REVIEW ARTICLE Neuropeptide Y and osteoblast differentiation – the balance between the neuro-osteogenic network and local control ´ Filipa Franquinho1,2,*, Marcia A Liz1,*, Ana F Nunes3, Estrela Neto4,5, Meriem Lamghari4 and ´ Monica M Sousa1 Nerve Regeneration Group, IBMC – Instituto de Biologia Molecular e Celular, Universidade Porto, Portugal ´ ´ ´ Departamento de Anatomia Patologica, Instituto Politecnico de Saude-Norte, Paredes, Portugal iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal ´ INEB – Instituto de Engenharia Biomedica, Divisao de Biomateriais, NewTherapies Group, Universidade Porto, Portugal ˜ Universidade Porto, Faculdade de Engenharia, Portugal 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 June 2010, accepted 12 July 2010) 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 receptor 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 mechanisms through which the balance between central and the peripheral NPY action might be achieved doi:10.1111/j.1742-4658.2010.07774.x 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 osteogenesis [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, autocrine ⁄ paracrine signals and mechanical loading [6–8] 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 F Franquinho et al 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 system and bone This concept was first described in the 1980s, but only recently have its molecular and mechanistic 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-osteogenic network, particularly the regulation of bone formation 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 classic histological methods, the presence of intense innervations of bone in animals and humans was shown [11–13] More details were unraveled as the technology advanced: in 1966, electron microscopy images of densely innervated cortical bone were published, and in 1969, myelinated and nonmyelinated nerve fibers associated 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 frequent after birth [15] These observations were confirmed 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 periosteum of the shaft, and the bone marrow With regard to NPY nerve fibers, their presence at postnatal day was shown in diaphyseal regions, and at postnatal days 6–8, these fibers were able to extend into the NPY and osteoblast differentiation 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 skeletal 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 immunohistochemistry analysis have shown that both myelinated and unmyelinated afferent (sensory) and efferent (autonomic) 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 innervations [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 dopamine hydroxylase immunoreactivity Moreover, in the calvarial periosteum and diploe, the presence of sensory 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 sensory, 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 orexigenic peptides NPY and agouti-related protein [26] Initially, the existence of multiple metabolic abnormalities 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] FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3665 NPY and osteoblast differentiation F Franquinho et al Table Summary of the bone phenotype in animal models for leptin and for the NPY system CBV, cortical bone volume; ND, not determined; TBV, trabecular bone volume Animal model Deficiency Bone phenotype Osteoblast activity Osteoclast activity Other observations References ob ⁄ ob Leptin Increased NPY levels 25,46 Leptin receptor Y2 Increased in trabecular bone Increased Increased Increased db ⁄ db Y2) ⁄ ) Increased TBV Decreased CBV Increased TBV Increased TBV and CBV Normal Normal 25 45,51 Y2) ⁄ )ob) ⁄ ) Leptin and Y2 Increased Increased Y1) ⁄ ) Y1 Decreased TBV and CBV in relation to Y2) ⁄ ) Increased TBV and CBV ND Increased NPY levels Normal leptin levels Increased NPY levels Increased Y4 Normal Normal Y2) ⁄ )Y4) ⁄ ) NPY) ⁄ ) TTR) ⁄ ) Y4 and Y2 NPY Transthyretin Increased TBV in relation to Y2) ⁄ ) Increased TVB and CBV Increased bone mineral density and TBV Increased Increased Increased Increased Normal Normal No inhibitory effects of NPY detected Normal NPY and leptin levels Increased NPY levels ND Increased amidated NPY Leptin levels not altered 62 Y4) ⁄ ) Increased in trabecular bone Normal (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 mediated 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 trabecular 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, particularly 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 depression) 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 3666 45,61 65 65 66 50 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 antiosteogenic 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 pathway 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 dramatically 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 marrow supported this vasoregulatory role [41,42] However, 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 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS F Franquinho et al 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 possibility that NPY might be directly involved in the regulation 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, chondrocytes 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 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 NPY and osteoblast differentiation NPY Peripheral nervous system Bone Circulating NPY in the blood NPY Bone microenvironment NPY ? Y1 Y2 NPY Osteoblasts Fig 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 regulated 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 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 mechanism and not by a direct mechanism in bone cells Supporting this hypothesis, just weeks following conditional 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 hypothalamus inhibits bone formation [51] It is important to note that obvious endocrine imbalances that would otherwise impact on bone homeostasis were not found FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3667 NPY and osteoblast differentiation F Franquinho et al 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 deficiency after menopause In this respect, it has been shown that the elevated osteoblast activity that characterizes the skeletal phenotype of Y2) ⁄ ) mice is maintained following gonadectomy in both female and male mice, and that the protection against gonadectomyinduced bone loss is also evident following hypothalamus-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 transiently increases NPY expression in the hypothalamus [54], which could contribute to the bone loss associated with this condition The topic of NPY and sex hormone interactions in bone and fat control has been recently reviewed [55] In summary, increased knowledge about the link between NPY and sex hormones in regulating bone biology could lead to better treatments 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 preosteoblasts 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 hypothalamic Y2, if the existence of Y2 in osteoblasts is further 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 section, 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 regarding 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 3668 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 pathways occur via distinct mechanisms, thereby showing diversity in the hypothalamic control of bone homeostasis To further investigate the consequences of the above findings, the effect of Y2 depletion on bone cell activity 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 therefore 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 background 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 indicates 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 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS F Franquinho et al 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 Therefore, 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 osteoprogenitor 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 osteoblasts [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] Nevertheless, despite the controversy, the above data support a direct role of Y1 signaling in the control of osteoblast 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 performed [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 mineralized 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 osteoblasts, 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 NPY and osteoblast differentiation 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 osteoblastic 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 downregulation 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 relationships 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 considered? FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS 3669 NPY and osteoblast differentiation F Franquinho et al Y4 – an additional player in bone remodeling? As described above, Y1 and Y2 have been clearly linked to bone biology No information existed, however, concerning the remaining Y receptors until germline deletion of Y4 was produced [65] Although bone mass was unaltered in Y4) ⁄ ) mice (Table 1), a synergistic relationship in the regulation of bone metabolism was described between the Y2 and Y4 pathways Deletion of both Y2 and Y4 increased cancellous bone volume 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 compared 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 redundancy 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 previously 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 signaling was evident throughout the skeleton, including 3670 cortical and cancellous bone [67] When NPY was specifically overexpressed in the hypothalamus of WT and NPY) ⁄ ) mice, a significant reduction in bone mass was produced, despite the development of an obese phenotype [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 status, 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 circuits 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 previous studies had already examined the effect of exogenous NPY administration on bone mass Whereas ICV infusion of NPY decreased bone mass [25], vectormediated 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 overexpression, 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-limiting 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 FEBS Journal 277 (2010) 3664–3674 ª 2010 The Authors Journal compilation ª 2010 FEBS F Franquinho et al NPY and osteoblast differentiation 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 differentiation 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 Fat tissue Hypothalamus NPY Leptin Y2 Circulating NPY Sympathic nervous system NPY NPY Y1 Y2 NPY Osteoblasts Fig NPY regulatory network NPY exerts its actions through both central and peripheral pathways 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 different Y receptors in this process is still obscure Additionally, 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 significance and what are the consequences of local NPY expression by different bone cell types? We should now not only concentrate on understanding the implications 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 several questions remain open, but also controversy still exists: how is the balance between the neuro-osteogenic network and local NPY control actually achieved? 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(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. .. 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... injury Fat tissue Hypothalamus NPY Leptin Y2 Circulating NPY Sympathic nervous system NPY NPY Y1 Y2 NPY Osteoblasts Fig NPY regulatory network NPY exerts its actions through both central and peripheral