts in the context of factors from the A204 rhabdomyosarcoma cells and/or the host stroma remains to be resolved.
In conclusion, we have characterized a novel form of secreted human ADAM 12, designated ADAM 12-S, and presented evidence that it provokes myogenesis in a nude mouse tumor model.
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ACKNOWLEDGEMENTS |
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We thank Dr. M. E. Durkin for helpful comments on the manuscript, Dr. J. T. Vilquin for advice on the neural cell adhesion molecule antiserum, and Dr. A. M. Mercurio for stimulating discussions. We thank Bent Børgesen for photographic assistance; and Brit Valentin, Aase Valsted, Annette Beth, and Nina Broholm for technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Danish Cancer Society and the Danish Medical Research Council (to U. M. W. and R. A.). Our laboratories were also supported by the VELUX, Novo-Nordisk, Munksholm, Haensch, Thaysen, Wærum, Bojesen, Beckett, Hartmann, and Meyer Foundations (to U. M. W. and R. A.) and by the National Institutes of Health (to E. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AF023476 and AF023477.
To whom correspondence should be addressed: the Institute of
Molecular Pathology, University of Copenhagen, Frederik V's vej 11, DK-2100, Copenhagen, Denmark. Tel.: 45 3532 6056; Fax: 45 3532 6081;
E-mail: molera{at}inet.uni-c.dk.
1 The abbreviations used are: ADAM(s), a disintegrin and metalloprotease; aa, amino acid(s); bp, base pair(s); EST, expressed sequence tag; DMEM, Dulbecco's modified Eagle's medium; kb, kilobase pair(s); mAb, monoclonal antibody; nt, nucleotides(s); PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RT, reverse transcriptase; SVMPs, snake venom metalloproteases.
2 F. Loechel and U. M. Wewer, manuscript in preparation.
3 Blast server, www.ncbi.nlm.nih.gov.
4 M. E. Durkin and U. M. Wewer, manuscript in preparation.
5 On-line address: www.ncbi.nlm.nih.gov/science96/
6 R. Albrechtsen and U. M. Wewer, unpublished observations.
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REFERENCES |
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