Expression and regulation of amphiregulin in Gs␣-mutated
human bone marrow stromal cells of fibrous dysplasia of
mandible
Shanshan Jia, MD, PhD,a Jianbin Yu, MD,b Dongsheng Zhang, MD, PhD,c
Peihui Zheng, MD, PhD,d Shizhou Zhang, MD, PhD,d Li Ma, MD,b Guijun Liu, MD,e and
Shengfeng Li, MD,e Ji’nan, China
PROVINCIAL HOSPITAL, AFFILIATED TO SHANDONG UNIVERSITY
Objectives: Fibrous dysplasia (FD) is a focal bone lesion composed primarily of immature bone marrow stromal cells
along with spicules of immature woven bone. However, cellular differentiation and proliferation in mutant human
bone marrow stromal cells (hBMSCs) of FD have not been fully elucidated. Therefore, the aim of this study was to
investigate the occurrence of Gs␣ mutation at the Arg201 codon in hBMSCs and human trabecular bone cells (hTBCs,
osteoblast-like cells). In addition, we evaluated the gene expression and protein secretion of amphiregulin from
hBMSCs and hTBCs and assessed the biologic activity and possible mechanism of amphiregulin in our system.
Study design. Mutant hBMSCs from FD patients and hTBCs from disease-free bone specimens of the same patient
were successfully cultured. We studied the Gs␣ mutations at the Arg201 codon by means of polymerase chain reaction
(PCR)–restriction fragment length polymorphism. Gene expression and protein secretion of amphiregulin in hBMSCs
and hTBCs was confirmed by reverse-transcription (RT) PCR and Western blotting analysis. The modulation
proliferation and possible mechanism of the exogenous addition of amphiregulin and epidermal growth factor receptor
tyrosine kinase inhibitor (AG-1478) were assessed by using Wst-1 assays.
Results. The Gs␣ mutations in clonal adherent mutant hBMSCs and hTBCs were successfully identified. We identified
amphiregulin to be highly expressed in hBMSCs compared with hTBCs. The growth of hBMSCs was stimulated by the
exogenous addition of amphiregulin and inhibited by AG-1478.
Conclusions. The Gs␣-mutant hBMSCs were successfully identified, and amphiregulin may play a significant role in
the proliferation of hBMSCs. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111:618-626)
Fibrous dysplasia (FD) is a focal bone lesion composed
primarily of immature bone marrow stromal cells along
with spicules of immature woven bone and, in some
cases, islands of hyaline cartilage.1 These lesions generally expand from the marrow cavity into the surrounding cortical bone and contain osteoclasts at the
periphery.2 The most frequently involved sites are the
craniofacial bones and the proximal femur. FD can
occur as an isolated monostotic or polyostotic disease
or as a part of the McCune-Albright syndrome (MAS),
which also includes skin hyperpigmentation and one or
more endocrinopathies. Histologically, FD is composed
a
Resident, Department of Orthodontics.
Student, Department of Oral and Maxillofacial Surgery.
c
Professor, Department of Oral and Maxillofacial Surgery.
d
Associate Professor, Department of Oral and Maxillofacial Surgery.
e
Resident, Department of Oral and Maxillofacial Surgery.
The first 2 authors contributed equally to this study.
Supported by grants from the National Natural Science Foundation of
China (no. 30973328).
Received for publication Sep. 26, 2010; returned for revision Dec. 4,
2010; accepted for publication Dec. 20, 2010.
1079-2104/$ - see front matter
© 2011 Mosby, Inc. All rights reserved.
doi:10.1016/j.tripleo.2010.12.017
b
618
of slender and curved trabeculae of bone and a cellular
proliferation of fibroblast-like cells, which are characteristically associated with long bones. Moreover, some
cases of FD also show sclerotic patterns, particularly in
craniofacial bones.3,4
Mutation of the subunit of signal-transducing G proteins (Gs␣) at the Arg201 codon (replacement of the
arginine by either cysteine or histidine), stimulating
cyclic adenosine monophosphate (cAMP) formation,
has been identified in various tissues in MAS5 and is
also thought to underlie the development of FD associated with a cellular retraction and deposition of abnormal bone matrix led by increased cAMP formation.6
These activating mutations occur postzygotically such
that the affected individuals are somatic mosaics and
represent a heterogeneous patient population depending
on the pattern of distribution of mutated cells throughout the body. The causative mutations inhibit the intrinsic guanosine triphosphatase activity of Gs␣ such
that it remains active in stimulating adenylyl cyclase
and leads to the overproduction of cAMP.1,7 Therefore,
the mutated cells constitutively generate high levels of
cAMP and have a high rate of proliferation.8 Activation
of the Gs␣/protein kinase A (PKA)/cAMP response
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Jia et al. 619
Fig. 1. Representative panoramic radiography, computerized tomography, and photomicrography of hematoxylin-eosin–stained
fibrous dysplasia. A, B, fibrous dysplasia is homogeneously radiopaque with a ground glass appearance and poorly defined
margins indicated by the arrows. C, Histologic section of FD lesion showing the typical pattern of spicules of woven bone
interspersed within whorls of fibrous tissue composed of immature osteoprogenitor cells.
element– binding (CREB) pathway also induces c-Fos
overexpression in FD lesions.9
Thus, FD is due to abnormal proliferation and differentiation of human bone marrow stromal cells
(hBMSCs) due to GNAS (G protein alpha stimulating
activity polypeptide) mutations. The histologic hallmark of this condition is extensive proliferation of
fibrous tissue within the bone marrow, produced by
these abnormally differentiated preosteoblastic cells.
Consequently, it is thought that increased mutant cell
proliferation plays an important role in the establishment and growth of some FD lesions. Therefore, the
investigation of inhibition of mutant cell proliferation
may be a potential therapy for FD/MAS patients.
Amphiregulin belongs to the epidermal growth factor (EGF) family which includes EGF, transforming
growth factor ␣, heparin-binding EGF, betacellulin,
and various heregulins. These factors mediate biologic
functions of epithelial and mesenchymal cells through
the EGF receptors (EGFRs).10 Several studies have
demonstrated that the EGF family and the EGFR signaling pathway play a crucial role in the osteoblast lineage,
fibrous cell, and cells associated with microvascular walls
in FD tissues with mosaic-activated mutation of
GNAS.11,12 Amphiregulin is a 252–amino acid transmembrane glycoprotein, and was originally isolated from human breast carcinoma cell line MCF-7.13 The mRNA
expression for amphiregulin can be detected in a variety of
carcinoma cell lines and in nontransformed epithelial and
mesenchymal cells from the colon, stomach, lung, breast,
ovary, and kidney.14,15 Also, amphiregulin stimulates the
proliferation of keratinocytes, fibroblasts, and epithelial
cells.14 Rencently, Shigeishi et al. demonstrated that amphiregulin stimulates mesenchymal cell proliferation of
human osseous dysplasia.16 Moreover, amphiregulin-null
mice displayed significantly less tibial trabecular bone
than wild-type mice.17
Taken together, mutant hBMSC differentiation and
proliferation of FD have not been fully elucidated. On
the other hand, amphiregulin can stimulate mesenchymal cell proliferation of human osseous dysplasia.
Therefore, in the present study, we investigated the
occurrence of Gs␣ mutation at the Arg201 codon in
hBMSCs derived from FD patient and human trabecular bone cells (hTBCs, osteoblast-like cells) derived
from disease-free bone specimens of the same patient.
In addition, we evaluated the gene expression and protein secretion of amphiregulin from hBMSCs and hTBCs.
To assess the biologic activity and possible mechanism
of amphiregulin in our system, we tested how amphiregulin modulates proliferation of hBMSCs by means
of Wst-1 assays.
MATERIAL AND METHODS
Patient
A 16-year-old girl was referred to the Department of
Oral and Maxillofacial Surgery at the Provincial Hospital, Affiliated Shandong University, for investigation
of a painless swelling of the right side of the jaw of 2
years’ duration. Oral examination showed a hard painless enlargement of the mandible posterior area. The
patient did not have any previous surgical interventions
in the area. Diagnosis of FD was confirmed based on
clinical history, radiographic findings (Fig. 1, A and B),
and histopathologic examination (Fig. 1, C). Also, the
patient did not exhibit endocrine hyperfunction or macular pigmented skin lesions. Under the approval of the
Ethical Committee, Provincial Hospital, Affiliated
Shandong University, fresh surgical fragments were
obtained from the patient after informed consent was
obtained.
Isolation of bone marrow stromal cells
hBMSC cultures were established as previously described.18,19 Briefly, cells were released by scraping
bone marrow of fresh specimens of the right inferior
border of the mandibular angle lesions into nutrient
medium consisting of alpha-modified minimum essen-
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Jia et al.
Table I. Description of the designed primers
Gene
G s␣
p53 Exon 8
Amphiregulin
GAPDH
Primer
Strand
Primer sequences
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
Sense
Antisense
5=-CCATTGACCTCAATTTTGTTTCAG-3=
5=-GGTAACAGTTGGCTTACTGGAAGTTG-3=
5=-TTTTGTTTCAGGACCTGCTTCGCGGC-3=
5=-ACTTTGTCCACCTGGAACTTGGTCTC-3=
5=-TGGTAATCTACTGGGACGGA-3=
5=-GCTTAGTGCTCCCTGGGGGC-3=
5=-ACGTTCGCACACCTGGGTGC-3=
5=-AGCAGCGGGGCTCTCATTGG-3=
5=-AGTGGTGGACCTGACCTGCCG-3=
5=-ATTCAGTGTGGTGGGGGACTGAGTG-3=
Genomic DNA extraction
Genomic DNA was extracted from hBMSCs and
hTBCs by using the Puregene DNA isolation kit according to the manufacturer’s instruction (Gentra, Minneapolis, MN, USA).
with strategy primers as reported previously,22,23 with
minor modifications. The primer sequences are presented in Table I. Approximately 50 ng genomic DNA
was amplified by PCR using primers P1 and P2, and 1
␮L of this amplification reaction was used as template
for subsequent nested PCR-RFLP assays to detect
GNAS Agr201 mutations (95°C for 30 seconds, 55°C for
30 seconds, and 75°C for 45 seconds). Then, 1 ␮L of
the amplified product was diluted 20-fold and used as a
template for a PCR reaction with the primer P2 and the
primer P3 which flanks the EagI restriction site, because the primer P3 creates a new restriction site for
EagI (CGGCCG) through the change to G in the first
position of codon 200 in the normal allele, thus enabling the detection of a mutation at the first and the
second positions of the Arg 201 codon. The thermocycling conditions were as follows: predenaturation at
95°C for 4 minutes, amplification (denaturation at 95°C
for 20 seconds, annealing at 55°C for 30 seconds, and
extension at 68°C for 30 seconds) for a duration of 30
cycles, with a final extension at 68°C for 10 minutes.
As a result, EagI cleaves amplicons from the normal
GNAS alleles into 79bp and 23-bp fragments. In contrast, the presence of any mutation at the Arg201 codon
eliminates this EagI site, and, therefore, amplification
products (102-bp fragment) from mutant alleles are
resistant to EagI digestion. Fragments (134 bp) of p53
gene exon 8 were amplified for 30 cycles (94°C for 45
seconds, 55°C for 1 minute, and 72°C for 30 seconds)
as a positive control to test for the suitability of the
respective material for PCR amplification. For each of
the digested products, 6 mL was electrophoresed on a
3% agarose gel in the presence of ethidium bromide
and visualized by the gel documentation system.
Polymerase chain reaction–restriction fragment
length polymorphism
We used the polymerase chain reaction–restriction
fragment length polymorphism (PCR-RFLP) procedure
to detect Gs␣ mutations at the Arg201 codon (CGT)
Reverse-transcription PCR analysis of
amphiregulin expression
The mRNA expression for amphiregulin in hBMSCs
and hTBCs was assessed by reverse-transcription (RT)
PCR analyses. Briefly, PCR reaction with 2 ␮L cDNA
tial medium (␣-MEM; Biofluids, Rockville, MD, USA)
plus 20% fetal bovine serum (Sijiqing Life Technologies, Hangzhou, China), glutamine, streptomycin, and
penicillin (Biofluids). Single-cell suspensions prepared
by serial passage through needles of decreasing diameter and a cell sieve (70 ␮m; Falcon; Becton Dickinson,
Lincoln Park, NJ, USA), were plated into 75-mL flasks
and incubated at 37°C in an atmosphere of 100% humidity and 5% CO2 to generate single colony– derived
strains. The medium used was the same as above,
which supports proliferation rather than differentiation
of stromal progenitors, as described previously.20 Cell
strains at the third passage were used for analysis of
Gs␣ mutation and amphiregulin expression.
Isolation of human trabecular bone cells
hTBCs derived from disease-free bone specimens
(left inferior border of the mandibular angle, confirmed
by histopathologic examination) of the same patient
were established as described previously.21 Briefly,
fragments of trabecular bone were immediately
washed, minced in phosphate-buffered saline solution,
and subsequently plated in low-calcium (0.02 mmol/L)
nutrient medium containing a 50:50 mixture of
DMEM/Ham F-12K (Biofluids) with 10% fetal bovine
serum (FBS), glutamine, penicillin, and streptomycin
(Biofluids) under 5% CO2 in air at 37°C. The medium
was changed every 3-4 days.
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was performed for 30 cycles of 1 minute at 94°C
(denaturation), 30 seconds at 59°C (annealing), and 45
seconds at 72°C (extension) in a Superscript First
Strand Synthesis System (Life Technologies, Gaithersburg, MD, USA) using the amphiregulin-specific
primer (Table I). After PCR amplification, the product
was separated on a 2% (w/v) agarose gel in TAE buffer
[Tris, 40 mmol/L; Na2EDTA, 1 mmol/L; and Na acetate, 40 mmol/L (pH 8.0)], stained with 0.5 ␮g/mL
ethidium bromide, and photographed under ultraviolet
light. GAPDH transcripts were used to control for the
RNA that was used to amplify the genes by RT-PCR.
Western blotting
Samples of hBMSCs and hTBCs were homogenized
on ice-cold Tris lysis buffer (pH 7.6) containing protease inhibitors. Lysates were then centrifuged for 10
minutes at 20,000g and the supernatant collected. Protein samples were resolved by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis. After gel
electrophoresis, proteins were transfered to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH,
USA). The membranes were blocked with 5% nonfat
milk in phosphate-buffered saline solution (PBS) containing 0.05% Tween 20 for 1 hour at room temperature
and incubated with primary antibodies overnight at
4°C. Anti– human amphiregulin rabbit polyclonal antibody (Sigma, St. Louis, MO, USA), anti– human EGF
receptor monoclonal antibody (Sigma), and ␤-actin
monoclonal antibody (Sigma) were applied at dilutions
of 1:500. Membranes were washed 3 times for 10
minutes in PBS with Tween before 1 hour of roomtemperature incubation with horseradish peroxidase
and conjugated secondary antibodies (goat anti-rabbit
IgG, 1:1,000; Santa Cruz Biotechnology, Santa Cruz,
CA, USA). After 6 ⫻ 5 minutes washes in PBS with
Tween and 1 ⫻ 20 minutes wash in PBS, the membranes were incubated with electrochemiluminescence
(ECL) for 1 minute before exposure to Kodak X-Omit
AR film (Eastman Kodak, Rochester, NY, USA). Membranes were reprobed with ␤-actin as a loading control.
Cell proliferation in the presence of different
concentration of amphiregulin
The cell proliferation after cultivation was determined quantitatively using Wst-1 colorimetric assay,
which measures the cellular mitochondrial dehydrogenase activity. The hBMSCs and hTBCs were seeded at
a density of 3 ⫻ 104 cells per well in 96-well cell
culture plates. The cells were then exposed to medium
containing different concentrations of recombinant amphiregulin (0.1, 1, 10, 100, 500, and 1,000 ng/mL) or no
amphiregulin. To ascertain the effect of amphiregulin,
the cells were exposed to the medium with 1% FBS.
Jia et al. 621
Cells were incubated for 6 days, and then 50 ␮L Tyrode
HEPES buffer and 5 ␮L Wst-1 solution (5 mmol/L
Wst-1 and 0.2 mmol/L 1-methoxy-5-methylphenazinium
methylsulfate in water) were added to each well and
incubated at 37°C for 2 hours. Then, 20 ␮L of each
reaction mixture was transfered to a new 96-well plate.
The absorbance at 450 nm was measured with a microplate reader (Infinite F200; Tecan, Salzburg, Austria). The
cell number was expressed as a percentage relative to the
cell number of control samples incubated with medium
alone.
Cell proliferation in the presence of amphiregulin
and EGF receptor tyrosine kinase inhibitor at
different times
The hBMSCs and hTBCs were seeded at a density of
3 ⫻ 104 cells per well in 96-well cell culture plates. The
cells were then exposed to medium containing recombinant amphiregulin (100 ng/mL) or no amphiregulin
for 2, 4, 6, 8, 10, and 12 days. To ascertain the effect of
amphiregulin, we exposed the cells to the medium with
1% FBS. The cells were also exposed to medium containing 5 ␮mol/L EGF receptor tyrosine kinase inhibitor (AG-1478; Calbiochem, San Diego, CA, USA).
After 2, 4, 6, 8, 10, and 12 days of culture, the cell
numbers were counted as described previously.
RESULTS
Cytology of hBMSCs and hTBCs
Using tissue culture techniques that have been established as previously described, we successfully cultured
mutant hBMSCs from FD patients and hTBCs from disease-free bone specimens of the same patients.18,19,21 The
hBMSCs derived from the FD tissue showed stable
growth and a spindle-shaped appearance. The population
doubling time of hBMSCs was 72 hours (data not shown).
Mutation analysis
PCR-RFLP analyses of genomic DNA from the mutant hBMSCs demonstrated the presence of an R201H
mutation in GNAS gene, thereby validating our assays.
Digestion of the genomic PCR products with the appropriate restriction enzyme showed the presence of
both normal and mutant alleles (Fig. 2), consistent with
the frequency of the mutant allele in this DNA sample,
as determined previously.24 On the other hand, hTBCs
used as a control showed no mutation in the Gs␣ gene
at the Arg201 codon (Fig. 2). Further PCR amplification
of EagI-digested products showed amplification only in
the mutant hBMSCs and not in any of the hTBCs (Fig.
2), confirming the absence of mutant alleles.
Expression of amphiregulin in hBMSCs
Gene expression of amphiregulin in hBMSCs and
hTBCs was confirmed by RT-PCR and Western blot-
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Jia et al.
Fig. 2. Analysis of Gs␣ mutations at the Arg201 codon using
PCR-RFLP. PCR reactions and restriction digests were performed as described in the Methods section and analyzed on
a 3% agarose gel. EagI digested the amplified DNA fragments
(102 bp), including codon 201 of the Gs␣ gene into 79-bp and
23-bp fragments in human trabecular bone cells (hTBCs). The
23-bp band was not visible in this gel, whereas mutant human
bone marrow stromal cells (hBMSCs) of fibrous dysplasia
remained undigested. The p53 gene exon 8 segments (134 bp)
were amplified in hBMSCs and hTBCs. The 100-bp ladder
was used as a size marker.
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Fig. 4. Effects of different concentration amphiregulin on
proliferation of human bone marrow stromal cells (hBMSCs)
and human trabecular bone cells (hTBCs). Cells were incubated with increasing concentrations of recombinant amphiregulin for 6 days, and total viable cell numbers were assessed
by Wst-1 assays. A control (no amphiregulin) was also performed. Maximal stimulation of growth in hBMSCs was
observed at a concentration of 100 ng/mL. hTBCs did not
show higher rates of growth in the addition of each concentration of amphiregulin as compared with no amphiregulin.
*P ⬍ .01.
ting analysis. The results showed that amphiregulin
mRNA was expressed at higher levels in hBMSCs
compared with hTBCs (Fig. 3, A). We also confirmed
the protein expressions of amphiregulin and EGFR in
hBMSCs (Fig. 3, B).
Fig. 3. Expression of amphiregulin in human bone marrow
stromal cells (hBMSCs) and human trabecular bone cells
(hTBCs). A, Total cellular RNA was extracted from hBMSCs
and hTBCs, and the mRNA expression of amphiregulin was
analyzed by means of RT-PCR. B, The protein expression of
amphiregulin and epidermal growth factor (EGF) receptor by
Western blotting.
Effects of amphiregulin on hBMSC and hTBC
proliferation
Amphiregulin stimulated an increase in the cell number in a dose-dependent manner (Fig. 4). Maximal
stimulation of growth in hBMSCs was observed at a
concentration of 100 ng/mL (Fig. 4). Then, hBMSCs
were exposed to medium containing recombinant amphiregulin (100 ng/mL) or no amphiregulin (control)
for 2-12 days. The hBMSCs had higher rates of growth
with the addition of amphiregulin compared with no
amphiregulin (Fig. 5, A). However, hTBCs did not
show higher rates of growth in the addition of each
concentration of amphiregulin compared with no amphiregulin (Fig. 5, B). These studies indicate that exogenous amphiregulin can affect the proliferation of
hBMSCs.
Inhibition of cell proliferation in hBMSCs
by AG-1478
If the amphiregulin-induced cell proliferation involves an interaction between amphiregulin and EGFR,
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Jia et al. 623
Fig. 6. Human bone marrow stromal cells (hBMSCs) were
treated with amphiregulin (100 ng/mL) in the presence or
absence of AG-1478 (5 ␮mol/L) for 6 days. Total viable cell
numbers were assessed by Wst-1 assays. Each column represents the mean of 3 independent experiments. Data represent mean ⫾ SD (n ⫽ 3). *P ⬍ .01.
Fig. 5. Human bone marrow stromal cells (hBMSCs) (A) and
human trabecular bone cells (hTBCs) (B) were incubated
with amphiregulin (100 ng/mL) or no amphiregulin for 2, 4,
6, 8, 10, and 12 days, and total viable cell numbers were
sequentially assessed by Wst-1 assay.
it should be possible to block this mitogenesis with an
inhibitor directed against the extracellular domain of
EGFR. Therefore, in the next experiment, hBMSCs
were treated with amphiregulin (100 ng/mL) in the
presence or absence of AG-1478 (5 ␮mol/L) for 6 days.
Total viable cell numbers were assessed by Wst-1 assays. As shown in Fig. 6, these effects were significantly inhibited by the addition of AG-1478, a specific
inhibitor of EGFR tyrosine kinase,25 suggesting that
biologic effects of amphiregulin are mediated through
EGFR-mediated signaling pathways. To further confirm the possible mediation of EGFR-mediated signaling pathways, amphiregulin mRNA expression and
protein secretion were analyzed by RT-PCR and Western blotting. As shown in Fig. 7, AG-1478 almost
completely blocked amphiregulin-induced amphiregulin mRNA and protein expression.
Fig. 7. Effects of the epidermal growth factor receptor tyrosine kinase inhibitor AG-1478 on mRNA expression for
amphiregulin. A, Cells were stimulated with amphiregulin
(100 ng/mL) in the presence (lanes 3, 4, 7, and 8) or absence
(lanes 1, 2, 5, and 6) of AG-1478. The total RNA was then
extracted, and RT-PCR was performed. B, The protein expression of amphiregulin was analyzed by Western blotting.
Lane 1: 100 ng/mL amphiregulin; lane 2: 100 ng/mL amphiregulin with AG-1478.
DISCUSSION
Clinically, FD is a benign fibrous-osseous lesion of
the bone. The spectrum of skeletal involvement varies
from asymptomatic monostotic lesions to polyostotic involvement resulting in progressive functional deficits and
esthetic problems. The polyostotic variants may be accompanied by skin pigmentation and a variety of endocrine disturbances, including sexual precocity, pituitary
adenoma, and hyperparathyroidism (MAS).1,2,26,27 Radiographically, the lesions present as unilocular or multiloc-
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Jia et al.
ular radiolucent lesions or as a mottled radiolucent/radiopaque pattern or dense radiopaque mass. The most
typical radiographic feature is that of a ground glass
opacification with ill-defined margins, blending imperceptibly into the surrounding bone. Histologically, fibrous
dysplasia shows irregularly shaped trabeculae of immature woven bone in a cellular fibroblastic stroma. In addition, a lamellar maturation can be present, especially in
older patients.28,29
Etiologically, activating missense mutations of the
GNAS gene, encoding the ␣-subunit of Gs, underlie all
forms of FD of bone and are thought to occur postzygotically, leading to a somatic mosaic state.30,31 Somatic mosaicism has been invoked as the mechanism
allowing for survival of mutant cells during development.31 The observation that the disease is never transmitted through the germ line is consistent with this
view. However, cells with both the normal and the
disease-associated genotype are found within an individual FD lesion, even at the level of individual clonogenic progenitors of bone-forming cells (colonyforming unit–fibroblasts).18 Transplantation of clonal
populations of normal hBMSCs into the subcutis of
immunocompromised mice resulted in normal ossicle
formation. In contrast, transplantation of clonal populations of mutant hBMSCs always led to the loss of
transplanted cells from the transplantation site and no
ossicle formation.18 This makes each FD lesion a somatic mosaic itself, rather than simply the “wrong”
piece of a macroscopic patchwork of phenotypically
abnormal tissue and normal tissue. However, the mutant stromal cell type, ranging from local mutational
load to stimuli instigating initiation of a lesion, could be
invoked to explain the development and the natural
prevention of an FD lesion at different sites.32
Therefore, in the present study, we investigated the
occurrence of GNAS mutations in clonal adherent mutant hBMSCs and hTBCs, respectively derived from
histologically abnormal and normal bone of the same
patient with FD. In addition, we used PCR-RFLP to
detect the occurrence of mutations at the Arg201 codon
in hBMSCs and hTBCs. PCR-RFLP analyses of positive control DNA from the mutant hBMSCs demonstrated the presence of an R201H mutation in GNAS
gene, consistent with the frequency of the mutant allele
in this DNA sample, as determined previously.24 On
the other hand, hTBCs used as a control showed no
mutation in the Gs␣ gene at the Arg201 codon. Our
results further confirmed that fibrous tissue in the abnormal marrow spaces of FD lesions is composed of
mutant cells with BMSCs. In addition, stromal cell
cultures from the abnormal FD bone marrow carry the
causative Gs␣ gene mutation.18,32,33
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In 1988, Shoyab et al. first discovered the glycoprotein amphiregulin in the concentrated conditioned medium of MCF-7 breast cancer cells treated with phorbol
12-myristate-13-acetate (PMA).13 Amphiregulin belongs to the EGF family which includes EGF, TGF␣,
HB-EGF, betacellulin, and various heregulins. These
factors mediate biological functions of epithelial and
mesenchymal cells through the EGFRs.10 Amphiregulin is bifunctional because it inhibits the growth of
many human tumor cells but stimulates the proliferation of other cells, such as normal fibroblasts, osteoblast
lineage, keratinocytes, and cells associated with microvascular walls in FD tissues with mosaic-activated mutation of GNAS.11,12,34 Similarly to EGF, amphiregulin
is produced as a precursor transmembrane protein that
undergoes proteolytic cleavage to yield the mature protein.17 So far, there have been no reports of amphiregulin production or function in mutant hBMSCs of FD.
However, EGF has been shown to have several effects
on FD or on mutant cells of FD: It stimulates osteoblast
proliferation, decreases alkaline phosphatase and collagen production, changes bone nodule formation, and,
yet, has catabolic effects on bone, i.e., similarly to
amphiregulin, bifunctional effects.
Therefore, in this study, we first detected the gene
expression of amphiregulin in hBMSCs and hTBCs by
means of RT-PCR and Western blotting. The results
showed that hBMSCs constitutively expressed amphiregulin and EGFR protein compared with hTBCs. The
growth of hBMSCs was stimulated by the exogenous
addition of amphiregulin and inhibited by AG-1478 (an
EGF receptor tyrosine kinase inhibitor). Our results
clearly demonstrated that amphiregulin is a growth
stimulator for hBMSCs. Taken together with the observation that an EGFR tyrosine kinase inhibitor inhibits
the mitogenic stimulation by amphiregulin, these findings suggest that amphiregulin can function through an
extracellular autocrine loop that involves EGFR in
hBMSCs. Rencently, Shigeishi et al. demonstrated that
amphiregulin stimulates mesenchymal cell proliferation of human osseous dysplasia.16 Moreover, amphiregulin-null mice significantly displayed less tibial trabecular bone than wild-type mice.17 To further confirm
the possible mediation of EGFR-mediated signaling
pathways, amphiregulin mRNA expression and protein
secretion were analyzed by RT-PCR and Western blotting. The results showed that AG-1478 almost completely blocked amphiregulin-induced amphiregulin
mRNA and protein expression.
However, the mechanism by which amphiregulin
stimulates hBMSCs differentiation is not well understood. The amphiregulin promoter contains a cAMPresponsive element, and several reports have demonstrated that expression of amphiregulin is regulated by
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cAMP.35,36 Qin and Partridge showed that the amphiregulin expression is regulated through the CREB phosphorylation pathway.37 Activation of the PKA/CREB
pathway, in which Gs␣ stimulates production of cAMP
and activation of PKA, leads to activation of multiple
transcription factors, including CREB, and Cbf␣1.38
Specifically, PKA is able to phosphorylate CREB at
Ser-133, this phosphorylated CREB is required for the
up-regulation of the AP family member, c-Fos, and the
c-Fos gene product Fos is overexpressed in FD lesions.39,40 Fos overexpression in transgenic mice results in bone lesions reminiscent of FD.41
In conclusion, by using PCR-RFLP, we successfully
identified the Gs␣ mutations in clonal adherent mutant
hBMSCs and hTBCs, respectively derived from histologically abnormal and normal bone of the same patient
with FD. Furthermore, we evaluated the gene expression and protein secretion of amphiregulin from hBMSCs
and hTBCs. The growth of hBMSCs was stimulated by
the exogenous addition of amphiregulin and inhibited
by AG-1478 (an EGFR tyrosine kinase inhibitor).
These results indicate that biologic effects of amphiregulin are at least partly mediated through EGFRmediated signaling pathways.
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Reprint requests:
Dongsheng Zhang, MD, PhD
Professor, Dean
Department of Oral and Maxillofacial Surgery
Provincial Hospital
Affiliated Shandong University
324 Jingwu Rd.
Ji’nan 250021
China
[email protected]