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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-09-2839.
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Blood, 15 April 2003, Vol. 101, No. 8, pp. 2983-2989
HEMATOPOIESIS
Stimulation of osteoprotegerin production is responsible for
osteosclerosis in mice overexpressing TPO
Hédia Chagraoui,
Micheline Tulliez,
Tarek Smayra,
Emiko Komura,
Stéphane Giraudier,
Theodore Yun,
Nathalie Lassau,
William Vainchenker, and
Françoise Wendling
From the IFR 54-INSERM U 362, Institut Gustave Roussy,
Villejuif, France; Service d'Anatomie et Cytologie
Pathologiques, Hôpital Cochin, Paris, France; LIPA,
IFR 54, Institut Gustave Roussy, Villejuif, France; and
Department of Immunology, University of Washington, Seattle.
 |
Abstract |
Myelofibrosis and osteosclerosis are prominent features arising in
mice overexpressing thrombopoietin (TPO). The pivotal role of
transforming growth factor  1 (TGF-1) in the pathogenesis of
myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to
determine whether osteosclerosis occurs through a deregulation of
osteoclastogenesis. Marrow cells from opg-deficient mice
(opg /) or wild-type (WT) littermates were
infected with a retrovirus encoding TPO and engrafted into an
opg/ or WT background for long-term
reconstitution. The 4 combinations of graft/host (WT/WT,
opg//opg/,
opg//WT, and
WT/opg/) were studied. Elevation of TPO and
TGF-1 levels in plasma was similar in the 4 experimental groups and
all the mice developed a similar myeloproliferative syndrome associated
with severe myelofibrosis. Osteosclerosis developed in WT hosts
engrafted with WT or opg/ hematopoietic
cells and was associated with increased OPG levels in plasma and
decreased osteoclastogenesis. In contrast,
opg/ hosts exhibited an osteoporotic
phenotype and a growth of bone trabeculae was rarely seen. These
findings suggest that osteosclerosis in mice with TPO overexpression
occurs predominantly via an up-regulation of OPG in host stromal cells
leading to disruption of osteoclastogenesis.
(Blood. 2003;101:2983-2989)
© 2003 by The American Society of Hematology.
| |
Introduction |
Bone remodeling depends on the tightly integrated
activity of 2 distinct cell types, the osteoblasts, which construct
bone, and the osteoclasts, which resorb bone (for reviews, see Ducy et
al1 and Teitelbaum2). Studies of spontaneous
mutations and the development of genetically manipulated mice have
improved the understanding of the complex processes involved in bone
remodeling. Formation of bone is dependent on the number, maturation,
and functions of the osteoblasts. Osteoblasts derive from mesenchymal progenitors through the regulatory action of cell-cell and cell-matrix interactions3 and by the actions of growth factors
produced locally or present in the circulation.4 Among
these growth factors, the most important are insulin-like growth factor
I (IGF-I), which increases the differentiation of
osteoblasts,5 members of the fibroblast growth factor
(FGF) family, which indirectly stimulate their
proliferation,6 and members of the transforming growth
factor (TGF-) multigene family (for a review, see Centrella et
al7). Bone morphogenetic proteins (BMPs) have the
ability to induce osteoblast differentiation, whereas the actions of
TGF- may be stimulatory or inhibitory.7-10 On the other
hand, bone resorption is controlled by monocyte-derived multinucleated,
giant osteoclasts.2 Extensive, recent studies have
demonstrated that the differentiation of osteoclasts requires 3 main
factors. Macrophage colony-stimulating factor/colony-stimulating factor
1 (M-CSF/CSF-1) controls the survival and proliferation of
monocytic progenitors.11,12 RANKL (also known as
OPGL/ODF/TRANCE) binds to the RANK receptor (receptor activator of
nuclear factor  B) expressed on the membrane of osteoclast progenitor
cells13 and promotes osteoclast differentiation and
maturation.14 Osteoprotegerin (OPG) is a secreted molecule that binds RANKL and strongly inhibits osteoclastogenesis by blunting the interaction RANKL/RANK.15 Genetically manipulated mice
have elegantly demonstrated complementing skeletal phenotypes of the OPG/RANKL system. Overexpression of OPG in transgenic
mice15 and targeted ablation of
RANKL16 or RANK17 in knockout mice resulted
in impaired osteoclastogenesis and severe osteopetrosis, whereas
administration of soluble RANKL18 or creation of OPG knockout mice14 resulted in enhanced osteoclastogenesis
and osteoporosis. Within the bone environment, both OPG and RANKL are
synthesized by osteoblast/stromal cells. Recent in vitro studies using
bone and bone marrow stromal cell lines have demonstrated that TGF-1
was a negative regulator of osteoclastogenesis, in part, through a
direct stimulation of the transcription and secretion of OPG and a
down-regulation of RANKL.19,20 On the other hand, it is
also reported that TGF- with a combination of soluble RANKL and
M-CSF strongly promotes the differentiation of osteoclast progenitors.21 Thus, the TGF- family appears to have
opposite functions in bone remodeling with direct stimulatory effects
on bone-forming and bone-resorbing cells and with indirect inhibitory effects on bone resorption.
Myelofibrosis and, more occasionally, osteosclerosis are major
complications occurring during the evolution of human idiopathic myelofibrosis (IM; also known as agnogenic myeloid metaplasia). IM is a
chronic myeloproliferative disorder with a clonal stem cell disorder
characterized by a trilineage myeloproliferation, splenomegaly, and
extramedullary hematopoiesis.22 The molecular mechanisms
underlying the clonal abnormal proliferation of hematopoietic cells23 are still unknown, but the involvement of a number
of fibrogenic cytokines derived from the megakaryocyte or monocyte hyperplasia has been repeatedly discussed in relation to the stromal reactive secondary response that leads to
myelofibrosis.24-30 Although less extensively studied, it
has also been suggested that elevated levels of TGF-1 and basic
fibroblastic growth factor (bFGF) could be implicated in the osteogenic
process.31
Overexpression of thrombopoietin (TPO), the physiologic regulator
of platelet production, in rodents has provided an experimental model
that recapitulates several characteristic features of human IM. High
systemic levels of TPO in mice invariably cause a myeloproliferative syndrome associated with marked megakaryocyte and granulocytic hyperplasia, splenomegaly, extramedullary hematopoiesis, splenic and
medullary fibrosis, and osteosclerosis.32-34 Recently, we
examined the role of TGF-1 in the promotion of myelofibrosis by
engrafting mutant TGF-1/ hematopoietic cells,
infected with a retrovirus encoding TPO, into lethally irradiated
wild-type (WT) hosts. Although myelofibrosis was systematically
observed in hosts repopulated with WT cells, no increase in reticulin
deposition was seen in mice reconstituted with
TGF-1/ donor cells, demonstrating that TGF-1 was
essential for the promotion of myelofibrosis.35 In
addition to the absence of myelofibrosis, we did not observe
significant neotrabecular bone growth in mice engrafted with
TGF-1/ cells at 4 months after transplantation, at a
time when osteopetrosis was systematic and prominent in the marrow
cavity from mice repopulated with WT cells. However, although
myelofibrosis did not occur at later times, bone growth in the femur
lumen was noted after 6 months in animals repopulated with
TGF-1/ cells.
To understand the mechanisms involved in the osteogenic response, we
investigated whether this was due to an impaired osteoclastogenesis through the OPG/RANKL axis. To that end, bone marrow stem cells from
opg knockout mice (opg/) or WT
littermates were infected with a retrovirus encoding the murine TPO
protein and engrafted into lethally irradiated
opg/ or WT mice for reconstitution in the
long term. The 4 combinations of graft/host were studied (WT/WT,
opg//opg/,
opg//WT, and
WT/opg/). We report here that, whatever the
graft/host combination, all the mice developed a comparable
myeloproliferative syndrome ending with severe myelofibrosis associated
with elevated levels of latent TGF-1 in the plasma. Severe
osteosclerosis was observed when transplantations were performed in a
WT background (WT/WT or opg//WT) and was
correlated with a marked elevation of OPG in plasma and a decrease in
the number of osteoclasts in femurs. In contrast, only rare bony
trabeculae merging from the cortical region were seen in hosts
lacking OPG in the microenvironment (WT/opg/
or opg//opg/).
Together, these observations suggest that the aberrant bone growth seen
in mice overexpressing TPO may be the consequence of an inhibition of
osteoclastogenesis via an increased production of OPG by stromal cells.
| |
Materials and methods |
Animals
Heterozygote opg+/ breeders
back-crossed on the C57Bl/6 background were kindly provided by E. Clark
(Seattle, WA).36 Mice were bred at the Institut Gustave
Roussy animal facility under specific pathogen-free conditions.
Genotyping was performed on DNA extracted from distal tail segments
from 15-day-old pups using standard techniques. An opg
locus-specific primer (primer A: 5'-GGTCCTCCTTGATTTTTCTAGCC-3') was
used in combination with primers specific for
neor (primer B: 5'-TGACCGCTTCCTCGTGGCTTT AC-3')
or opg (primer C: 5'-TGCCCTGACCACTCTTATACGGGGAC-3'). Primers
A and B amplify a 500-bp product from the targeted allele; primers A
and C amplify a 200-bp product from the WT allele. Polymerase chain
reaction (PCR) conditions were as described.36
Transduction of BM cells and transplantation
Eight- to 10-week-old male and female
opg/ and WT littermates were used as bone
marrow (BM) donors or recipients. Four groups of 10 animals each were
constituted: opg/ BM engrafted into
opg/ hosts
(opg// opg/),
opg/ BM into WT hosts
(opg//WT), WT BM into
opg/ hosts
(WT/opg/), and WT BM into WT hosts (WT/WT).
The infection procedure was performed as previously
described.34 Briefly, 4 days after 5-fluorouracil treatment (150 mg/kg administered intraperitoneally), marrow cells were
cocultured with 1 × 106 MPZenTPO virus-producing
GP+E-86 cells in Dulbecco Modified medium (DMEM; Sigma Aldrich,
Saint Quentin Fallavier, France) containing 10% heat-inactivated fetal
bovine serum (FBS; Gibco BRL, Paisley, United Kingdom),
penicillin (100 U/mL), streptomycin (100 µg/mL), glutamine, (2 mM)
and supplemented with murine FLT3 ligand (muFLT3-L; 20 ng/mL),
murine interleukin 3 (muIL-3; 100 U/ml), muIL-6 (20 ng/ml), and
murine stem cell factor (muSCF; 20 ng/mL). All cytokines were
purchased from R & D Systems (Oxon, United Kingdom). After 4 days,
nonadherent cells were harvested. An aliquot was used immediately in
clonogenic progenitor assays to determine the percentage of infected
colony-forming cells (CFCs). The remaining were inoculated intravenously via the retro-orbital sinus into lethally irradiated hosts (9.5 Gy, x-ray apparatus, single dose) in a ratio of one donor
per one recipient.
In vitro progenitor assay for transduction efficiencies
At the end of the infection protocol, cells were seeded in
standard methylcellulose culture (Methocult M3134; Stem Cell
Technologies, Vancouver, BC, Canada) supplemented with 1 mM
L-glutamine (Gibco BRL) and 104 M
2-mercaptoethanol. Medium contained 20% FBS and a combination of
recombinant growth factors including muIL-3 (100 U/mL); pegylated human
recombinant megakaryocyte growth and differentiation factor (PEG-rHuMGDF; 10 ng/mL), muSCF (50 ng/mL), and human
erythropoietin (huEPO; 2 U/mL). Seeding densities were
2 × 104 cells/mL. Cultures were plated in triplicate and
incubated at 37°C in a humidified incubator containing 5%
CO2 in air. Seven days following initiation of culture,
colonies (> 50 cells) were scored under an inverted microscope and 30 colonies were picked at random. The integrated retroviral sequence was
detected by PCR analysis. Primer sets corresponding to the TPO cDNA
were sense 5'-ACTTTAGCCTGGAGAATGGAAA-3' and antisense
5'-CCAGGAGTAATCTTGACTCTGA-3' allowing the amplification of a 499-bp
product. Actin was used as an internal control: sense
5'-GTACCACAGGCATTGTGATG-3' and antisense 5'-GCAACATAGCACAGCTTCTC-3'.
PCR conditions were previously described.35
Hematologic evaluation and histopathology
Orbital plexus blood was collected in citrated tubes at
monthly intervals from the anesthetized mice. Nucleated blood cells and
differential cell counts, hematocrit level, and platelet counts were
determined using an automated blood coulter calibrated for mouse blood
(MS9, Schloessing Melet, Cergy-Pontoise, France). Platelet-poor plasma
(PPP) was prepared and stored at  20°C for determination of TPO,
OPG, and TGF-1 levels. Twelve weeks after transplantation, 3 mice in
each group were humanely killed under anesthesia. Femurs were excised,
cleaned of soft tissue, fixed in Glyo-Fixx fixative (CML, Nemours,
France), decalcified, and embedded in paraffin. Sections (4-5 µm)
were stained with hematoxylin and eosin or Gomori stain for overall
cytology and according to Gordon-Sweet for reticulin.
Adenovirus encoding biologically active TGF-1
WT mice were injected intravenously with 2 × 108
plaque-forming units (pfu) of a recombinant adenovirus vector encoding
a mutated TGF-1 cDNA. Cysteines at amino acids 223 and 225 (Ad-TGF-1s223/s225) were changed to serine
resulting in the secretion of a fraction of biologically active
TGF-1 forms.37,38 The empty vector was used as a
control. Blood was collected 1 month after the injection and plasma was
used for the determination of TGF-1 and OPG levels.
Cytokine enzyme-linked immunosorbent assay
TPO and OPG levels in plasma were determined with the
murine TPO or murine OPG Quantikine Kits from R & D Systems, according to manufacturer's instructions. The sensitivity limits of the assays
were 62.5 pg/mL and 31.2 pg/mL, respectively. The human TGF-1 immunoassay (R & D Systems), which detects only active forms
of TGF-1, was used for determination of circulating TGF-1 levels.
Samples were assayed before (spontaneously active TGF-1) and after
acidification (active and latent forms). For acidification, the
protocol recommended by the manufacturer was followed without modification. The sensitivity of the assay was 31.2 pg/mL active TGF-1.
Statistical analysis
The results are presented as mean ± SD. The data were
analyzed with the 2-tailed Student t test.
| |
Results |
Hematopoietic changes in WT and opg/
hosts repopulated with TPO-overexpressing hematopoietic cells
To discern whether OPG was directly implicated in
osteosclerosis that develops in mice overexpressing TPO, BM cells from
WT or mutant opg/ littermates were infected
with the MPZen-TPO retrovirus and engrafted into lethally irradiated WT
or opg/ recipients. Transduction efficiency
in progenitor cells was evaluated at the end of the infection protocol
by a colony assay as described in "Materials and methods." No
significant differences were observed between the 2 cellular genotypes
with more than 80% transduced progenitor cells (range, 80%-92%; 2 repeated experiments with WT and opg/ BM
cells). Lethally irradiated hosts were engrafted with 2 to 4 × 106 cells and peripheral blood was analyzed at
monthly intervals. Whatever the graft-to-host combination, TPO levels
in plasma were more than 3000-fold increased 1 month after
transplantation (1667 ± 125 ng/mL, n = 24, as compared to
0.464 ± 0.053 ng/mL in normal controls, n = 6) and these values
remained elevated during the follow-up (Figure
1A). Accordingly, platelet counts rose
rapidly peaking at approximately 3.5 × 109/mL 1 and 2 months after transplantation. Although TPO levels remained high,
platelet counts slowly declined in each group at 3 months (Figure 1B).
Nucleated blood cells were 5- to 7-fold augmented over normal values at
2 months after transplantation and these high numbers were maintained
at 3 months (Figure 1C). Whatever the time of examination or the group
of mice, no major differences were seen in the differential cell
counts. Leukocytosis was mainly due to an increase in polymorphonuclear
granulocytes representing about 46% ± 8% of the total nucleated
cell population at 3 months (n = 6 mice in each group), monocytes
making up 9% ± 3% of white blood cells, and immature myeloid
precursor and blast cells reaching values of 12% ± 6% as compared
to 12% ± 3%, 1.2% ± 0.3%, and 0% in control mice,
respectively (data not shown). In contrast to platelet and nucleated
cell counts, hematocrit values in mice that received
transplants in the 4 groups rapidly declined and animals
became severely anemic with hematocrit values below 25% ± 3% at 3 months after transplantation (Figure 1D). These changes were comparable
to those previously described in C57Bl/6 mice overexpressing
TPO.34
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| Figure 1.
TPO overexpression causes a comparable elevation in
platelet and nucleated blood cell numbers and an anemia in hosts
engrafted with TPO virus-infected BM cells from WT or
opg/ donors. Results are
presented as the mean ± SD of 7 to 10 animals per experimental
group. WT hosts engrafted with WT BM cells are shown with open
triangles ( ); WT hosts engrafted with
opg/ BM cells are shown with filled
triangles ( ); opg/ hosts engrafted with
WT BM cells are shown with open squares ( ); and
opg/ hosts engrafted with
opg/ BM cells are shown with filled squares
( ). (A) TPO in plasma was measured with an enzyme-linked
immunosorbent assay (ELISA) at 1, 2, and 3 months after
transplantation. Values in normal WT or opg/
controls were 0.46 ± 0.05 ng/mL (n = 12). (B) Platelet numbers
during the 3 months of follow-up. (C) Total numbers of nucleated blood
during the follow-up. (D) Hematocrit values.
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Elevated levels of latent TGF-1 in plasma
As previously reported,34,35 only insignificant
amounts of spontaneously immunoreactive TGF-1 were measured in PPP
at any time during the follow-up and samples were acidified to
determine levels of latent TGF-1 forms. One month after
transplantation, latent TGF-1 levels were augmented 3- to 4-fold
over baseline levels measured in controls (15.8 ± 1.2 ng/mL
versus 4.1 ± 0.3 ng/mL, respectively). These levels remained 3 times
higher than in controls during the 3 months of follow-up. No
significant difference was seen between the 4 groups of reconstituted
mice (Figure 2).
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| Figure 2.
Sustained elevation in plasmatic TGF-1 levels in
hosts repopulated with WT or
opg/ TPO virus-infected BM
cells. TGF-1 was measured in PPP by ELISA after acidification
of the samples. Each bar represents the mean ± SD of 7 to 10 animals per experimental group as indicated under the x-axis. The mice
receiving transplants were analyzed at 1 month (open bars), 2 months
(hatched bars), and 3 months (dotted bars), except for the
WT/opg/ group where samples were lost
accidentally. Independently of the graft/host combination,
levels of latent TGF-1 were significantly (P < .01)
increased over control levels (black bars) in the 4 experimental
groups.
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Development of myelofibrosis in WT and
opg/ hosts
Three mice in each group that received transplants were humanely
killed 3 months after transplantation. All mice displayed splenomegaly
with spleen weights increased up to 10-fold over controls
(1000 ± 220 mg versus 122 ± 35 mg, respectively). Examination of
histologic sections stained with hematoxylin and eosin revealed that
the spleen red pulp was markedly expanded by a massive proliferation of
often dysmorphic or apoptotic megakaryocytes found in large clusters
associated with numerous neutrophil granulocytes displaying all stages
of maturation. Eosinophils were rarely seen. Erythroid cells were
poorly represented. Similar cytologic changes were seen in femur
sections with an accumulation of megakaryocytes displaying nuclear and
cytologic alterations. When sections were stained with silver to reveal
reticulin deposition, a prominent densification of the reticulin
network was seen in spleen (Figure 3) and
marrow (not shown) from all the mice. Spleen sections stained according
to Gomori revealed occasional accumulation of collagen fibers (not
shown).
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| Figure 3.
Development of myelofibrosis in the spleen of
TPO-overexpressing mice.
Representative spleen sections from WT and
opg/ hosts hematologically repopulated with
TPO virus-infected BM cells from WT or opg/
donors at 3 months after transplantation. Sections were stained
according to Gordon-Sweet to reveal reticulin fiber deposition. (A) WT
host engrafted with WT BM cells. (B) opg/
host engrafted with opg/ BM cells. (C) WT
host engrafted with opg/ BM cells. (D)
opg/ host engrafted with WT BM
cells. Sections show severe accumulation of reticulin fibers
in the 4 experimental groups. Original magnification for all panels,
× 200.
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Osteosclerosis in WT hosts engrafted with WT or
opg/ BM cells
At 3 months after transplantation, femurs of WT hosts
reconstituted with TPO virus-infected hematopoietic cells from either WT or opg/ donors appeared
radiographically abnormal. Femurs were usually more radiodense than WT
controls (Figure 4A) and showed a
disappearance of the distinct cortical margin with a blurring of the
medullary compartment (Figure 4G,M). Osteosclerosis was confirmed by
histologic examination. The femoral cortical region was thickened and
the medullary cavity was filled with interconnecting newly formed bone
trabeculae merging from the endosteal side of the cortical region
(compare Figure 4B-C to H-I and N-O). Osteoclasts were rarely seen in
any sections examined on the endosteal or periosteal bone
surfaces.
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| Figure 4.
Radiographic and histologic sections of the femurs of WT and
opg/ hosts engrafted with WT or
opg/ TPO-overexpressing BM cells.
Representative photographs from a WT control (A-C) and a 3-month-old
opg/ control (D-F) are shown for comparison.
The middle column shows longitudinal sections of femurs
stained according to Gordon-Sweet (original magnification × 25).
Details of the femoral cortical area are illustrated in the right
column (original magnification × 60). Histologic sections are stained
according to Gordon-Sweet. Note decreased bone mineral radiodensity (D)
and severe cortical bone porosity in the
opg/ control (E-F). (G) Radiograph of the
femur of a WT host engrafted with WT donor cells. Note significant
radiodensification of the bone. (H-I) Representative histologic section
of the femur shows the presence of numerous bone trabeculae (arrow)
occluding the femoral cavity. (J) Radiograph of the femur of an
opg/ host engrafted with
opg/ donor cells shows poorly defined
cortical region and significant loss of radiodensity. (K-L) Severe
osteoporosis is confirmed histologically. (M) Radiograph of the femur
of a WT host engrafted with opg/ donor
cells. Increased bone density is seen. (N-O) Representative histologic
section of the femur shows osteosclerosis with accumulation of bone
trabeculae in the shaft (arrow). (P) Radiograph of the femur of an
opg/ host engrafted with WT donor cells.
Note thinning of the cortical region and decreased bone density. (Q-R)
Histologically, the femur is profoundly osteoporotic with a porous
cortical region. Radiography and histology were from the same animals
examined 3 months after transplantation. Similar results were observed
in 3 mice per experimental groups.
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Osteoporosis in opg-deficient hosts repopulated with
opg/ or WT BM cells
In contrast to WT repopulated hosts, radiographic analysis of
opg/ recipients reconstituted with TPO
virus-infected hematopoietic cells from
opg/ (Figure 4J) or WT (Figure 4P) donors
showed an overall decrease in bone density comparable to control
opg/ mice (Figure 4D) of a matched
age.18 Histologic analysis of the femurs at 3 months after
transplantation showed severe osteoporosis of the femoral cortical
margin (Figure 4K,Q) as displayed in aging opg/ hosts (Figure 4E-F). The cortical
region appeared porous (Figure 4L,R) with numerous vessels filled with
hematopoietic cells and several resorption pits on the bone surface.
When compared with control unmanipulated
opg/ mice of a matched age, the main
difference noticed was the presence of small lamellar structure growing
in the marrow space. Abundant osteoblasts and multinucleated
osteoclasts lining the endosteal bone surface were seen in
opg/ hosts that received transplants of
either WT or opg/ hematopoietic cells.
OPG levels in plasma
The amount of OPG protein in plasma was measured at monthly
intervals in each experimental group. The average OPG level in WT
controls from this colony was 2.3 ± 0.2 ng/mL (n = 8) and
undetectable in the control opg/ mutants.
One month after engraftment, levels of circulating OPG were about
6-fold augmented (11.6 ± 0.6 ng/mL, n = 12) in WT repopulated hosts. No significant difference was seen between WT mice repopulated with WT or opg/ cells
(P < .1). At 2 and 3 months, levels were decreased but remained 3 to 4 times more elevated than in controls (Figure
5). Whatever the time of examination, no
correlation was found between TGF-1 and OPG levels (data not shown).
In contrast, no circulating OPG was detected in the
opg/ hosts reconstituted with a WT or an
opg/ transplant at any time.
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| Figure 5.
OPG levels in plasma from hosts repopulated with
WT or
opg/ TPO virus-infected BM
cells. OPG levels were measured in PPP by ELISA. Each bar
represents the mean ± SD of 7 to 10 animals per experimental
group as indicated under the x-axis. The mice receiving transplants
were analyzed at 1, 2, and 3 months, except for the
WT/opg/ group where samples were lost
accidentally. The level of circulating OPG in WT controls (n = 8) is
shown by black bars. One month after transplantation (open bars), OPG
levels were 6-fold increased in WT hosts engrafted with TPO
virus-infected BM cells from WT or opg/
donors. Levels remained about 3-fold elevated at 2 months (hatched
bars) and 3 months (dotted bars) with no significant difference
(P < .5) between the 2 groups. OPG was undetectable in
plasma from control and engrafted opg/
hosts.
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TGF-1 has been demonstrated to be a potent cytokine-regulating OPG
transcription and secretion factor in the in vitro stromal/osteoblastic cell lines.19,20 To investigate whether the increased OPG
secretion was related to the elevation in systemic TGF-1 levels in
an animal context, WT mice were inoculated with an adenovirus vector
encoding spontaneously bioactive forms of TGF-1 or the empty
vector.38 One month after infection, detectable levels of
transgene-encoded bioactive TGF-1 was measured in plasma
(0.41 ± 0.13 ng/mL) in association with a marked elevation of latent
forms (21.5 ± 6.2 ng/mL versus 5.1 ± 2.1 ng/mL in controls
inoculated with the empty vector). Under these conditions,
immunodetectable levels of OPG remained at baseline (Figure
6). These data suggest that the rapid onset of osteosclerosis in mice with TPO overexpression is mediated through an up-regulation of endogenous stroma-derived OPG by a mechanism independent from TGF-1.
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| Figure 6.
Effect of bioactive TGF-1 on OPG secretion in
vivo.
Immunoreactive TGF-1 (), latent TGF-1 (), and OPG
( ) were measured in PPP from WT mice infected with
2 × 108 pfu of an adenovirus encoding a mutated TGF-1
cDNA (Ad-TGF-1s223/s225) or the empty adenovirus
vector as a control. Plasma was collected 1 month after infection. Each
bar represents the mean ± SD of 6 animals. For TGF-1
determination, samples were assayed prior to acidification (bioactive
transgene-encoded, TGF-1) and after acidification (active + latent forms). Black bar indicates spontaneously active TGF-1; open
bar, latent + active TGF-1; hatched bar, OPG. Levels of OPG in
mice treated with the empty or Ad-TGF-1 vector are not statistically
different ( P < .15).
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| |
Discussion |
The systematic development of myelofibrosis and
osteosclerosis in mice that received transplants of marrow cells
infected with a TPO-encoding retrovirus represents a suitable model to study the underlying causes of the pathologic stromal reaction and the
abnormal bone growth.32-34 We have previously documented the pivotal role of TGF-1 secreted by hematopoietic cells in the
promotion of myelofibrosis.35 In this report, we present evidence that the aberrant bone construction is mainly mediated by an
up-regulation of stroma-derived OPG leading to an inhibition of
bone resorption.
Hematopoietic stem cells from mutant homozygote
opg-deficient mice (opg/) and WT
littermates36 were infected with a retrovirus to
overexpress TPO and engrafted into lethally irradiated hosts. OPG is a
secreted protein highly expressed in stromal/osteoblastic
cells15 and in various immune and hematopoietic tissues
from adult mice.39-42 To discriminate the effects of OPG
derived from the host microenvironment and the hematopoietic
transplant, the 4 different combinations of graft/host were analyzed.
Whatever the combinations, all the repopulated mice showed comparable
TPO elevation in plasma and rapidly developed the typical
myeloproliferative syndrome characterized by thrombocytosis,
leukocytosis, severe anemia, splenomegaly with an accumulation of
dysmorphic megakaryocytes, and extramedullary hematopoiesis.32,34 In accordance with previous
reports,32,35,43 TGF-1 levels in plasma were 3- to
4-fold elevated over controls (Figure 2) and each recipient displayed
prominent myelofibrosis in spleen and femurs within 3 months. Femoral
radiographs and histologic analysis of WT hosts engrafted with either
WT or opg/ hematopoietic cells revealed
severe osteosclerosis and a rarity of multinucleated osteoclasts. In
contrast, rare bone trabeculae, porous bone cortical areas, and active
osteoclastogenesis were seen in opg/
recipients whether they were engrafted with cells originating from WT
or opg/ donors. These observations suggested
a role for endogenous host-derived OPG in the induction of
osteosclerosis. Because osteosclerosis developing in WT reconstituted
hosts was reminiscent of the phenotype of mice overexpressing
OPG,15,44 we evaluated OPG plasmatic levels during the
time course. Independently of the graft origin (WT or
opg/), OPG levels in WT hosts were 6-fold
increased over controls at 1 month and remained more than 3 times
elevated during the follow-up (Figure 5). In contrast, OPG was
undetectable in the circulation of opg/
hosts, even when the animals received transplants of WT bone marrow
cells. These findings indicate that an up-regulation of stroma-derived
OPG is implicated in the pathogenesis of osteosclerosis developing in
mice with TPO overexpression.
The mechanism of OPG up-regulation in this in vivo model is not
defined. Previous reports have demonstrated a stimulatory effect of
TGF-1 on OPG secretion in primary osteoblasts and
stromal/osteoblastic cell lines.19,20,45 Because TGF-1
levels are increased in mice with osteomyelofibrosis, it was tempting
to postulate a TGF-1-dependent mechanism. Our findings are not in
favor of this hypothesis. First, inoculation of WT mice with an
adenovirus encoding biologically active TGF-1 failed to stimulate
OPG secretion (Figure 6). Second, in WT mice developing osteosclerosis,
no relationship between levels of OPG and TGF-1 was found (not
shown). Third, in WT mice repopulated with hematopoietic cells
originating from TGF-1/ donors,35
TGF-1 levels remained at baseline, whereas significant elevations in
plasmatic OPG levels were found (data not shown). These data suggest
that regulation of OPG secretion might occur in vivo via a mechanism
independent of TGF-1. Cells involved in the pathogenesis of
osteomyelosclerosis secrete and release platelet-derived growth
factor (PDGF) and IL-1,28,46 2 cytokines that have
the ability to stimulate OPG synthesis by stromal
cells.47-50 Although we cannot formally exclude a possible
role for TGF-1 bound in the bone extracellular matrix, the present
data suggest that the mechanisms leading to in vivo stimulation of OPG
secretion still remain to be clarified.
A high level of endogenous TGF-1 may lead to defective
osteoclastogenesis through an inhibition of RANKL
expression.19,51 We did not investigate the expression
level of RANKL in our model, but such a mechanism appears unlikely
because osteoclastogenesis remained active in the
opg/ hosts. On the other hand, it is known
that TGF- family has both positive and negative impacts on
osteoblast proliferation and differentiation.7,8,10 The
low growth of bone trabeculae in the marrow lumen of
opg/ hosts that received transplants of WT
or opg/ suggests that TGF-1 may exert
direct stimulatory effects on bone construction.9
Nevertheless, stimulation of bone construction appears to be far less
potent that inhibition of resorption because none of the
opg/ hosts showed osteosclerosis at 3 months.
Collectively, the data confirm the prominent impact of TGF-1 in the
development of myelofibrosis in mice hyperstimulated with
TPO35 and demonstrate the important role of OPG in the pathogenesis of osteosclerosis. The cellular types involved in the
increased and sustained secretion of TGF-1 are still a matter of
debate.28,30 Nevertheless, the observation that
osteomyelofibrosis develops at a slow rate in GATA-1low
mutant mice, whereas expression levels of megakaryocyte-derived cytokines are reduced,52 further highlights the major
implication of megakaryocytes in the pathogenesis of
myelofibrosis/osteosclerosis. In patients with IM, accumulation of
dysplastic megakaryocytes in marrow and increased TGF-1 levels have
been linked to the pathogenesis of myelofibrosis.27 Future
studies aiming to analyze plasmatic levels of TGF-1 and OPG during
the progression of IM are needed to evaluate whether development of
osteosclerosis correlates with increased OPG secretion, as demonstrated
by our data. Experimentally induced myelofibrosis/osteosclerosis
represent valuable models for understanding the nature of the
pathologic events leading to the complex stroma and bone modifications
and for devising therapeutic strategies for preventing the more severe
manifestations of the disease.
| |
Acknowledgments |
We are grateful to Dr Edward Clark for kindly providing the
opg+/ breeders and to Dr J.-L. Villeval for
the MPZenTPO virus-producing GP+E-86 cells. We acknowledge
A. M. Hagnere for expert assistance with the histopathologic
studies and A. Rouchès for help with the animals.
| |
Footnotes |
Submitted September 17, 2002; accepted December 13, 2002.
Prepublished
online as Blood First Edition Paper, December 27, 2002; DOI
10.1182/blood-2002-09-2839.
Supported by grants from the Institut National de la Santé
et de la Recherche Médicale, the Institut Gustave Roussy, the Ministère de la Recherche, and the Ligue Nationale contre le Cancer (Equipe labellisée 2000). H.C. and E.K. are supported by a
fellowship from the Ministère de la Recherche and the Ligue Nationale contre le Cancer, respectively.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: William Vainchenker, LIPA, IFR 54, Institut
Gustave Roussy, Villejuif, France; e-mail:
verpre{at}igr.fr.
| |
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Abstract
Introduction