From the
This paper reports the molecular cloning of a novel gene in the
mouse that shows structural similarities to the microfibril protein
fibrillin and to the latent transforming growth factor-
Transforming growth factor-
In certain lines of cultured
cells, small latent growth factor complexes may contain additional high
molecular weight proteins. The best characterized of these high
molecular weight proteins is the latent TGF-
Two
LTBPs have been isolated to date. The deduced human LTBP-1 amino acid
sequence is composed of a signal peptide, 16 epidermal growth
factor-like repeats with the potential to bind calcium (EGF-CB
repeats), two copies of a unique motif containing 8 cysteine residues,
an RGD cell attachment motif, and an 8-amino acid motif identical to
the cell binding domain of the laminin B2 chain (AGPHCEKC) (Kanzaki
et al., 1990). There is evidence that LTBP-1 binds calcium,
which, in turn, induces a structural change that protects LTBP from
proteolytic attack (Colosetti et al., 1993). LTBP-2 shows 41%
sequence identity to LTBP-1, and its structural domains show a similar
overall organization (Moren et al., 1994).
While the
functions of LTBP-1 and LTBP-2 presently are unknown, several ideas
have been put forward in the literature. First, LTBP may regulate the
intracellular biosynthesis of latent TGF-
In this paper, we report the
molecular cloning of a new gene in the mouse that is related to LTBP-1
and LTBP-2. This conclusion is supported by (i) comparative
analysis of DNA sequence identity, (ii) an evaluation of gene
expression in developing tissues, and (iii)
immunoprecipitation studies that show that that new mouse gene product
forms specific complex with TGF-
Sequence analysis of cDNA
clones revealed an open reading frame of 3,753 nucleotides. A
methionine codon in a favorable context for translation initiation was
provisionally designated the translation start site
(CCTGCGATGC; see Kozak (1991)). The initiator methionine was
followed by a characteristic signal sequence of 21 amino acids. Beyond
this, the conceptual amino acid sequence appeared to be organized into
five structurally distinct domains (Fig. 2). Domain 1 was a
28-amino acid segment (amino acids 22-49) with a net basic charge
(estimated pI, 12.36). Domain 2 (amino acids 50-439) consisted of
an EGF-like repeat, a 135-amino acid segment that was proline-rich
(20.7%) and glycine-rich (11.8%) but not cysteine-rich, a Fib-motif
(Pereira et al., 1993), an EGF-CB repeat, and a TGF-bp repeat.
Domain 3 (amino acids 440-552) was characterized by its high
proline content (21%). Domain 4 (amino acids 553-1229) consisted of 14
consecutive cysteine-rich repeats. Based on structural homologies,
12/14 repeats were EGF-CB motifs (Handford et al., 1990),
whereas 2/14 were TGF-bp motifs (Kanzaki et al., 1990). Domain
5 was a unique 22-amino acid segment at the carboxyl terminus (amino
acids 1230-1251).
A total of 19 cysteine-rich repeats
were found in domains 2 and 4 of the new mouse gene product. Thirteen
were EGF-like and 11/13 showed the calcium binding consensus sequence:
(D/N)(I/V)(D/N)(E/D)C
We (Pereira et
al., 1993; Zhang et al., 1994; Yin et al., 1995)
and others (Kanzaki et al., 1990; Taipale et al.,
1994) have previously noted that similarities exist in the structural
organization of LTBP and fibrillin. Fibrillin-1 and fibrillin-2 are
secreted extracellular matrix proteins with five structurally distinct
domains (cysteine-rich repeats are the dominant motif in two of five
domains). Like the fibrillins, LTBP also can be divided into five
structurally distinct domains. As shown in Fig. 2for LTBP-1,
these include a relatively short domain downstream of the signal
peptide with an estimated pI of 11.14 (amino acids 21-33; domain
1); a domain consisting of EGF-like, EGF-CB, TGF-bp, and Fib motifs
plus a proline-rich and glycine-rich sequence (amino acids
34-407; domain 2); a proline-rich domain (amino acids
408-545; domain 3); a large, domain consisting of EGF-CB, TGF-bp,
and TGF-bp-like repeat motifs (amino acids 546-1379; domain 4); and a
relatively short domain at the carboxyl terminus (amino acids
1380-1394; domain 5). These similarities in fibrillin and LTBP
likely explain the initial isolation of the low homology mouse PCR
fragment ( i.e. the human fibrillin-1 oligonucleotide primers
used to amplify mouse cDNA were designed to direct the synthesis of a
EGF-CB repeat).
Recent evidence
suggests that the fibrillin and LTBP genes can be distinguished on the
basis of their expression patterns during development. Whereas the
fibrillins are exclusively expressed by connective cells (Zhang et
al., 1994),
An overview of
the expression pattern is presented in Fig. 3. Approximate
midsagittal sections of normal mouse embryos at days 8.5-9.0,
13.5, and 16.5 p.c. of development were hybridized with a
We first asked whether or not MC3T3-E1 cells
co-express the mouse gene product and TGF-
We then prepared an affinity-purified
antibody
(274) capable of immunoprecipitating the new mouse
gene product (see ``Experimental Procedures''). A full-length
mouse cDNA was assembled into the pcDNA3 mammalian expression vector
(Invitrogen) and expressed following transient transfection of 293T
cells. Nascent polypeptides, radiolabeled by addition of
[
This paper reports the molecular cloning of a novel gene in
the mouse that encodes a single transcript of
No previous study has presented a mouse
LTBP sequence, but it seems clear that the gene reported in this paper
is not simply the mouse homologue of human LTBP-1 or LTBP-2. First,
domain 4 of the LTBP-3 coding sequence has a smaller number of EGF-like
repeat motifs than reported for either LTBP-1 and LTBP-2 (LTBP-3
= 8; LTBP-2 = 13; and LTBP-1 = 11). Second, we
have isolated several fragments of the human LTBP-3 gene and shown that
the coding sequence is distinct from that of LTBP-1 and LTBP-2 (data
not shown). Third, the human LTBP-1, LTBP-2, and LTBP-3 genes are
localized to separate chromosomes. The LTBP-1 gene was assigned to
human chromosome 2 based on the analysis of human x rodent
somatic cell hybrid lines (Stenman et al., 1994); the LTBP-2
gene was assigned to human chromosome 14, band q24 based on the
analysis of human x rodent somatic cell hybrid lines and
fluorescent in situ hybridization studies (Moren et
al., 1994); and the LTBP-3 gene was localized to chromosome 11,
band q12 (while the mouse gene was mapped to mouse chromosome 19, band
B, a region of conserved synteny) from the analysis of human x rodent somatic cell hybrid lines and fluorescent in situ hybridization studies (Li et al., 1995).
If LTBP-3 is like LTBP-1, it has the potential to function as
a secreted, extracellular structural protein. As demonstrated here,
domain 1 of LTBP-3 appears to be a unique sequence that likely has a
globular conformation. Domain 1 also is highly basic and may facilitate
LTBP-2 binding to acidic molecules ( e.g. acidic proteoglycans)
within the extracellular space. Sequences rich in basic amino acids
have also been shown to function as endoproteolytic processing signals
for several peptide hormones (Barr, 1991; Steiner et al.,
1992). It is possible, therefore, that the NH
Our data
indicate that MC3T3-E1 preosteoblasts co-express LTBP-3 and TGF-
The idea that LTBP may regulate
TGF-
We offer two explanations for the apparent
difference between our results and those of Dallas et al. (1994). On one hand, the differences may reflect trivial species
differences in reagents used by the two groups ( i.e. mouse
versus rat/human). Alternatively, expression of large latent
TGF-
It will be important for future studies to also
determine if LTBP-3 binds calcium, since EGF-CB repeats have been shown
to mediate high affinity calcium binding in LTBP-1 and other proteins
(Colosetti et al., 1993). Calcium binding, in turn, may
contribute to molecular conformation and the regulation of its
interactions with other molecules. The presence of dibasic amino acids
suggests that LTBP-3 may also undergo cell- and tissue-specific
proteolysis. Finally, it will be important for future studies to
identify LTBP isoforms and their functional role during tissue
morphogenesis and wound healing. TGF-
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) L40459.
The authors are grateful to F. Ramirez for advice and
discussion of results prior to publication.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-
) binding protein (LTBP), a component of the latent TGF-
complex. The gene was initially isolated during a low stringency
polymerase chain reaction screen of a NIH 3T3 cell cDNA library using
primers that amplify a human fibrillin-1 epidermal growth factor-like
repeat. Three lines of evidence suggest that the mouse gene is a third
member of the LTBP gene family, which we designate LTBP-3. First, the
deduced polypeptide, which consists of 15 epidermal growth factor-like
repeats, 3 TGF binding protein repeats, and 2 proline- and glycine-rich
sequences, shows 38.4% identity with LTBP-1 but only 27% identity with
fibrillin-1. Second, the gene appears to be co-expressed in developing
mouse tissues with TGF-
. Third, immunoprecipitation studies using
mouse preosteoblast MC3T3-E1 cells and a specific anti-peptide
polyclonal antiserum reveal that the mouse polypeptide forms a complex
with the TGF-
1 precursor. Finally, we note that the LTBP-3 gene
was recently localized to a distinct genetic locus (Li, X., Yin, W.,
Perez-Jurado, L., Bonadio, J., and Francke, U. (1995) Mamm. Genome 6, 42-45). Identification of a third binding protein
provides further insight into a mechanism by which latent TGF-
complexes can be targeted to connective tissue matrices and cells.
(TGF-
s)
(
)
represents a family of structurally related molecules with
diverse effects on mammalian cell shape, growth, and differentiation
(Roberts and Sporn, 1990). Initially synthesized as a precursor
consisting of an amino-terminal propeptide followed by mature
TGF-
, two chains of nascent pro-TGF-
associate in most
tissues to form a M
106,000 inactive
disulfide-bonded dimer. Homodimers are most common, but heterodimers
have also been described (Cheifetz et al., 1987; Ogawa et
al., 1992). During biosynthesis, the mature TGF-
dimer is
cleaved from the propeptide dimer. TGF-
latency results in part
from the noncovalent association of propeptide and mature TGF-
dimers (Pircher et al. (1984, 1986), Wakefield et al. (1987), Millan et al. (1991), and see also Miyazono and
Heldin (1989)). Consequently, the propeptide dimer is often referred to
as the latency-associated protein, and this protein plus the
disulfide-bonded TGF-
dimer are also known as the small latent
complex. In the extracellular space, small latent complexes must be
dissociated to activate mature TGF-
. The mechanism of activation
of the latent complex is thought to be one of the most important steps
governing TGF-
effects (Lyons et al., 1988;
Antonelli-Orlidge et al., 1989; Twardzik et al.,
1990; Sato et al., 1993).
binding protein, or
LTBP (Miyazono et al., 1988; Kanzaki et al., 1990;
Tsuji et al., 1990; Olofsson et al., 1992; Taketazu
et al., 1994). LTBP produced by different cell types is
heterogeneous in size, perhaps because of alternative splicing or
because of tissue-specific proteolytic processing (Miyazono et
al., 1988; Wakefield et al., 1988; Kanzaki et
al., 1990; Tsuji et al., 1990). Latent TGF-
complexes that contain LTBP are known as large latent complexes. LTBP
has no known covalent linkage to mature TGF-
, but rather it is
linked by a disulfide bond to the latency-associated protein.
precursors. Cultured
erythroleukemia cells efficiently assemble and secrete large latent
TGF-
complexes, whereas they slowly secrete small latent TGF-
complexes that contain anomalous disulfide bonds (Miyazono et
al., 1991, 1992). Therefore, LTBP may facilitate the normal
assembly and secretion of latent TGF-
complexes. Second, LTBP may
target latent TGF-
to specific types of connective tissue. Recent
evidence suggests that the large latent TGF-
complex is covalently
bound to the extracellular matrix via LTBP (Taipale et al.,
1994). Based on these observations, LTBP has been referred to as a
``matrix receptor'', i.e. a secreted protein that
targets and stores latent growth factors such as TGF-
to the
extracellular matrix. Third, LTBP may modulate the activation of latent
complexes. This idea is based in part on recent evidence that suggests
that mature TGF-
is released from extracellular storage sites by
proteases such as plasmin and thrombin and that LTBP may protect small
latent complexes from proteolytic attack (Falcone et al.,
1993; Benezra et al., 1993; Taipale et al., 1994),
i.e. protease activity may govern the effect of TGF-
in
tissues, but LTBP may modulate this activity. Fourth, LTBP may play an
important role in targeting the latent TGF-
complex to the cell
surface, allowing latent TGF-
to be efficiently activated
(Flaumenhaft et al., 1993).
1. A recent study has also shown
that the new mouse gene is localized to a distinct genetic locus (Li
et al., 1994).
Taken together, these results
suggest that LTBP is a gene family of at least three members and that
an appropriate designation for the new gene is LTBP-3. Identification
of a third binding protein family member provides further insight into
the mechanism by which latent TGF-
complexes can be targeted to
connective tissue matrices and cells.
cDNA Cloning
Aliquots (typically 40-50,000
plaque-forming units) of phage particles from a cDNA library in the
ZAP II vector made from NIH 3T3 cell mRNA (Stratagene) and fresh
overnight XL1-Blue cells (grown in Luria broth supplemented with 0.4%
maltose in 10 mM MgSO
) were mixed, incubated for
15 min at 37 °C, mixed again with 9 ml of liquid (50 °C) top
layer agarose (NZY broth plus 0.75% agarose), and then spread evenly
onto freshly poured 150-mm NZY-agar plates. Standard methods were used
for the preparation of plaque-lifts and filter hybridization (42
°C, in buffer containing 50% formamide, 5
SSPE, 1
Denhardt's solution, 0.1% SDS, 100 mg/ml salmon sperm DNA, 100
mg/ml heparin). Filters were washed progressively to high stringency
(0.1
SSC, 0.1% SDS, 65 °C). cDNA probes were radiolabeled
by the nick translation method using commercially available reagents
and protocols (Sure Site kit, Novagen). Purified phage clones were
converted to pBluescript plasmid clones, which were sequenced using
Sequenase (version 2.0) as described previously (Chen et al.,
1993). (
)Sequence alignment and identity was determined using
sequence analysis programs from the Genetics Computer Group
(MacVector).
Tissue In Situ Hybridization
To prepare normal
sense and antisense probes, a unique 342-base pair fragment from the
3`-untranslated region (+3973 to +4314, counting the A of the
initiator Met codon as +1; see Fig. 1, ish) was
subcloned into the pBluescript KS+ plasmid (Stratagene, Inc.).
Template DNA was linearized with either EcoRI or
BamHI, extracted, and precipitated with ethanol. Sense and
antisense transcripts were generated from 1 µg of template with T3
and T7 polymerases in the presence of [S]UTP at
>6 mCi/ml (Amersham Corp., >1200 Ci/mmol) and 1.6 units/ml RNasin
(Promega), with the remaining in vitro transcription reagents
provided in a kit (SureSite, Novagen Inc.). After transcription at 37
°C for 1 h, DNA templates were removed by a 15-min digest at 37
°C with 0.5 units/ml RNase-free DNase I, extracted and precipitated
with ethanol. Riboprobes were hydrolyzed to an average final length of
150 base pairs by incubating in 40 mM NaHCO
, 60
mM Na
CO
, 80 mM dithiothreitol
for
40 min at 60 °C. Hydrolysis was terminated by the addition
of sodium acetate, pH 6.0, and glacial acetic acid to 0.09 M and 0.56% (v/v), respectively, and the probes were then
ethanol-precipitated, dissolved in 0.1 M dithiothreitol,
counted, and stored at
20 °C until use. Day 8.5-9.0,
day 13.5, and day 16.5 mouse embryo tissue sections (Novagen) and the
in situ hybridization protocol were exactly as described (Chen
et al., 1993).
Figure 1:
Overlapping
mouse cDNA clones representing the mouse LTBP-3 gene sequence. A
partial representation of restriction sites is shown. N,
NcoI; P, PvuII; R, RsaII;
B, BamHI; H, HindIII. The numbering
system at the margin of each cDNA fragment and at the bottom of the figure assumes that the A of the initiator Met codon is
nucleotide 1.
Northern Analysis
MC3T3-E1 cell
poly(A) RNA (2-10-µg aliquots) was
electrophoresed on a 1.25% agarose, 2.2 M formaldehyde gel and
then transferred to a nylon membrane (Hybond-N, Amersham Corp.). The
RNA was cross-linked to the membrane by exposure to a UV light source
(1.2
10
mJ/cm
, UV Stratalinker 2400,
Stratagene) and then prehybridized for >15 min at 65 °C in
Rapid-Hyb buffer (Amersham, Corp.). A specific cDNA probe consisting
solely of untranslated sequence from the 3` end of the transcript was
P-labeled by random priming and used for hybridization (2
h at 65 °C). Blots were washed progressively to high stringency
(0.1
SSC, 0.1% SDS, 65 °C), and then placed against x-ray
film with intensifying screens (XAR, Kodak) at
86 °C.
Antibody Preparation
LTBP-3 antibodies were raised
against a unique peptide sequence found in domain 2 (amino acids
155-167). Peptide 274 (GESVASKHAIYAVC) was synthesized using an
ABI model 431A synthesizer employing FastMoc chemistry. The sequence
was confirmed using an ABI 473 protein sequencer. A cysteine residue
was added to the carboxyl terminus to facilitate cross-linking to
carrier proteins. For antibody production, the synthetic peptide was
coupled to rabbit serum albumin using m-maleimidobenzoic acid
N-hydroxysuccinimide ester at a substitution of 7.5 mg of
peptide/mg of rabbit serum albumin. One mg of the peptide-rabbit serum
albumin conjugate in 1 ml of Freund's complete adjuvant was
injected subcutaneously at 10 different sites along the backs of
rabbits. Beginning at 3 weeks after initial immunization, the rabbits
were given biweekly booster injections of 1 mg of peptide-rabbit serum
albumin in 100 µl of Freund's incomplete adjuvant. IgG was
prepared by mixing immune serum with caprylic acid (0.7 ml caprylic
acid/ml of serum), stirring for 30 min, and centrifuging at 5000
g for 10 min. The supernatant was decanted and
dialyzed against two changes of phosphate-buffered saline (PBS)
overnight at 4 °C. The antibody solution was then affinity purified
by passing it over a column containing the immunizing peptide coupled
to Affi-Gel 10 affinity support. Bound antibodies were eluted with 0.2
M glycine, pH 2.3, immediately dialyzed against PBS, and
concentrated to 1 mg/ml prior to storage at
70 °C.
Transfection
Transient transfection was performed
using standard protocols (Sambrook et al., 1989). Briefly,
subconfluent cells (covering 20% of a 100-mm plastic tissue
culture dish) were washed 2 times in Dulbecco's modified
Eagle's medium tissue culture medium (Life Technologies, Inc.)
and then incubated for 3 h at 37 °C in a sterile mixture of
DEAE-dextran (0.25 mg/ml), chloroquine (55 mg/ml), and 15 µg of
plasmid DNA (Courey and Tjian, 1988). Cells then were shocked by
incubation with 10% dimethyl sulfoxide in sterile PBS for 2 min at 37
°C, washed 2
with Dulbecco's modified Eagle's
medium (Sambrook et al., 1989), and incubated in
Dulbecco's modified Eagle's medium plus 10% fetal calf
serum and antibiotics for 72 h at 37 °C.
Immunoprecipitation
For immunoprecipitation, 1 ml
of antibody (1:400 final concentration, in PBS-TDS buffer (0.38 mM NaCl, 2.7 mM KCl, 8.1 mM NaHPO
, 1.5 mM KH
PO
, 1% Triton X-100, 0.5% deoxycholic
acid, and 0.1% SDS) was added to 1 ml of radiolabeled medium proteins.
The mixture was incubated with shaking at 4 °C for 1 h, protein
A-Sepharose CL-4B beads were added (200 µl, 10% suspension), and
this mixture was incubated with shaking for 1 additional h at 4 °C.
Immunoprecipitated proteins were pelleted by brief centrifugation, the
pellet was washed 6 times with PBS-TDS buffer, 2
protein
loading dye was added, and the samples were boiled for 5 min and then
fractionated on 4-18% gradient SDS-polyacrylamide gel
electrophoresis (Bonadio et al., 1985). Cold molecular weight
markers (200-14.3 kDa, Rainbow mix, Amersham Corp.) were used to
estimate molecular weight. The gel was dried and exposed to film for
the indicated time at room temperature.
Western Analysis
Fractionated proteins within
SDS-polyacrylamide gels were transferred to a nitrocellulose filter for
2 h using Tris-glycine-methanol buffer, pH 8.3, at 0.5
mA/cm. The filter was blocked, incubated with nonfat milk
plus antibody (1:1000 dilution) for 2 h, and washed. Antibody staining
was visualized using the ECL Western blotting reagent (Amersham Corp.)
according to the manufacturer's protocols.
Molecular Cloning of a Mouse Gene Related to Human
Fibrillin-1 and to LTBP
While cloning mouse fibrillin-1 (Yin
et al., 1995), a 3T3 cell cDNA library was PCR amplified using
human fibrillin-1 oligonucleotide primers. A PCR fragment with low
homology to human fibrillin-1 was obtained. This result was surprising
because the human and mouse fibrillin-1 genes share >95% coding
sequence identity. To investigate the possibility that the low homology
mouse PCR fragment was derived from a related (fibrillin-like) cDNA,
the 3T3 cell cDNA library was screened at high stringency using the PCR
fragment as the probe. A walking strategy eventually yielded a series
of overlapping cDNA clones (Fig. 1).
Figure 2:A, schematic showing the
structure of mouse fibrillin-1 ( top), the LTBP-3 gene product
( middle), and human LTBP-1 ( bottom). Structural
domains are denoted below each diagram. Note that regions A-E
of mouse fibrillin-1 (Yin et al., 1995) correspond to domains
1-5 of the mouse LTBP-3 gene product and LTBP. Symbols designate
the following structural elements: EGF-CB repeats, open rectangles;
TGF-bp repeats, open ovals; Fib motif, open circle; TGF-bp-like repeat, patterned oval; cysteine-rich sequences, patterned rectangles; proline/glycine-rich region, thin curved line, domain 2; proline-rich region,
thick curved line, domain #3. Symbols
designating the signal peptide have been deleted for simplicity.
B, cDNA and deduced amino acid sequence of the mouse LTBP-2
gene. The boundaries of domains 1-5 are denoted by upward pointing arrows. Note that the signal peptide
precedes domain 1. Sites of potential N-linked glycosylation
are double underlined. Sites of potential dibasic
endoproteolytic cleavage are in
boldface.
The conceptual amino acid sequence consisted
of 1251 amino acids with an estimated pI of 5.92, a predicted molecular
mass of 134,710 Da, and five potential N-linked glycosylation
sites. RGD and laminin B2 chain cell adhesion sequences were not
identified. Northern blot analysis of mouse embryo RNA using a
3`-untranslated region probe identified a transcript band of 4.6
kilobases (data not shown). In this regard, 4310 nucleotides have been
isolated by cDNA cloning, including a 3`-untranslated region of 401
nucleotides and a 5`-upstream sequence of 156 nucleotides. The apparent
discrepancy between the Northern analysis result and the cDNA sequence
analysis suggested that the 5`-upstream sequence may include
300
nucleotides of additional upstream sequence. This estimate was
consistent with preliminary primer extension mapping studies indicating
that the 5`-upstream sequence is 400-500 nucleotides in
length.
(
)
. This consensus was derived from an
analysis of 154 EGF-CB repeats in 23 different proteins and from
structural analyses of the EGF-CB repeat, both bound and unbound to
calcium ion (Selander-Sunnerhagen et al., 1992). Variations on
the consensus have been noted previously (Yin et al., 1995),
and one of these, DL(N/D)EC
, was identified in the
third EGF-like repeat of domain 4. In addition, a potential calcium
binding sequence, which has not previously been reported
(ET(N/D)EC
), was identified in the first EGF-like
repeat of domain 4. Ten of 13 EGF-CB repeats also contained a second
consensus sequence,
C
X(D/N) XXXX(Y/F) XC
,
which represents a recognition sequence for an Asp/Asn hydroxylase that
co- and posttranslationally modifies D/N residues (Stenflo et
al., 1987; Gronke et al., 1989).
Evidence That the Mouse Gene Codes for a New Latent
TGF-
An initial goal was to determine if
the mouse gene we had cloned was a third member of the fibrillin gene
family or a third LTBP. Comparison of sequence identity using the GAP
and BESTFIT programs (Genetics Computer Group) revealed only 27% amino
acid homology between the new mouse gene product and fibrillin-1. A
similar level of sequence identity (22%) existed between the mouse gene
product and human fibrillin-2. These values are low for a putative
fibrillin family member, since fibrillin-1 and fibrillin-2 share
Binding Protein
50% identity (Zhang et al., 1994). A search of available
data bases revealed that the mouse gene product was most similar to
human and rat LTBP-1 and, especially, to human LTBP-1. Amino acid
sequence comparison with human LTBP-1 showed 60% identity for domain 1;
52% identity for domain 2; 30% identity for domain 3; 43% identity for
domain 4; and 7% identity for domain 5. The average identity over the
five domains was 38.4%. Significantly, cysteine residues in the mouse
gene product and human LTBP-1 were highly conserved.
LTBP is co-expressed with TGF-
in
epithelial, parenchymal, and connective cells (Tsuji et al.,
1990). If the sequence identity comparison was correct in suggesting
that the new mouse gene was a LTBP family member, it followed that all
three cell types would express this gene. Tissue in situ hybridization was used to test this hypothesis.
S-labeled single-stranded antisense riboprobe synthesized
from a 382-base pair cDNA coding for the 3`-untranslated region of the
mouse gene. The probe sequence showed less than 30% identity when
compared with the corresponding sequence of human LTBP-1 and LTBP-2,
which is too low to give spurious hybridization signals with the level
of stringency we employed. Uterine and placental tissues were also
present in the day 8.5-9.0 embryo tissue sections. As a negative
control, a
S-labeled single stranded normal sense
riboprobe from the same cDNA construct was used. At day 8.5-9.0
of development, expression of the new mouse gene above the background
of the experiment was observed in mouse embryo tissues; the mesometrial
and anti-mesometrial uterine tissues; and the ectoplacental cone,
placenta, placental membranes. Expression of the transcript above the
background of the experiment was also observed in mouse embryo tissues
at days 13.5 and 16.5 p.c. Gene expression was particularly
intense in the liver.
Figure 3:
Overview of expression of the LTBP-3
gene during development, as determined by tissue in situ hybridization. The figure consists of autoradiograms made by
direct exposure of tissue sections to film after hybridization with
radiolabeled probes but before dipping slides in radiographic emulsion.
Day 8.5-9.0 sections contained embryos surrounded by intact
membranes, uterine tissues, and the placental disk, cut in random
planes. Day 13.5 and 16.5 p.c. sections contain isolated whole
embryos sectioned in the sagittal plane near or about the mid-line.
Arrowheads point to liver (day 13.5 p.c. embryo on
the left) and to brain and spinal cord (day 13.5 p.c.
embryo on the right and day 16.5 p.c. embryo).
Identical conditions were maintained throughout autoradiography and
photography, thereby allowing a comparison of the overall strength of
hybridization in all tissue sections (days 8.5 versus 13.5
versus 16.5 p.c., and antisense versus sense
negative control probes).
Microscopy of day 8.5-9.0 embryos
p.c. (which was taken from the same slides used to prepare
whole mount sections shown in Fig. 3) confirmed the widespread
expression of the new mouse gene by mesenchymal cells (data not shown).
Significant expression in the developing central nervous system,
somites, and cardiovascular tissue (myocardium plus endocardium) was
observed (Fig. 4, A and B). This pattern of
expression contrasts sharply with that of fibrillin-1, which is
expressed only in mouse endocardium at this stage of development.
Figure 4:
Selected microscopic views of mouse LTBP-3
gene expression in day 8.5-9.0 p.c. mouse developing
tissues. All photographs were taken from the same slides used to
prepare whole mount sections shown in Fig. 3 (after dipping slides in
radiographic emulsion). A, neural tube; note the expression by
neuroepithelial cells and by surrounding mesenchyme. B, heart.
The figure demonstrates expression by myocardial and endocardial
( arrowheads) cells. Darkfield photomicrographs were taken
after exposure of tissues to photographic emulsion for 2 weeks. In all
darkfield photographs, red blood cell and other plasma membranes give a
faint white signal that contributes to the background of the
experiment. 1 cm = 20 µm (all
figures).
Microscopy of day 13.5 and day 16.5 p.c. embryos
(also taken from the same slides used to prepare whole mount sections
shown in Fig. 3) demonstrated expression of the new mouse gene by
osteoblasts and by periosteal cells of the calvarium, mandible, and
maxilla (Fig. 5 A). The transcript was also identified in
both cartilage and bone of the lower extremity (data not shown). A
positive signal was detected in perichondrial cells and chondrocytes of
articular cartilage, the presumptive growth plate, the cartilage model
within the central canal, blood vessel endothelial cells within the
mid-diaphysis, and the surrounding skeletal muscle cells (not shown).
The new mouse gene was also expressed by respiratory epithelial cells
lining developing small airways and connective tissue cells in the
pulmonary interstitium (Fig. 5 B, upper panel) and by myocardial cells (atria and ventricles) and
endocardial cushion tissue (Fig. 5 B, lower panel). A positive signal was also expressed by cells
within the walls of large arteries (data not shown). The transcript was
also expressed by acinar cells of the exocrine pancreas
(Fig. 5 C, upper panel). Mucosal
epithelial cells lining the upper and lower digestive tract also
expressed the transcript (Fig. 5 C, lower panel), as did the smooth muscle and connective
tissue cells found in the gut submucosa and the parenchymal cells
(hepatocytes) and extramedullary hematopoietic cells of the liver (not
shown). The new mouse gene was also expressed by cells of developing
nephrons, the ureteric bud, kidney blastema, and the kidney
interstitium (Fig. 5 D, upper panel),
by epidermal and adnexal keratinocytes, dermal connective tissue cells
(Fig. 5 D, lower panel), and brown fat
cells within the dorsal subcutis (data not shown) and by ganglion cells
of the developing mouse retina (Fig. 6 E). The transcript
was also intensely expressed by cells within the cerebrum, brainstem,
spinal cord, and peripheral nerves (not shown). In summary, Figs.
3-5 clearly demonstrated expression of the new mouse gene by
epithelial, parenchymal, and connective tissue cells.
Figure 5:
Microscopy of mouse LTBP-3 gene expression
in day 13.5 and day 16 p.c. mouse developing tissues. All
photographs were taken from the same slides used to prepare whole mount
sections shown in Fig. 3 (after dipping slides in radiographic
emulsion). A, cartilage model of developing long bone from
lower extremity. Note expression by chondrocytes and by perichondrial
cells. B, lung and heart. In the upper panel (lung), note expression by epithelial cells of developing airway
and by the surrounding parenchymal cells. The lower panel (heart) demonstrates continuing expression by myocardial cells.
C, digestive system. In the upper panel (pancreas), note expression by acinar epithelial cells. The
lower panel demonstrates expression by intestinal
epithelial and subepithelial cells. D, kidney and skin. In the
upper panel (kidney), note expression by blastemal
cells beneath the kidney capsule, epithelial cells of developing
nephrons and tubules, and the interstitial mesenchyme. The lower panel demonstrates expression by epidermal, adnexal, and
dermal cells of developing skin. E, retina. Note expression by
retinal epithelial cells and by adjacent connective tissue cells.
Darkfield photomicrographs were taken after exposure of tissues to
photographic emulsion for 2 weeks. In all darkfield photographs, red
blood cell and other plasma membranes give a faint white signal that
contributes to the background of the experiment. Note the absence of
spurious hybridization signal in areas of the slide that lack cellular
elements. 1 cm = 20 µm (all
figures).
Figure 6:
Time-dependent expression of the LTBP-3
gene by MC3T3-E1 cells. mRNA preparation and Northern blotting were
performed as described under ``Experimental Procedures.''
Aliquots of total RNA as determined by UV spectroscopy were loaded in
each lane of the Northern gel (not shown). As demonstrated by methylene
blue staining (Sambrook et al., 1989), equal amounts of RNA
were transferred to the nylon membrane (not shown). The results
demonstrate a clear, strong peak in LTBP-3 gene expression by 14 days
in culture. Weaker signals denoting LTBP-3 gene expression also can be
observed after 5 days and 28 days in
culture.
Collectively,
the sequence identity and gene expression data favor the conclusion
that the new mouse gene product was an LTBP family member. To further
support this conclusion, MC3T3-E1 mouse preosteoblasts were used to
demonstrate that the mouse gene product was capable of binding
TGF-. MC3T3-E1 cells were chosen because previous studies have
shown that they synthesize and secrete TGF-
, which may act as an
autocrine regulator of osteoblast proliferation (Amarnani et
al., 1993; Van Vlasselaer et al., 1994; Lopez-Casillas
et al., 1994).
. Cells were plated on
100-mm dishes under differentiating conditions (Franseochi et al. 1992; Quarles et al., 1992) and the medium was replaced
twice weekly. Parallel dishes were plated and assayed for cell number
and alkaline phosphatase activity, which confirmed that osteoblast
differentiation was indeed taking place. Aliquots of total cellular RNA
was prepared from these MC3T3-E1 cells after 5, 14, and 28 days in
culture for Northern blot analysis. As shown in Fig. 6,
expression of the new mouse gene peaked on day 14 of culture. This
result has been independently reproduced (data not shown). Since
MC3T3-E1 cells also show a peak in alkaline phosphatase activity on day
14 of culture (Quarles et al., 1992), the results suggest for
the first time that LTBP-2 gene expression is an early marker of
osteoblast differentiation.
S]Cys to the medium of transfected cells, were
immunoprecipitated using affinity-purified antibody 274. As shown in
Fig. 7
, the new mouse polypeptide was estimated to be
180-190 kDa. To ensure the specificity of antibody 274 binding,
we showed that preincubation of antibody with 10 µg of synthetic
peptide blocks immunoprecipitation of the 180-190 kDa band.
Figure 7:
Antisera 274 specifically binds LTBP-3
epitopes. Transfection of 293T cells with a full-length mouse LTBP-3
expression plasmid followed by radiolabeling, preparation of medium
samples, immunoprecipitation, and 4-18% gradient
SDS-polyacrylamide gel electrophoresis were performed as described
under ``Experimental Procedures.'' The figure presents a
SDS-polyacrylamide gel electrophoresis autoradiogram of medium samples
following a 2-day exposure to film. Lane assignments
are as follows: lane 1, radiolabeled 293T medium
(prior to transfection) immunoprecipitated with preimmune serum;
lane 2, radiolabeled 293T medium (prior to
transfection) immunoprecipitated with antibody 274; lane 3, radiolabeled 393T medium (following transfection and
preincubation with 10 µg of LTBP-3 synthetic peptide mixture)
immunoprecipitated with antibody #274; lane 4,
radiolabeled 293T medium (following transfection) immunoprecipitated
with antibody 274. As indicated by the bar, the
full-length LTBP-3 molecule migrated at 180-190
kDa.
Finally, MC3T3-E1 cells were cultured for 7 days under
differentiating conditions and double-labeled with 30 µCi/ml
[S]cysteine and
[
S]methionine in deficient media. Radiolabeled
media was dialyzed into cold PBS with protease inhibitors. Aliquots of
the dialyzed medium sample (10
incorporated CPM) were
analyzed by a combined immunoprecipitation/Western analysis protocol.
The mouse polypeptide was clearly and reproducibly secreted by MC3T3-E1
cells, migrating under reducing conditions as a single band of
180-190 kDa (Fig. 8, top panel).
Consistent with the results of previous studies ( e.g. Miyazono
et al. (1988), Dallas et al. (1994), Moren et al. (1994)), bands of 70 and 50 kDa corresponding to the TGF-
1
precursor were co-immunoprecipitated with the 180-kDa LTBP-3 protein.
Weak bands of 40 and 12 kDa were also identified in experiments in
which only immunoprecipitation was performed (data not shown). The
latter were not included in the top panel of
Fig. 8
because they migrated within that portion of the gel
included in the Western analysis. Protein bands of 70-12.5 kDa
are not variant forms of LTBP-3; Fig. 7demonstrates that LTBP-3
migrates as a single band of 180-190 kDa following transient
transfection of 293T cells, which fail to make TGF-
1 (data not
shown). As shown in Fig. 8( bottom panel), we
also found a unique band consistent with monomeric mature TGF-
1 in
the LTBP-2 immunoprecipitate. Antibody 274 is incapable of binding
TGF-
1 as determined by radioimmunoassay using commercially
available reagents (R& Systems) and the manufacturer's
suggested protocols.
These results have been reproduced in
six independent experiments that utilized three separate lots of
MC3T3-E1 medium. Altogether, the results presented in Figs. 6 and 8
demonstrate that the new mouse LTBP-like polypeptide binds TGF-
in vitro.
Figure 8:
Co-immunoprecipitation of LTBP-3 and
TGF-1 produced by MC3T3-E1 cells. Top panel,
aliquots (
10
incorporated counts/min) of radiolabeled
media produced by MC3T3-E1 cells after 7 days in culture were
immunoprecipitated as described under ``Experimental
Procedures.'' Bars indicate the position of cold
molecular mass standards used to estimate molecular mass: 200, 97.4,
69, 46, 30, 21.5, and 14.3 kDa (Rainbow mix, Amersham Corp.).
Immunoprecipitates were separated using 4-18% gradient
SDS-polyacrylamide gel electrophoresis and reducing conditions. The
figure shows a negative control ( left lane)
consisting of MC3T3-E1 medium immunoprecipitated using preimmune sera.
In contrast, the right lane consists of MC3T3-E1
medium immunoprecipitated with anti-LTBP-3 antibody #274. Bottom panel, Western blotting was performed as described (see
``Experimental Procedures'') using the lower portion of the
gradient gel and a commercially available antibody to TGF-
1 (Santa
Cruz Biotechnology, Inc.). Antibody staining was detected using
commercially available reagents and protocols (ECL Western blotting
Reagent, Amersham Corp.). The figure shows the negative (preimmune
sera) control ( left lane). The right lane consists of MC3T3-E1 medium immunoprecipitated with
anti-LTBP-2 antibody 274. The intense band at the top of the
photo ( right and left lanes) represents
antibody light chains detected by the ECL
reagent.
4.6 kilobases and a
deduced polypeptide of 1251 amino acids. Although it is similar to
fibrillin, the amino acid sequence of the new mouse gene product most
resembles the latent TGF-
binding protein. Additionally, the mouse
gene is expressed more widely during development than either
fibrillin-1 or fibrillin-2. The fibrillin-1 and fibrillin-2 genes are
expressed by mesenchymal cells of developing connective tissue (Zhang
et al., 1994),
while the TGF-
genes and
proteins are expressed by developing epithelial, parenchymal, and
stromal cells (Heine et al., 1987; Lehnert and Akhurst, 1988;
Pelton et al., 1989, 1990a, 1990b; Millan et al.,
1991). Tsuji et al. (1990) and others have suggested that the
expression of TGF-
binding proteins should mirror that of
TGF-
itself. As shown in Figs. 3-5, the mouse gene is
intensely expressed by epithelial, parenchymal, and stromal cells
during murine development, a pattern that is consistent with the
co-expression hypothesis. Finally, the new mouse gene product also
binds TGF-
produced by MC3T3-E1 cells. Collectively, these data
indicate that we have isolated a new member of the LTBP gene family,
which we designate LTBP-3.
terminus of
LTBP-3 is proteolytically processed in a tissue-specific manner.
Domains 2 and 4 consist of consecutive cysteine-rich repeats, the
majority of which are of the EGF-CB type. Besides binding calcium,
these repeats may provide LTBP-3 with regions of extended conformation
capable of interacting with other matrix macromolecules (Engel, 1989).
Domain 3 is proline-rich and may be capable of bending (or functioning
like a hinge) in three-dimensional space (MacArthur and Thornton,
1991). (In this regard, domain 2 is of interest because it has a
similar stretch of 135 amino acids that is both proline- and
glycine-rich. Since glycine-rich sequences are also thought to be
capable of bending or functioning like a hinge in three-dimensional
space, this amino acid sequence may interrupt the extended conformation
of domain 2, thereby providing it with a certain degree of
flexibility.) Domain 5 also appears to be a unique sequence having a
globular conformation. The absence of a known cell attachment motif may
indicate that, in contrast to LTBP-1, the LTBP-3 molecule may have a
more limited role in the extracellular matrix ( i.e. that of a
structural protein) in addition to its ability to target latent
TGF-
complexes to specific connective tissues.
1
and that these proteins form a complex in the culture medium. While the
nature of the complex is currently under investigation, these results
are particularly interesting because bone represents one of the largest
known repositories of latent TGF-
(200 µg/kg bone; Seyedin
et al., 1986, 1987), and because this growth factor plays a
critical role in the determination of bone structure and function. For
example, TGF-
is thought to (i) provide a powerful
stimulus to bone formation in developing tissues, (ii)
function as a possible ``coupling factor'' during bone
remodeling (a process that coordinates bone resorption and formation),
and (iii) exert a powerful bone inductive stimulus following
fracture. Activation of the latent complex may be an important step
governing TGF-
effects, and LTBP may modulate the activation
process ( e.g. it may ``protect'' small latent
complexes from proteolytic attack); therefore, further characterization
of LTBP in skeletal tissues should provide important new insights into
bone structure and function.
activity by modulating the intracellular assembly and
extracellular storage of latent growth factor complexes must remain
speculative, however, because the function of LTBP in bone is uncertain
at present. Bonewald et al. (1991) showed that the major form
of TGF-
in conditioned media from bone organ cultures is a 100-kDa
(small) latent complex lacking LTBP. (Chinese hamster ovary cells also
produce only the 100-kDa latent complex.) More recently, Dallas et
al. (1994) demonstrated that cultured osteoblast-like cells
derived from osteosarcomas of various types secrete two major forms of
latent TGF-
1, namely, a 290-kDa complex that contains 190-kDa
LTBP-1 and a 100-kDa complex lacking LTBP-1. A second high molecular
mass complex that contained latent TGF-
2 and LTBP was also
identified. Even though the presence and relative amount of low and
high molecular mass complexes varied with cell type, the 100-kDa latent
TGF-
complex appeared to be the physiologically important form in
bone cells because TGF-
1 and LTBP were not coordinately expressed,
and LTBP was not required for the assembly and secretion of small
latent complexes.
complexes bearing LTBP may be physiologically relevant to,
i.e. may be part of the mechanism of, the preosteoblast
osteoblast differentiation cascade. We base the second possibility on
our evidence that MC3T3-E1 cells express large latent TGF-
1
complexes bearing LTBP-2 precisely at the time of transition from the
preosteoblast to osteoblast phenotype (
day 14 in culture or at the
onset of alkaline phosphatase expression; see Quarles et al. (1992)). Dallas et al. (1994) may not have found large
latent complexes bearing LTBP because their cell and organ culture
systems do not truly model osteoblast differentiation. The organ
culture model, for example, likely is composed of differentiated
osteoblasts but few bone progenitors, making it a difficult model at
best in which to study the differentiation cascade. It is also well
known that MG63, ROS17/2.8, and UMR 106 cells are rapidly dividing
and they express the osteoblast phenotype. Thus, these
osteoblast-like cell lines do not show the uncoupling of cell
proliferation and cell differentiation that characterizes the normal
(physiologically relevant) preosteoblast
osteoblast transition
(Gerstenfeld et al., 1987; Stein and Lian, 1993). Therefore,
the production of small versus large latent TGF-
complexes may be associated with specific stages in the maturation of
bone cells.
regulates extracellular
matrix production by suppressing matrix degradation (through a decrease
in the expression of proteases such as collagenase, plasminogen
activator, and stromelysin plus an increase in the expression of
proteinase inhibitors such as plasminogen activator inhibitor-1 and
tissue inhibitor of metalloproteinase-1) and by stimulating matrix
macromolecule synthesis (for recent reviews, see Lyons and Moses
(1990), and Miyazono et al. (1993)). Conversely, production of
extracellular matrix has been shown to down regulate TGF-
gene
expression (Streuli et al., 1993). TGF-
may therefore
regulate extracellular matrix production through a sophisticated
feedback loop that influences the expression of a relatively large
number of genes. LTBP-1, LTBP-2, and LTBP-3 may contribute to this
regulation by facilitating the assembly and secretion of large latent
growth factor complexes and then targeting the complex to specific
connective tissues (Taipale et al., 1994).
,
transforming growth factor-
; LTBP, latent TGF-
binding
protein; EGF, epidermal growth factor; EGF-CB, EGF-like repeats with
the potential to bind calcium; TGF-bp, a cysteine-rich motif initially
identified in the transforming growth factor-
1-binding protein;
Fib motif, a cysteine-rich motif initially identified in fibrillin-1;
PCR, polymerase chain reaction; nt, nucleotide(s); PBS,
phosphate-buffered saline; p.c, postcoital.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.