From the Department of Orthopaedics and Cell Biology,
Yale University School of Medicine, New Haven, Connecticut 06510 and
the ¶ Cellular Biochemistry and Biophysics Program,
Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New
York, New York 10021
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ABSTRACT |
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Here we report the cloning and initial
biochemical characterization of the mouse
metalloprotease/disintegrin/cysteine-rich (MDC) protein meltrin and
the analysis of the mRNA expression of four MDC genes
(meltrin
, meltrin
, mdc9, and mdc15) in
bone cells, including osteoclasts and osteoblasts. Like most other MDC
proteins, the predicted meltrin
protein consists of a signal sequence, prodomain, metalloprotease domain with a predicted catalytic site, disintegrin domain, cysteine-rich region, epidermal growth factor
repeat, transmembrane domain, and cytoplasmic domain with putative
signaling motifs, such as potential SH3 ligand domains. Northern blot
analysis indicates that meltrin
is widely expressed, with the highest expression in bone, heart, and lung. RNase protection studies revealed expression of all four MDC genes analyzed here in
osteoblasts, whereas only mdc9 and mdc15
mRNAs were detectable in osteoclast-like cells generated in
vitro. Treatment of primary osteoblasts with 10 nM
calcitriol increased meltrin
expression more than
3-fold, and both meltrin
and meltrin
expression is apparently regulated in a differentiation-associated
manner in a mouse osteoblastic cell line, MC3T3E1. Collectively, these results suggest that meltrin
and meltrin
may play a role in osteoblast differentiation and/or function but
are not likely to be involved in osteoclast fusion.
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INTRODUCTION |
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Proteins containing a metalloprotease domain, a disintegrin
domain, and a cysteine-rich region
(MDC1 proteins, also referred
to as ADAMs (1)) are a family of membrane-anchored glycoproteins that
are related to soluble snake venom metalloproteases and integrin
ligands. MDC proteins have been implicated in a variety of important
cellular processes, including sperm-egg and muscle cell membrane
binding and fusion (2-6); in neuronal induction, lateral inhibition,
and neuronal outgrowth in Drosophila (7-9); and in the
release of the membrane-anchored cytokine tumor necrosis factor (10, 11). The finding that the tumor necrosis factor
convertase is
an MDC protein (10, 11) and that Drosophila Kuzbanian may be
involved in the intracellular processing Notch (9, 12) raises the
intriguing possibility that other membrane-anchored proteins are also
processed and/or released by metalloprotease-disintegrin proteins (13).
Because several MDC proteins contain potential cytoplasmic signaling
motifs, including SH3 ligand domains (1, 14-16), MDC proteins may also
play a role in signaling events, or they may be regulated through
interactions with cytoplasmic proteins. At present, over 20 genes
encoding MDC proteins have been identified in different species,
including Caenorhabditis elegans (17), Drosophila
melanogaster (7, 8), Xenopus laevis (18, 19), mice (1,
6, 14, 20, 21), guinea pigs (1, 4, 21, 22), and humans (10, 11, 14, 15, 23). Although the function of many of these proteins remains to be
determined, it is clear that different MDC proteins are capable of
performing a variety of important tasks.
To date, MDC proteins have been implicated in two distinct membrane
binding and fusion events. Fertilin, which functions in sperm-egg
binding and fusion, is a heterodimeric complex of two MDC proteins,
fertilin and fertilin
. A monoclonal antibody against fertilin
blocks sperm-egg fusion (2), as do peptides corresponding to the
predicted integrin binding sequence of fertilin (5, 24-26). On the
egg, an
6
1 integrin has been identified as a receptor for sperm, and thus as a candidate receptor for fertilin
(25). In C2C12 mouse muscle cells, an MDC protein called meltrin
has been found to play a role in the membrane fusion event that gives
rise to multinucleated myotubes (6). The involvement of MDC proteins in
sperm-egg fusion and in muscle cell fusion suggests that MDC proteins
are closely linked to the process of membrane fusion, either by
mediating a prerequisite binding or signaling step, or by directly
triggering the membrane fusion event via a predicted fusion peptide
(27).
In this study, we report the cDNA cloning, sequencing, and initial
biochemical characterization of the mouse MDC protein meltrin .
Because the highest expression of meltrin
in adult mice
was previously reported in bone (6), we further investigated the expression of meltrin
and of the three other
metalloprotease-disintegrins, meltrin
, mdc9,
and mdc15 (metargidin), in osteoblasts and osteoclasts. Our
results demonstrate that all four MDC genes are expressed in
osteoblasts and that the expression of meltrin
is
inducible by 1
,25-dihydroxycholecalciferol (calcitriol).
Furthermore, expression of meltrin
and
meltrin
appears to be regulated during osteoblast differentiation. Neither meltrin
, which has been
implicated in muscle fusion, nor meltrin
is detectable
in in vitro-generated osteoclast-like cells (OCLs), which
arise from the fusion of mononuclear precursors of the
monocyte/macrophage/osteoclast lineage. Therefore, we conclude that
Meltrin
and Meltrin
may not play a role in osteoclast
fusion but instead are more likely involved in osteoblast differentiation and/or osteoblast activity in bone.
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MATERIALS AND METHODS |
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cDNA Cloning of Meltrin and Meltrin
--
mRNA
from ~20% fused mouse C2C12 cells (28) was used to synthesize a
cDNA library, utilizing the Stratagene ZAP cDNA library synthesis kit (according to the manufacturer's instructions; see also
Ref. 29). The unamplifed cDNA library was screened under high
stringency conditions with a [
-32P]dCTP-labeled
cDNA probe for meltrin
, which was generated as follows: nested reverse transcription-PCR was performed on C2C12 cDNA (see above) with the following primers designed from the published partial meltrin
sequence (6): primer 1 (sense
primer), GAGGAATGT GACTGTGGAGA; primer 2 (sense primer), GTGACCTCCC
CGAGTTCTGC; and primer 3 (antisense primer), CAGTTTCCAT AGGTGTCACC.
Primers 1 and 3 were used in a primary reaction, which was diluted
1:400 and used as template for a secondary reaction with primers 2 and 3, performed in the presence of [
-32P]dCTP. Using this
probe, 20 positive cDNA clones were identified from 106
plaques screened. A PCR analysis with the pBluescript vector-specific T3 primer (Stratagene), and a 5' antisense primer (TTCAGAGCCA CGTTGGAGGC), designed from the previously published partial
meltrin
sequence (6), was carried out to find the clone
with the longest 5' extension. The longest clone thus identified was
subjected to in vitro excision, sequenced completely on both
strands using a primer walk approach, and found to have a cDNA
insert of 6413 base pairs. The initial methionine residue of the
deduced meltrin
protein sequence is followed by a hydrophobic
predicted signal sequence and an apparently full-length open reading
frame encoding a protein with 920 amino acid residues. Analysis of the
mouse meltrin
cDNA and translated protein sequences was
performed using MacVector sequence analysis software (Kodak Scientific
Imaging Systems), and the alignment and hydrophobicity plot was
generated using DNASTAR software (Megalign and Protean program
modules).
RNA Analysis--
Mouse tissue Northern blots were obtained from
CLONTECH and probed with a PCR-generated
32P-labeled mouse meltrin cDNA under
high stringency conditions as described previously (20). For RNase
protection assays, total RNA was extracted from various bone cells
according to the previously described method (30), and the
concentrations were determined by spectrophotometry. To prepare cRNA
probes, cDNA fragments of mouse mdc9 (an
XhoI/EcoRI fragment from nucleotide 106 to
nucleotide 309 (14)), meltrin
(an
AflII/HindIII fragment from nucleotide 842 to
nucleotide 1091 (6)), meltrin
(a
HindIII/BamHI fragment from nucleotide 2883 to
nucleotide 3210), mouse mdc15 (an
AflII/EcoRI fragment from nucleotide 727 to
nucleotide 1074; GenBank accession no. AF006196), the calcitonin
receptor (from nucleotide 1156 to nucleotides 1677 (31)) and GAPDH
(from nucleotide 521 to nucleotides 660) were subcloned into the
appropriate sites in a pBluescript II vector (Stratagene) and
transcribed in vitro in the antisense direction by T7 or T3
RNA polymerase in the presence of [
-32P]UTP. Cold UTP
was also added to the reaction for GAPDH to reduce the specific
activity of the probe to approximately one tenth. Multiprobe RNase
protection assays were performed using RPA II kit (Ambion, Inc.,
Woodward, TX) according to the protocol suggested by the supplier. 10 µg of total RNA was incubated with two or more labeled cRNA probes
(20,000-50,000 cpm per reaction for each probe) in a single tube at
42 °C for 16 h. After digesting single-stranded RNA with a
mixture of RNase A (5 units/ml) and RNase T (200 units/ml) at 37 °C
for 30 min, protected RNA fragments were precipitated, resolved on a
5% denaturing polyacrylamide gel, and visualized by
autoradiography.
Antibodies--
Two New Zealand White rabbits were immunized
using a GST fusion protein with the N-terminal half of the
meltrin cytoplasmic domain (amino acid residues
728-817), and two rabbits were immunized with a GST fusion protein
with the C-terminal half of the cytoplasmic tail (corresponding to
amino acid residues 818-920). The fusion protein expression constructs
were assembled using PCR primers with added restriction sites to allow
in-frame cloning into the pGEX/4T-3 GST fusion vector (Pharmacia LKB).
Antibodies were raised by Animal Pharm (Healdsburg, CA) according to
established protocols (32).
Cell Cultures--
A murine macrophage cell line, IC21, was
obtained from American Type Culture Collection (Rockville, MD) and
maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum. C2C12 cells (28) were grown in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum and 2% chick embryo extract
(growth medium) for 2 days, and myogenic differentiation and fusion
were induced by adding Dulbecco's modified Eagle's medium with 2%
horse serum (Life Technologies, Inc.). A murine osteoblastic cell line, MC3T3E1 (33), was a generous gift from Dr. R. T. Franceschi (University of Michigan, Ann Arbor, MI), and was grown in -minimum Eagle's medium containing 10% fetal bovine serum. Primary
osteoblastic cells were isolated from calvariae of newborn CD1 mice
(Charles River Laboratories, Wilmington, MA) by digestion with 0.1%
collagenase type IA (Sigma) and 0.2% dispase (Boehringer Mannheim) as
described previously (34). Bone marrow cells were obtained from tibiae and femurs of 8-12-week-old CD1 mice (Charles River Laboratories). To
generate OCLs in vitro, 2 × 107 marrow
cells were cocultured with 2 × 106 primary
osteoblasts in a 10-cm culture dish in the presence of 10 nM 1
,25-dihydroxycholecalciferol (calcitriol) (34).
After 6 days, cells were treated sequentially with 0.1%
collagenase/0.2% dispase and 5 mM EDTA to remove
osteoblastic cells; OCLs remained strongly attached to the culture
dish. These OCLs have virtually all of the characteristics of authentic
osteoclasts, such as the ability to resorb bone, and the expression of
lineage-specific markers, including tartrate-resistant acid
phosphatase, a specific marker for cells of the osteoclast lineage
(35), and the calcitonin receptor (36). In this coculture system,
formation of OCLs is totally dependent on the presence of calcitriol.
In every preparation, more than 80% of the OCL-enriched population was
positively stained for tartrate-resistant acid phosphatase.
Expression of Meltrin in COS-7 Cells--
A pcDNA3
expression vector (Invitrogen) for meltrin
was
constructed by subcloning an EcoRI/HindIII
fragment, which contains the full-length meltrin
open
reading frame. The EcoRI site is part of the 5' linker
sequence in a
ZAP library, and the HindIII site is at
nucleotide 2883, just downstream of the meltrin
stop codon at nucleotide 2808. The meltrin
pcDNA3
expression vector, or an identical control vector without an insert,
were used to transfect COS-7 cells using LipofectAMINE reagent as a
delivery vehicle (Life Technologies, Inc.). The transiently transfected cells were harvested after 3 days in tissue culture, and expression of
meltrin
was monitored by Western blot analysis as
described below.
Western Blot Analysis--
Protein A-purified IgG was isolated
from antisera raised against GST fusion proteins with either the
C-terminal or the N-terminal part of the meltrin cytoplasmic domain (see above). Meltrin
-specific
antibodies were affinity-purified using the appropriate fusion protein
coupled to CNBr-activated Sepharose CL4B (Pharmacia LKB, coupling
performed according to the manufacturer's instructions). The IgG
sample that remained after affinity purification and had effectively
been depleted of meltrin
-specific antibodies was used as
a control for some Western blots. The affinity-purified IgG, the
control IgG, and the complete antiserum and preimmune antiserum were
tested by Western blot analysis on COS-7 cells expressing
meltrin
from a pcDNA3 vector or on control COS-7 cells transfected with pcDNA3 vector lacking an insert. The
affinity-purified and control antibodies were also used on a lysate of
C2C12 mouse myoblasts that included about 20% fused multinucleated
cells. The Western blot of a primary osteoblast (POB) lysate was probed with immune or preimmune serum at a 1:500 dilution, which produced a
stronger signal than the affinity-purified antibodies under the
conditions used here. All cells were lysed at a concentration of 4 × 106/ml in cell lysis buffer (1% Nonidet P-40 in PBS and
protease inhibitors (3)). Following lysis, nuclei were removed by
spinning at 13,000 rpm in a Sorvall tabletop centrifuge for 15 min at
4 °C. COS-7 cell lysates devoid of nuclei were mixed with 2× sample loading buffer immediately, whereas C2C12 and POB lysates were incubated with 100 µl of Concanavalin A-Sepharose (Pharmacia, LKB)
per 1 ml extract for 1 h at 4 °C to enrich for glycoproteins. Bound glycoproteins were eluted in 1× sample buffer after three washes
in cell lysis buffer. Prior to electrophoresis, all samples were
denatured and reduced by heating at 95 °C for 5 min in the presence
of 50 mM dithiothreitol and subsequently alkylated by incubation with 100 mM iodo-acetic acid. Proteins were
separated by SDS-polyacrylamide gel electrophoresis (37) and
transferred to nitrocellulose (Schleicher & Schuell) using a semidry
blotting apparatus (E&K, Saratoga, CA). Blotted protein samples were
blocked in 5% nonfat dry milk dissolved in PBS, incubated in primary
and secondary antibody in PBS, 0.05% Tween 20, washed in PBS, 0.5% Tween. Bound antibodies were visualized using a horseradish
peroxidase-coupled secondary antibody according to the manufacturer's
instructions (Promega) in combination with a chemiluminescence
detection system (ECL, Amersham Corp.) and Kodak XAR autoradiography
film (Eastman Kodak Co.). In initial tests for specificity on Western
blots of meltrin
-transfected COS-7 cells, all four
antisera (two each raised against the C-terminal and the N-terminal
half of the meltrin
cytoplasmic tail) recognized a band
of 115 kDa (data not shown), in addition to other, faster migrating
bands. These bands were not visible on blots of COS-7 cells transfected
with the vector alone. For all Western blots presented here, antibodies
raised against the C-terminal part of the meltrin
cytoplasmic tail from one of the two rabbits were used, because these
antibodies reacted with the least number of nonspecific bands (defined
as bands that were also present in nontranfected COS-7 cell lysates or
as bands that were not recognized by all four antisera).
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RESULTS |
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cDNA Cloning and Sequence of Meltrin --
A cDNA clone
coding for full-length mouse Meltrin
was first isolated from a
mouse C2C12 muscle cell library, using a PCR-generated cDNA
fragment as a probe (see "Materials and Methods") and then sequenced using a primer walk approach. This clone had an insert of
6413 base pairs, with an open reading frame of 2760 base pairs, 35 base
pairs of 5' untranslated sequence, and 3618 base pairs of 3'
untranslated sequence. The meltrin
protein sequence, as deduced
from the open reading frame, predicts a protein of 920 amino acid
residues with four sites of N-linked glycosylation. The
sequence of Meltrin
contains all protein modules that are characteristic of members of the MDC protein family: a signal sequence
is followed by a prodomain, a metalloprotease domain with a predicted
catalytic site consensus sequence
(HEIGHNFGMSHD), a disintegrin
domain, a cysteine-rich region, an epidermal growth factor-like repeat,
a transmembrane domain, and a cytoplasmic tail. Like several other MDC
proteins, such as MDC9 (14), meltrin
(6), MDC15 (15, 16), and the
tumor necrosis factor
convertase (10, 11), the cytoplasmic tail
harbors potential signaling motifs that include proline-rich predicted
SH3 ligand sequences. The sequence of Meltrin
is shown in Fig.
1A in an alignment with the
two most closely related MDC proteins presently known, meltrin
and
ADAM 13. The hydrophilicity plot of Meltrin
(Fig. 1B)
predicts an N-terminal hydrophobic signal sequence and a transmembrane
domain between amino acid residues 707 and 725. After removal of the
predicted signal sequence, Meltrin
has a deduced molecular mass of
99.23 kDa.
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Analysis of the Tissue Distribution of Meltrin , Meltrin
,
mdc9, and mdc15 and of the Expression in Bone Cell Cultures in
Vitro--
By Northern blot analysis, meltrin
mRNA
expression can be detected in all adult mouse tissues examined, with
the highest expression in heart and lung, followed by brain and spleen
and then by relatively low expression in liver, skeletal muscle,
kidney, and testis (Fig. 2). Analysis by
RNase protection assays revealed that meltrin
mRNA
is also expressed in various tissues, including brain, heart, kidney,
liver, lung, and skeletal muscle (see Fig. 4; some data not shown).
Because the highest expression of meltrin
and
meltrin
in adult mouse tissues was observed in bone (see Fig. 4; some data not shown), confirming the data from a previous report (6), we attempted to determine which cell types in bone express
the mRNA coding for these proteins by RNase protection analysis.
For comparison, we also examined mRNA levels of two other MDC
genes, mdc9 and mdc15, which are both widely
expressed. As primary cell materials, we utilized POBs isolated from
newborn mouse calvariae and OCLs generated in vitro by
cocultures of POBs and bone marrow cells. First, to investigate whether
expression of meltrin
and meltrin
is
associated with osteoclast differentiation, we examined the expression
in cocultures in the absence or presence of calcitriol, which is a
strong inducer of osteoclast differentiation (36). As shown in Fig.
3A, an osteoclast-specific
marker, the calcitonin receptor, was only detectable in cocultures
treated with calcitriol and in purified OCLs, thus verifying the
integrity and fidelity of the mRNA samples. Interestingly, mRNA
expression of meltrin
, and to a lesser extent of
meltrin
, in cocultures was up-regulated by treatment
with calcitriol. However, neither meltrin
nor
meltrin
was detectable in purified mature OCLs. When we
analyzed mRNA from POBs cultured alone in the absence or presence
of calcitriol, we found abundant expression of both meltrin
and meltrin
in the POB cultures and a 3.6-fold
induction in the steady state level of meltrin
mRNA
by calcitriol (Fig. 3A and Table
I). Consistent with the absence of
meltrin
and meltrin
messages in purified
OCLs, a macrophage cell line, IC21, also lacked the expression of
meltrin
and meltrin
. These results suggest that meltrin
and meltrin
are not
appreciably expressed in the monocyte/macrophage/osteoclast lineage and
that their expression is restricted to cells of mesenchymal origin such
as osteoblasts and myoblasts. The fact that the level of
meltrin
and meltrin
expression in
cultured POBs is much higher than that in the whole bone (Fig.
4) also supports the idea that the
osteoblast is the major cell type expressing these two MDC genes.
However, because heterogeneous primary cultures were used in this
study, we cannot exclude the possibility that meltrin
and meltrin
are expressed in early osteoclast precursors
and other hematopoietic cells. Taken together with the calcitriol
effects on POBs, these results indicate a correlation between
meltrin
and meltrin
expression and
osteoblast differentiation.
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Western Blot Analysis of Meltrin --
One of the questions
raised by the domain structure of MDC proteins is how the domains are
processed to yield a functional protein. To assess any potential
proteolytic processing of Meltrin
, we raised antibodies in rabbits
against GST fusion proteins with different parts of the Meltrin
cytoplasmic tail (see "Materials and Methods"). Probing Western
blots of reduced and alkylated extracts from COS-7 cells transfected
with meltrin
with affinity-purified anti-Meltrin
cytoplasmic tail antibodies (see "Materials and Methods") revealed
bands of 115, 87, 42, and 35 kDa (Fig. 5,
lane 1). These bands appear to be specific for Meltrin
,
as they are not recognized by control antibodies depleted of Meltrin
reactive IgG (Fig. 5, lane 2) and are not seen when
COS-7 cells that have been transfected with the expression vector alone
are probed with the affinity-purified antibodies (Fig. 5, lane
3). On a Western blot of extracts from primary osteoblasts probed
with the Meltrin
antiserum, bands of 115, 87, and 42 kDa and some
minor bands close to 35 kDa are visible (Fig. 5, lane 4);
these bands are not recognized by preimmune serum (Fig. 5, lane
5). This band pattern closely resembles the pattern observed in
COS-7 cells expressing Meltrin
(Fig. 5, lane 1) When an
extract of ~20% fused C2C12 mouse myoblasts was probed with the
affinity-purified Meltrin
IgG, a band of 115 kDa and weaker bands
of 110 and 42 kDa were visible (Fig. 5, lane 6); these bands
were not present in an identical sample probed with the control IgG
(Fig. 5, lane 7). These results suggest that a majority of
Meltrin
is present in a form that most likely includes the
prodomain, although POB cells apparently contain a significant amount
of an 87-kDa Meltrin
band that presumably lacks the
prodomain. The faster migrating bands likely correspond to processed
forms of the protein in all three cell types examined here, although
these bands could conceivably also arise due to cross-reactivity of the
antibodies with other proteins, including related MDC proteins.
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DISCUSSION |
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Proteins containing a metalloprotease domain, a disintegrin
domain, and a cysteine-rich region (MDC proteins) have been implicated in a variety of important cellular processes, including neurogenesis (7, 8), protein ectodomain shedding (10, 11), and cell-cell binding and
fusion (2, 4, 6). According to a previous report, an MDC protein termed
meltrin (also referred to as ADAM 12 (1)) has been shown
to play a critical role in C2C12 myoblast fusion (6). Furthermore, both
meltrin
, and a second MDC protein of unknown function
termed meltrin
were shown to be most abundantly expressed in adult bone, and in neonatal muscle (6), which was
confirmed in the present study. Because bone also contains fusing
cells, i.e. multinucleated osteoclasts, these observations raised the possibility that meltrin
and/or
meltrin
may be expressed in cells of the osteoclast
lineage and may therefore be involved in the fusion process of these
cells. However, our results indicate that in bone, both
meltrin
and meltrin
are primarily
expressed in cells of the osteoblast lineage but cannot be detected in
mature osteoclasts by the relatively sensitive method of RNase
protection. We also found that meltrin
and
meltrin
are widely expressed in other tissues at various
levels, implying that both meltrins may exert divergent functions in
different types of cells, including myoblasts and osteoblasts.
In this study, the full-length cDNA of mouse meltrin has been cloned and sequenced. Of the presently known MDC proteins, X. laevis ADAM 13 (18) and Meltrin
(6) are most closely related to Meltrin
. X. laevis ADAM 13 is localized to
somatic mesoderm and cranial neural crest cells from gastrulation
through neurulation and tail bud stages and has therefore been
suggested to function in neural crest adhesion and migration and in
myoblast differentiation (18). From the sequence similarity between
Meltrin
and X. laevis ADAM 13 (41.9%) it can not be
judged whether these two proteins are orthologues, and further study
will be necessary to compare the developmental expression of Meltrin
to that of ADAM 13. A PCR sequence tag generated from X. laevis was found to be 74% identical to Meltrin
, making it
unlikely that Meltrin
and ADAM 13 are orthologues (18). Because a
similarly high level of sequence conservation has also been observed
between mouse and X. laevis Mdc9 (75% sequence similarity)
and between human MDC11 and X. laevis Mdc11 (72% sequence
similarity),2 other potential
orthologues may be equally well conserved. For this reason, Meltrin
was considered to likely be a novel metalloprotease-disintegrin protein
and was therefore assigned the number 19 in the ADAM numbering system
(see Ref. 1).3
To obtain some insight into the potential functions of meltrin
, meltrin
, mdc9, and mdc15 in bone, we analyzed
the expression levels of these four MDC genes in bone cells. The
full-length sequences of mouse Mdc9, human MDC9 (14), mouse Meltrin
(6), and human MDC15 (15) have been previously reported. Because mouse
bone cells were used here, mouse Mdc15
cDNA4 was used for RNase
protection experiments. Mouse Mdc15 is 79.7% identical to human MDC15
at the amino acid level, is widely expressed, and, interestingly,
contains the sequence TDD instead of RGD in the predicted integrin
binding sequence. All four MDC proteins analyzed here contain a
metalloprotease domain with a predicted catalytic site, a disintegrin
domain, and cytoplasmic domains with predicted signaling motifs,
including SH3 ligand domains. Other features of potential functional
significance shared by the four MDC proteins include a predicted
subtilisin type proprotein convertase cleavage site
(RX(K/R)R) between the prodomain and the metalloprotease
domain (38, 39) and an odd-numbered cysteine in the prodomain that most
likely is involved in a cysteine switch regulation of the protease (40,
41).
Our results demonstrate that in bone, the major cell type expressing
meltrin and meltrin
is the osteoblast. It
is now well established that osteoblasts originate from mesenchymal
stem cells, which also give rise to myocytes, chondrocytes, and
adipocytes. For example, several clonal cell lines of mesenchymal
origin have been reported to be able to differentiate into two or more
distinct cell types under appropriate conditions (42, 43). Although it
is yet to be determined whether or not meltrin
and
meltrin
are expressed in chondrocytes and adipocytes,
the relatively restricted pattern of expression in myoblasts and
osteoblasts implies that they may play key roles in differentiation
and/or functions of mesenchymal cells in connective tissues. Further evidence for potential functions of the meltrins in bone can be derived
from our findings that the expression of meltrin
and meltrin
is regulated during osteoblast differentiation
in vitro and that expression of meltrin
in
primary osteoblasts is inducible by calcitriol, an active metabolite of
vitamin D3. Osteoblasts are known to express receptors for vitamin D,
and calcitriol has been shown to have various direct effects on both
primary osteoblasts and osteoblastic cell lines (44). Vitamin D is one
of the major regulators of bone and mineral metabolism, as evidenced by
the fact that lack of vitamin D action causes clinical disorders such as osteomalacia and rickets due to a mineralization defect (reviewed in
Ref. 44). Collectively, our results suggest a link between osteoblast
differentiation and the expression of meltrin
and meltrin
. It should, however, be noted that the cells
used in this study, mouse calvarial cells and MC3T3E1 cells, may not
represent normal osteoblast populations in every aspect. For example,
calcitriol has inhibitory effects on osteocalcin expression in mouse
calvarial cells, as opposed to stimulatory effects observed in rat and
human osteoblasts (45). Furthermore, it is known that in
vitro differentiation of MC3T3E1 cells may differ from primary
osteoblasts because they express very little, if any, of the
transcription factor Msx-2 during the proliferative stage (46).
Based on what is known about other metalloprotease-disintegrin
proteins, we predict that the meltrins may function in cell adhesion
and/or signaling between osteoblasts or may be involved in protein
ectodomain cleavage or shedding of osteoblast proteins that may require
processing for activity (13). Preliminary studies with MC3T3E1 cells
that were stably transfected with meltrin failed to
reveal any changes in the expression of osteoblastic genes, such as
type 1 collagen and osteocalcin, when compared with vector-transfected
control cells (data not shown). Clearly, further study, such as the
analysis of the function of individual protein domains of the meltrins,
and targeted gene knockout experiments will be necessary to clarify the
physiological role of meltrin
and meltrin
in osteoblasts.
Osteoclasts, another major type of bone cells, are terminally
differentiated, multinucleated bone-resorbing cells that originate in
multipotent hematopoietic stem cells and belong to the
monocyte/macrophage lineage (reviewed in Refs. 36 and 47). RNase
protection assays suggest that osteoclasts express only mdc9
and mdc15, but not meltrin or
meltrin
, whereas osteoblasts express all four MDC genes.
This would argue against a role of meltrin
and
meltrin
in osteoclast fusion, although an expression in
fusing osteoclasts that is down-regulated immediately after fusion
cannot be ruled out with the assay used here. Although the level of
mdc9 and mdc15 expression observed in purified
mature OCLs was considerably lower than that found in osteoblasts and
cocultures, it seems unlikely that the expression of meltrin
and meltrin
observed here is due to contamination
with other cells because we were not able to detect meltrin
and meltrin
expression in the OCL RNA samples, even
after longer exposure (data not shown). Thus, our results suggest that
mdc9 and mdc15 are expressed in cells of the
osteoclast lineage, although their expression in osteoclast precursors
remains to be verified by single-cell level analyses.
For an initial biochemical characterization of Meltrin , antibodies
against the cytoplasmic tail were raised and used in a Western blot
analysis of COS-7 cell expressing Meltrin
, of mouse C2C12 cells,
and of mouse POB cells. In all three cases, the predominant form of
Meltrin
has a molecular mass of 115 kDa, which is ~15 kDa larger
than the predicted molecular mass of 99.72 kDa of Meltrin
after
removal of the signal sequence. This band most likely corresponds to a
glycosylated form of Meltrin
, in which the prodomain and
metalloprotease domain are membrane-anchored. A band of 85 kDa, which
is relatively more abundant in POBs, probably corresponds to a form of
Meltrin
in which the prodomain has been removed but the
metalloprotease domain is still attached. The less prominent bands of
42 and 35 kDa most likely represent shorter proteolytically processed
forms of Meltrin
. Although the functional significance of these
processed forms remains to be determined, for fertilin
and for
Meltrin
, there is evidence that removal of the metalloprotease
domain may regulate a role in membrane binding and/or fusion (3, 6,
13).
In summary, we have cloned the full-length cDNA of mouse
meltrin and demonstrated that meltrin
and
three other members of the MDC gene family, meltrin
,
mdc9, and mdc15, are expressed in bone cells. The
finding that meltrin
and meltrin
are
highly expressed in osteoblasts and are regulated by vitamin D and
during osteoblast differentiation suggests that these two proteins may play a role in bone formation. Our results further show that
meltrin
, which was previously implicated in muscle
fusion, is apparently not expressed in mature osteoclasts. Further
study will be necessary to dissect the role of the metalloprotease
domain, the disintegrin domain, and the cytoplasmic signaling motifs of
these MDC gene products in bone metabolism.
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ACKNOWLEDGEMENT |
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We thank J. Leibow for excellent technical assistance.
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FOOTNOTES |
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* This work was funded in part by grants to C. P. B. (National Institutes of Health R55GM51988 and National Science Foundation MCB-9631601), by Memorial Sloan-Kettering Cancer Center Support Grant NCI-P30-CA-08748, and by a grant from Hoechst Marion Roussel (Roussel-Uclaf, France), to R. B.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) AF019887.
§ Present address: University of Tokushima, School of Medicine, First Department of Internal Medicine, 3-18-15 Kuramoto-cho Tokushima-shi, Tokushima 770, Japan.
Present address: Institute of Cellular and Molecular
Biology, Research Laboratories of Schering AG,
D-13342 Berlin, Germany.
** To whom correspondence should be addressed: Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Ave., New York, NY 10021. Tel.: 212-639-2915; Fax: 212-717-3047; E-mail: c-blobel{at}ski.mskcc.org.
1 The abbreviations used are: MDC, metalloprotease, disintegrin, and cysteine-rich domains; OCL, osteoclast-like cell; PCR, polymerase chain reaction; GST, glutathione S-transferase; POB, primary osteoblast.
2 H. Cai, J. Krätzschmar, and C. Blobel, manuscript in preparation.
3 J. White, personal communication.
4 L. Lum and C. Blobel, manuscript in preparation.
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REFERENCES |
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