Bone Marrow Oxytocin Mediates the Anabolic Action of Estrogen on the Skeleton

19 Pages·2012·9.15 MB·English

JBC Papers in Press. Published on July 2, 2012 as Manuscript M112.365049
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.365049
Oxytocin is downstream of estrogen action on bone
Bone Marrow Oxytocin Mediates the Anabolic Action of Estrogen on the Skeleton*

Graziana Colaianni1,2*, Li Sun1*, Adriana Di Benedetto1, Roberto Tamma2, Ling-Ling Zhu1,
Jay Cao3, Maria Grano2, Tony Yuen1, Sylvia Colucci2, Concetta Cuscito2, Lucia Mancini2,
Jianhua Li1, Katsuhiko Nishimori4, Itai Bab5, Heon Jin-Lee6, Jameel Iqbal1, W. Scott
Young III7, Clifford Rosen8, Alberta Zallone2, Mone Zaidi1 (*equal contributions)

From The 1Mount Sinai Bone Program, Mount Sinai School of Medicine, New York, USA;
2Department of Human Anatomy and Histology, University of Bari Medical School, Bari, Italy;
3USDA, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand
Forks, ND, USA; 4Graduate School of Agricultural Science, Tohoku University, Aoba-ku,
Sendai, Japan; 5Bone Laboratory, The Hebrew University of Jerusalem, Jerusalem, Israel;
6Department of Oral Microbiology, School of Dentistry, Kyungpook National University, Daegu,
Korea; 7Section on Neural Gene Expression, National Institute of Mental Health, NIH, DHHS,
Bethesda, MD; and 8Maine Medical Research Institute, Scarborough, ME, USA.

*Running title: Oxytocin is downstream of estrogen action on bone

To whom Correspondence should be addressed: Mone Zaidi, The Mount Sinai Bone Program,
Mount Sinai School of Medicine, New York, USA, Tel.: [phone]; Fax: [phone];
E-mail: [email protected]; Alberta Zallone, Department of Human Anatomy and Histology,
University of Bari Medical School, Bari, Italy, Tel.: [phone]; Fax: [phone]; E-
mail: [email protected]

Keywords: Osteoporosis, oxytocin, bone remodeling, estrogen, pituitary gland

Background: The mechanism underlying the kinase JNK, the mechanism of its effect on
anabolic effect of estrogen on the skeleton is the osteoblast remains unclear. Here we
unclear. report that the anabolic action of estrogen
Results: We report that estrogen-induced in mice occurs, at least in part, through
bone formation in mice occurs through oxytocin (OT) produced by osteoblasts in
oxytocin (OT) produced by osteoblasts in bone marrow. We show that the absence of
bone marrow. OTRs in OTR-/- osteoblasts or attenuation
Conclusion: Feed-forward OT release in the of OTR expression in silenced cells both
bone marrow by a rising estrogen level may inhibit estrogen-induced osteoblast
facilitate rapid skeletal recovery after lactation. differentiation, transcription factor up-
Significance: The study highlights a novel regulation, and/or OT production in vitro.
mechanism for estrogen action on bone. In vivo, OTR-/- mice, known to have a bone
formation defect, fail to display increases in
SUMMARY trabecular bone volume, cortical thickness,
Estrogen uses two mechanisms to and bone formation in response to estrogen.
exert its effect on the skeleton: it inhibits Furthermore, osteoblast-specific Col2.3-
bone resorption by osteoclasts and, at Cre+/OTRfl/fl mice, but not TRAP-
higher doses, can stimulate bone formation. Cre+/OTRfl/fl mice, mimic the OTR-/-
While the anti-resorptive action of estrogen phenotype and also fail to respond to
arises from the inhibition of the MAP estrogen. These data attribute the
1

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxytocin is downstream of estrogen action on bone
phenotype of OTR deficiency to an differentiation to a mature mineralizing
osteoblastic, rather than an osteoclastic phenotype (6). Consistent with this anabolic
defect. Physiologically, feed-forward OT action, the genetic deletion of OT or the OTR
release in the bone marrow by a rising in mice decreased osteoblast differentiation
estrogen concentration may facilitate rapid and bone formation, resulting in osteopenia
skeletal recovery during the latter phases of (6). Furthermore, the increased bone
lactation. formation noted normally during pregnancy
was attenuated in OT-/- mice, as was fetal
Oxytocin (OT) is a nanopeptide skeletal mineralization in OT-/- pups (8).
synthesized in the hypothalamus and released Together, these findings suggest that the
into circulation by the posterior pituitary. Its anabolic action of OT may, in fact, facilitate
primary function is to mediate the milk maternal skeletal recovery and fetal
ejection reflex in nursing mammals (1). It skeletogenesis.
also augments uterine contraction during OT is known to be produced in
parturition (2). Serum OT levels peak during peripheral tissues, such as the testes, ovary,
late pregnancy and are maintained during heart, lung, and vascular tissue (9-14). In
lactation. These periods correspond, certain of these organs, estrogen regulates OT
respectively, to maximal fetal and post-natal and/or OTR expression (15, 16). Likewise,
bone growth, which in turn leads to maternal we find that OT is produced in abundance by
losses from the skeleton of ~120g of calcium bone marrow osteoblasts in response to
(3, 4). Hormonal adaptations, including low estrogen, with the potential of exerting an
estrogen and persistently elevated PTHrP anabolic effect on the skeleton (17). Estrogen
levels, as well as increased sympathetic tone at high doses is a skeletal anabolic (18), but its
all lead to a state of maternal hyper-resorption precise mechanism remains unknown. Here,
and intergenerational calcium transfer (3-5). we show that the osteoblastic OTR is required
However, the profound bone loss of lactation for the full anabolic action of estrogen. We
recovers fully within a relatively short period postulate that an autocrine feed-forward
even as estrogen levels are slowly returning to OT/OTR loop exists in which estrogen
normal (5). The mechanism(s) responsible for releases OT from osteoblasts, which then acts
this dramatic anabolic skeletal recovery upon osteoblastic OTRs to further amplify
remain poorly understood. estrogen action. This could be particularly
To determine whether OT mediates advantageous for maternal skeletal
any or all of these functions during pregnancy replenishment during the post-partum period,
and/or lactation, we studied whether OT when estrogen levels are returning to normal
directly affects the skeleton. While and OT is high (19).
intracerebroventricular OT did not affect bone
remodeling, peripheral administration did EXPERIMENTAL PROCEDURES
affect bone remodeling and bone mass (6). Mouse Models − OTR-/- mice,
However, instead of recapitulating the originally created on a C57BL/6Jx129Sv
elevated resorption and bone loss that occurs background6, have been back-crossed with
during pregnancy and lactation, peripherally C57BL/6J mice for >15 generations. OTRfl/fl
injected OT markedly increased bone mass in mice were developed as described previously
both wild typeand ovariectomized mice (6, 7). on a C57BL/6J background (33); the mice

In line with its peripheral effect, we found that have subsequently been backcrossed further to
osteoblasts possess abundant OT receptors C57BL/6J mice >10 generations. All mice
(OTRs) and that OT triggers osteoblast were group-housed (5 mice per cage) upon
2
Oxytocin is downstream of estrogen action on bone
weaning at ~3 weeks of age. Col2.3-Cre and FCS in the presence of 50 µg/ml ascorbic acid
TRAP-Cre mice were generous gifts from Drs. and 10 mM β-glycerophosphate (Sigma) for
Simon Mendez-Ferera (Mount Sinai) and mRNA and protein extraction, and Von Kossa
David Roodman (University of Pittsburgh), staining. MC3T3-E1 cells were kindly
respectively. The two Cre transgenic lines provided by Dr. Anna Teti (University of
were crossed initially with OTRfl/fl mice, L’Aquila, Italy). These cells were transfected
following which the Cre+/OTRfl/+ genotypes with OTR siRNA or vector-only duplexes (50
were crossed with OTRfl/fl mice to yield the nM) using lipofectamine 2000 (Invitrogen).
respective osteoblast- and osteoclast-specific Western blotting and qPCR were performed as
Cre+/OTRfl/fl mice. Absence of the OTR was described (6).
confirmed by PCR and Western blotting in It is difficult to determine the sex of
isolated cells, as before (34). the mice before 2 to 3 weeks. However, we
Skeletal Phenotyping − Protocols utilized 5 day-old pups to isolate calvarial
approved by Mount Sinai School of osteoblasts for PCR and Western blotting,
Medicine’s IACUC and University of Bari’s rather than bone marrow-derived osteoblasts
Animal Welfare Committee were used for because of the possible contamination of these
injecting, bleeding, ovariectomizing, and cultures. Older mice cannot be used for
sacrificing female mice. Areal BMD was calvarial osteoblast cultures due to difficulties
measured at various sites representing in getting highly pure osteoblast fractions.
trabecular and cortical bone (Lunar Piximus, We also show that estrogen induces robust OT
GE, Wauwatosa, WI). Micro-CT was and OTR expression in male osteoblasts
performed on trabecular bone (µCT40, (Supplementary Figure 1); the time course of
Scanco). For histomorphometry, groups of the effect is concordant with that seen in
female mice received either calcein (15 mg/kg, female cells (17).
i.p.) 10 and 2 days prior to sacrifice, or for
specific experiments, calcein and xylenol RESULTS
orange (90 mg/kg, i.p.) 6 and 1 days prior to Estrogen is known to exert its anabolic
sacrifice, respectively. Standard protocols action in rodents by enhancing osteoblast
were utilized for sectioning the spine and differentiation, although an effect on apoptosis
femur and for dynamic and static has also been described (20). To study if OT
histomorphometry (24, 35). In separate mediates estrogen effects on
experiments, female mice were injected either osteoblastogenesis, we questioned whether
with a single dose or biweekly with 50 µg/kg OTR-/- calvarial osteoblasts were less sensitive
of 17β-estradiol or 0.9% w/v NaCl (vehicle) to estrogen-induced differentiation. We found
i.p. (cumulative weekly dose 100 µg/kg). In that 17β-estradiol increased mineralizing
certain instances, blood ( 1 ml) was collected osteoblast colony (CFU-Ob) formation in wild
by intracardiac puncture. Osteocalcin and OT type bone marrow stromal cell cultures, but
were measured using a mouse-based not in cultures from OTR-/- mice (Figure 1A).
Osteocalcin (DRG Inc., NY) and OT (Enzo Likewise, 17β-estradiol-induced expression of
Inc., PA) ELISA kits, respectively. the differentiation gene osteocalcin in wild
Cell Experiments − Osteoblasts were type osteoblasts, albeit modest, was abolished
isolated from OTR-/- and wild type female in OTR-/- cells (Figure 1Ba). These data show
mouse bone marrow-adherent fraction or from that the augmentation of osteoblast
calvaria of 5 day-old pups (gender unknown). differentiation by estrogen requires OTRs.
Cells were cultured in phenol-red-free α- To understand why estradiol failed to
MEM (Gibco) with 10% charcoal-stripped increase the differentiation of OTR-/-
3
Oxytocin is downstream of estrogen action on bone
osteoblasts, we studied the expression of the Our previous data showing that 17β-
critical transcription factors Runx2, Osterix, estradiol induces OT and OTR expression in
and ATF4 in cultures of calvarial osteoblasts osteoblasts (17) prompted us to further

isolated from 5 day-old male and female pups. determine whether the effects of estrogen on
Western blotting revealed that 17β-estradiol osteoblast OT expression were dependent on
significantly increased Runx2 and ATF4 OTRs. We found that OT mRNA expression
protein expression in wild type cells, whereas in response to 17β-estradiol was abrogated in
these increases were blunted in OTR-/- cells up OTR-/- (female) and OTR-silenced MC3T3.E1
to 12 hours (Figure 1C). qPCR confirmed the osteoblasts (Figure 2C), suggesting that OTRs
up-regulation of ATF4 mRNA (Figure 1Bb). are required for estrogen-induced OT
Likewise, 17β-estradiol modestly but production. In line with this, OTR-/- mice
significantly increased Osterix mRNA at both failed to show an increase in serum OT levels
6 and 12 hours in wild type osteoblasts, but in response to 17β-estradiol (Figure 2D).
not in OTR-/- cells (Figure 1Bc). We did not Knowing that OTRs are required for estrogen-
observe parallel changes in Osterix protein induced OT expression, we further
with estrogen. However, levels remained hypothesized that OT acts on its receptor to
lower in OTR-/- cells for up to 12 hours after induce its own production. We found using
17β-estradiol treatment (Figure 1C). qPCR that OT did induce OT mRNA in
To confirm these ex vivo findings from MC3T3.E1osteoblasts at 6 hours (Figure 2E).
primary bone marrow and calvarial osteoblast This putative autocrine stimulation was
cultures, we used siRNA to knock down OTR abolished by the OTR antagonist atosiban
expression in a pre-osteoblastic cell line, (Figure 2E), establishing that the OT-induced
MC3T3.E1. Western blotting and qPCR OT expression required OTR activation.
showed a dramatic reduction in OTR protein Data from this and our previous study
and mRNA, respectively, in OTR-silenced (17) support a hypothesis whereby estrogen
cells (Figure 2A). Whereas 17β-estradiol induces the expression of both OT and OTR.
significantly increased Runx2, Osterix, ATF4 The induced OT then acts upon the OTR in an
and osteocalcin mRNA in vector-treated cells, autocrine loop to amplify the anabolic effects
this effect was profoundly attenuated in OTR- on estrogen. To test this hypothesis in vivo,
silenced cells (Figure 2B). The data suggest we treated two month-old female OTR-/- mice
that the pro-osteoblastic actions of 17β- or wild type littermates with 17β-estradiol (50
estradiol require OTR expression, and occur µg/kg) or vehicle, biweekly, for 30 days
mainly through the up-regulation of Runx2 (cumulative weekly dose of 100 µg/kg). This
and ATF4, although other pathways may also is consistent with the cumulative weekly dose
be involved. of between 70 and 280 µg/week (10 to 40
As is evident, all genes are not equally µg/kg/day) used by others (21, 22).
responsive to estrogen. Furthermore, estrogen Histomorphometry showed lower trabecular
responsiveness can vary depending on BV/TV, Tb.Th, MAR and BFR in placebo-
osteoblast source, differentiation state, and treated OTR-/- mice compared with placebo-
culture conditions. However, it is important to treated wild type littermates, confirming
note that the results from bone marrow previous results(6) (Figure 3). 17β-estradiol

osteoblast cultures showed overall significantly increased trabecular and cortical
concordance with results from calvarial bone parameters, importantly BV/TV, Tb.N,
osteoblast cultures in terms of the lack of MAR, BFR, Tb.N.Ob/B.Pm, Ct.Th and
responsiveness of OTR-/- cells to 17β-estradiol. Ct.N.Ob/B.Pm (periosteal), in wild type mice,
but not in OTR-/- mice (Table 1). Interestingly,
4
Oxytocin is downstream of estrogen action on bone
Tb.N, MAR and Ct.Th further declined in resulted in a significant reduction in BMD and
OTR-/- mice despite 17β-estradiol treatment. BV/TV, with a trend towards reduced Tb.N
Osteoclast parameters in trabecular and and increased Tb.Sp (Figures 5A to 5C). That
cortical bone were not significantly different the phenotype of the osteoblast-specific
between wild type and OTR-/- mice, consistent Col2.3-Cre+/OTRfl/fl mouse recapitulated, in
with previous results (6), and did not full, the global OTR-/- phenotype suggests that
significantly change in either group upon 17β- an osteoblast defect is the predominant, if not
estradiol treatment (Table 1). Concordant the sole, determinant of the osteopenia of
with a bone formation defect, while 17β- OTR deficiency. Figure 5D shows that
estradiol elevated serum osteocalcin, a bone osteoclast-selective, TRAP-Cre+/OTRfl/fl mice
formation marker in OTR-/- and wild type did not display osteopenia at either trabecular
mice, the response was significantly (p < 0.01) or cortical sites, confirming that OTR
attenuated in OTR-/- mice (Figure 3D). In sum, deficiency in the osteoclast does not alter bone
the overall failure of OTR-/- mice to display a mass6. These mice were therefore not further
full anabolic response to 17β-estradiol phenotyped using micro-CT or
suggests that the bone-forming action of histomorphometry. Reduced bone formation
estrogen is, at least in large part, OTR- was however confirmed in Col2.3-
dependent. Cre+/OTRfl/fl mice by dynamic
We next tested whether OT signalling histomorphometry. Namely, both MAR and
also played a role in mediating the effect of BFR were reduced significantly, expectedly
estrogen on bone mass in hypogonadal mice. without effects on MS (mineralizing surface)
For this, mice were ovariectomized or sham or resorbed surface (Figures 6A, 6B and 6C).
operated and given either 17β-estradiol or We used 16-week-old Col2.3-
vehicle, as above. Areal bone mineral density Cre+/OTRfl/fl mice to determine whether
measurements 30 days after ovariectomy osteoblast, rather than osteoclast OTRs
revealed significant decrements in wild type mediate the skeletal anabolic effect of 17β-
and OTR-/- mice at the lumbar spine and femur estradiol. Serial BMD measurements revealed
(Figure 4A and 4B). However, when that while 17β-estradiol expectedly increased
compared to zero-dose controls, both sham- BMD over 3 weeks of treatment in Col2.3-
operated and wild type mice treated with 17β- Cre-/OTRfl/fl (control) mice, it did not do so in
estradiol showed increases in BMD, whereas Col2.3-Cre+/OTRfl/fl mice (Figure 6D). At the
the respective OTR-/- mice did not (Figure 4C 3 week time point, there was a significant
and 4D). This suggested that, as in the BMD difference between 17β-estradiol-
eugonadal state, an intact OTR axis treated Col2.3-Cre+/OTRfl/fl and control mice
contributes to the action of estrogen in (Figure 6D). That the anabolic effect of 17β-
hypogonadism. estradiol on bone mass was abolished in mice
We sought to determine definitively selectively lacking OTRs in osteoblasts,
whether the effect of 17β-estradiol in suggests that neither the osteoclast nor the
increasing bone mass was osteoblast-mediated nervous system contributes to the effect, at
or whether there was an osteoclastic least, in large part.
component. For this, we generated osteoblast-
or osteoclast-specific OTR-deficient mice by DISCUSSION
crossing OTRfl/fl mice with Col2.3-Cre or We hypothesize that estrogen acts to
TRAP-Cre mice, respectively (see ‘Methods’). enhance osteoblast differentiation and bone
The absence of OTR in osteoblasts, but not in formation, at least, in part, by stimulating the
osteoclasts, in Col2.3-Cre+/OTRfl/fl mice production of OT, which then acts on its own
5
Oxytocin is downstream of estrogen action on bone
receptor to trigger further OT release. This, OT circuit in which locally produced OT
we hypothesize, constitutes a local autocrine activates its own receptor. This role appears
circuit that amplifies bone formation triggered consistent with the historic function of OT in
by estrogen. mineral homeostasis observed in lower
Our hypothesis is based upon several mammals (9, 28).
observations. First, there is evidence from Second, we have injected 17β-estradiol
others (7) and us (6) that OT enhances at a dose of 50 µg/kg, biweekly, which
osteoblastogenesis, bone formation, and/or corresponds to a cumulative dose of 100
bone mass through a peripheral action on µg/kg per week. This is consistent with doses
osteoblast OTRs. In this respect, OT mimics between 10 and 40 µg/kg/day (or cumulative
the estrogen, which, at higher-than-anti- weekly doses of 70 to 280 µg/kg) used by
resorptive doses, stimulates osteoblastogenesis others to prevent bone loss due to aging or
with some effects on osteoblast apoptosis (20). ovariectomy (21, 22). Ovariectomy is
Second, this study provides in vitro evidence associated with increased resorption and
that OT is downstream of estrogen. Thus, the formation, at least in the acute setting, and the
deficiency of OTRs in OTR-/- osteoblasts or effects of estrogen in restoring bone mass
OTR-silenced cells markedly reduces the arise mainly from its anti-osteoclastic action
effect of 17β-estradiol on mineralized colony (29) in a hyper-resorptive state. Here, we
formation, as well as the expression of show that the effects of 17β-estradiol on bone
osteoblast certain differentiation genes and mass in ovariectomized mice are also
transcription factors. Third, we have shown dependent on an intact OTR axis. However,
that OT is produced by osteoblasts and that in order to obviate confounding effects of
17β-estradiol stimulates OT expression within elevated resorption and its inhibition by
2 hours (17). 17β-estradiol also stimulates estrogen, and short of using chronically
OTR expression, but with a slower time estrogen-deprived rodents (30), we have
course (17). Here, we show that estrogen instead used wild type mice with normal bone
action on OT production is dependent upon resorption and bone formation. As has been
OTR expression; the absence of OTRs in noted by others (21) in non-ovariectomized
OTR-/- osteoblasts or OTR down-regulation in settings, we find that 17β-estradiol does not
silenced cells thus abolishes estrogen-induced affect osteoclast parameters, whether or not
OT expression. Finally, the effects of 17β- the mice are devoid of OTRs. In contrast,
estradiol on bone mass and bone formation are osteoblast numbers, bone formation, and
abolished/attenuated in OTR-/- mice, and cortical and trabecular bone mass were all
importantly, in osteoblast-specific Col2.3- enhanced significantly with 17β-estradiol in
Cre+/OTRfl/fl mice. In both models, no wild type mice. This anabolic action was
appreciable effects on osteoclast (resorption) attenuated in OTR-/- mice, suggesting that the
parameters were noted. effects of estrogen on bone mass were, at least
Several implications arise from these in part, dependent on OTRs.
studies. First, is the recurring notion that not Third, our study definitively rules out
only do primitive pituitary hormones, such as mediation of OT effects on the skeleton
TSH, FSH and ACTH, have receptors in through the osteoclast or the central nervous
extra-endocrine targets, such as bone (23-25), system. We have shown previously that
but also that certain of these ligands, including intracerebroventricular OT does not affect
TSHβ, ACTH and OT, themselves can be certain acute bone formation parameters (6),
expressed in a host of tissues (17, 26, 27). We despite the known presence of OT-ergic
suggest that estrogen stimulates an autocrine neurones. We now show that the osteopenia
6
Oxytocin is downstream of estrogen action on bone
of global OTR deficiency in OTR-/- mice is expression locally and/or systemically, and a
mimicked in its entirety by osteoblastic OTR reduction in osteocyte osteolysis (31).
deficiency in Col2.3-Cre-/OTRfl/fl mice. However, skeletal recovery is relatively quick
Furthermore, Col2.3-Cre-/OTRfl/fl mice fail to suggesting that estrogen restoration alone
display increases in bone mass in response to cannot be the sole explanation. Similarly,
17β-estradiol. This confirms that not only is although skeletal PTHrP is up-regulated with
the osteoblastic OTR critical for skeletal weaning, it is not essential for bone mass
homeostasis, but that it mediates selectively recovery (4). On the other hand, in the post-
and solely the anabolic action of estrogen on partum period during lactation, as estrogen
the mouse skeleton. production gradually returns, OT levels
Overall, our findings may have remain elevated, probably due to chronic
implications both biologically and clinically. suckling. A feed-forward autocrine network
The parallel actions of estrogen and OT on in the marrow niche, whereby rising estrogen
bone formation, and particularly a feed- levels enhance local OT production ultimately
forward loop for estrogen-mediated OT leading to greater bone formation through the
release from the osteoblast, may be of OTR, appears to be a plausible additional
significance. For example, OT levels are high mechanism that could explain the “anabolic”
late in pregnancy, during parturition, and post- phase of skeletal recovery post-lactation.
partum (19). These time periods also Finally, the repletion of osteoblast OT
correspond to a period of relentless pressure signaling may represent a non-estrogenic
on the maternal skeleton for bio-available means of restoring bone in those forms of
calcium, initially to support prenatal fetal bone loss that are characterized mainly by
mineralization, and subsequently, for post- osteoblast dysfunction (32). Indeed, human
natal nutrition from lactation (3, 4). Not OT is in use for inducing labor, and it is not
surprisingly, significant cortical and trabecular inconceivable that bone-specific OT analogs
bone loss are a consequence of lactation, as may spare the reproductive organs. Finally,
well as suppressed estrogen concentrations (4). and more broadly, future studies are needed to
However, with the onset of menses, and evaluate if the feed-forward OT loop, as well
immediately post-weaning, there is a rapid as short loops of other pituitary hormones,
restoration of bone mass. This “anabolic” operate in tissues other than the skeleton and
phase is likely to be multifactorial in origin, traditional endocrine targets.
and has been attributed to return of pre-
pregnancy estrogen levels, a change in PTHrP

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FOOTNOTES

*This study was supported by grants to MZ from the National Institute on Aging (AG023176
and AG040132); a grant to MZ and LS from the National Institute of Diabetes, Digestive and
Kidney Diseases (DK080459), and grants to AZ from Italian Space Agency (Osteoporosis and
Muscular Atrophy Project), European Space Agency (European Research in Space and
Terrestrial Osteoporosis Microgravity Application Promotion Project), and the Ministero
dell’istruzione, dell’Universita della Ricerca. HJ-L and WSY were supported by the National
Institutes of Mental Health Intramural Research Program (Z01-MH-002498-23). JC was
suppoted by the USDA ARS CRIS program 5450-51000-046-00D. J.I. acknowledges the
support of the American Federation for Aging Research.
1To whom Correspondence should be arressed: Mone Zaidi, The Mount Sinai Bone Program,
Mount Sinai School of Medicine, New York, USA, Tel.: [phone]; Fax: [phone];
E-mail: [email protected]; Alberta Zallone, 2Department of Human Anatomy and
Histology, University of Bari Medical School, Bari, Italy, Tel.: [phone]; Fax: 39 080
5478308; E-mail: [email protected]

FIGURE LEGENDS

TABLE 1. Static and dynamic estimates from bone histomorphometry, namely bone
volume/trabecular volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th),
trabecular spacing (Tb.Sp), mineral apposition rate (MAR), bone formation rate (BFR),
trabecular osteoblast numbers (Tb.N.Ob/B.Pm), trabecular osteoclast numbers (Tb.N.Oc/B.Pm),
cortical thickness (Ct.Th), cortical osteoblast numbers (Ct.N.Ob/B.Pm), and cortical osteoclast
numbers (Ct.N.Oc/B.Pm) (units as shown). Results are represented as mean + SEM; n = 3-10.
Statistics: Students t-test with Bonferroni correction, comparing E treatment versus 0-dose, as
2
well as wild type versus OTR-/- mice (p values as shown).

FIGURE 1. Estrogen-induced osteoblastogenesis is attenuated in OTR-/- cells. (A)
Representative pictures (a) and colony counts (b) of ex vivo Von Kossa-positive mineralizing
colonies, CFU-ob, arising from wild type (OTR+/+) or OTR-/- bone marrow stromal cells with
either 17β-estradiol (10-8 M) or vehicle for 21 days. (B) Quantitative PCR (qPCR) for
osteocalcin (OCN) gene expression in OTR+/+ and OTR-/- osteoblasts for 0, 6, and 12 hours;
expression was normalized to GAPDH and plotted as fold-increase from the 0-hour wild type
sample. (C) OTR+/+ and OTR-/- calvarial osteoblasts were evaluated for changes in their
expression of the osteoblast transcription factors ATF4, Runx2, and Osterix by Western blotting
after treatment with 17β-estradiol (10-8 M) for 6, 12, and 24 hours (a). The band density was
quantitated corrected for the loading control β-actin (b). (D) qPCR of wild type (red) and OTR-/-
10