Tài liệu Báo cáo khoa học: Neuropeptide Y and osteoblast differentiation – the balance between the neuro-osteogenic network and local control ppt

REVIEW ARTICLE
Neuropeptide Y and osteoblast differentiation – the
balance between the neuro-osteogenic network and local
control
Filipa Franquinho
1,2,
*, Ma
´
rcia A. Liz
1,
*, Ana F. Nunes
3
, Estrela Neto
4,5
, Meriem Lamghari
4
and
Mo
´
nica M. Sousa
1
1 Nerve Regeneration Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal
2 Departamento de Anatomia Patolo
´
gica, Instituto Polite
´
cnico de Sau
´
de-Norte, Paredes, Portugal
3 iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal
4 INEB – Instituto de Engenharia Biome

´
dica, Divisa˜o de Biomateriais, NewTherapies Group, Universidade do Porto, Portugal
5 Universidade do Porto, Faculdade de Engenharia, Portugal
Introduction
For correct bone development, the coordinated growth,
differentiation, function and interaction of different cell
types is needed. In the normal adult bone, constant
turnover occurs, driven by three major cell types: the
osteoclasts, which are responsible for bone resorption
at multiple discrete sites; the osteoblasts, which are
responsible for the synthesis and mineralization of bone
matrix, forming new bone following resorption; and
the osteocytes, which are known to sense variations in
mechanical forces acting on bone and to respond to
this by signaling, via sclerotin, to coordinate osteogene-
sis [1–5]. This bone remodeling is essential to maintain
ion homeostasis, to respond to stimuli (such as
mechanical loading), and to replace damaged bone.
Moreover, this process has to be very tightly regulated,
such that a constant bone mass is maintained, i.e. so
that the amount of bone resorbed equals the amount of
bone formed. The regulation of bone remodeling has
been conventionally linked to hormones, auto-
crine ⁄ paracrine signals and mechanical loading [6–8].
Keywords
bone innervation; leptin; NPY; NPY
receptors; osteoblasts
Correspondence
M. Mendes Sousa, IBMC, Rua Campo
Alegre 823, 4150-180 Porto, Portugal

Fax: +351 22 6099157
Tel: +351 22 6074900
E-mail:
Website: />*These authors contributed equally to this
work
(Received 29 March 2010, revised 2 June
2010, accepted 12 July 2010)
doi:10.1111/j.1742-4658.2010.07774.x
Accumulating evidence has contributed to a novel view in bone biology:
bone remodeling, specifically osteoblast differentiation, is under the tight
control of the central and peripheral nervous systems. Among other players
in this neuro-osteogenic network, the neuropeptide Y (NPY) system has
attracted particular attention. At the central nervous system level, NPY
exerts its function in bone homeostasis through the hypothalamic Y2 recep-
tor. Locally in the bone, NPY action is mediated by its Y1 receptor.
Besides the presence of Y1, a complex network exists locally: not only there
is input of the peripheral nervous system, as the bone is directly innervated
by NPY-containing fibers, but there is also input from non-neuronal cells,
including bone cells capable of NPY expression. The interaction of these
distinct players to achieve a multilevel control system of bone homeostasis
is still under debate. In this review, we will integrate the current knowledge
on the impact of the NPY system in bone biology, and discuss the mecha-
nisms through which the balance between central and the peripheral NPY
action might be achieved.
Abbreviations
CGRP, calcitonin gene-related peptide; ICV, intracerebroventricular; NPY, neuropeptide Y; PAM, peptidylglycine a-amidating monooxygenase;
SP, substance P; TTR, transthyretin; VIP, vasoactive intestinal peptide; WT, wild-type.
3664 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS
However, as we will discuss throughout this review,
in the last decade several reports provided evidence

that bone homeostasis is also under the influence of
central and peripheral neural control, creating a new,
previously unsuspected, link between the nervous sys-
tem and bone. This concept was first described in the
1980s, but only recently have its molecular and mecha-
nistic details been unraveled, transforming this issue in
one of the most stimulating areas of research in bone
biology. In this research line, particular emphasis has
been given to osteoblasts. The topic of a neuro-osteo-
genic network, particularly the regulation of bone for-
mation by neuropeptide Y (NPY), will be discussed in
detail in the following paragraphs.
The neuro-osteogenic network – proof
of concept
Clear evidence of bone innervation is the observation
that bone injury is often accompanied by both acute
and chronic pain. The first demonstration that the
bone tissue is innervated, i.e. nerve fibers entering and
leaving the bone, was provided by Estienne in 1545 [9].
Almost four centuries later, De Castro described nerve
fibers associated with blood vessels near osteoblasts
and osteoclasts [10]. Subsequently, with the use of clas-
sic histological methods, the presence of intense inner-
vations of bone in animals and humans was shown
[11–13]. More details were unraveled as the technology
advanced: in 1966, electron microscopy images of den-
sely innervated cortical bone were published, and in
1969, myelinated and nonmyelinated nerve fibers asso-
ciated with bone blood vessels were described [14].
In relation to neural control of bone development,

most of the reports addressing this issue are based on
studies of bone innervation at different stages of
embryogenesis. During development, autonomic fibers
immunoreactive to protein gene product 9.5 and
ubiquitin C-terminal hydrolase (specific markers for
neural and neuroendocrine tissues) were found in rat
long bones at embryonic day 15, in the diaphyseal and
metaphyseal perichondrium, and became more fre-
quent after birth [15]. These observations were con-
firmed in later studies [16,17]. A detailed analysis of
bone innervation during development was also
provided [16]. In this study, sensory fiber-associated
neuropeptides, calcitonin gene-related peptide (CGRP)
and substance P (SP) were first observed at embryonic
day 21 in the epiphyseal perichondrium, the perios-
teum of the shaft, and the bone marrow. With regard
to NPY nerve fibers, their presence at postnatal day 4
was shown in diaphyseal regions, and at postnatal
days 6–8, these fibers were able to extend into the
metaphyseal region [15]. In developing calvaria, nerve
fibers were observed traversing the bone through the
periosteum, diploe, endosteum, dura, arachnoid and
pia at multiple locations with no particular pattern
[18].
In adult bones, sensory fibers derived from primary
afferent neurons present in the dorsal root and some
cranial nerve ganglia represent the majority of the skel-
etal innervation system, whereas the other nerve fiber
populations are adrenergic and cholinergic in nature,
and originate from paravertebral sympathetic ganglia

[16]. Experimental nerve deletion and immunohisto-
chemistry analysis have shown that both myelinated
and unmyelinated afferent (sensory) and efferent (auto-
nomic) fibers are present in the bone marrow and the
periosteum [16,19]. Their phenotyping revealed the
presence of several neurotransmitter fibers, specifically
vasoactive intestinal peptide (VIP), CGRP, SP and
NPY. Bones of the calvaria also receive a rich supply
of sensory, sympathetic and parasympathetic innerva-
tions [20–24]. In adult rats, the calvarial periosteum
and diploe were found to be innervated by sympathetic
fibers immunoreactive to VIP and NPY, originating
from postganglionic neurons in the superior cervical
ganglion, whose fibers exhibited VIP, NPY or dopa-
mine hydroxylase immunoreactivity. Moreover, in the
calvarial periosteum and diploe, the presence of sen-
sory innervation (CGRP or SP) was also reported,
with higher concentrations in the sutures [18,22].
The impact of the nervous system in
bone biology
As described above, several histological studies have
revealed the presence in bone of neuropeptides of sen-
sory, sympathetic and glutaminergic types. However,
despite these early descriptions linking the bone to the
nervous system, the first clear evidence supporting
the concept of a nervous system–bone network was the
finding that leptin-deficient mice (ob ⁄ ob mice) had a
high bone mass despite their hypogonadism [25]
(Table 1). Leptin is an adipocyte-derived hormone that
acts on the brain to reduce food intake, by regulating

the activity of neurons in the hypothalamic arcuate
nucleus. To exert its function in this brain region,
leptin stimulates neurons that express anorexigenic
peptides, and inhibits neurons that coexpress the orexi-
genic peptides NPY and agouti-related protein [26].
Initially, the existence of multiple metabolic abnormali-
ties in ob⁄ ob mice made it experimentally challenging
to determine the mechanism by which leptin deficiency
led to increased bone mass [27–29]. As there are no
leptin receptors detectable on mouse osteoblasts [30]
F. Franquinho et al. NPY and osteoblast differentiation
FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3665
(ruling out the possibility of an autocrine, paracrine or
endocrine mechanism of regulation in the ob ⁄ ob
model), and given that the majority of leptin receptors
exist in the arcuate nucleus of the hypothalamus, the
hypothesis that leptin controls bone formation via a
central mechanism was raised. The most convincing
evidence supporting this hypothesis was the rescue
of the bone mass phenotype of the ob ⁄ ob mice by
intracerebroventricular (ICV) infusion of leptin in the
hypothalamic region, clearly demonstrating that the
inhibitory action of leptin on bone formation is medi-
ated by a central circuit [25]. Further supporting the
importance of leptin in the control of bone formation,
mice lacking the leptin receptor (db ⁄ db mice), similarly
to ob ⁄ ob mice, showed a three-fold increase in trabecu-
lar bone volume, owing to increased osteoblast activity
[25] (Table 1).
As referred to above, a major target of leptin in the

hypothalamus is NPY. It is noteworthy that the level
of NPY is increased in ob ⁄ ob mice, as leptin inhibits
its expression in arcuate neurons [31]. NPY is one of
the most evolutionarily conserved peptides, and is
abundantly expressed in numerous brain regions, par-
ticularly in the hypothalamus [32], but also in the
periphery. Since the discovery of NPY [33], a robust
body of literature has developed around the potential
functions of this peptide [34]. NPY actions range from
stress-related behaviors (such as anxiety and depres-
sion) to the regulation of energy homeostasis and
memory, among others. The role of the NPY system,
particularly in the regulation of food intake and energy
homeostasis, has been well established. To determine
whether the overexpression of NPY in ob ⁄ ob mice
could contribute to their high bone mass, ICV infusion
of NPY in wild-type (WT) mice was performed [25].
Similarly to leptin, NPY inhibited bone formation,
strongly suggesting that the increased NPY expression
in ob ⁄ ob mice does not mediate their increased bone
density [25]. Moreover, NPY ablation in ob ⁄ ob mice
further demonstrated that NPY acts as an antiosteo-
genic factor [35]. Given the well-described interaction
between NPY and leptin in the regulation of energy
homeostasis, it was suggested that their regulation of
osteoblast activity occurred through a common path-
way. However, as will be discussed latter in this
review, the current evidence clearly demonstrates that
NPY regulates bone formation through a mechanism
distinct from the pathway mediated by leptin [36].

The presence of nerve fibers immunoreactive to
NPY in the bone, mostly distributed alongside blood
vessels, was demonstrated in early studies [22,37].
Moreover, this NPY immunoreactivity was dramati-
cally reduced in sympathectomized animals, indicating
the sympathetic origin of these nerve endings [22].
Given the distribution of the NPY-positive nerve
fibers, it was initially proposed that this neuropeptide
had a vasoregulatory role in the bone, rather than
being a regulator of bone cell activity [15,38–40]. The
fact that NPY was produced by megakaryocytes and
mononuclear hematopoietic cells within the bone mar-
row supported this vasoregulatory role [41,42]. How-
ever, NPY-immunoreactive fibers were also identified
in the periosteum and cortical bone [41,43], raising the
possibility that NPY could play a role in bone biology
Table 1. Summary of the bone phenotype in animal models for leptin and for the NPY system. CBV, cortical bone volume; ND, not deter-
mined; TBV, trabecular bone volume.
Animal
model Deficiency Bone phenotype
Osteoblast
activity
Osteoclast
activity Other observations References
ob ⁄ ob Leptin Increased TBV
Decreased CBV
Increased in
trabecular bone
Increased Increased NPY levels 25,46
db ⁄ db Leptin receptor Increased TBV Increased Normal ND 25

Y2
) ⁄ )
Y2 Increased TBV and CBV Increased Normal Increased NPY levels
Normal leptin levels
45,51
Y2
) ⁄ )
ob
) ⁄ )
Leptin and Y2 Decreased TBV and CBV
in relation to Y2
) ⁄ )
Increased Increased Increased NPY levels 45,61
Y1
) ⁄ )
Y1 Increased TBV and CBV Increased Increased in
trabecular bone
No inhibitory effects
of NPY detected
62
Y4
) ⁄ )
Y4 Normal Normal Normal Normal NPY and
leptin levels
65
Y2
) ⁄ )
Y4
) ⁄ )
Y4 and Y2 Increased TBV in relation to Y2

) ⁄ )
Increased Increased Increased NPY levels 65
NPY
) ⁄ )
NPY Increased TVB and CBV Increased Normal ND 66
TTR
) ⁄ )
Transthyretin Increased bone mineral
density and TBV
Increased Normal Increased amidated
NPY Leptin
levels not altered
50
NPY and osteoblast differentiation F. Franquinho et al.
3666 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS
besides the putative vasoregulation. Previous studies
had already demonstrated that osteoblasts are sensitive
to treatment with NPY [44,45], suggesting the presence
of NPY receptors in bone cells and raising the possibil-
ity that NPY might be directly involved in the regula-
tion of osteoblast activity. NPY is able to act through
five different receptors (Y1, Y2, Y4, Y5 and y6) that
vary in their binding profiles and in their distribution
in the central nervous system and periphery. Y1, Y2
and Y5 are the best characterized NPY receptors, and
the majority of NPY functions are associated with
them. Supporting the assumption that Y receptors are
present in bone cells, one of the NPY receptors, Y1,
was shown to be present in human osteoblastic and
osteosarcoma-derived cell lines and in mouse cultured

bone marrow stromal cells and osteoblasts [40,46–48],
despite the absence of the other Y receptors (Y2, Y4,
Y5 and y6) [40,46]. In addition to the presence of
NPY-immunoreactive fibers and the presence of
NPY receptors, local NPY production in bone cells,
both at embryonic stages and in the adult, has been
reported recently in osteoblasts, osteocytes, chondro-
cytes and bone marrow stromal cells [49,50]. These
reports have opened a new window in which NPY
may additionally function as an autocrine ⁄ paracrine
factor. A summary of the anatomical structures with
NPY ⁄ NPY receptors is provided in Fig. 1. The current
view on the role of NPY in bone biology will be
discussed below.
A definite role for NPY in bone
regulation – the Y2 knockout mouse
As mentioned above, evidence for an important role of
the NPY system has emerged in the regulation of bone
formation. The lack of a complete range of selective
pharmacological tools for the Y receptors has made it
challenging to assign a specific Y receptor to a given
NPY effect. To overcome this problem, germline and
conditional knockouts have been generated for the
Y receptors. These animals, together with germline
and conditional knockouts lacking leptin or the leptin
receptor, have revealed not only that the hypothalamus
controls osteoblast activity, but also that two main
central pathways are implicated in bone turnover,
namely Y2 and leptin [51,52]. Another seminal finding
that came from the analysis of these animal models

was that the actions of the NPY system in bone
biology are more complex than simple downstream
mediation of leptin. The studies that allowed these
conclusions are summarized and discussed below.
A definite role for the NPY receptors in the regulation
of bone turnover was demonstrated following germline
deletion of Y2 [51]. Y2
) ⁄ )
mice had a two-fold increased
bone volume, as indicated by the increased trabecular
bone volume and thickness (Table 1). This augmented
bone volume resulted from increased bone formation
i.e. from elevated osteoblast activity. Moreover, in vitro
analysis of Y2
) ⁄ )
mesenchymal stem cells revealed an
increased number of osteoprogenitor cells, which may
additionally underlie the increase in bone formation in
the absence of Y2 in vivo [46].
Whereas, in WT bone marrow stromal cells, Y1
expression is detected (and expression of Y2, Y4, Y5
or y6 is absent), in Y2
) ⁄ )
bone marrow stromal cells
the expression of all five known Y receptors is absent
[46]. Therefore, the effect observed in Y2
) ⁄ )
mice was
thought to be mediated by a centrally controlled mech-
anism and not by a direct mechanism in bone cells.

Supporting this hypothesis, just 5 weeks following con-
ditional deletion of hypothalamic Y2 in adult mice, a
bone phenotype similar to that of germline Y2
) ⁄ )
mice
was achieved, indicating that Y2 signaling in the hypo-
thalamus inhibits bone formation [51]. It is important
to note that obvious endocrine imbalances that would
otherwise impact on bone homeostasis were not found
Y2Y1
NPY
Peripheral nervous
system
Bone
Osteoblasts
NPY
?
NPY
Circulating NPY
in the blood
NPY
Bone microenvironment
Fig. 1. Anatomical structures with NPY ⁄ NPY receptors. Peripheral
nerve fibers derived from basal, dorsal root and sympathetic ganglia
innervate the bone and release NPY in the sites of innervation.
Besides peripheral innervation, bone biology is also centrally regu-
lated by NPY (highly expressed in the hypothalamus) and probably
also by autocrine mechanisms, as osteoblasts (expressing Y1 and
possibly Y2) are themselves capable of producing and secreting
NPY.

F. Franquinho et al. NPY and osteoblast differentiation
FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3667
in either germline or hypothalamus-specific Y2
) ⁄ )
mice
[51]. The rapid increase in bone mass in adult mice
after hypothalamic deletion of Y2 raises the prospect
of new possibilities in the prevention and treatment of
osteoporosis, a major concern following estrogen defi-
ciency after menopause. In this respect, it has been
shown that the elevated osteoblast activity that charac-
terizes the skeletal phenotype of Y2
) ⁄ )
mice is main-
tained following gonadectomy in both female and male
mice, and that the protection against gonadectomy-
induced bone loss is also evident following hypothala-
mus-specific deletion of Y2 in both male and female
mice [53]. Further supporting a link between estrogen
and NPY, it is known that estrogen deficiency tran-
siently increases NPY expression in the hypothalamus
[54], which could contribute to the bone loss associated
with this condition. The topic of NPY and sex hor-
mone interactions in bone and fat control has been
recently reviewed [55]. In summary, increased knowl-
edge about the link between NPY and sex hormones
in regulating bone biology could lead to better treat-
ments for osteoporosis.
Despite the initial consensus that Y2 is not
expressed locally by osteoblasts, a recent study

addressed the expression of Y2 in MC3T3-E1 preos-
teoblasts derived from mouse calvaria bone, and
showed that, at least in this cell line, and in agreement
with previous findings [56], Y2 mRNA expression
occurs under osteoblast differentiation conditions [57].
Besides central control of bone formation by hypotha-
lamic Y2, if the existence of Y2 in osteoblasts is fur-
ther demonstrated, the complexity of the regulation of
bone homeostasis by the NPY system will certainly
increase.
Evidence for a distinct mechanism of
action of leptin and Y2 antiosteogenic
pathways
The bone phenotype of conditional hypothalamic
Y2
) ⁄ )
mice reported above was similar to the one
reported for mice deficient in leptin action (ob ⁄ ob and
db ⁄ db mice) [25]. Yet, as will be discussed in this sec-
tion, it is now well accepted that the antiosteogenic
pathways of leptin and of the Y receptor proceed via
distinct mechanisms.
The similarity between ob ⁄ ob and Y2
) ⁄ )
mice regard-
ing their bone phenotype, together with the increased
NPY levels in the hypothalamus of both models
[58,59], suggested a link between the mechanisms of
action of NPY and leptin in the regulation of bone
mass. Moreover, it led to the hypothesis that NPY

might be a common mediator underlying the high
bone mass in these two mouse models [40]. However,
on comparison of the long bones of male Y2
) ⁄ )
and
ob ⁄ ob mice, an opposite effect between cortical and
trabecular bone is observed under conditions of leptin
deficiency, whereas in Y2
) ⁄ )
mice, both cortical and
trabecular bone mass are increased [60]. These findings
suggest that the Y2 and leptin antiosteogenic path-
ways occur via distinct mechanisms, thereby showing
diversity in the hypothalamic control of bone homeo-
stasis.
To further investigate the consequences of the above
findings, the effect of Y2 depletion on bone cell activ-
ity was studied under conditions of elevated leptin and
NPY by overexpressing NPY in the hypothalamus of
Y2
) ⁄ )
mice [25]. These animals had a marked increase
in leptin levels, and thereby an increase in body weight
and adipose mass. As expected, this increase in NPY
and leptin levels led to a decrease in bone formation
[25]. This was observed when NPY was overexpressed
in both Y2
) ⁄ )
and WT mice. However, Y2
) ⁄ )

mice
maintained a two-fold increase in osteoblast activity as
compared with WT mice [25], demonstrating that the
osteogenic activity of Y2
) ⁄ )
was preserved, and there-
fore clearly suggesting distinct actions of Y2 and leptin
in the regulation of osteoblast activity: whereas
increased leptin levels decrease bone formation, Y2
deletion activates osteoblast activity.
More recently, to further investigate the link between
the anabolic pathways of leptin and Y2 deficiencies,
genetic studies were performed to assess the effect of
specific Y receptor deletions on a leptin-deficient back-
ground. Interestingly, Y2
) ⁄ )
ob
) ⁄ )
double-knockout
mice had a decrease in bone volume relative to the single
knockout Y2
) ⁄ )
mice (Table 1), suggesting that some
interaction between leptin and the Y2 pathway might
occur [61]. In fact, future studies are still needed to
further understand the interaction between leptin, NPY
and bone.
Nonhypothalamic control of bone – Y1
In addition to the presence of NPY-immunoreactive
fibers, local NPY production in bone cells has been

reported recently [49,50]. This local production indi-
cates the possibility of an alternative pathway to the
central regulation of bone homeostasis. However,
the two independent in vitro studies showing local
NPY production in bone cells gave conflicting results
concerning the implications of NPY for osteoblast
differentiation. This discrepancy is probably related
to the different approaches used and the distinct
questions addressed. Igwe et al. [49,50] analyzed the
role of NPY in osteoblast differentiation with the use
NPY and osteoblast differentiation F. Franquinho et al.
3668 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS
of mouse calvarial osteoblasts in the presence of
NPY, whereas Nunes et al. [49,50] used primary bone
marrow stromal cells isolated from transthyretin (TTR)
knockout mice (which display high levels of NPY in
the brain and bone), without NPY treatment. There-
fore, the direct effect of local NPY on bone cells
remains poorly understood and requires additional
analysis.
The in vitro actions of NPY on osteoblasts suggested
the existence of Y receptors in this cell type [37,43]. In
fact, Y1 was found to be already highly expressed in
bone marrow stromal cells and bone marrow osteopro-
genitor cells differentiating to the osteoblast lineage
[40,46–48,62]. Its expression is downregulated in Y2
) ⁄ )
mice, given the elevated NPY levels in these animals
[46]. This finding is consistent with in vitro studies
showing that NPY treatment results in a significant

decrease of the Y1 transcript in differentiating osteo-
blasts [58]. Moreover, osteoblastic differentiation in
cultured osteoprogenitor cells was recently shown to
be enhanced following NPY treatment, probably
owing to downregulation of Y1 expression [58]. These
data are in contrast to recent findings, which have led
to NPY being described as the factor responsible for
decreased osteoblast differentiation in vitro [62]. Never-
theless, despite the controversy, the above data support
a direct role of Y1 signaling in the control of osteo-
blast biology.
Besides the central control exerted by Y2, there are
increasing data suggesting the importance of Y1 in
bone homeostasis [46,57,62]. To test this hypothesis,
germline deletion of Y1 in mice was recently per-
formed [62]. These animals were shown to have high
bone mass, with increased osteoblast activity on both
cancellous and cortical bone [62] (Table 1). Moreover,
Y1
) ⁄ )
bone marrow stromal cells formed more miner-
alized nodules, osteoprogenitor cells showed increased
proliferation and osteogenesis, and Y1
) ⁄ )
mature
osteoblasts had increased mineral-producing ability
[63]. In summary, these data suggests that NPY, via
Y1, directly inhibits the differentiation of mesenchymal
progenitor cells as well as the activity of mature osteo-
blasts, providing a likely mechanism for the high bone

mass phenotype of Y1
) ⁄ )
mice [63]. Additionally,
when targeted deletion of Y1 was performed in the
hypothalamus, bone density was not altered, further
supporting the specific role of Y1 in the local control
of bone remodeling [62].
As detailed above, the presence Y1 in osteoblasts
and other peripheral tissues suggests that, in addition
to a neural circuit, systemic factors may also interact
with Y1. It is therefore possible that these factors
converge on Y1 to modulate peripheral processes. To
test this possibility, the interaction of Y1 with several
known regulators of bone, including leptin, sex
steroids and NPY, was assessed in in vivo models [64].
This study demonstrated that androgens are required
for activation of the bone anabolic response in Y1
) ⁄ )
mice. Interestingly, an increased hypothalamic NPY
level was able to reduce osteoblast activity in WT and
Y1
) ⁄ )
mice, but Y1
) ⁄ )
mice retained higher osteoblast
activity. In consequence, it was suggested that other
signals (probably acting through androgens), and not
only changes in NPY activity, are needed for the
anabolic activity of Y1
) ⁄ )

mice.
In summary, deletion of either Y1 or Y2 results in
increased bone formation. Whereas the Y2 response is
mediated centrally, the Y1 response is mediated by
osteoblastic Y1. Thus, hypothalamic signals sustain a
systemic regulatory influence via Y2, whereas osteoblas-
tic Y1 enables additional local control of the systemic
response. However, it is debatable whether the effect of
Y1 results only from local production of NPY. Thus,
further studies are needed to fully assess the direct role
of NPY and Y1 in bone remodeling.
The NPY–Y2–Y1 crosstalk
As referred to above, deletion of Y2 downregulates Y1
expression in bone marrow stromal cells, suggesting
that impaired Y1 signaling might contribute to the
high bone mass phenotype of Y2
) ⁄ )
mice [46]. Alone,
this would suggest a common signaling pathway for
the regulation of bone homeostasis. Furthermore, no
additive effects were observed in mice lacking both Y1
and Y2 [62]. However, whereas the increased bone
volume in Y2
) ⁄ )
mice is caused by increased bone
formation, the increased bone volume in Y1
) ⁄ )
mice
results from altered bone turnover, with enhancements
of both osteoblast and osteoclast activity [62]. In view

of these findings, it was suggested that Y1 and Y2
might act at different points along a common signaling
pathway. In this respect, it has been recently shown
that NPY induces Y2 upregulation and Y1 downregu-
lation in osteoblasts, stimulating the differentiation of
bone marrow stromal cells [57]. Therefore, given the
complexity of the NPY–Y2–Y1 crosstalk, further
research is needed to explore in more detail the rela-
tionships among the signaling evoked by Y1 and Y2
and osteoblast activity. Also, several questions remain
to be answered concerning the direct action of NPY
on osteoblasts, as well as in relation to the mechanisms
underlying the regulation of bone homeostasis via Y2:
can the effects of Y2 be exclusively attributed to the
hypothalamus, or should a peripheral pathway be con-
sidered?
F. Franquinho et al. NPY and osteoblast differentiation
FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3669
Y4 – an additional player in bone
remodeling?
As described above, Y1 and Y2 have been clearly
linked to bone biology. No information existed, how-
ever, concerning the remaining Y receptors until germ-
line deletion of Y4 was produced [65]. Although bone
mass was unaltered in Y4
) ⁄ )
mice (Table 1), a syner-
gistic relationship in the regulation of bone metabolism
was described between the Y2 and Y4 pathways. Dele-
tion of both Y2 and Y4 increased cancellous bone vol-

ume in male mice to a greater level than that observed
in Y2
) ⁄ )
mice [65]. This increase in the bone volume
of Y2
) ⁄ )
Y4
) ⁄ )
double knockouts was associated with
a general increase in bone turnover. It is noteworthy
that this was associated with a significant reduction in
serum leptin level in male Y2
) ⁄ )
Y4
) ⁄ )
mice as com-
pared with WT mice or single-knockout Y2
) ⁄ )
mice
[60]. This synergistic effect and the decreased leptin
levels are absent in female mice, suggesting a gender
specificity of the bone response.
Further assessment of the role of NPY
in the control of bone homeostasis –
the NPY knockout and NPY
overexpressor models
As discussed above, despite the actions reported for
NPY and Y receptors in the control of bone biology,
the role of NPY in this process remains to be defined
precisely. In this respect, the initial report on NPY

) ⁄ )
mice, by showing no changes in bone volume in this
animal model, raised important doubts concerning the
control of bone activity by this neuropeptide [66].
However, one should bear in mind that, although
NPY is their main ligand, the Y receptors can also be
activated by peptide YY and pancreatic polypeptide.
Consequently, it was hypothesized that this redun-
dancy may underlie the lack of a bone phenotype in
NPY
) ⁄ )
mice [67]. In contrast to the observations in
NPY
) ⁄ )
mice, the same group showed a significant
increase in bone mass following loss of arcuate nucleus
NPY-producing neurons [66]. To further substantiate
the role of NPY in the control of bone homeostasis, a
recent study employed several NPY mutant mouse
models including specific reintroduction of NPY into
the hypothalamus of adult NPY
) ⁄ )
mice [67]. In this
more recent study, and in contrast to what was previ-
ously reported, NPY
) ⁄ )
mice were described as having
significantly increased bone mass resulting from an
enhanced osteoblast activity (Table 1). This generalized
bone anabolic response resulting from loss of NPY sig-

naling was evident throughout the skeleton, including
cortical and cancellous bone [67]. When NPY was spe-
cifically overexpressed in the hypothalamus of WT and
NPY
) ⁄ )
mice, a significant reduction in bone mass was
produced, despite the development of an obese pheno-
type [67]. This hypothalamic NPY-induced loss of
bone mass agrees with models that mimic the effects of
fasting, as they also show increased hypothalamic
NPY levels. Thus, the authors concluded that their
data support the hypothesis that the skeletal tissue also
responds to hypothalamic perception of nutritional sta-
tus, independently of body weight. It is, however,
important to note that the reduction in bone mass
caused by NPY administration in the hypothalamus
did not totally reverse the high bone mass of NPY
) ⁄ )
mice, suggesting that peripheral NPY may also be an
important regulator of bone mass. In conclusion, this
study further reinforced the hypothesis that central cir-
cuits alone fail to explain NPY signaling in the bone;
that is, local paracrine⁄ autocrine control of osteoblast
activity by NPY needs to be considered. Several previ-
ous studies had already examined the effect of exoge-
nous NPY administration on bone mass. Whereas ICV
infusion of NPY decreased bone mass [25], vector-
mediated overexpression of NPY in the hypothalamus
of WT mice resulted in no alteration in cancellous
bone volume, although osteoblast activity, estimated

by osteoid width, was markedly reduced following
adeno-associated virus (AAV)–NPY injection [61,64].
However, with regard to this central NPY overexpres-
sion, the consequential increase in leptin levels [68,69],
was not excluded as the cause of the effects observed.
Besides delivery of NPY, the TTR knockout mouse
(TTR
) ⁄ )
) has been described as a model of increased
NPY, given the overexpression of peptidylglycine
a-amidating monooxygenase (PAM) [70], the rate-limit-
ing enzyme in the process of neuropeptide maturation
[71]. As NPY requires PAM-mediated a-amidation for
biological activity [72], PAM overexpression in TTR
) ⁄ )
mice results in increased levels of processed amidated
NPY, without an increase in NPY expression [50]. As
expected, this strain has increased NPY content in the
brain and bone, and this finding was related to
increased bone mineral density and trabecular volume,
arguing against the generalized antiosteogenic activity
of NPY. In agreement with these observations, TTR
) ⁄ )
bone marrow stromal cells had increased NPY levels
and exhibited enhanced competence in undergoing
osteoblastic differentiation. In the case of TTR
) ⁄ )
mice, one should, however, bear in mind that it is
possible that, as a consequence of PAM overexpression,
increased levels of other amidated neuropeptides may

produce some complexity. Despite this concern, the use
of TTR
) ⁄ )
mice as a model of increased NPY offers
NPY and osteoblast differentiation F. Franquinho et al.
3670 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS
the advantage that, in addition to the increased NPY
levels, the level of leptin is not altered in this animal
model [73], excluding its interference in the bone
phenotype observed. In summary, the TTR
) ⁄ )
mouse,
an additional model displaying increased NPY levels,
suggests that increased levels of NPY locally in the
bone might be related to increased bone mass and
increased osteoblast activity, in agreement with the
recent report showing enhanced osteoblastic differentia-
tion in vitro in the presence of NPY [58]. However, the
limitation introduced by the fact that, in TTR-deficient
mice, the resulting bone phenotype can be attributable
to increases in other amidated neuropeptides, rather
than NPY, stresses the need to use additional
approaches and models to understand the role of NPY
signaling in bone.
Conclusions
There is now increasing evidence that the NPY system
is a player in the regulation of bone homeostasis, and
more specifically of osteoblast activity, through central
and peripheral mechanisms (Fig. 2). Most of this body
of knowledge has been derived from the analysis of

Y receptor knockout mice. Therefore, the majority of
the studies discussed in this review regarding the
involvement of NPY in bone metabolism have been
generated with mice as a model. The relevance of this
network in humans has not yet been addressed. There
is an urgent need to complement these studies with
clinical research, to further confirm their relevance and
to prepare for the future design of new therapeutic
strategies for bone disease ⁄ injury.
Y1 and Y2 have been shown to be independently
involved in the control of bone formation, whereas a
possible synergistic interaction between Y4 and Y2 has
been described. However, it remains to be established
whether other Y receptors are also involved in bone
remodeling. Moreover, the crosstalk between the dif-
ferent Y receptors in this process is still obscure. Addi-
tionally, the direct effect of local NPY on bone cells
remains controversial. What would be the effects of
direct NPY injection into the bone? What is the signifi-
cance and what are the consequences of local NPY
expression by different bone cell types? We should
now not only concentrate on understanding the impli-
cations of these novel findings, but also explore them
with new experimental designs to better understand
them.
In summary, the biology of the control of bone mass
by NPY still needs to be further explored, as not only
do several questions remain open, but also controversy
still exists: how is the balance between the neuro-osteo-
genic network and local NPY control actually

achieved?
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