ACCELERATED PUBLICATION
Domain-specific Mutations of a Transforming Growth Factor
(TGF)-
1 Latency-associated Peptide Cause Camurati-Engelmann Disease
Because of the Formation of a Constitutively Active Form of
TGF-
1*
Takashi
Saito
,
Akira
Kinoshita§,
Koh-ichiro
Yoshiura§,
Yoshio
Makita¶,
Keiko
Wakui
,
Koichi
Honke
,
Norio
Niikawa§, and
Naoyuki
Taniguchi
**
From the
Department of Biochemistry, Osaka University
Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, the
§ Department of Human Genetics, Nagasaki University School
of Medicine, 12-4 Sakamoto, Nagasaki 852-8102, the ¶ Department of
Pediatrics and Public Health, Asahikawa Medical College, 2-1-1-1 Midorigaokahigashi, Asahikawa, Hokkaido 078-8510, and the
Department of Medical Genetics and Division of Clinical and
Molecular Genetics, Shinshu University School of Medicine, 3-1-1 Asahi,
Matsumoto, Nagano 390-8621, Japan
Received for publication, December 5, 2000, and in revised form, February 2, 2001
 |
ABSTRACT |
Transforming growth factor
(TGF)-
1 is secreted as a latent form, which consists of its mature
form and a latency-associated peptide (
1-LAP) in either the presence
or the absence of additional latent TGF-
1-binding protein. We
recently reported that three different missense mutations (R218H,
R218C, and C225R) of
1-LAP cause the Camurati-Engelmann disease
(CED), an autosomal dominant disorder characterized by hyperosteosis
and sclerosis of the diaphysis of the long bones. Pulse-chase
experiments using fibroblasts from CED patients and expression
experiments of the mutant genes in an insect cell system suggest that
these mutations disrupt the association of
1-LAP and TGF-
1 and
the subsequent release of the mature TGF-
1. Furthermore, the cell
growth of fibroblasts from a CED patient and mutant gene-transfected
fibroblasts was suppressed via TGF-
1. The growth suppression
observed was attenuated by neutralizing antibody to TGF-
1 or by
treatment of dexamethasone. On the other hand, the proliferation of
human osteoblastic MG-63 cells was accelerated by coculture with CED
fibroblasts. These data suggest that the domain-specific
mutations of
1-LAP result in a more facile activation of TGF-
1,
thus causing CED.
 |
INTRODUCTION |
Transforming growth factor-
1
(TGF-
1)1 is a
multifunctional protein acting on cell growth, differentiation, and
morphogenesis of many different-type cells. In skeletal tissue,
TGF-
1 serves as a systematic regulator that couples bone formation
and resorption by regulating the function of the osteoblasts and
osteoclasts (1-5).
The mature form of TGF-
1 is proteolytically cleaved from the
N-terminal remnant of the TGF-
1 precursor, designated as TGF-
1 latency-associated peptide (
1-LAP) but remains non-covalently associated with the rest of the complex, which plays a role in latency
of TGF-
1 (6). TGF-
1 is ubiquitously distributed, and the
activation of the latent form is likely an important step, because it
exists as either a large latent form, composed of
1-LAP, TGF-
1,
and latent TGF-
1-binding protein (LTBP), or a small latent form,
which is devoid of LTBP (7). Under normal conditions, activation of the
latent TGF-
1 is strictly controlled as follows. The large latent
form is temporarily converted to the small latent form, which is then
cleaved by plasmin or the plasmin-like protease to give the mature
TGF-
1 (6, 8, 9).
Camurati-Engelmann disease (CED) or progressive diaphyseal dysplasia
(DPD1) is an autosomal dominant disorder that is characterized by
hyperosteosis and sclerosis of the diaphysis of the long bones (10).
The onset of CED is often during early childhood with severe pain in
the legs, muscle weakness, a waddling gait, and easy fatigability. The
patients occasionally suffer from systemic manifestations, such as
anemia, leukopenia, or hepatosplenomegaly (11). We, as well as two
other groups, previously assigned the locus for CED to chromosome
19q13.1-q13.3 (12-14). By a positional candidate gene approach and
haplotype analyses, three different missense mutations (R218H, R218C,
and C225R), which are located near the C terminus of
1-LAP, were
found in nine CED families (15, 16). In this paper, we report on
the biochemical mechanism by which the domain-specific mutations of
1-LAP cause CED.
 |
EXPERIMENTAL PROCEDURES |
Pulse-Chase Analysis--
R218H CED skin fibroblasts and normal
skin fibroblasts were grown in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal bovine serum (FBS), inoculated in
60-mm dishes, and then grown to near confluency. Metabolic labeling and
immunoprecipitation procedures were performed as previously described
(7), with slight modifications. Cells were placed in a cysteine- and
methionine-free medium containing 10% fetal calf serum and incubated
overnight. For pulsing, the cell culture medium was replaced with a
medium containing [35S]cysteine (100 µCi/ml) and
[35S]methionine (100 µCi/ml), incubated for 15 min,
washed with fresh DMEM, and then incubated for various periods.
Media were collected, and 10 µl/ml of normal rabbit serum was
added, followed by a further incubation for 2 h at 4 °C, and 15 µl/ml of rProtein A-Sepharose (Amersham Pharmacia Biotech) was
then added. The resulting mixture was incubated for an additional 45 min at 4 °C with gentle mixing. The beads were spun down by
centrifugation, and the supernatants were collected.
Immunoprecipitation of these samples was carried out by adding 15 µl
of LT-1 (
1-LAP antibody)-conjugated rProtein A-Sepharose, followed
by an overnight incubation at 4 °C with gentle mixing. The beads
were collected by centrifugation, washed three times with 1% Triton
X-100 in PBS, twice with 0.2% Triton X-100 in PBS, and once with PBS.
The proteins bound to the beads were eluted by the addition of 15 µl
of SDS sample buffer, and the eluates were heated to boiling for 5 min
at 95 °C. Samples were then subjected to SDS-PAGE using 5-20%
polyacrylamide gradient gels (Bio-Rad) under non-reducing conditions.
The gels were dried and exposed to an imaging plate (Fuji Film).
35S radioactivity was detected using a BAS2500 system (Fujix).
Enzyme-linked Immunosorbent Assay (ELISA)--
To activate the
latent TGF-
1 to the immunoreactive form, conditioned media were
treated with 1N HCl for acidic activation and then
neutralized with NaOH, according to the manufacturer's instructions (R
& D Systems Inc.).
Preparation of Recombinant Latent TGF-
1--
A 1195-base pair
EcoRI-XbaI cDNA fragment containing a
full-length of R218H-, R218C-, C225R-, and wild-type-TGF-
1 were
ligated into pFast Bac vector (Life Technologies, Inc.) and transfected to DH10Bac competent cells for recombination. Recombined Bacmid DNAs
were transfected into Sf21 insect cells, and after 72 h, the resultant virus was amplified for 4 days on Sf21 monolayers in Grace medium (Life Technologies, Inc.) that contained 10% FBS. Virus stocks were then reamplified in Sf21 monolayers for 4 days. Fresh Sf21 cells were infected with reamplified virus and
grown in serum-free Sf-900 (Life Technologies, Inc.). After 4 days, the
recombinant protein was harvested from the
Sf21-conditioned medium.
Samples of each conditioned medium were concentrated to 3 mg/ml of
protein using a Centricon
10 (Amicon; Millipore), and 10 µl
of the concentrated conditioned media were subjected to SDS-PAGE in an
8% gel under non-reducing conditions.
Effect of the Mutant-gene Transfection on Cell
Proliferation--
Each construct was recombined in a pRc/CMV
vector. These vectors were transiently expressed in normal skin
fibroblasts. The resulting cells were inoculated on a 96-well plate
(2 × 103 cells/well) with or without a neutralizing
antibody against TGF-
1 (R & D Systems Inc.). R218H CED skin
fibroblasts were also cultured in the presence or absence of
dexamethasone. After 48-h, 5 µCi/ml of [3H]thymidine
was added, and 6 h later the cells were collected. [3H]Thymidine incorporation was determined with a
-plate counter.
Human Osteoblastic MG-63-cell Proliferation Assay--
MG-63
cells, a transformed human osteoblastic cell line, were seeded at
1 × 104 cells/well (24-well plate) as the bottom
wells, and R218H CED and normal fibroblasts were cocultured in DMEM
supplemented with 2% FBS at 5 × 103 cells/well using
the cell culture inserts (pore size 3 µm; Falcon) as the upper wells.
After 36 h, 5 µCi/ml of [3H]thymidine was added
and incubated for 6 h. The MG-63 cells were then collected, and
[3H]thymidine incorporation was determined with a
-plate counter.
 |
RESULTS AND DISCUSSION |
A hydropathy plot analysis of the domain involved in the mutations
(R218H, R218C, and C225R) of
1-LAP suggested a working model in
which CED mutations make conformational abnormalities of the C terminus
of
1-LAP (data not shown). This domain is closely associated with
the formation of intermolecular disulfide bonds between two
1-LAPs
at Cys-223 and Cys-225 that are needed to form a latent TGF-
1. To
investigate the possibility that conformational abnormalities play a
role in the processing of latent TGF-
1 in CED, the conditioned
medium of skin fibroblasts bearing the R218H mutation, which had been
metabolically labeled, was immunoprecipitated with an antibody for
1-LAP (7). Analysis of the samples by SDS-PAGE under non-reducing
conditions revealed that a 100-kDa TGF-
1/
1-LAP complex
(i.e. the small latent form) disappeared within
30 min, and only an 86-kDa
1-LAP homodimer band was detected in the
R218H medium. Both bands were present up to 120 min in the normal
medium (Fig. 1). However, neither the
intracellular assembly nor its ability to secrete TGF-
1 appeared to
be affected in the mutant fibroblasts, as judged from the manner in
which the large latent form was processed. Moreover, no differences in
plasmin or plasmin like-proteolytic activity, in terms of the conversion of the small latent TGF-
1 into the mature TGF-
1 (6, 9), were noted in either types of cells (data not shown). These observations suggest that the R218H mutation leads to the vulnerability of the small latent TGF-
1 and the subsequent release of the mature TGF-
1.

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Fig. 1.
A pulse-chase analysis of latent
TGF- 1 forms of R218H CED and normal
fibroblasts. Both R218H CED and normal fibroblasts were
pulse-labeled for 15 min with [35S]cysteine and
[35S]methionine and then chased for the indicated times.
Conditioned media were reacted with an antibody against 1-LAP (7),
and the immunoprecipitates were subjected to SDS-PAGE in a 5-20%
gradient gel under non-reducing conditions.
|
|
To assess this hypothesis, the amount of mature TGF-
1 protein in the
conditioned media of R218H and normal fibroblasts was determined by an
enzyme-linked immunosorbent assay, which detects only the mature form
(R & D Systems Inc.). Although there were no differences in secretion
of the large latent form (Fig. 1) or the expression level of TGF-
1
mRNA (Fig. 2A) between
R218H and normal fibroblasts, the medium from the R218H fibroblasts contained higher levels of mature TGF-
1 protein after a 48-h incubation than the normal fibroblasts (Fig. 2B). Because
the mature TGF-
1 has a half-life of only about 2 min (17), its concentration was very small. When the latent TGF-
1 was partially activated to give the immunoreactive form by acid treatment (9), the
detectable TGF-
1 in the R218H medium was more than five times that
in the normal one (Fig. 2C). These results strongly suggest that the R218H mutation facilitates the dissociation of the mature TGF-
1 from
1-LAP, thus leading to the accumulation of mature TGF-
1.

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Fig. 2.
The amounts of mature
TGF- 1 protein in the conditioned media from
R218H CED and normal fibroblasts. Conditioned media were collected
at 48-h. The amounts of TGF- 1 protein in these samples were assayed
using a TGF- 1-ELISA kit (R & D Systems Inc.). A, Northern
blot analysis to confirm the expression level of TGF- 1 in R218H CED
and normal fibroblasts. B, the amount of mature TGF- 1
protein in HCl non-treated conditioned media. N.D.,
not detected. C, the amount of mature TGF- 1 protein in
HCl pre-treated conditioned media (9).
|
|
To study further the effect of mutations on the formation of the small
latent form of TGF-
1, recombinant latent TGF-
1 proteins were
prepared using a baculovirus expression system. In the conditioned media of Sf21 cells that had been infected with viruses
containing the R218H, R218C, and the C225R mutant TGF-
1, the 100-kDa
small latent complex was undetectable, and only the 86-kDa
1-LAP
homodimer was observed (Fig. 3), whereas
a recombinant wild-type TGF-
1 produced the small latent form. These
results suggest that conformational changes of
1-LAP as a result of
these domain-specific mutations lead to instability of the small latent
form, which is consistent with the results observed in the CED cells
(Fig. 1). An alteration in the activation of latent TGF-
1 in the CED
cells is schematically presented in Fig. 5.

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Fig. 3.
Effect of the mutations on the expression of
recombinant latent TGF- 1. Samples were
subjected to Western blot analysis using an anti-LAP monoclonal
antibody (R & D Systems Inc.). Lane 1, conditioned medium
from uninfected Sf21 cells; lanes 2-4, wild-type
TGF- 1 baculovirus clones; lanes 5-7, R218H-TGF- 1
baculovirus clones; lanes 8-10, R218C-TGF- 1 baculovirus
clones; lanes 11-13, C225R-TGF- 1 baculovirus
clones.
|
|
Finally, to study the effects of the
1-LAP mutations on TGF-
1
activity, the cell growth of the CED and normal fibroblasts and the
transfectants that produce mutant
1-LAPs was determined by
[3H]thymidine incorporation into DNA (18). The
growth of R218H CED fibroblasts was slower than that of the
normal cells. Although the mRNA expression levels of the
introduced TGF-
1 gene were similar (data not shown), the
proliferation rates of R218H-, R218C-, and the C225R-TGF-
1
gene-transfected cells were slower than the empty vector or
wild-type-TGF-
1 gene-transfected cells (Fig. 4A). Furthermore, the
suppression of growth in the CED cells and the cells to which mutant
genes had been introduced were attenuated by a neutralizing antibody
against TGF-
1 (Fig. 4A).
There are some reports that experiential administration of
dexamethasone and other steroids is found to be effective to CED
patients (19). In addition, in vivo and in vitro
studies have indicated that treatment of dexamethasone brings about
decrease of mRNA level of TGF-
1 (20, 21) and that
glucocorticoids alter bone cell activity and decrease bone mass by
changing the local TGF-
1 actions in skeletal tissues (1, 2).
Therefore, we studied the effect of dexamethasone on the mRNA level
of TGF-
1 and the proliferation of R218H CED fibroblasts. The
mRNA levels of TGF-
1 in R218H CED and normal fibroblasts were
reduced (Fig. 4B), and the suppression of growth in the CED
fibroblasts was attenuated by the treatment of dexamethasone in a
dose-dependent manner (Fig. 4C). On the other
hand, the proliferation of human osteoblastic MG-63 cells (22), which
were cocultured with R218H CED fibroblasts, was accelerated, and this
acceleration was attenuated by treatment of dexamethasone (Fig.
4D). Dexamethasone did not affect the growth of MG-63 cells
when they were cultured alone (data not shown). These results suggest
that TGF-
1 released from the CED fibroblast stimulated the cell
growth of osteoblasts. Overall, these data indicate that the
1-LAP/TGF-
1 mutations observed in CED patients modulate TGF-
1
activity (Fig. 5). It is conceivable that a similar situation exists in
bone tissues.

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Fig. 4.
Effect of the domain-specific mutations
in 1-LAP on the suppression of cell
proliferation via TGF- 1. A, R218H
CED and normal fibroblasts and plasmid-transfected cells were cultured
for 48 h, and their proliferation rates were determined by
[3H]thymidine incorporation (18). A neutralizing
antibody against TGF- 1 was added to the samples in a final
concentration of 5 µg/ml. B, Northern blot analysis of the
expression level of TGF- 1 mRNA. R218H CED and normal fibroblasts
were treated with dexamethasone (10 nM). C,
R218H CED and normal fibroblasts were treated with dexamethasone at the
indicated dose for 48-h, and their proliferation rates were determined
by [3H]thymidine incorporation. These data (A
and C) are depicted as the mean ± S.E.
(n = 8). D, the proliferation of MG-63 cells
was determined by [3H]thymidine incorporation under the
coculture system with CED fibroblast in the presence or absence of
dexamethasone (10 nM). These data show the mean ± S.E. (n = 4).
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Fig. 5.
Schematic illustration of the activation of
latent TGF- 1. The activation of latent
TGF- 1 normally occurs via a three-step mechanism (large latent form
small latent form mature form). In the mutants of CED, TGF- 1
does not form the small latent form. Therefore, the mutant TGF- 1 may
undergo rapid activation and/or rapid turnover.
|
|
Previous studies have indicated a strict regulation of osteoblasts and
osteoclasts in bone tissues and that many factors are involved in this
process (23-25). TGF-
1 plays an important role in promoting bone
matrix synthesis and in reducing matrix degradation and resorption in
the diaphysis of long bones by inhibiting both the formation and
activity of osteoclasts (1-5). The observations herein point to the
importance of factors that regulate the activation of TGF-
1 in the
bone matrix.
In conclusion, the domain-specific mutations in
1-LAP/TGF-
1 play
a role in Camurati-Engelmann disease by modifying the activation of
TGF-
1. The present study may provide a new stimulus for
investigating the pathophysiology and treatment of CED, as well as
related disorders such as osteoporosis.
 |
ACKNOWLEDGEMENTS |
We thank Drs. K. Miyazono, K. Hashimoto,
and H. Yoshikawa for kindly providing the antibodies against
1-LAP,
normal skin fibroblast, and MG-63 cells, respectively. We also thank
Drs. H. F. Deutsch and K. Suzuki for critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by Grant-in-aid for Scientific
Research on Priority Area 10178104 from the Ministry of Education, Science, Sports and Culture, Japan.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.
**
To whom correspondence should be addressed. Tel.:
81-6-6879-3421; Fax: 81-6-6879-3429; E-mail:
proftani@biochem.med.osaka-u.ac.jp.
Published, JBC Papers in Press, February 13, 2001, DOI
10.1074/jbc.C000859200
 |
ABBREVIATIONS |
The abbreviations used are:
TGF, transforming
growth factor;
LAP, latency-associated peptide;
LTBP, latent
TGF-
1-binding protein;
CED, Camurati-Engelmann disease;
DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
ELISA, enzyme-linked immunosorbent assay.
 |
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