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
Trang 1Neuropeptide Y and osteoblast differentiation – the
balance between the neuro-osteogenic network and local control
Filipa Franquinho1,2,*, Ma´rcia A Liz1,*, Ana F Nunes3, Estrela Neto4,5, Meriem Lamghari4 and Mo´nica M Sousa1
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: msousa@ibmc.up.pt
Website: http://www.ibmc.up.pt/nerve
*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 receprecep-tor 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.
Trang 2However, 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]
Trang 3(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
Osteoblast activity
Osteoclast activity Other observations References
Decreased CBV
Increased in trabecular bone
Increased Increased NPY levels 25,46
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) ⁄ )
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
NPY Leptin levels not altered
50
Trang 4besides 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 mechmech-anism 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
Y2 Y1
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.
Trang 5in 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
Trang 6of 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?
Trang 7Y4 – 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
Trang 8the 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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