Takashi Saito, Akira Kinoshita, Koh-ichiro
Yoshiura, Yoshio Makita, Keiko Wakui,
Koichi Honke, Norio Niikawa and Naoyuki
Taniguchi
J. Biol. Chem. 2001, 276:11469-11472.
doi: 10.1074/jbc.C000859200 originally published online February 13, 2001

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ACCELERATED PUBLICATION:
Domain-specific Mutations of aTransforming Growth Factor (TGF)- β1
Latency-associated Peptide Cause
Camurati-Engelmann Disease Because of
the Formation of a Constitutively Active
Form of TGF-β1

THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 276, No. 15, Issue of April 13, pp. 11469 –11472, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.

Accelerated Publication
Domain-specific Mutations of aTransforming Growth Factor(TGF)-␤1 Latency-associatedPeptide Cause CamuratiEngelmann Disease Because of
the Formation of a Constitutively
Active Form of TGF-␤1*

Takashi Saito‡, Akira Kinoshita§,
Koh-ichiro Yoshiura§, Yoshio Makita¶,
Keiko Wakui储, 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 ofPediatrics and Public Health, Asahikawa Medical
College, 2-1-1-1 Midorigaokahigashi, Asahikawa,
Hokkaido 078-8510, and the 储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

Transforming growth factor (TGF)-␤1 is secreted as alatent form, which consists of its mature form and alatency-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 thesubsequent release of the mature TGF-␤1. Furthermore,
the cell growth of fibroblasts from a CED patient andmutant 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 proliferationof human osteoblastic MG-63 cells was accelerated bycoculture with CED fibroblasts. These data suggest thatthe domain-specific mutations of ␤1-LAP result in amore facile activation of TGF-␤1, thus causing CED.
* 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. This 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.: 81-6-68793421; Fax: 81-6-6879-3429; E-mail: proftani@biochem.med.osakau.ac.jp.
This paper is available on line at http://www.jbc.org

EXPERIMENTAL PROCEDURES

Pulse-Chase Analysis—R218H CED skin fibroblasts and normal skinfibroblasts 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 andimmunoprecipitation procedures were performed as previously described (7), with slight modifications. Cells were placed in a cysteineand methionine-free medium containing 10% fetal calf serum and incubated overnight. For pulsing, the cell culture medium was replacedwith a medium containing [35S]cysteine (100 ␮Ci/ml) and [35S]methionine (100 ␮Ci/ml), incubated for 15 min, washed with fresh DMEM,
and then incubated for various periods. Media were collected, and 10
␮l/ml of normal rabbit serum was added, followed by a further incubation for 2 h at 4 °C, and 15 ␮l/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 ␮lof LT-1 (␤1-LAP antibody)-conjugated rProtein A-Sepharose, followedby an overnight incubation at 4 °C with gentle mixing. The beads werecollected 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
1
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.

11469

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Received for publication, December 5, 2000, and in revised form,
February 2, 2001
Published, JBC Papers in Press, February 13, 2001,
DOI 10.1074/jbc.C000859200

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 boneformation and resorption by regulating the function of theosteoblasts and osteoclasts (1–5).
The mature form of TGF-␤1 is proteolytically cleaved fromthe N-terminal remnant of the TGF-␤1 precursor, designatedas TGF-␤1 latency-associated peptide (␤1-LAP) but remains
non-covalently associated with the rest of the complex, whichplays 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 isstrictly controlled as follows. The large latent form is temporarily converted to the small latent form, which is then cleavedby 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 thatis characterized by hyperosteosis and sclerosis of the diaphysisof the long bones (10). The onset of CED is often during earlychildhood with severe pain in the legs, muscle weakness, awaddling gait, and easy fatigability. The patients occasionally
suffer from systemic manifestations, such as anemia, leukopenia, or hepatosplenomegaly (11). We, as well as two othergroups, 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.

11470

Domain-specific Mutations in ␤1-LAP Result in CED

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 competentcells 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 Sf21conditioned medium.
Samples of each conditioned medium were concentrated to 3 mg/ml ofprotein using a Centricon⫺10 (Amicon; Millipore), and 10 ␮l of theconcentrated conditioned media were subjected to SDS-PAGE in an 8%
gel under non-reducing conditions.
Effect of the Mutant-gene Transfection on Cell Proliferation—Eachconstruct was recombined in a pRc/CMV vector. These vectors weretransiently 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 ␮Ci/ml of [3H]thymidine was added, and6 h later the cells were collected. [3H]Thymidine incorporation wasdetermined with a ␤-plate counter.
Human Osteoblastic MG-63-cell Proliferation Assay—MG-63 cells, atransformed human osteoblastic cell line, were seeded at 1 ⫻ 104
cells/well (24-well plate) as the bottom wells, and R218H CED andnormal fibroblasts were cocultured in DMEM supplemented with 2%
FBS at 5 ⫻ 103 cells/well using the cell culture inserts (pore size 3 ␮m;
Falcon) as the upper wells. After 36 h, 5 ␮Ci/ml of [3H]thymidine wasadded 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

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 inthese 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., notdetected. C, the amount of mature TGF-␤1 protein in HCl pre-treatedconditioned media (9).
proteins bound to the beads were eluted by the addition of 15 ␮l 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 gelswere 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 weretreated with 1N HCl for acidic activation and then neutralized with
NaOH, according to the manufacturer’s instructions (R & D Systems
Inc.).

A hydropathy plot analysis of the domain involved in themutations (R218H, R218C, and C225R) of ␤1-LAP suggested aworking 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, theconditioned medium of skin fibroblasts bearing the R218H
mutation, which had been metabolically labeled, was immunoprecipitated with an antibody for ␤1-LAP (7). Analysis of thesamples by SDS-PAGE under non-reducing conditions revealed
that a 100-kDa TGF-␤1/␤1-LAP complex (i.e. the small latentform) disappeared within 30 min, and only an 86-kDa ␤1-LAP
homodimer band was detected in the R218H medium. Bothbands 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

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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 werereacted 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.

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.

Domain-specific Mutations in ␤1-LAP Result in CED

11471

FIG. 5. Schematic illustration of theactivation of latent TGF-␤1. The activation of latent TGF-␤1 normally occurs
via a three-step mechanism (large latentform 3 small latent form 3 matureform). 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.

form was processed. Moreover, no differences in plasmin orplasmin like-proteolytic activity, in terms of the conversion of
the small latent TGF-␤1 into the mature TGF-␤1 (6, 9), werenoted 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.
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 thelarge latent form (Fig. 1) or the expression level of TGF-␤1
mRNA (Fig. 2A) between R218H and normal fibroblasts, themedium 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 verysmall. When the latent TGF-␤1 was partially activated to givethe 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.
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 infectedwith 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 thesmall latent form. These results suggest that conformationalchanges of ␤1-LAP as a result of these domain-specific mutations lead to instability of the small latent form, which isconsistent 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.
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

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FIG. 4. Effect of the domain-specificmutations 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 thesamples in a final concentration of 5 ␮g/
ml. B, Northern blot analysis of the expression level of TGF-␤1 mRNA. R218H
CED and normal fibroblasts were treatedwith dexamethasone (10 nM). C, R218H
CED and normal fibroblasts were treatedwith dexamethasone at the indicated dosefor 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, theproliferation 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 themean ⫾ S.E. (n ⫽ 4).

Domain-specific Mutations in ␤1-LAP Result in CED

11472
3

treatment of CED, as well as related disorders such as
osteoporosis.
Acknowledgments—We thank Drs. K. Miyazono, K. Hashimoto, andH. Yoshikawa for kindly providing the antibodies against ␤1-LAP, normal skin fibroblast, and MG-63 cells, respectively. We also thankDrs. H. F. Deutsch and K. Suzuki for critically reading the manuscript.
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determined by [ H]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 theintroduced TGF-␤1 gene were similar (data not shown), theproliferation 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 bya neutralizing antibody against TGF-␤1 (Fig. 4A). There aresome 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 indicatedthat treatment of dexamethasone brings about decrease ofmRNA level of TGF-␤1 (20, 21) and that glucocorticoids alterbone 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 andthe proliferation of R218H CED fibroblasts. The mRNA levelsof 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 dosedependent 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 thisacceleration 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 conceivablethat a similar situation exists in bone tissues.
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 importantrole 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