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Regular Article
HEMATOPOIESIS AND STEM CELLS
Myelopoiesis is regulated by osteocytes through Gs␣-dependent signaling
*Keertik Fulzele,1 *Daniela S. Krause,2 Cristina Panaroni,1 Vaibhav Saini,1 Kevin J. Barry,1 Xiaolong Liu,1 Sutada Lotinun,3
Roland Baron,1,3 Lynda Bonewald,4 Jian Q. Feng,5 Min Chen,6 Lee S. Weinstein,6 Joy Y. Wu,1 Henry M. Kronenberg,1
David T. Scadden,2 and Paola Divieti Pajevic1
1Endocrine Unit and 2Center of Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 3Harvard School of Dental
Medicine, Boston, MA; 4Department of Oral Biology, University of Missouri at Kansas City, Kansas City, MO; 5Department of Biomedical Sciences, Texas A&M
Health Science Center, Baylor College of Dentistry, Dallas, TX; and 6Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, MD
Hematopoietic progenitors are regulated in their respective niches by cells of the bone
marrow microenvironment. The bone marrow microenvironment is composed of a
• Deletion of Gs␣ in osteocytes
variety of cell types, and the relative contribution of each of these cells for hematopoietic
induces severe osteopenia
lineage maintenance has remained largely unclear. Osteocytes, the most abundant yet
least understood cells in bone, are thought to initiate adaptive bone remodeling
and a dramatic expansion of
responses via osteoblasts and osteoclasts. Here we report that these cells regulate
cells of the myeloid lineage.
hematopoiesis, constraining myelopoiesis through a Gs␣-mediated mechanism that
• Osteocytes regulate hematoaffects G-CSF production. Mice lacking Gs␣ in osteocytes showed a dramatic increase
poiesis and specifically conin myeloid cells in bone marrow, spleen, and peripheral blood. This hematopoietic
tribute to myelopoiesis by
phenomenon was neither intrinsic to the hematopoietic cells nor dependent on
secreting proliferative factors
osteoblasts but was a consequence of an altered bone marrow microenvironment
such as G-CSF.
imposed by Gs␣ deficiency in osteocytes. Conditioned media from osteocyte-enriched
bone explants significantly increased myeloid colony formation in vitro, which was
blocked by G-CSF–neutralizing antibody, indicating a critical role of osteocyte-derived G-CSF in the myeloid expansion. (Blood.
2013;121(6):930-939)
Key Points
Introduction
Under normal physiologic conditions, bone marrow (BM) serves as
the primary site for hematopoiesis, where the daily replenishment
of mature hematopoietic cells is sustained by hematopoietic stem
cells (HSCs) residing in the niche. The hematopoietic hierarchy
begins with a small population of life-long self-renewing long-term
HSCs giving rise to short-term HSCs, which gradually lose certain
lineage differentiation potential to become multipotent progenitors.
Further lineage restriction of these progenitors gives rise to either
common lymphoid progenitors or common myeloid progenitors,
which generate the 2 main lineages, lymphoid and myeloid,
respectively.1 In addition to cell-intrinsic factors,2 extrinsic factors
from cells in the niche regulate the self-renewal, differentiation,
and migration of HSCs.3 Primary components of the niche include
osteoblasts,4,5 osteoclasts,6 vascular endothelial cells,7 mesenchymal stem cells,8 and cells of the sympathetic nervous system.8,9 The
BM harbors specialized niches for lineage-restricted progenitors
conducive to the development of specific hematopoietic cell
lineages. These include BM stromal cell niches for B-lymphopoiesis
through expression of CXCL12 and IL-7,10,11 and BM endothelial
cell niches for megakaryocyte progenitors.12 Therefore, the signals
from cells of the BM microenvironment appear to be able to direct
hematopoietic lineage commitment and also contribute to the
pathogenesis of hematopoietic disorders.
Among the cells of the skeletal and BM microenvironment,
bone-forming osteoblasts are the most versatile regulators of
hematopoiesis. Constitutive activation of the parathyroid hormone
(PTH)/PTHrP receptor in osteoblasts increases the numbers of
HSCs via increased expression of Notch1 ligand jagged1,4 whereas
conditional deletion of Gs␣ in preosteoblasts leads to decreased
B-cell precursors because of decreased IL-7 expression by the
osteoblasts.11 Induced osteoblast deficiency results in an early loss
of HSCs and subsequent loss of lymphoid, erythroid, and myeloid
progenitors in the BM.13
Osteocytes are the most abundant bone cells (95%),14 which are
terminally differentiated from osteoblasts and are embedded deep
within the bone matrix during bone formation. Compared with the
short-lived osteoblasts and osteoclasts, osteocytes can live for
years, making them ideal candidates to survey bone quality and
initiate a bone-remodeling cycle when necessary. Recent studies
demonstrated that these cells coordinate bone remodeling by
Submitted June 13, 2012; accepted October 24, 2012. Prepublished online
as Blood First Edition paper, November 16, 2012; DOI 10.1182/blood-201206-437160.
The online version of this article contains a data supplement.
*K.F. and D.S.K. contributed equally to this study.
There is an Inside Blood commentary on this article in this issue.
930
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 USC section 1734.
BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
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BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
secreting RANKL, the critical osteoclastogenic factor,15,16 and
Sclerostin, a Wnt inhibitor and suppressor of osteoblast proliferation and functions.17 One class of important signaling pathways in
osteocytes and osteoblasts are the G protein–coupled receptor
(GPCR) signaling pathways that act through heterotrimeric G proteins.18 The best characterized subunit of G-proteins, Gs␣, activates
adenylyl cyclase that, in turn, catalyzes the production of cAMP
and activates protein kinase A, which ultimately regulates gene
expression.19 Osteocytes express several Gs␣-coupled receptors,
including the PTH/PTHrP receptor,20 prostaglandin receptors (EP2
and EP4),21 and other receptors.22 Given the profound influence of
osteoblasts and osteoclasts in regulating hematopoiesis, we hypothesized that osteocytes could regulate hematopoiesis through Gs␣
signaling.
To test this hypothesis, we engineered mice lacking Gs␣
specifically in osteocytes (OCY-Gs␣KO). These mice display
severe osteopenia because of a decrease in osteoblast numbers and
a dramatic expansion of cells of myeloid lineage in BM, spleen,
and peripheral blood (PB). Interestingly, the lymphoid cells were
not affected in BM and PB. Transplantation of BM from control to
OCY-Gs␣KO mice rapidly recapitulated the myeloproliferation,
whereas the converse experiment abolished it, demonstrating that
the defect is not intrinsic to the hematopoietic cells but is an effect
of an altered BM microenvironment imposed by Gs␣-deficient
osteocytes. Treatment of these mice with antisclerostin antibody
restored the numbers of osteoblasts and normalized bone mineral
density; however, the myeloproliferative phenotype persisted,
suggesting that osteocytes directly regulate hematopoiesis through
a Sclerostin/Wnt signaling independent pathway. In vitro assays
revealed that osteocytes secrete several myeloproliferative factors,
including G-CSF, capable of regulating myelopoiesis. Our findings
show, for the first time, that osteocytes are a major regulator of
myelopoiesis, most likely through regulation of myeloid progenitor
cell niches. We also show that the osteocyte-myeloid cell interaction is dependent on Gs␣-signaling in osteocytes and is partially
mediated by osteocyte-derived G-CSF.
Methods
Mice
All procedures involving mice were approved by the Institutional Animal
Care and Use Committee of Massachusetts General Hospital. Mice lacking
Gs␣ in osteocytes were generated by crossing Gs␣flox/flox mice23 with mice
expressing Cre-recombinase driven by a 10-kb DMP1 promoter.24 Mice
lacking PTH-PTHrP receptor (PPR) in osteocytes were generated by mating
PPRflox/flox mice25 with DMP1-Cre mice. The genotype of the mice and
tissue-specific DNA recombination were performed as described in supplemental Methods (available on the Blood Web site; see the Supplemental
Materials link at the top of the online article).
For sclerostin neutralization, control and OCY-Gs␣KO mice were
injected subcutaneously with antisclerostin antibody (25 mg/kg, 2 times per
week; kindly provided by Michaela Kneissel, Novartis) from 3 days until
4 weeks old.
Complete blood counts and flow cytometry
Complete blood counts from PB were performed by a VetScan HM5
Hematology System (Abaxis). For immunophenotyping of terminally
differentiated hematopoietic cells, BM and spleen cells were stained for
15 minutes on ice with fluorescent antibodies to B lymphocytes (B220,
IgM, and CD93), T lymphocytes (CD4 and CD8a), granulocytes (CD11b
and Gr-1), erythrocytes (Ter119 and CD45), megakaryocytes (CD41 and
CD42d), and monocytes/macrophages (F4/80 and CD11b). For hematopoi-
OSTEOCYTES AND MYELOPOIESIS
931
etic progenitor cell analysis, BM and spleen cells were lineage depleted and
then stained with fluorescent antibodies to hematopoietic stem cells (LKS:
Lin⫺ c-Kit⫹ Sca-1⫹ and LKS SLAM: Lin⫺ c-Kit⫹ Sca-1⫹ CD150⫹
CD48⫺), multipotent progenitors (MPP: Lin⫺ c-kit⫹ Sca-1⫹ Thy1.1⫺
Flk-2⫹), common myeloid progenitors (CMP: IL-7R␣⫺ Lin⫺ c-kit⫹ Sca-1⫺
Fc␥Rlo CD34⫹), granulocyte macrophage progenitors (GMP: IL-7R␣⫺
Lin⫺ c-kit⫹ Sca-1⫺ Fc␥R⫹ CD34⫹), and megakaryocyte erythroid progenitors (MEP: IL-7R␣⫺ Lin⫺ c-kit⫹ Sca-1⫺ Fc␥Rlo CD34⫺). All antibodies
were obtained from eBioscience. The fluorescently labeled cells were then
analyzed on a BD LSRII (BD Biosciences).
BM transplantation
Recipient mice were lethally irradiated with a single dose of 9.5 Gy and
then transplanted with lineage depleted mononuclear BM cells from donor
mice by tail vein injection. Complete blood cell counts were measured in
donor and recipient mice before transplantation and in recipient mice 4 and
9 weeks after transplantation.
OEBE generation and in vitro culture
Osteocyte-enriched bone explants (OEBEs) were generated from long
bones of 7-week-old OCY-Gs␣KO or control mice. After removing the
epiphyses and flushing out BM, the explants were digested with collagenase
and EDTA to remove endosteal and periosteal osteoblasts and BM cells as
described previously.22 For myeloid colony generation, 2 ⫻ 104 BM cells
from 8- to 10-week-old OCY-Gs␣KO or control mice were cultured in
duplicate in myeloid colony supporting methylcellulose-based complete
medium (M3434) containing 3 U/mL erythropoietin, 10 ng/mL recombinant murine IL-3 (rmIL-3), 10 ng/mL rmIL-6, and 50 ng/mL recombinant
murine stem cell factor (StemCell Technologies). The cells were cultured in
35-mm culture dishes with a 2-mm grid (174926, Nalge Nunc) and
incubated for 10 days at 37°C. Total myeloid colonies per plate were
counted by morphologic scoring. For megakaryocyte colony formation,
1 ⫻ 105 BM cells from 7-week-old OCY-Gs␣KO or control mice were
cultured in duplicate in MegaCult-C media supplemented with 50 ng/mL
thrombopoietin, 10 ng/mL IL-3, and 20 ng/mL IL-6 (StemCell Technologies). The cells were cultured in double-chamber slides and stained for
acetylcholinesterase as per the manufacturer’s protocol (StemCell Technologies). For the coculture assay, OEBE from 2 tibia and 2 femurs from control
and OCY-Gs␣KO mice were cultured (in IMDM with 10% FBS) on
transwells with 2 ⫻ 104 BM cells from a control mouse cultured in M3434
methylcellulose medium at the bottom of the 35-mm culture dish. To assess
the direct contribution of osteocyte-produced growth factors and cytokines
in supporting myeloid or megakaryocytic cell growth, conditioned media
was obtained by culturing OEBEs from OCY-Gs␣KO or control mice for
7 days in 1.5 mL of ␣-MEM with 10% FBS (Invitrogen). The conditioned
media was mixed in a 1:3 proportion with incomplete methylcellulose
medium (M3231) or MegaCult-C medium containing FBS but lacking
other growth factors. A total of 2 ⫻ 104 bone BM cells from a control
mouse were cultured in this mixed medium in 12-well low-cell binding
plates (145385, Nalge Nunc), incubated at 37°C for 10 days. Colonies were
counted at the end.
Bioassays
cAMP was measured in cultured calvarial osteoblasts26 as described
previously.20 G-CSF in serum and OEBE conditioned media was measured
by ELISA (Raybiotech). SDF1␣, TNF-␣, and IL-12 levels were measured
in OEBE conditioned media by ELISA (Raybiotech). G-CSF and IL-12 in
OEBE conditioned media were normalized to OEBE dry weight.
Statistical analysis
All data are presented as mean ⫾ SEM. Statistical significance of differences between groups was determined by Student t test. P values ⱕ .05
were accepted as significant.
Additional methods are described in supplemental Methods.
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932
FULZELE et al
BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
Figure 1. DMP1-Cre–mediated loss of Gs␣ in osteocytes. (A) Breeding strategy for generating mice with osteocyte-specific disruption of Gs␣. (B) Top panel: Schematic
representation (not to scale) showing the relative location of loxP sites (triangles), Gs␣-exon 1 (rectangle), multiplex PCR primers (F1, R1, R2), and the expected PCR product
sizes. Bottom panel: PCR analysis showed Gs␣ allele recombination in skeletal (tibia and calvaria) tissue but not in hematopoietic (BM, spleen, and liver) tissues from
OCY-Gs␣KO mice (n ⫽ 3). (C) Relative expression of Gs␣ in femur and tibia (n ⫽ 4) from control (Con) and OCY-Gs␣KO (KO) mice. Gs␣ mRNA expression in (D) osteoblasts,
BM, spleen, (E) Gr1⫹ flow-sorted granulocytes, and (F) F4/80⫹ CD11b⫹ flow-sorted monocytes/macrophages (n ⫽ 4). (G) cAMP synthesis in calvarial osteoblasts isolated
from control and OCY-Gs␣KO mice in response to PTH and forskolin (FSK; n ⫽ 4). (H) von Kossa staining of femurs showing osteopenia in OCY-Gs␣KO (KO) mice compared
with control (Con) mice. (I-J) Whole-body dual-emission x-ray absorptiometry showing (I) bone mineral density (BMD; g/cm2) and (J) bone mineral content (BMC; g) in young
(7 weeks old) and adult (21 weeks old) mice (n ⱖ 7 mice). Error bars represent mean ⫾ SEM. *P ⬍ .05 by t test.
Results
Mice with conditional ablation of Gs␣ in osteocytes display
severe osteopenia
We conditionally ablated the ubiquitously expressed stimulatory
subunit of G-proteins, Gs␣, in osteocytes (OCY-Gs␣KO) by
mating Gs␣E1flox/flox mice23 with DMP1-Cre mice24 (Figure 1A).
Cre-loxP–mediated DNA recombination was observed in skeletal,
but not in hematopoietic tissues of OCY-Gs␣KO mice (Figure 1B).
Skeletal and PB phenotypes were not different between wild-type
and DMP1-Cre mice matched by genetic background (supplemental Figure 1A-F). Therefore, littermates lacking the DMP1-Cre
transgene but carrying the Gs␣E1 floxed alleles (Gs␣E1flox/flox)
were used as controls. OCY-Gs␣KO mice were viable, fertile, and
indistinguishable from littermate controls at birth. Gs␣ mRNA
expression in osteoblast- and BM-depleted osteocyte-enriched long
bones (supplemental Figure 3A) from OCY-Gs␣KO mice was
decreased by 70% (Figure 1C) but was unchanged in osteoblasts,
BM, spleen, monocytes/macrophages, or granulocytes (Figure 1D-F).
Moreover, the functional capacity of Gs␣ to synthesize cAMP was not
altered in calvarial osteoblasts from OCY-Gs␣KO and control mice
(Figure 1G), confirming osteocyte-specific disruption of Gs␣. OCYGs␣KO mice displayed osteopenia, characterized by a dramatic decrease in trabecular and cortical bone as revealed by von Kossa staining
(Figure 1H), bone mineral density (Figure 1I), and bone mineral content
(Figure 1J) by dual-energy x-ray absorptiometry. Examination of
osteocyte microstructure showed increased osteocyte density (supplemental Figure 4) but a disorganized and reduced lacunar-canalicular network
(supplemental Figure 4).
Myelopoiesis, but not lymphopoiesis, is increased in mice
lacking Gs␣ in osteocytes
Examination of hematopoietic organs in OCY-Gs␣KO mice showed
marked splenomegaly (Figure 2A) and focal areas of hypercellularity in the BM (Figure 2B). Normalizing for decreased body weight
(Figure 2C), spleen weights were increased 4-fold (Figure 2D) in
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BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
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Figure 2. Osteocyte-specific loss of Gs␣ results in expansion of myeloid cells in BM and spleen. (A) Splenomegaly in OCY-Gs␣KO (KO) mice compared with controls (Con).
(B) Representative areas of hypercellularity in BM of OCY-Gs␣KO femurs. (C) Decreased whole-body weight and (D) increased normalized spleen weights in OCY-Gs␣KO mice (n ⱖ 7).
(E-H) PB counts showing (E) number of leukocytes, (F) % neutrophils, (G) number of platelets, and (H) number of lymphocytes in 7- and 21-week-old control and OCY-Gs␣KO female mice
(n ⱖ 9). (I-L) Immunophenotypic analysis of hematopoietic cells in BM and spleen showing absolute counts for B cells (B220⫹ IgM⫹), granulocytes (Gr1⫹ CD11b⫹), erythroid cells (Ter119⫹
CD45⫺), monocytes (F4/80⫺ CD11b⫹), macrophages (F4/80⫹ CD11b⫹), T cells (CD4⫹), and cytotoxic T cells (CD8a⫹) in BM (I-J) and spleens (K-L) of 7-week-old (I,K) and 21-week-old
(J,L) control and OCY-Gs␣KO female mice (n ⱖ 6). Error bars represent mean ⫾ SEM. *P ⬍ .05 by t test.
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934
FULZELE et al
OCY-Gs␣KO mice compared with controls. We performed complete blood counts in PB and flow cytometry analysis on BM and
spleens in young (7-week-old) and adult (21-week-old) mice. PB
from young and adult OCY-Gs␣KO mice showed a significant
increase in leukocytes (Figure 2E) and platelets (Figure 2G) and a
5-fold increase in the percentage of neutrophils (Figure 2F).
Lymphocyte numbers were not changed at either age (Figure 2H) in
the PB. Hematocrit fraction, red blood cells, and hemoglobin were
unchanged at 7 weeks of age in OCY-Gs␣KO mice compared with
controls (HCT, 53.4% ⫾ 5.07% vs 53.9% ⫾ 2.82%; RBC,
10.2 ⫾ 1.13 vs 10.6 ⫾ 0.6 ⫻ 106 cells/␮L; hemoglobin, 15.0 ⫾ 0.83
vs 16.1 ⫾ 0.59 g/dL; mean ⫾ SEM, n ⫽ 10). Immunophenotypic
analyses of mature hematopoietic cells in the BM showed a
significant increase in the absolute number of Gr1⫹/CD11b⫹
granulocytes and F4/80⫺/CD11b⫹ monocytes in 7-week-old OCYGs␣KO mice which were further increased in 21-week old
OCY-Gs␣KO mice compared with controls (Figure 2I-J), suggesting that the hematopoietic abnormalities progress with aging. There
was a modest but significant decrease in Ter119⫹/CD45⫺ erythroid
cells and an increase in F4/80⫹/CD11b⫹ macrophages in the BM of
OCY-Gs␣KO mice. Significant increases in the absolute numbers
of granulocytes, monocytes, and macrophages were observed in
spleens of OCY-Gs␣KO mice compared with controls (Figure
2K-L). B220⫹/IgM⫹ mature B cells, CD4⫹ or CD8a⫹ mature
T cells, and CD41⫹/CD42d⫹ megakaryocytes were unchanged in
the OCY-Gs␣KO BM and spleen (Figure 2I-L; supplemental
Figure 5A). However, the frequency of mature B cells and T cells
was modestly, but significantly, decreased in the spleen OCYGs␣KO mice compared with controls (supplemental Figure 2).
These findings indicate that osteocyte-specific disruption of Gs␣
results in an expansion of myeloid cells in the BM, spleen, and PB,
suggesting that Gs␣ signaling in osteocytes is required for normal
hematopoiesis.
Alteration of the BM microenvironment by Gs␣-deficient
osteocytes is necessary to induce the myeloproliferation
To determine whether the hematologic abnormalities present in the
OCY-Gs␣KO mice were intrinsic to the hematopoietic cells or
imposed by an osteocyte-dependent altered BM microenvironment,
we performed BM transplantation experiments. BM transplantation
from control donor mice into lethally irradiated OCY-Gs␣KO
recipient mice rapidly induced leukocytosis, neutrophilia, and
thromobocytosis in recipient mice beginning 4 weeks after transplantation and continuing at 9 weeks after transplantation (Figure
3A-D). Reciprocal transplantation of BM from OCY-Gs␣KO
donor mice with severe myeloproliferation into lethally irradiated
control recipients completely normalized the hematopoietic profile
already at 4 weeks after transplantation (Figure 3E-H). These
results indicate that the altered BM microenvironment of Gs␣deficient osteocytes is required for initiation and maintenance of
the myeloproliferation and that the hematopoietic abnormality is
not intrinsic to the hematopoietic cells. To identify the defect in
progenitor cells that may lead to myeloid proliferation, we enumerated HSCs (LKS and LKS SLAM) and myeloid progenitor cells
(multipotent progenitor cell [MPP], common myeloid progenitor
cell [CMP], granulocyte macrophage progenitor cell [GMP], and
megakaryocyte erythroid progenitor cell [MEP])1 in BM and
spleen of these animals. None of the progenitors was significantly
different in BM of control and OCY-Gs␣KO mice (Figure 3K). In
spleens from OCY-Gs␣KO mice, LKS and LKS-SLAM HSCs
were significantly increased, although there was no difference in
myeloid progenitors (Figure 3L). Similarly, in vitro methylcellu-
BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
lose colony assays (Figure 3M) showed a 3-fold increase in the
number of myeloid colonies generated by splenocytes from OCYGs␣KO mice over controls (Figure 3O), indicating an increase in
HSCs in OCY-Gs␣KO spleens. There was no difference in the
number of myeloid or megakaryocytic colonies formed by BM cells
from control and OCY-Gs␣KO mice (Figure 3N; supplemental
Figure 5B), demonstrating the requirement of an altered BM microenvironment to induce myeloproliferation in OCY-Gs␣KO mice.
Myeloproliferation in Gs␣-deficient osteocytes is independent
of osteoblasts and the Wnt/␤-catenin signaling pathway
Osteocytes may influence hematopoiesis directly by secreting
cytokines or chemokines or indirectly by acting via intermediate
cells, such as osteoblasts. The dramatic decrease in the numbers of
osteoblasts in OCY-Gs␣KO mice (Figure 4E), could, per se, alter
the BM microenvironment and cause the myeloproliferation.
Mature osteocytes are the primary source of sclerostin, a potent
suppressor of Wnt/␤-catenin signaling, osteoblast numbers, and
activity.27 Expression of SOST, the gene encoding sclerostin, was
increased 3-fold in mRNA from osteoblast- and BM-depleted
osteocyte-enriched long bones from OCY-Gs␣KO mice compared
with controls (Figure 4A). Sclerostin protein was also increased in
tibia sections from OCY-Gs␣KO mice (Figure 4B) as assessed by
immunohistochemistry. Moreover, mRNA expression of Axin2,
downstream target of Wnt signaling, was significantly decreased in
the CD11b⫹ FACS-sorted myeloid cells from OCY-Gs␣KO mice
(Figure 4C), indicating suppressed Wnt/␤-catenin signaling. To
investigate whether the myeloproliferation seen in the OCYGs␣KO mice was a consequence of reduced osteoblast number
(Figure 4B) and/or suppressed Wnt/␤-catenin signaling (Figure
4C), we treated control and OCY-Gs␣KO mice with a sclerostinneutralizing antibody. We used a 25 mg/kg body weight dose of
antisclerostin antibody, which has previously been shown to
effectively neutralize circulating sclerostin.28 Antisclerostin antibody treatment partially restored the suppressed Wnt signaling in
OCY-Gs␣KO mice, as shown by a significant increase in Axin2
gene expression in CD11b⫹ myeloid cells (Figure 4C). As expected, and as previously reported,28 treatment with antisclerostin
antibody significantly increased trabecular bone fraction and
osteoblast numbers both in control and OCY-Gs␣KO mice (Figure
4D-E). However, the bone volume fraction and osteoblast numbers
were still significantly decreased in antisclerostin treated OCYGs␣KO mice compared with nontreated controls, suggesting that
additional factors, besides sclerostin might be responsible for the
decreased osteoblast numbers and osteopenia in these mice.
Despite the significant increase in osteoblast numbers, antisclerostintreated OCY-Gs␣KO mice continued to exhibit leukocytosis,
neutrophilia, and thromobocytosis (Figure 4F-H), indicating that
the myeloproliferation is not dependent on reduced osteoblast
numbers or suppression of Wnt/␤-catenin signaling.
Myeloid expansion in OCY-Gs␣KO mice is not driven by
the PPR
Numerous GPCRs signal through the common downstream effector Gs␣. Osteocytes express hundreds of GPCRs, as recently
reported.22 Among these receptors, the PPR, which signals primarily through Gs␣, has been well studied, and its effect on hematopoiesis has been previously reported. Constitutive activation of
PTH/PTHrP receptor in osteoblasts increases the numbers of HSCs
via increased expression of Notch1 ligand jagged1.4 To test
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Figure 3. Alteration of the BM microenvironment by Gs␣-deficient osteocytes is necessary to induce myeloproliferation. (A) BM transplantation schema from control
to lethally irradiated OCY-Gs␣KO mice or (F) vice versa. (B-E) PB cell counts of 7-week-old control donor and OCY-Gs␣KO recipient mice before transplantation and recipient
mice 4 and 9 weeks after transplantation showing (B) number of leukocytes, (C) % neutrophils, (D) number of platelets, and (E) number of lymphocytes. (G-J) PB cell counts
from 7-week-old OCY-Gs␣KO donor and control recipient mice before transplantation and recipient mice 4 and 9 weeks after transplantation showing (G) number of
leukocytes, (H) % neutrophils, (I) number of platelets, and (J) number of lymphocytes (n ⫽ 3 for donors and n ⫽ 8 for recipients). (K-L) LKS (Lin⫺ c-Kit⫹ Sca-1⫹), LKS SLAM
(Lin⫺ c-Kit⫹ Sca-1⫹ CD150⫹ CD48⫺), MPPs (Lin⫺ c-kit⫹ Sca-1⫹ Thy1.1⫺ Flk-2⫹), CMPs (IL-7R␣⫺ Lin⫺ c-kit⫹ Sca-1⫺ Fc␥Rlo CD34⫹), GMPs (IL-7R␣⫺ Lin⫺ c-kit⫹ Sca-1⫺ Fc␥R⫹
CD34⫹), and MEPs (IL-7R␣⫺ Lin⫺ c-kit⫹ Sca-1⫺ Fc␥Rlo CD34⫺) in (K) BM and (L) spleen from control and OCY-Gs␣KO female mice (n ⫽ 6). (M) Strategy for methylcellulose
colony formation assay. (N-O) Myeloid colonies from (N) 2 ⫻ 104 BM cells and (O) 1 ⫻ 105 spleen cells from 8- to 10-week-old control and OCY-Gs␣KO mice (n ⫽ 4). Error
bars represent mean ⫾ SEM. *P ⬍ .05 by t test compared with donor.
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FULZELE et al
BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
Figure 4. Antisclerostin antibody normalizes osteoblast numbers but does not normalize myeloid cell
expansion. (A) Relative expression of sclerostin (Sost)
mRNA from femur and tibia from 7-week-old control (Con)
and OCY-Gs␣KO (KO) mice (n ⫽ 4). (B) Immunohistochemical staining for sclerostin on tibia sections from
7-week-old control and OCY-Gs␣KO mice (n ⫽ 3).
(C) mRNA expression of Axin2 in the CD11b⫹ FACSsorted myeloid cells of 7-week-old control (Con) and
OCY-Gs␣KO (KO) mice (n ⫽ 4) in saline or antisclerostin
antibody-treated mice. (D) Trabecular bone fraction,
BV/TV (%), and (E) numbers of osteoblasts per bone
perimeter, N.Ob/B.Pm (/mm) in antisclerostin antibodytreated control (Con) and OCY-Gs␣KO (KO) mice (n ⫽ 4).
(F-H) PB counts showing (F) number of leukocytes, (G) %
neutrophils, and (H) number of platelets in antisclerostin
antibody-treated control (Con) and OCY-Gs␣KO (KO)
mice (n ⫽ 6). Error bars represent mean ⫾ SEM. *P ⬍ .05
by t test.
whether the PPR signaling is responsible for the osteocytemediated myeloid cell expansion, we engineered mice lacking PPR
in osteocytes (OCY-PPRKO) by mating PPR-floxed mice with
DMP1-Cre mice. Successful PPR gene ablation in the OCYPPRKO mice was demonstrated by a significantly reduced PPR
mRNA expression in osteocyte-enriched long bones from OCYPPRKO compared with littermate controls (Figure 5A). In contrast
to the complete blood count profile of PB from OCY-Gs␣KO mice
(Figure 2E-H), OCY-PPRKO mice showed no changes in the
numbers of leukocytes, neutrophils, platelets, and lymphocytes
compared with controls (Figure 5B-E). Immunophenotypic
analysis of mature hematopoietic cells in BM and spleens of
control and OCY-PPRKO mice showed no difference in the
percentage of granulocytes (Gr1⫹/CD11b⫹), monocytes (F4/80⫺/
CD11b ⫹ ), macrophages (F4/80 ⫹ /CD11b ⫹ ), erythrocytes
(Ter119⫹/CD45⫺), B cells (B220⫹/IgM⫹), and T cells (CD4⫹ or
CD8a⫹; Figure 5F-G). Taken together, these results demonstrate
that PPR signaling in osteocytes is not driving the myeloproliferation present in the OCY-Gs␣KO mice.
Osteocytes secrete myeloproliferative factors and G-CSF that
partially drives the myeloproliferation.
To investigate whether osteocytes influence hematopoiesis directly
by secreting myeloproliferative factors, we developed an ex vivo
coculture method in which osteoblast- and BM-depleted OEBEs
from control or OCY-Gs␣KO mice are cocultured with BM cells
from control mice in standard growth factor-supplemented methylcellulose media (M3434, StemCell Technologies; Figure 6A). After
10 days of coculture, the number of myeloid colonies was
significantly increased in OEBE from control mice and was further
significantly increased in the presence of OEBEs derived from
OCY-Gs␣KO mice (Figure 6B). We then used conditioned media
collected from control and OCY-Gs␣KO OEBE cultures (7-day
cultures) to generate myeloid colonies from control BM cultured in
growth factor-deficient methylcellulose media (M3231). As shown
in Figure 6C, conditioned medium induced colony formation,
indicating that osteocytes secrete factor(s) that promote myeloproliferation. These factors were further increased in Gs␣-deficient
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BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
OSTEOCYTES AND MYELOPOIESIS
937
Figure 5. Disruption of PPR in osteocytes shows no hematologic abnormalities. (A) Relative expression of PPR mRNA in femur and tibia (n ⫽ 4) from control (Con) and
OCY-PPR (KO) mice. (B-D) PB counts showing (B) number of leukocytes, (C) % neutrophils, (D) number of platelets, and (E) number of lymphocytes in OCY-PPRKO and
littermate controls (n ⱖ 16). (F-G) Immunophenotypic analysis of hematopoietic cells in BM and spleen showing B cells (B220⫹ IgM⫹), granulocytes (Gr1⫹ CD11b⫹), erythroid
cells (Ter119⫹ CD45⫺), monocytes (F4/80⫺ CD11b⫹), macrophages (F4/80⫹ CD11b⫹), T cells (CD4⫹), and cytotoxic T cells (CD8a⫹) in (F) BM and (G) spleens of 12-week-old
OCY-PPRKO and littermate controls (n ⱖ 16). Error bars represent mean ⫾ SEM. *P ⬍ .05 by t test.
osteocytes as the number of myeloid colonies was significantly
increased in the presence of conditioned media from OCY-Gs␣KO
OEBE compared with control (Figure 6C). Conditioned media
from both control and OCY-Gs␣KO OEBE also modestly increased the numbers of megakaryocytic colonies in growth factordeficient MegaCult-C media (supplemental Figure 5C).
To identify the putative factor(s) secreted by control and
Gs␣-deficient osteocytes, we examined the expression of several
cytokines known to promote granulopoiesis and thrombopoiesis.29,30 mRNA expression for IL-4, IL-6, GM-CSF, TNF-␣, and
thrombopoietin was not different between OEBEs from control and
OCY-Gs␣KO mice (supplemental Figure 3C). RNA expression of
SDF-1 and IL-12 was significantly increased in OEBE from
OCY-Gs␣KO mice compared with controls (supplemental Figure
3C), although we could not detect SDF-1 in conditioned media
from either OEBE. In accordance with the increased IL-12 gene
expression, IL-12 secretion into conditioned media by OCYGs␣KO OEBE was also significantly increased over controls
(supplemental Figure 3D). Interestingly, mRNA expression for
G-CSF, a potent promoter of production, maturation, function, and
mobilization of granulocytes both in vitro and in vivo,30 was
increased 2-fold in OEBE from OCY-Gs␣KO compared with
controls (Figure 6D). G-CSF was also increased in serum of
OCY-Gs␣KO mice (Figure 6E) and 7-day conditioned media from
Gs␣KO OEBE (Figure 6F). Pretreatment of 7-day conditioned
media from control and OCY-Gs␣KO OEBE with a G-CSF
neutralizing antibody significantly decreased the numbers of methylcellulose colonies compared with isotype-matched IgG-treated
conditioned media (Figure 6G), indicating that increased G-CSF
production by Gs␣-deficient osteocytes partially drives the myeloproliferation in these mice. G-CSF–neutralizing antibody treatment also decreased megakaryocytic colonies; however, the changes
were not statistically different from the corresponding groups
(supplemental Figure 5C). To investigate whether Gs␣-signaling
could directly regulate G-CSF expression in bone, we treated
OEBE with forskolin and measured G-CSF mRNA expression after
4 hours. Interestingly, forskolin significantly increased G-CSF
mRNA expression, rather than suppressing it (supplemental Figure
6), suggesting that the increase in G-CSF present in the OCYGs␣KO mice is not a direct but rather a mediated mechanism.
Discussion
Here we report, for the first time, that disruption of Gs␣-signaling
specifically in osteocytes results in a dramatic expansion of cells of
myeloid lineage in BM, spleen, and PB. The hematopoietic defect
is not intrinsic to the hematopoietic cells or dependent on osteoblasts but is dependent on a BM microenvironment altered by Gs␣
deficiency in osteocytes. The direct regulation of hematopoiesis by
osteocytes is supported by several evidences. In our mouse model,
Gs␣ expression and activity is normal in hematopoietic cells and
osteoblasts, thereby excluding the possibility of an osteoblastic- or
hematopoietic-cell autonomous defect resulting from off-target
Cre-recombinase activity. Moreover, transplantation experiments
with control BM into lethally irradiated OCY-Gs␣KO mice fully
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938
FULZELE et al
BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
Figure 6. Osteocyte-dependent in vitro myeloproliferation. (A) Schematic representation of cocultures of OEBEs or conditioned media from OEBEs with BM cells in
methylcellulose media. (B) Number of myeloid colonies in growth factor-supplemented methylcellulose media (M3434) from coculture of BM cells from control mice with
OEBEs from control or OCY-Gs␣KO mice (n ⫽ 4). (C) Number of colonies in growth factor-deficient methylcellulose media (M3231) after coculture of BM cells from control
mice with conditioned media from OEBEs from control or OCY-Gs␣KO mice (n ⫽ 4). (D) G-CSF mRNA expression in OEBEs (n ⫽ 5). G-CSF in (E) serum (n ⱖ 7) and
(F) OEBE-conditioned media (n ⫽ 6) from control and OCY-Gs␣KO mice. (G) Number of colonies in growth factor-deficient methylcellulose media (M3231) formed after
coculture of BM cells from control mice with conditioned media from OEBEs from control or OCY-Gs␣KO mice and in the presence or absence of a G-CSF neutralizing antibody
(n ⫽ 4). Error bars represent mean ⫾ SEM. *P ⬍ .05 by t test.
recapitulate the hematopoietic abnormalities, demonstrating a
dependence of the myeloproliferation on a defective BM environment. OCY-Gs␣KO mice have a dramatic reduction in the number
of osteoblasts, in part as a consequence of the elevated sclerostin
expression. To investigate whether the hematopoietic defect was
dependent on the reduced osteoblast number, we treated OCYGs␣KO and control with antisclerostin antibodies. Antibody treatment increased the numbers of osteoblasts in control and OCYGs␣KO mice but did not abolish the myeloproliferation in the
OCY-Gs␣KO mice. Interestingly, there was a 2-fold increase in the
percentage of neutrophils in sclerostin antibody-treated control
mice, suggesting that sclerostin per se might be a tonic inhibitor of
neutrophils. Therefore, in OCY-Gs␣KO mice, the decrease in
osteoblasts or the suppression of Wnt-␤catenin signaling in BM is
not sufficient to drive the myeloproliferation. To investigate
whether the myeloid expansion was associated with stem cell
dysregulation, we analyzed HSC and myeloid progenitors both in
BM and spleen of these OCY mice. Interestingly, there was a
significant increase in HSCs in spleens from OCY-Gs␣KO mice,
whereas there was no difference in myeloid progenitors.
Several GPCRs are expressed on osteocytes, including the PPR,
EP2, and EP4 prostaglandin receptors, and many more, as recently
reported.22 Interestingly, ablation of the PPR in osteocytes (OCYPPRKO) did not induce hematopoietic abnormalities, indicating
that other GPCRs might contribute to the myeloproliferation
observed in OCY-Gs␣KO mice. Alternatively, the OCY-Gs␣KO
phenotype might be the result of lack of signaling from multiple
Gs␣-coupled receptors, and single receptors deletion in osteocytes
might not fully recapitulate the phenotype of the OCY-Gs␣KO mice.
In this study, we have developed an in vitro model to coculture
osteoblast- and BM-depleted OEBE with BM cells that allowed us
to explore the relative contribution of osteocytes on the differentiation and function of hematopoietic cells. As shown in Figure 6B,
osteocytes directly affected the number of myeloid cells formed in
this coculture system. Conditioned media from either control or
OCY-Gs␣KO OEBEs induced a significant increase in myeloid
colonies, demonstrating, for the first time, that osteocytes secrete
myeloproliferative factors through Gs␣-dependent and -independent
(CM from control OEBEs) mechanisms. One such factor is G-CSF,
whose expression and secretion are significantly increased in
Gs␣-deficient osteocytes. Osteoblasts have been shown to produce
G-CSF through which they can support hematopoiesis.31 Here we
show that osteocytes also produce G-CSF and that its increased
production by Gs␣-deficient osteocytes partially drives the myeloproliferation, as demonstrated by our neutralizing antibody
experiments.
A previous study found that an increase in TNF-␣ in the BM
microenvironment led to MPS in RAR␥-deficient mice32 and
TNF-␣ promoted granulopoiesis by suppressing the expression of
SDF-1.33 However, TNF-␣ or SDF-1 proteins could not be detected
in conditioned media from either control or OCY-Gs␣KO OEBE.
In conclusion, we have demonstrated that osteocytes can
regulate myelopoiesis through the secretion of several factors,
including G-CSF, via a Gs␣-dependent mechanisms. Using antisclerostin antibodies, we demonstrated that the myeloproliferation
is independent of osteoblasts or the Wnt-␤catenin pathway. Lastly,
phenotypical characterization of the OCY-PPRKO mice revealed
that PPR receptor signaling is not involved in the myeloid
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BLOOD, 7 FEBRUARY 2013 䡠 VOLUME 121, NUMBER 6
OSTEOCYTES AND MYELOPOIESIS
proliferation, suggesting that other Gs␣-dependent receptors might
be involved in osteocytic regulation of hematopoiesis.
Acknowledgments
The authors thank Michaela Kneissel and Novartis for providing
the antisclerostin antibody and David Dombkowski for assistance
with FACS.
This work was supported by the National Institutes of Health
(grants AR060221 and DK079161, P.D.P.; grant K08CA138916,
D.S.K.), the Intramural Research Program of National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health (L.S.W.), and the Harvard Stem Cell Institute (J.Y.W.).
939
Authorship
Contribution: P.D.P., K.F., and D.S.K designed and interpreted the
experiments and wrote the manuscript; K.F., D.S.K., P.D.P., V.S.,
C.P., and L.B. performed experiments; K.J.B. and X.L. provided
technical help; S.L. and R.B. performed histomorphometric analysis; M.C., L.S.W., and J.Q.F. provided material; and J.Y.W.,
H.M.K., and D.T.S. provided scientific input and critically revised
the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Paola Divieti Pajevic, Endocrine Unit, Massachusetts General Hospital, 50 Blossom St, Thier 1101, Boston, MA
02114; e-mail: [email protected].
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2013 121: 930-939
doi:10.1182/blood-2012-06-437160 originally published
online November 16, 2012
Myelopoiesis is regulated by osteocytes through Gsα-dependent
signaling
Keertik Fulzele, Daniela S. Krause, Cristina Panaroni, Vaibhav Saini, Kevin J. Barry, Xiaolong Liu,
Sutada Lotinun, Roland Baron, Lynda Bonewald, Jian Q. Feng, Min Chen, Lee S. Weinstein, Joy Y.
Wu, Henry M. Kronenberg, David T. Scadden and Paola Divieti Pajevic
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