Moreover, the higher ami-dated neuropeptide levels in TTR KO mice were related to increased bone mineral density and trabecular volume.. In summary, this work contributes to a better und
Trang 1differentiation – insights from transthyretin knockout
mice
Ana F Nunes1,2,*, Ma´rcia A Liz1, Filipa Franquinho1, Liliana Teixeira3, Vera Sousa1, Chantal
Chenu4, Meriem Lamghari3, and Mo´nica M Sousa1,
1 Nerve Regeneration, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
2 ICBAS, Universidade do Porto, Portugal
3 INEB – Instituto de Engenharia Biome´dica, Divisa˜o de Biomateriais, Universidade do Porto, Portugal
4 Department of Veterinary Basic Sciences, The Royal Veterinary College, London, UK
Introduction
The regulation of bone remodeling has been
conven-tionally linked to local factors, hormones, and
mechanical loading [1–3] However, in the last decade,
several reports have provided evidence that bone
homeostasis is also under the influence of central and peripheral neural control [4–8] This concept is sup-ported by a number of histological studies revealing the existence of neuropeptide fibers and neuropeptide
Keywords
amidated neuropeptide; bone marrow
stromal cells; bone mass; NPY; osteoblastic
differentiation
Correspondence
M M Sousa, IBMC, Rua Campo Alegre
823, 4150-180 Porto, Portugal
Fax: +351 22 6099157
Tel: +351 22 6074900
E-mail: msousa@ibmc.up.pt
Website: http://www.ibmc.up.pt/nerve
*Present address
iMed.UL, Faculty of Pharmacy, University of
Lisbon, Portugal
These authors contributed equally to this
work
(Received 12 November 2009, revised
3 November 2009, accepted 5
November 2009)
doi:10.1111/j.1742-4658.2009.07482.x
To better understand the role of neuropeptide Y (NPY) in bone homeosta-sis, as its function in the regulation of bone mass is unclear, we assessed its expression in this tissue By immunohistochemistry, we demonstrated, both at embryonic stages and in the adult, that NPY is synthesized by osteoblasts, osteocytes, and chondrocytes Moreover, peptidylglycine a-am-idating monooxygenase, the enzyme responsible for NPY activation by amidation, was also expressed in these cell types Using transthyretin (TTR) KO mice as a model of augmented NPY levels, we showed that this strain has increased NPY content in the bone, further validating the expression of this neuropeptide by bone cells Moreover, the higher ami-dated neuropeptide levels in TTR KO mice were related to increased bone mineral density and trabecular volume Additionally, RT-PCR analysis established that NPY is not only expressed in MC3T3-E1 osteoblastic cells and bone marrow stromal cells (BMSCs), but is also detectable by RIA in BMSCs undergoing osteoblastic differentiation In agreement with our
in vivoobservations, in vitro, TTR KO BMSCs differentiated in osteoblasts had increased NPY levels and exhibited enhanced competence in undergo-ing osteoblastic differentiation In summary, this work contributes to a better understanding of the role of NPY in the regulation of bone forma-tion by showing that this neuropeptide is expressed in bone cells and that increased amidated neuropeptide content is related to increased bone mass
Abbreviations
ALP, alkaline phosphatase; BMD, bone mineral density; BMSC, bone marrow stromal cell; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; KO, knockout; microCT, micro computed tomography; NF200, neurofilament 200; NPY, neuropeptide Y; PAM, peptidylglycine a-amidating monooxygenase; PGP9.5, protein gene product 9.5; RANK, receptor activator of nuclear factor-jB; T4,thyroxine; TTR, transthyretin.
Trang 2receptors in bone [9] Neuropeptide Y
(NPY)-immuno-reactive fibers have been found to be mostly
distri-buted in association with blood vessels and in the
periosteum [10–12] NPY immunoreactivity was
dra-matically reduced in sympathectomized animals,
indi-cating the sympathetic origin of these nerves [11]
Despite the fact that NPY-containing nerve fibers have
been described in the bone, no data exist concerning
the expression of this neuropeptide in bone cells
How-ever, NPY has been detected in the periosteum and
bone marrow by RIA [13], particularly in
megakaryo-cytes [14] Recently, it was additionally reported that
the NPY receptor Y1, but not Y2, Y4, Y5 or Y6, was
expressed in cultured bone marrow stromal cells
(BMSCs) and osteoblasts [15]
Despite the existence of NPY fibers and one of its
receptors in the bone, NPY knockouts (KOs) have
normal bone mass, questioning a role for NPY control
in bone activity [8] On the other hand, different
mouse models that have in common the fact that they
present increased NPY levels, the Y2 receptor-KO and
the leptin-deficient and leptin receptor-deficient mouse
(ob⁄ ob and db ⁄ db mice, respectively), display a high
cancellous bone mass phenotype associated with
increased osteoblast activity [5,7,16], supporting a role
for NPY in bone biology In the case of ob⁄ ob mice
and db⁄ db mice, there is increased NPY activity in the
hypothalamus, owing to the lack of leptin-induced
inhibition of NPY expression [16] Y2 receptor KO
and leptin-deficient mice share key characteristics, with
similar increases in cancellous bone mass and NPY
levels in the hypothalamus, suggesting a commonality
of mechanism However, it was recently shown that
leptin and Y2 receptor pathways independently
modu-late cancellous bone homeostasis [17] With regard to
Y2 receptor-deficient mice, both germline and
condi-tional hypothalamic Y2 receptor KO mice share the
same high bone mass phenotype [5], demonstrating
that central hypothalamic Y2 receptors are crucial for
this process Interestingly, although germline Y1
recep-tor KO mice also display increased bone formation,
conditional deletion of hypothalamic Y1 receptors did
not alter bone homeostasis, suggesting a
nonhypotha-lamic control of bone mass [6] The Y1 receptor being
the only NPY receptor identified in the bone, these
results suggest that absence of NPY signaling in the
bone (as occurs in Y1 receptor-deficient mice) results
in increased bone mass
NPY effects in bone mass have been further
inves-tigated by exogenous administration Whereas
intra-cerebroventricular infusion of NPY decreased bone
mass [7], vector-mediated overexpression of NPY in
the hypothalamus of wild-type mice resulted in no
alteration in cancellous bone volume, although osteo-blast activity, estimated using osteoid width, was markedly reduced following adeno-associated virus NPY injection [17,18] These results are not in accor-dance with the cancellous bone phenotype of the above-mentioned mouse models of elevated NPY lev-els All of these opposing results make necessary a closer look at the role of NPY in the regulation of bone mass
Transthyretin (TTR) KO mice show increased levels
of amidated neuropeptides, owing to overexpression of peptidylglycine a-amidating monooxygenase (PAM) [19], the only enzyme that a-amidates peptides, and which is rate-limiting in the process of neuropeptide maturation, as its substrates exist in excess [20,21] Among the neuropeptides that are amidated by PAM, NPY is the most abundant in both the central and the peripheral nervous systems As NPY requires PAM-mediated a-amidation for biological activity [22], PAM overexpression in TTR KO mice results in increased levels of processed amidated NPY, without an increase
in NPY expression [19] As a consequence of the increased amidated NPY levels, TTR KO mice show a significant NPY overexpressor phenotype
Given the lack of information on the expression of NPY in the bone, together with the controversy con-cerning its function in bone homeostasis, we aimed at gaining a better understanding of the role of this neu-ropeptide in the control of bone mass by making use
of TTR KO mice, a model of increased NPY
Results
In bone, NPY is detected in chondrocytes, osteoblasts, and osteocytes
NPY expression was investigated in wild-type (WT) and TTR KO bone tissue by immunohistochemistry, using an antibody specific for the amidated form of NPY NPY immunolabeling was observed in bone marrow cells, including megakaryocytes (Fig 1Aa), as already described in the literature [14] The periosteum (Fig 1Ab) also showed NPY immunoreactivity, as already reported for mice and rats [10–12] However,
we observed NPY immunostaining in chondrocytes, osteoblasts, and osteocytes (Fig 1Ac–f, respectively, arrows) No NPY immunoreactivity was found in osteoclasts (data not shown) Similar to our observa-tions in the adult bone, NPY immunoreactivity was detected starting at embryonic day 16 in megakaryo-cytes, osteoblasts, and chondrocytes; this NPY detec-tion pattern was maintained at embryonic day 18 (Fig 1B) No immunoreactivity was detected when the
Trang 3A F
C
B
D
E
d
b
a
c
a
b
c
d
e
BM
C
O
C
C C
BM
BM
Anti-osteocalcin Anti-NPY
P M
Os
Os
O
O
O
AC
BM
M
Fig 1 NPY immunohistochemistry in the bone tissue BM, bone marrow; C, chondrocytes; O, osteoblasts; AC, articular chondrocytes; Os, osteocytes; M, megakaryocytes; P, periosteum Scale bar: 50 lm (A) NPY immunoreactivity in bone cells, namely bone marrow cells and megakaryocytes (a), periosteum (b), articular cartilage chondrocytes (c), late proliferating chondrocytes (d), osteoblasts (e), and osteocytes (f) Arrows indicate labeled cells, and fibers in the case of the periosteum (B) NPY immunoreactivity in the bone at embryonic day 18, show-ing NPY stainshow-ing in megakaryocytes (a), chondrocytes (b), and osteoblasts (c) (C) Immunohistochemistry in bone sections where the anti-body against NPY was replaced by mouse IgG (D) NPY immunohistochemistry in NPY KO bone sections (E) Comparison between NPY (right) and osteocalcin (left) immunolabeling in the bone tissue Arrows indicate labeled osteoblasts (F) Nerve fiber (NF200 and PGP9.5) immunohistochemistry: articular cartilage chondrocytes (a), proliferating chondrocytes (b), osteoblasts (c), bone marrow cells (d), and perios-teum (e).
Trang 4NPY antibody was replaced by mouse IgGs (Fig 1C).
Moreover, in NPY KO mouse bone sections, none of
the different bone cells showed NPY immunostaining
(Fig 1D), suggesting that the immunoreactivity
observed in WT and TTR KO bone tissue was
NPY-specific To further demonstrate NPY synthesis in
os-teoblasts, osteoblast-specific staining was performed
with an antibody against osteocalcin (Fig 1E, left
panel, arrow) The results obtained revealed that the
pattern of staining was comparable to that obtained
for NPY, as shown in the right panel of Fig 1E, thus
confirming NPY expression in osteoblasts To further
demonstrate that NPY is synthesized in these bone
cells, additional negative controls were performed
Using antibody against neurofilament 200 (NF200) or
antibody against protein gene product 9.5 (PGP9.5),
two nerve fiber markers, no staining was observed in
chondrocytes, osteoblasts, or bone marrow cells
(Fig 1Fa–d, respectively), whereas in the periosteum
typical nerve fiber labeling was detected (Fig 1Fe)
TTR KO bone tissue has increased amidated NPY levels
From the comparison between WT and TTR KO NPY immunoreactivity in bone sections, we observed that TTR KO bone tissue displayed increased amidated NPY levels when compared to the wild type (Fig 2A, arrows), further demonstrating the expression of this neuropep-tide by bone cells NPY immunostaining was increased
in chondrocytes, osteoblasts, osteocytes, bone marrow cells and megakaryocytes (Fig 2a–e, respectively) from TTR KO mice when compared to the same WT cells This result is in accordance with the increased NPY lev-els reported in the nervous system of TTR KOs [19], sug-gesting that the increased NPY levels in this strain are not nervous system-restricted Given the increased NPY levels in TTR KO bones, PAM expression was subse-quently evaluated in this tissue by immunohistochemis-try; PAM was detected in bone marrow cells, including megakaryocytes (Fig 2Ba, arrows), osteoblasts, and
A
a
a
b
c
M BM
d
e
B
C
Fig 2 Analysis of NPY and PAM in WT and TTR KO bone sections Scale bar: 50 lm (A) Comparison of NPY immunostaining in
WT and TTR KO bone sections Arrows indicate the different cell types in evidence
in each panel, namely articular cartilage chondrocytes (a), proliferating chondrocytes (b), osteoblasts (c), osteocytes (d), bone marrow cells (BM) and megakaryocytes (M) (e) (B) PAM immunostaining in the bone marrow (a; arrows indicate megakaryo-cytes), osteocytes (b; arrows), osteoblasts (b;p arrowheads), and chondrocytes (c) (C) Quantification of the density of PAM immunostaining in the bone marrow of WT and TTR KO mice a P < 0.05.
Trang 5osteocytes (Fig 2Bb, arrowheads and arrows,
respec-tively), as well as in chondrocytes (Fig 2Bc) The major
difference in PAM expression among WT and TTR KO
bones was found in the bone marrow, where PAM
immunostaining was approximately two-fold higher in
TTR KO mice (Fig 2C) Despite the fact that NPY and
PAM expression were not observed in osteoclasts, the
hypothesis that increased NPY levels in the bone of
TTR KO mice may have an indirect effect on osteoclasts
existed To address this hypothesis, preosteoclasts and
mature osteoclasts in WT and TTR KO bones were
detected by OSCAR staining Following quantification,
no differences in osteoclast number were detected
between strains (data not shown)
TTR KO mice have increased bone mineral
density (BMD) and trabecular volume
To address whether the increased NPY levels observed
in TTR KO femurs have physiological consequences in
the bone, we started by comparing bone histology in
WT and TTR KO mice The femur length did not
dif-fer significantly between strains (wild type,
15.6 ± 1.4 mm; TTR KO, 15.9 ± 1.0 mm) To
fur-ther analyze in detail the bone phenotype, micro
com-puted tomography (microCT) scanning analysis of
femurs, including measurement of BMD, was
per-formed As shown in Fig 3A (left and middle panels),
two-dimensional trabecular number and thickness were
increased in TTR KO femurs when compared with
WT femurs Furthermore, three-dimensional trabecular
bone volume in the proximal metaphysis was also
higher in TTR KO animals (Fig 3A, right panel)
From the statistical analysis of WT (n = 9) and TTR
KO (n = 10) femurs, the results obtained demonstrate
an increased trabecular volume (bone volume⁄
trabecu-lar volume) and BMD in TTR KO mice when
com-pared with WT littermates (Fig 3B) These results
suggest that increased amidated neuropeptide levels are
related to increased bone density and volume The
increase in bone volume was, however, detected only
in trabeculae, whereas the bone cortex was unaffected
This result suggested that the process of endochondral
ossification might be specifically affected To assess
this hypothesis, the growth plates of WT and TTR
KO mice were analyzed As can be seen in Fig 3C, no
differences were detectable by histological analysis of
growth plates from WT and TTR KO mice
NPY is expressed in osteoblasts
To further address NPY expression in bone cells,
namely in the osteoblastic cell line MC3T3-E1, and in
primary cultures of BMSCs throughout osteoblastic differentiation, we performed RT-PCR analysis of NPY expression Using brain as the positive control of NPY expression, we detected NPY in MC3T3-E1 cells and in both WT and TTR KO BMSCs (Fig 4A) Fur-thermore, both WT and TTR KO BMSCs on days 3,
7 and 14 of culture in osteogenic differentiation media showed NPY expression; no statistical differences were observed between WT and TTR KO BMSC cultures throughout the differentiation period (data not shown) To determine whether TTR KO mice BMSCs undergoing osteoblastic differentiation recapitulate our findings in the nervous system, i.e show increased PAM transcription and increased levels of amidated NPY, without increased NPY mRNA expression, we quantified PAM expression and the levels of the bio-logically active neuropeptide in differentiating WT and TTR KO BMSC cultures As expected, TTR KO mice BMSCs displayed increased amidated NPY levels (approximately 2.4-fold at day 3) when compared to
WT cells (Fig 4B) Despite the fact that the NPY con-tent decreased over the 14 days of differentiation, indi-cating that undifferentiated BMSCs have higher levels
of NPY than differentiated osteoblasts, these still expressed amidated neuropeptide One should, how-ever, note that in WT BMSCs, NPY levels were not altered throughout the course of BMSC differentiation (days 3–14; Fig 4B) Therefore, NPY should not be regarded as either a marker of osteoblast differentia-tion or a marker of mature osteoblasts In agreement with the increased NPY levels, PAM expression in TTR KO BMSCs was increased, with a similar fold change as that observed for the levels of amidated NPY (Fig 4C) Y1 expression was detected by RT-PCR in differentiating WT and TTR KO BMSCs, with no Y2 or Y5 receptor amplification (data not shown), in accordance with recently published results [15] However, no statistical difference was observed between the two strains regarding Y1 expression (data not shown)
TTR KO BMSCs show increased osteoblast differentiation
To examine whether WT and TTR KO BMSCs differ
in their capability to undergo osteoblast differentia-tion, as a possible consequence of their differential am-idated NPY content, isolated BMSCs from WT and TTR KO mice were cultured under osteoblast differen-tiation conditions Osteoblast phenotype markers such
as alkaline phosphatase (ALP) activity and osteocalcin expression were determined In both cultures, ALP activity increased in a time-dependent manner and
Trang 6B
C
Fig 3 MicroCT in WT and TTR KO mouse femurs (A) Bone microarchitecture in WT and TTR KO mice Left and middle panels: 2D microCT images of metaphyseal bone, showing reconstructed longitudinal sections (left panel) and transverse sections taken
1 mm from the growth plate (middle panel) The line crossing the transversal sections indicates the orientation of the longitudinal sections Right panel: 3D mi-croCT images of metaphyseal trabecular bone in WT and TTR KO mice (B) Quantifi-cation of trabecular volume [bone vol-ume ⁄ trabecular volume (BV ⁄ TV)] and BMD
in WT and TTR KO mice Results are pre-sented as average ± standard error of the mean a P < 0.05 (C) Hematoxylin ⁄ eosin staining of the growth plate (femur) of WT and TTR KO mice (3 months old) Scale bars: 50 lm.
A B
C
Fig 4 NPY and PAM expression in bone cells from WT and TTR KO mice (A) NPY RT-PCR analysis in brain, MC3T3-E1 cells, and BMSCs (B) NPY quantification in BMSCs from WT and TTR KO mice at days 1, 3, 7 and 14 of differentiation into osteoblasts (C) Semiquantitative RT-PCR analysis of PAM expression normalized for b-actin (left) or HPRT (right) expression in BMSCs from WT and TTR KO mice at days 3 and 14 of osteoblast differentiation Results are presented as average ± stan-dard error of the mean; a P < 0.05.
Trang 7peaked on day 7, with significantly increased levels (ranging from two-fold to three-fold) being seen in TTR KO osteogenic cultures at days 3 and 7 when compared to WT cultures (Fig 5A) Regarding osteo-calcin expression, WT cultures displayed a time-depen-dent increase in osteocalcin levels, with a peak of expression on day 14 (Fig 5B), which is characteristic
of the osteoblastic differentiation process in vitro In the case of TTR KO BMSCs, no increase in osteocal-cin expression was observed from day 3 to day 7 of differentiation, probably because those cells already showed high osteocalcin levels at day 3 of differentia-tion (Fig 5B) Nonetheless, at day 14, TTR KO cul-tures showed a significant increase in osteocalcin expression when compared with the WT cultures (Fig 5B) To further confirm these data, RT-PCR was performed using additional housekeeping genes [those encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyl-transferase (HPRT)] as well as osteopontin, an extra marker of osteoblastic differentiation Day 3 of BMSC differentiation was chosen for performance of the con-firmation because, at this time point, not only ALP activity but also osteocalcin expression are increased in TTR KO BMSCs The expression levels of both osteo-calcin (Fig 5C) and osteopontin (Fig 5D) were always increased in TTR KO BMSCs, irrespective of the housekeeping gene used to perform the normalization Taken together, these data suggest that TTR KO BMSCs show enhanced competence in undergoing osteoblast differentiation in vitro
Discussion The data presented in this study demonstrate that NPY is expressed in several types of bone cell, with both in vitro and in vivo evidence Moreover, we show that increased NPY levels are related to increased bone density, as well as to augmented competence in BMSC differentiation into osteoblasts In agreement with our findings, a recent report further supports the contribution
18
16
14
12
10
8
–1 )
6
4
2
0
25
20
15
10
10
8
6
4
2
0
0
2
4
6
8
10
12
14
b
a
a
5
0
Day 3
Day 3
Day 7
Day 7 c
b
b
TTR KO
TTR KO
WT
Day 14
Day 14 WT
WT
TTR KO
b
b WT
TTR KO WT
osteocalcin
β-actin
B
A
C
D
Fig 5 Osteoblast differentiation of WT and TTR KO BMSCs as assessed by ALP, osteocalcin and osteopontin levels (A) ALP activ-ity of WT and TTR KO BMSCs under osteoblast differentiation con-ditions at days 3, 7 and 14 (B–D) Semiquantitative RT-PCR analysis
in WT and TTR KO BMSCs of (B) osteocalcin expression, normal-ized for the expression of b-actin, at days 3, 7 and 14, (C) osteocal-cin expression normalized for the expression of GAPDH and HPRT
at day 3, and (D) osteopontin expression, normalized for the expression of b-actin, GAPDH and HPRT at day 3 under osteoblast differentiation conditions Results are presented as average ± stan-dard error of the mean; a P < 0.05; b P < 0.005; c P < 0.0005.
Trang 8of the NPY pathway in bone homeostasis via a direct
action on osteoblasts [23] In that report, it was shown
that chronically elevated NPY levels modulate the
lev-els of Y2 receptor expression (according to the stage
of osteoblast differentiation) and that NPY is a
nega-tive regulator of Y1 receptor expression Moreover,
functional analysis revealed the osteogenic potential of
NPY, with osteoblast phenotype markers being
signifi-cantly enhanced in osteoprogenitor cells stimulated by
NPY, probably owing to downregulation of the Y1
receptor
Until now, NPY expression has only been detected
in bone marrow cells, including megakaryocytes [14]
Here, we show for the first time that BMSCs also
con-tribute to NPY in the bone marrow, as NPY is
expressed both in BMSCs and in BMSCs undergoing
osteoblastic differentiation Moreover, this article is
the first to report NPY expression in chondrocytes,
osteoblasts, osteocytes and the osteoblastic cell line
MC3T3-E1 In relation to chondrocytes, no studies
were performed regarding the role of NPY in the
dif-ferentiation of this cell type This could probably be
the aim of a subsequent study, where possible
differ-ences in articular cartilage or growth plate between
WT and TTR KO bones should be addressed In the
case of osteoclasts, although NPY expression was not
detected in this cell type, the elevated NPY levels in
TTR KO bones might have some indirect effect on
osteoclasts In fact, we recently reported that NPY
modulates receptor activator of nuclear factor-jB
(RANK) ligand and osteoprotegerin, two key factors
regulating bone remodeling [23] The inhibitory effect
of NPY on RANK ligand production by BMSCs was
also investigated by Amano et al [24], who suggested
that the inhibitory effect of NPY on osteoclastogenesis
was caused by suppression of isoprenaline-induced
RANK ligand production by stromal cells, upstream
of RANK ligand mRNA expression
It is known that central NPY regulates bone mass, as
conditional ablation of hypothalamic Y2 receptors
results in increased bone formation [5] Moreover,
lep-tin-deficient mice, in which NPY is increased in the
hypothalamus, show high cancellous bone mass, but
reduced cortical production [25] Central NPY can also
influence peripheral tissues through alterations in
auto-nomic neuronal activity This is probably mediated by
NPY projections from the hypothalamus to the
brain-stem areas where sympathetic neuronal activity is
mod-ulated [26] Thus, to achieve its functions, NPY may act
centrally on hypothalamic receptors and⁄ or
peripher-ally on its osteoblastic receptor Y1 after being released
from sympathetic nerve terminals supplying the skeletal
tissue With this work, we have opened a new window
in which NPY may additionally function as an auto-crine factor, as it is expressed by osteoblasts as well
We further demonstrate that TTR KO bone tissue displays increased amidated NPY levels, when com-pared to WT tissue, further demonstrating the expres-sion of this neuropeptide in bone cells In theoretical terms, the major TTR ligands, thyroxine (T4) and reti-nol, could be responsible, at least in part, for the bone phenotype observed in TTR KO mice Retinol defi-ciency is known to increase BMD [27]; additionally, reti-noic acid inhibits osteogenic differentiation of BMSCs [28,29] Despite the fact that TTR KO mice have retinol plasma levels below the level of detection [30], symp-toms of vitamin A deficiency are absent in these ani-mals In agreement with this, their total retinol tissue levels are not significantly different from those of WT mice [31] Moreover, retinoic acid plasma levels are two-fold to three-two-fold higher in TTR KO mice, probably compensating for their low retinol levels [31] Taking the above into account, it is highly unlikely that, with normal retinol levels in tissues and increased retinoic acid levels in the plasma, an impairment in retinol homeostasis would be responsible for the increased BMD in TTR KO mice Regarding thyroid hormones,
it is well known that hyperthyroidism in adult patients leads to decreased BMD [32] As expected, both total T4 and tri-iodothyronine serum levels are decreased in TTR KO mice [32,33] However, similar to what is described above for retinol, this decrease is unrelated to symptoms of hypothyroidism or thyroid gland abnor-malities [34] Again, in terms of tissue content, TTR KO mice show no differences in T4 levels from WT mice [35,36] This euthyroid status probably arises as a conse-quence of the high free T4 serum pool in the TTR KO mice [34] Such a euthyroid status is essential for normal skeletal development and maintenance, and therefore it
is hard to see how the bone phenotype of TTR KO mice could be related to thyroid hormones
It is additionally possible that in TTR KO mice, as
a consequence of PAM overexpression, increased levels
of other amidated neuropeptides may produce some complexity In this respect, although contradictory results have been reported for the action in bone of some amidated neuropeptides, such as substance P, others, such as pancreatic polypeptide and calcitonin gene-related peptide, have been described as stimulat-ing the differentiation of MC3T3-E1 cells [37] or increasing the number of bone colonies formed from bone marrow stromal cells (MSC) in vitro [38], simi-larly to what is reported here in the absence of TTR However, although not discarding the possible influ-ence of the putative increases in the levels of other amidated neuropeptides in this model, which should be
Trang 9addressed in future experiments, TTR KO mice not
only show increased NPY levels when compared with
other NPY overexpression models, but also present
an accompanying NPY overexpression phenotype This
phenotype includes decreased energy expenditure,
decreased depressive-like behavior, and increased
car-bohydrate consumption and preference, and most of
these features are not commonly observed in other
NPY overexpression models [19] It is noteworthy that
the increased NPY levels in TTR KO mice are
unre-lated to increased NPY mRNA expression, and result
from increased processing and amidation by PAM,
which is upregulated in TTR KO animals In fact,
although TTR is not expressed in BMSCs, PAM
expression is increased in TTR KO BMSCs, suggesting
that TTR KO osteoblasts have intrinsically augmented
PAM expression in relation to WT cells, as a
conse-quence of their physiological TTR-free environment
A similar finding was reported for TTR KO neurons
(like BMSCs, neurons lack TTR expression), as these
cells were also shown to display intrinsically decreased
neurite outgrowth, as a consequence of their
physio-logical TTR-free environment [39]
NPY control of bone mass is still controversial On
the one hand, there are two different mouse models
with increased NPY expression that show high
cancel-lous bone mass, the Y2 receptor KO mice [5] and mice
lacking leptin (ob⁄ ob mice) [7,16] Although sharing a
similar high cancellous bone phenotype, both models
differ in cortical bone regulation, with increased
corti-cal bone mass in Y2 receptor KO mice and decreased
cortical density in ob⁄ ob mice [23] On the other hand,
no NPY signaling in the bone, as is the case in Y1
receptor KO mice, leads to high bone mass [6], and
central NPY overexpression yields decreased osteoblast
activity [18] and bone mass [7], with no alteration in
cancellous bone volume [17,18] With regard to this
central NPY overexpression, the consequential increase
in leptin levels [40,41] cannot be excluded as the cause
of the effects observed Furthermore, the apparent
dis-crepancy between Y1 and Y2 receptor KO models
regarding NPY signaling and bone phenotype was
recently clarified by the hypothesis that the increased
central NPY levels observed in the Y2
receptor-defi-cient mice lead to Y1 receptor downregulation on bone
cells, which would explain their increased bone mass
phenotype [15] The fact that deletion of both Y1 and
Y2 receptors did not produce additive effects on
increased bone mass further supports this hypothesis,
as it suggests a common pathway from the
hypothala-mus to the bone involving both Y2 and Y1 signaling
[6], with probable central Y2 and peripheral Y1 effects
on bone tissue The NPY KO mouse is not very
help-ful in this matter, as its bone mass is normal [8] Here
we show that in TTR KO mice, an additional model showing increased NPY levels, an increased cancellous bone mass phenotype is observed, in agreement with the Y2 receptor KO and ob⁄ ob mouse phenotypes, fur-ther suggesting that increased NPY content might be related to increased cancellous bone mass Despite all the concerns discussed above regarding the use of TTR KO mice as a model of increased NPY levels, the main advantage of these animals over other NPY over-expression models is that, in addition to the increase in NPY levels, the leptin level is not altered [42], exclud-ing its interference in the bone phenotype observed
In summary, we provide evidence that NPY is expressed in bone cells, namely in osteoblasts Further-more, we report that in a model of increased amidated neuropeptide levels, showing an NPY overexpression phenotype, an increased bone mass phenotype is pres-ent Finally, on the basis of these findings, further work is needed to determine the localization of NPY and NPY receptors during bone injury, disease, and aging, and thereby elucidate the possible role of NPY
in the bone regeneration process
Experimental procedures Animals
Mice were handled according to the European Communi-ties Council Directive (86⁄ 609 ⁄ EEC) and national rules, and all studies performed were approved by the Portuguese General Veterinarian Board Male WT and TTR KO [33] littermate offspring of heterozygous breeding pairs, in the
129⁄ Sv background, were maintained at 24 ± 1 C under a
12 h light⁄ dark cycle and fed regular chow and tap water
ad libitum Prior to all experimental procedures, animals were anesthetized with ketamine (1 mgÆg)1body weight)⁄ mede-tomidine (0.02 lgÆg)1 body weight) Animals were killed with an overdose of anesthetic
Immunohistochemistry
Femurs from 3 month old male WT (n = 6) and TTR KO (n = 5) littermates were fixed in 4% paraformaldehyde
in NaCl⁄ Pi, decalcified in TBD-1 commercial solution (Thermo Electron Corporation), and embedded in paraffin; serial 4 lm thick longitudinal sections were then cut For studies during embryonic development, 16 day or 18 day
WT pregnant females were killed by cervical dislocation, and the fetuses were collected by cesarian section Sections were then deparaffinized, dehydrated in a modified alcohol series, and blocked for the endogenous peroxidase activity NPY immunohistochemistry was performed with the MOM Kit (Vector, Peterborough, UK), following the manufacturer’s
Trang 10instructions Briefly, bone sections from WT and TTR KO
mice, as well as sections from NPY KO mice (prepared
simi-larly to WT and TTR KO mouse samples; a kind gift from
H Herzog, Garvan Institute, Australia) were incubated in
the MOM kit blocking reagent for 1 h at room temperature,
prior to incubation with the monoclonal NPY antibody
NPY05 (generously provided by E Grouzmann, University
Hospital, Lausanne, Switzerland; diluted 1 : 2000 in MOM
diluent) for 1 h at room temperature NPY05 is specific for
the amidated form of NPY [30] Antigen visualization was
performed with the MOM avidin–biotinylated peroxidase
complex reagent (Vector), using 3-amino-9-ethyl carbazole
(Sigma, Lisbon, Portugal) as substrate On parallel control
sections, the primary antibody was replaced by mouse IgG
(Sigma) Immunohistochemical investigations for NF200
and PGP9.5, both markers of nerve fibers, osteocalcin (a
positive control for osteoblast staining), PAM and OSCAR
(a marker of preosteoclasts and mature osteoclasts) were
also performed Briefly, sections were incubated in blocking
buffer (1% BSA and 4% bovine serum in NaCl⁄ Pi) for
30 min at 37C in a moist chamber, and then incubated with
primary antibodies at the appropriate dilution in blocking
buffer, overnight at 4C The dilutions used were 1 : 2500
for rabbit NF200 IgG (Sigma), 1 : 4000 for rabbit
PGP9.5 IgG (Serotec, Kidlington, UK), 1 : 500 for goat
anti-osteocalcin IgG (Biomedical Technologies Inc., Stoughton,
MA, USA), 1 : 500 for rabbit anti-PAM IgG (a kind gift
from R Mains, University of Connecticut Health Center),
and 1 : 100 for mouse anti-OSCAR IgG (Santa Cruz
Bio-technology, Heidelberg, Germany) Antigen visualization
was performed with the biotin–extravidin–peroxidase kit
(Sigma), using 3-amino-9-ethylcarbazole (Sigma) as
sub-strate On parallel control sections, the primary antibody was
replaced with blocking buffer Immunohistochemical
analy-sis was performed independently by two observers For
quantification of PAM immunohistochemistry, the number
of labeled cellsÆmm)2 was scored in three nonoverlapping
micrographs with a magnification of· 40
Bone histology
Femurs were harvested from 3 month old male WT
(n = 6) and TTR KO (n = 5) mice After their length had
been measured, bones were fixed in 4% paraformaldehyde
in NaCl⁄ Pi, decalcified as described above, and embedded
in paraffin Serial 10 lm thick longitudinal sections were
cut Sections were then deparaffinized, dehydrated in a
modified alcohol series, and stained for hematoxylin⁄ eosin
MicroCT analysis
Dissected hindlimbs (femur plus tibia from WT and TTR
KO littermates, n = 9 and n = 10, respectively) were
scanned with high resolution (5 lm pixel size) microCT
(Skyscan 1172; Skyscan, Kontich, Belgium) The whole
mouse femur and tibia were reconstructed, and the trabecu-lar bone in the proximal metaphysis, comprising a region starting 0.25 mm from the growth plate and extending 1.5 mm (or 300 tomograms) distally, was analyzed Histo-morphometric analysis in two and three dimensions was performed with Skyscan software (ct-analyser v 1.5.1.3, Skyscan) For analysis of trabecular bone, cortical bone including the trabecular compartment was excluded by operator-drawn regions of interest, and 3D algorithms were used to determine the bone volume percentage (bone volume⁄ trabecular volume)
BMD measurement by microCT
Volumetric BMD values of the trabecular bone compart-ment within the femural and tibial metaphysis were mea-sured from the same regions of interest used to derive the microarchitectural parameters, using the manufacturer’s instructions Briefly, two calibration phantoms (Skyscan) with densities of 0.25 and 0.75 gÆcm)3and a sample of water were scanned and reconstructed using the same settings used for the femurs and tibiae The gray scale density values were converted into Hounsfield units, which were then used to compute the mean volumetric BMD of each femur and tibia
Cell cultures MC3T3-E1 mouse osteoblastic cell line culture
MC3T3-E1 cells, established as an osteoblastic cell line from normal mouse calvaria, were grown in alpha-MEM (Invitro-gen, Carlsbad, CA, USA) supplemented with 10% (v⁄ v) fetal bovine serum (Invitrogen), 0.5% (v⁄ v) gentamicin (Invitro-gen), 1% (v⁄ v) fungizone (Invitrogen), 50 lgÆmL)1vitamin C (Sigma) and 10 mm b-glycerophosphate (Sigma) in a humidi-fied 5% CO2incubator at 37C The medium was changed twice weekly At confluence, the cells were trypsinized and seeded in 24-well plates at a cell seeding density of
4· 104cells per well
BMSC culture
Primary BMSCs were obtained according to the method developed by Maniatopoulos et al [43] Briefly, femurs and tibias from 1 month old male WT and TTR KO littermates were aseptically excised from the hindlimbs, the epiphyses were cut off, and the marrow was flushed with standard culture medium, which consisted of alpha-MEM supple-mented with 10% fetal bovine serum, 50 lgÆmL)1 gentami-cin sulfate, and 2.5 lgÆmL)1 amphotericin B (Invitrogen) Cells were seeded in 75 cm2plastic culture flasks, and incu-bated in a humidified incubator (37C and 5% CO2) The medium was changed after the first 24 h to remove nonad-herent cells Subsequently, the adnonad-herent cells were cultured for 10 days, the medium being renewed every 3 days