Neuropeptide Y expression and function during osteoblast differentiation – insights from transthyretin knockout mice Ana F. Nunes 1,2, *, Ma ´ rcia A. Liz 1 , Filipa Franquinho 1 , Liliana Teixeira 3 , Vera Sousa 1 , Chantal Chenu 4 , Meriem Lamghari 3,  and Mo ´ nica M. Sousa 1,  1 Nerve Regeneration, IBMC – Instituto de Biologia Molecular e Celular, Universidade do Porto, Portugal 2 ICBAS, Universidade do Porto, Portugal 3 INEB – Instituto de Engenharia Biome ´ dica, Divisa˜o de Biomateriais, Universidade do Porto, Portugal 4 Department of Veterinary Basic Sciences, The Royal Veterinary College, London, UK Introduction The regulation of bone remodeling has been conven- tionally linked to local factors, hormones, and mechanical loading [1–3]. However, in the last decade, several reports have provided evidence that bone homeostasis is also under the influence of central and peripheral neural control [4–8]. This concept is sup- ported by a number of histological studies revealing the existence of neuropeptide fibers and neuropeptide Keywords amidated neuropeptide; bone marrow stromal cells; bone mass; NPY; osteoblastic differentiation Correspondence M. M. Sousa, IBMC, Rua Campo Alegre 823, 4150-180 Porto, Portugal Fax: +351 22 6099157 Tel: +351 22 6074900 E-mail: msousa@ibmc.up.pt Website: http://www.ibmc.up.pt/nerve *Present address iMed.UL, Faculty of Pharmacy, University of Lisbon, Portugal These authors contributed equally to this work (Received 12 November 2009, revised 3 November 2009, accepted 5 November 2009) doi:10.1111/j.1742-4658.2009.07482.x To better understand the role of neuropeptide Y (NPY) in bone homeosta- sis, as its function in the regulation of bone mass is unclear, we assessed its expression in this tissue. By immunohistochemistry, we demonstrated, both at embryonic stages and in the adult, that NPY is synthesized by osteoblasts, osteocytes, and chondrocytes. Moreover, peptidylglycine a-am- idating monooxygenase, the enzyme responsible for NPY activation by amidation, was also expressed in these cell types. Using transthyretin (TTR) KO mice as a model of augmented NPY levels, we showed that this strain has increased NPY content in the bone, further validating the expression of this neuropeptide by bone cells. Moreover, the higher ami- dated neuropeptide levels in TTR KO mice were related to increased bone mineral density and trabecular volume. Additionally, RT-PCR analysis established that NPY is not only expressed in MC3T3-E1 osteoblastic cells and bone marrow stromal cells (BMSCs), but is also detectable by RIA in BMSCs undergoing osteoblastic differentiation. In agreement with our in vivo observations, in vitro, TTR KO BMSCs differentiated in osteoblasts had increased NPY levels and exhibited enhanced competence in undergo- ing osteoblastic differentiation. In summary, this work contributes to a better understanding of the role of NPY in the regulation of bone forma- tion by showing that this neuropeptide is expressed in bone cells and that increased amidated neuropeptide content is related to increased bone mass. Abbreviations ALP, alkaline phosphatase; BMD, bone mineral density; BMSC, bone marrow stromal cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; KO, knockout; microCT, micro computed tomography; NF200, neurofilament 200; NPY, neuropeptide Y; PAM, peptidylglycine a-amidating monooxygenase; PGP9.5, protein gene product 9.5; RANK, receptor activator of nuclear factor-jB; T 4, thyroxine; TTR, transthyretin. FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 263 receptors in bone [9]. Neuropeptide Y (NPY)-immuno- reactive fibers have been found to be mostly distri- buted in association with blood vessels and in the periosteum [10–12]. NPY immunoreactivity was dra- matically reduced in sympathectomized animals, indi- cating the sympathetic origin of these nerves [11]. Despite the fact that NPY-containing nerve fibers have been described in the bone, no data exist concerning the expression of this neuropeptide in bone cells. How- ever, NPY has been detected in the periosteum and bone marrow by RIA [13], particularly in megakaryo- cytes [14]. Recently, it was additionally reported that the NPY receptor Y1, but not Y2, Y4, Y5 or Y6, was expressed in cultured bone marrow stromal cells (BMSCs) and osteoblasts [15]. Despite the existence of NPY fibers and one of its receptors in the bone, NPY knockouts (KOs) have normal bone mass, questioning a role for NPY control in bone activity [8]. On the other hand, different mouse models that have in common the fact that they present increased NPY levels, the Y2 receptor-KO and the leptin-deficient and leptin receptor-deficient mouse (ob ⁄ ob and db ⁄ db mice, respectively), display a high cancellous bone mass phenotype associated with increased osteoblast activity [5,7,16], supporting a role for NPY in bone biology. In the case of ob ⁄ ob mice and db ⁄ db mice, there is increased NPY activity in the hypothalamus, owing to the lack of leptin-induced inhibition of NPY expression [16]. Y2 receptor KO and leptin-deficient mice share key characteristics, with similar increases in cancellous bone mass and NPY levels in the hypothalamus, suggesting a commonality of mechanism. However, it was recently shown that leptin and Y2 receptor pathways independently modu- late cancellous bone homeostasis [17]. With regard to Y2 receptor-deficient mice, both germline and condi- tional hypothalamic Y2 receptor KO mice share the same high bone mass phenotype [5], demonstrating that central hypothalamic Y2 receptors are crucial for this process. Interestingly, although germline Y1 recep- tor KO mice also display increased bone formation, conditional deletion of hypothalamic Y1 receptors did not alter bone homeostasis, suggesting a nonhypotha- lamic control of bone mass [6]. The Y1 receptor being the only NPY receptor identified in the bone, these results suggest that absence of NPY signaling in the bone (as occurs in Y1 receptor-deficient mice) results in increased bone mass. NPY effects in bone mass have been further inves- tigated by exogenous administration. Whereas intra- cerebroventricular infusion of NPY decreased bone mass [7], vector-mediated overexpression of NPY in the hypothalamus of wild-type mice resulted in no alteration in cancellous bone volume, although osteo- blast activity, estimated using osteoid width, was markedly reduced following adeno-associated virus NPY injection [17,18]. These results are not in accor- dance with the cancellous bone phenotype of the above-mentioned mouse models of elevated NPY lev- els. All of these opposing results make necessary a closer look at the role of NPY in the regulation of bone mass. Transthyretin (TTR) KO mice show increased levels of amidated neuropeptides, owing to overexpression of peptidylglycine a-amidating monooxygenase (PAM) [19], the only enzyme that a-amidates peptides, and which is rate-limiting in the process of neuropeptide maturation, as its substrates exist in excess [20,21]. Among the neuropeptides that are amidated by PAM, NPY is the most abundant in both the central and the peripheral nervous systems. As NPY requires PAM- mediated a-amidation for biological activity [22], PAM overexpression in TTR KO mice results in increased levels of processed amidated NPY, without an increase in NPY expression [19]. As a consequence of the increased amidated NPY levels, TTR KO mice show a significant NPY overexpressor phenotype. Given the lack of information on the expression of NPY in the bone, together with the controversy con- cerning its function in bone homeostasis, we aimed at gaining a better understanding of the role of this neu- ropeptide in the control of bone mass by making use of TTR KO mice, a model of increased NPY. Results In bone, NPY is detected in chondrocytes, osteoblasts, and osteocytes NPY expression was investigated in wild-type (WT) and TTR KO bone tissue by immunohistochemistry, using an antibody specific for the amidated form of NPY. NPY immunolabeling was observed in bone marrow cells, including megakaryocytes (Fig. 1Aa), as already described in the literature [14]. The periosteum (Fig. 1Ab) also showed NPY immunoreactivity, as already reported for mice and rats [10–12]. However, we observed NPY immunostaining in chondrocytes, osteoblasts, and osteocytes (Fig. 1Ac–f, respectively, arrows). No NPY immunoreactivity was found in osteoclasts (data not shown). Similar to our observa- tions in the adult bone, NPY immunoreactivity was detected starting at embryonic day 16 in megakaryo- cytes, osteoblasts, and chondrocytes; this NPY detec- tion pattern was maintained at embryonic day 18 (Fig. 1B). No immunoreactivity was detected when the NPY is expressed in osteoblasts A. F. Nunes et al. 264 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS A F CB D E ab c d b a c e f a b c d e BM M C C O C C C BM BM Anti-osteocalcin Anti-NPY P M Os Os O O O AC BM M Fig. 1. NPY immunohistochemistry in the bone tissue. BM, bone marrow; C, chondrocytes; O, osteoblasts; AC, articular chondrocytes; Os, osteocytes; M, megakaryocytes; P, periosteum. Scale bar: 50 lm. (A) NPY immunoreactivity in bone cells, namely bone marrow cells and megakaryocytes (a), periosteum (b), articular cartilage chondrocytes (c), late proliferating chondrocytes (d), osteoblasts (e), and osteocytes (f). Arrows indicate labeled cells, and fibers in the case of the periosteum. (B) NPY immunoreactivity in the bone at embryonic day 18, show- ing NPY staining in megakaryocytes (a), chondrocytes (b), and osteoblasts (c). (C) Immunohistochemistry in bone sections where the anti- body against NPY was replaced by mouse IgG. (D) NPY immunohistochemistry in NPY KO bone sections. (E) Comparison between NPY (right) and osteocalcin (left) immunolabeling in the bone tissue. Arrows indicate labeled osteoblasts. (F) Nerve fiber (NF200 and PGP9.5) immunohistochemistry: articular cartilage chondrocytes (a), proliferating chondrocytes (b), osteoblasts (c), bone marrow cells (d), and perios- teum (e). A. F. Nunes et al. NPY is expressed in osteoblasts FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 265 NPY antibody was replaced by mouse IgGs (Fig. 1C). Moreover, in NPY KO mouse bone sections, none of the different bone cells showed NPY immunostaining (Fig. 1D), suggesting that the immunoreactivity observed in WT and TTR KO bone tissue was NPY- specific. To further demonstrate NPY synthesis in os- teoblasts, osteoblast-specific staining was performed with an antibody against osteocalcin (Fig. 1E, left panel, arrow). The results obtained revealed that the pattern of staining was comparable to that obtained for NPY, as shown in the right panel of Fig. 1E, thus confirming NPY expression in osteoblasts. To further demonstrate that NPY is synthesized in these bone cells, additional negative controls were performed. Using antibody against neurofilament 200 (NF200) or antibody against protein gene product 9.5 (PGP9.5), two nerve fiber markers, no staining was observed in chondrocytes, osteoblasts, or bone marrow cells (Fig. 1Fa–d, respectively), whereas in the periosteum typical nerve fiber labeling was detected (Fig. 1Fe). TTR KO bone tissue has increased amidated NPY levels From the comparison between WT and TTR KO NPY immunoreactivity in bone sections, we observed that TTR KO bone tissue displayed increased amidated NPY levels when compared to the wild type (Fig. 2A, arrows), further demonstrating the expression of this neuropep- tide by bone cells. NPY immunostaining was increased in chondrocytes, osteoblasts, osteocytes, bone marrow cells and megakaryocytes (Fig. 2a–e, respectively) from TTR KO mice when compared to the same WT cells. This result is in accordance with the increased NPY lev- els reported in the nervous system of TTR KOs [19], sug- gesting that the increased NPY levels in this strain are not nervous system-restricted. Given the increased NPY levels in TTR KO bones, PAM expression was subse- quently evaluated in this tissue by immunohistochemis- try; PAM was detected in bone marrow cells, including megakaryocytes (Fig. 2Ba, arrows), osteoblasts, and A a a bc b c BM M M BM d e B C Fig. 2. Analysis of NPY and PAM in WT and TTR KO bone sections. Scale bar: 50 lm. (A) Comparison of NPY immunostaining in WT and TTR KO bone sections. Arrows indicate the different cell types in evidence in each panel, namely articular cartilage chondrocytes (a), proliferating chondrocytes (b), osteoblasts (c), osteocytes (d), bone marrow cells (BM) and megakaryocytes (M) (e). (B) PAM immunostaining in the bone marrow (a; arrows indicate megakaryo- cytes), osteocytes (b; arrows), osteoblasts (b;p arrowheads), and chondrocytes (c). (C) Quantification of the density of PAM immunostaining in the bone marrow of WT and TTR KO mice. a P < 0.05. NPY is expressed in osteoblasts A. F. Nunes et al. 266 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS osteocytes (Fig. 2Bb, arrowheads and arrows, respec- tively), as well as in chondrocytes (Fig. 2Bc). The major difference in PAM expression among WT and TTR KO bones was found in the bone marrow, where PAM immunostaining was approximately two-fold higher in TTR KO mice (Fig. 2C). Despite the fact that NPY and PAM expression were not observed in osteoclasts, the hypothesis that increased NPY levels in the bone of TTR KO mice may have an indirect effect on osteoclasts existed. To address this hypothesis, preosteoclasts and mature osteoclasts in WT and TTR KO bones were detected by OSCAR staining. Following quantification, no differences in osteoclast number were detected between strains (data not shown). TTR KO mice have increased bone mineral density (BMD) and trabecular volume To address whether the increased NPY levels observed in TTR KO femurs have physiological consequences in the bone, we started by comparing bone histology in WT and TTR KO mice. The femur length did not dif- fer significantly between strains (wild type, 15.6 ± 1.4 mm; TTR KO, 15.9 ± 1.0 mm). To fur- ther analyze in detail the bone phenotype, micro com- puted tomography (microCT) scanning analysis of femurs, including measurement of BMD, was per- formed. As shown in Fig. 3A (left and middle panels), two-dimensional trabecular number and thickness were increased in TTR KO femurs when compared with WT femurs. Furthermore, three-dimensional trabecular bone volume in the proximal metaphysis was also higher in TTR KO animals (Fig. 3A, right panel). From the statistical analysis of WT (n = 9) and TTR KO (n = 10) femurs, the results obtained demonstrate an increased trabecular volume (bone volume ⁄ trabecu- lar volume) and BMD in TTR KO mice when com- pared with WT littermates (Fig. 3B). These results suggest that increased amidated neuropeptide levels are related to increased bone density and volume. The increase in bone volume was, however, detected only in trabeculae, whereas the bone cortex was unaffected. This result suggested that the process of endochondral ossification might be specifically affected. To assess this hypothesis, the growth plates of WT and TTR KO mice were analyzed. As can be seen in Fig. 3C, no differences were detectable by histological analysis of growth plates from WT and TTR KO mice. NPY is expressed in osteoblasts To further address NPY expression in bone cells, namely in the osteoblastic cell line MC3T3-E1, and in primary cultures of BMSCs throughout osteoblastic differentiation, we performed RT-PCR analysis of NPY expression. Using brain as the positive control of NPY expression, we detected NPY in MC3T3-E1 cells and in both WT and TTR KO BMSCs (Fig. 4A). Fur- thermore, both WT and TTR KO BMSCs on days 3, 7 and 14 of culture in osteogenic differentiation media showed NPY expression; no statistical differences were observed between WT and TTR KO BMSC cultures throughout the differentiation period (data not shown). To determine whether TTR KO mice BMSCs undergoing osteoblastic differentiation recapitulate our findings in the nervous system, i.e. show increased PAM transcription and increased levels of amidated NPY, without increased NPY mRNA expression, we quantified PAM expression and the levels of the bio- logically active neuropeptide in differentiating WT and TTR KO BMSC cultures. As expected, TTR KO mice BMSCs displayed increased amidated NPY levels (approximately 2.4-fold at day 3) when compared to WT cells (Fig. 4B). Despite the fact that the NPY con- tent decreased over the 14 days of differentiation, indi- cating that undifferentiated BMSCs have higher levels of NPY than differentiated osteoblasts, these still expressed amidated neuropeptide. One should, how- ever, note that in WT BMSCs, NPY levels were not altered throughout the course of BMSC differentiation (days 3–14; Fig. 4B). Therefore, NPY should not be regarded as either a marker of osteoblast differentia- tion or a marker of mature osteoblasts. In agreement with the increased NPY levels, PAM expression in TTR KO BMSCs was increased, with a similar fold change as that observed for the levels of amidated NPY (Fig. 4C). Y1 expression was detected by RT-PCR in differentiating WT and TTR KO BMSCs, with no Y2 or Y5 receptor amplification (data not shown), in accordance with recently published results [15]. However, no statistical difference was observed between the two strains regarding Y1 expression (data not shown). TTR KO BMSCs show increased osteoblast differentiation To examine whether WT and TTR KO BMSCs differ in their capability to undergo osteoblast differentia- tion, as a possible consequence of their differential am- idated NPY content, isolated BMSCs from WT and TTR KO mice were cultured under osteoblast differen- tiation conditions. Osteoblast phenotype markers such as alkaline phosphatase (ALP) activity and osteocalcin expression were determined. In both cultures, ALP activity increased in a time-dependent manner and A. F. Nunes et al. NPY is expressed in osteoblasts FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 267 A B C Fig. 3. MicroCT in WT and TTR KO mouse femurs. (A) Bone microarchitecture in WT and TTR KO mice. Left and middle panels: 2D microCT images of metaphyseal bone, showing reconstructed longitudinal sections (left panel) and transverse sections taken  1 mm from the growth plate (middle panel). The line crossing the transversal sections indicates the orientation of the longitudinal sections. Right panel: 3D mi- croCT images of metaphyseal trabecular bone in WT and TTR KO mice. (B) Quantifi- cation of trabecular volume [bone vol- ume ⁄ trabecular volume (BV ⁄ TV)] and BMD in WT and TTR KO mice. Results are pre- sented as average ± standard error of the mean. a P < 0.05. (C) Hematoxylin ⁄ eosin staining of the growth plate (femur) of WT and TTR KO mice (3 months old). Scale bars: 50 lm. A B C Fig. 4. NPY and PAM expression in bone cells from WT and TTR KO mice. (A) NPY RT-PCR analysis in brain, MC3T3-E1 cells, and BMSCs. (B) NPY quantification in BMSCs from WT and TTR KO mice at days 1, 3, 7 and 14 of differentiation into osteoblasts. (C) Semiquantitative RT-PCR analysis of PAM expression normalized for b-actin (left) or HPRT (right) expression in BMSCs from WT and TTR KO mice at days 3 and 14 of osteoblast differentiation. Results are presented as average ± stan- dard error of the mean; a P < 0.05. NPY is expressed in osteoblasts A. F. Nunes et al. 268 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS peaked on day 7, with significantly increased levels (ranging from two-fold to three-fold) being seen in TTR KO osteogenic cultures at days 3 and 7 when compared to WT cultures (Fig. 5A). Regarding osteo- calcin expression, WT cultures displayed a time-depen- dent increase in osteocalcin levels, with a peak of expression on day 14 (Fig. 5B), which is characteristic of the osteoblastic differentiation process in vitro.In the case of TTR KO BMSCs, no increase in osteocal- cin expression was observed from day 3 to day 7 of differentiation, probably because those cells already showed high osteocalcin levels at day 3 of differentia- tion (Fig. 5B). Nonetheless, at day 14, TTR KO cul- tures showed a significant increase in osteocalcin expression when compared with the WT cultures (Fig. 5B). To further confirm these data, RT-PCR was performed using additional housekeeping genes [those encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine-guanine phosphoribosyl- transferase (HPRT)] as well as osteopontin, an extra marker of osteoblastic differentiation. Day 3 of BMSC differentiation was chosen for performance of the con- firmation because, at this time point, not only ALP activity but also osteocalcin expression are increased in TTR KO BMSCs. The expression levels of both osteo- calcin (Fig. 5C) and osteopontin (Fig. 5D) were always increased in TTR KO BMSCs, irrespective of the housekeeping gene used to perform the normalization. Taken together, these data suggest that TTR KO BMSCs show enhanced competence in undergoing osteoblast differentiation in vitro. Discussion The data presented in this study demonstrate that NPY is expressed in several types of bone cell, with both in vitro and in vivo evidence. Moreover, we show that increased NPY levels are related to increased bone density, as well as to augmented competence in BMSC differentiation into osteoblasts. In agreement with our findings, a recent r eport further supports the contribution 18 16 14 12 10 8 ALP activity (nmolPNP·mg·h –1 ) 6 4 2 0 25 20 15 10 10 8 6 4 2 0 0 actin GAPDH HPRT GAPDH HPRT 2 4 6 8 10 12 14 b a a 5 0 Day 3 Day 3 Day 7 Day 7 c b b TTR KO TTR KO WT Day 14 Day 3 Day 7 Day 14 Day 14 WT WT TTR KO b b WT TTR KO WT osteocalcin osteocalcin/actin Day 3 osteocalcin/house keeping gene Day 3 osteopontin/house keeping gene β-actin KO WT KO WT KO B A C D Fig. 5. Osteoblast differentiation of WT and TTR KO BMSCs as assessed by ALP, osteocalcin and osteopontin levels. (A) ALP activ- ity of WT and TTR KO BMSCs under osteoblast differentiation con- ditions at days 3, 7 and 14. (B–D) Semiquantitative RT-PCR analysis in WT and TTR KO BMSCs of (B) osteocalcin expression, normal- ized for the expression of b-actin, at days 3, 7 and 14, (C) osteocal- cin expression normalized for the expression of GAPDH and HPRT at day 3, and (D) osteopontin expression, normalized for the expression of b-actin, GAPDH and HPRT at day 3 under osteoblast differentiation conditions. Results are presented as average ± stan- dard error of the mean; a P < 0.05; b P < 0.005; c P < 0.0005. A. F. Nunes et al. NPY is expressed in osteoblasts FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 269 of the NPY pathway in bone homeostasis via a direct action on osteoblasts [23]. In that report, it was shown that chronically elevated NPY levels modulate the lev- els of Y2 receptor expression (according to the stage of osteoblast differentiation) and that NPY is a nega- tive regulator of Y1 receptor expression. Moreover, functional analysis revealed the osteogenic potential of NPY, with osteoblast phenotype markers being signifi- cantly enhanced in osteoprogenitor cells stimulated by NPY, probably owing to downregulation of the Y1 receptor. Until now, NPY expression has only been detected in bone marrow cells, including megakaryocytes [14]. Here, we show for the first time that BMSCs also con- tribute to NPY in the bone marrow, as NPY is expressed both in BMSCs and in BMSCs undergoing osteoblastic differentiation. Moreover, this article is the first to report NPY expression in chondrocytes, osteoblasts, osteocytes and the osteoblastic cell line MC3T3-E1. In relation to chondrocytes, no studies were performed regarding the role of NPY in the dif- ferentiation of this cell type. This could probably be the aim of a subsequent study, where possible differ- ences in articular cartilage or growth plate between WT and TTR KO bones should be addressed. In the case of osteoclasts, although NPY expression was not detected in this cell type, the elevated NPY levels in TTR KO bones might have some indirect effect on osteoclasts. In fact, we recently reported that NPY modulates receptor activator of nuclear factor-jB (RANK) ligand and osteoprotegerin, two key factors regulating bone remodeling [23]. The inhibitory effect of NPY on RANK ligand production by BMSCs was also investigated by Amano et al. [24], who suggested that the inhibitory effect of NPY on osteoclastogenesis was caused by suppression of isoprenaline-induced RANK ligand production by stromal cells, upstream of RANK ligand mRNA expression. It is known that central NPY regulates bone mass, as conditional ablation of hypothalamic Y2 receptors results in increased bone formation [5]. Moreover, lep- tin-deficient mice, in which NPY is increased in the hypothalamus, show high cancellous bone mass, but reduced cortical production [25]. Central NPY can also influence peripheral tissues through alterations in auto- nomic neuronal activity. This is probably mediated by NPY projections from the hypothalamus to the brain- stem areas where sympathetic neuronal activity is mod- ulated [26]. Thus, to achieve its functions, NPY may act centrally on hypothalamic receptors and ⁄ or peripher- ally on its osteoblastic receptor Y1 after being released from sympathetic nerve terminals supplying the skeletal tissue. With this work, we have opened a new window in which NPY may additionally function as an auto- crine factor, as it is expressed by osteoblasts as well. We further demonstrate that TTR KO bone tissue displays increased amidated NPY levels, when com- pared to WT tissue, further demonstrating the expres- sion of this neuropeptide in bone cells. In theoretical terms, the major TTR ligands, thyroxine (T 4 ) and reti- nol, could be responsible, at least in part, for the bone phenotype observed in TTR KO mice. Retinol defi- ciency is known to increase BMD [27]; additionally, reti- noic acid inhibits osteogenic differentiation of BMSCs [28,29]. Despite the fact that TTR KO mice have retinol plasma levels below the level of detection [30], symp- toms of vitamin A deficiency are absent in these ani- mals. In agreement with this, their total retinol tissue levels are not significantly different from those of WT mice [31]. Moreover, retinoic acid plasma levels are two- fold to three-fold higher in TTR KO mice, probably compensating for their low retinol levels [31]. Taking the above into account, it is highly unlikely that, with normal retinol levels in tissues and increased retinoic acid levels in the plasma, an impairment in retinol homeostasis would be responsible for the increased BMD in TTR KO mice. Regarding thyroid hormones, it is well known that hyperthyroidism in adult patients leads to decreased BMD [32]. As expected, both total T 4 and tri-iodothyronine serum levels are decreased in TTR KO mice [32,33]. However, similar to what is described above for retinol, this decrease is unrelated to symptoms of hypothyroidism or thyroid gland abnor- malities [34]. Again, in terms of tissue content, TTR KO mice show no differences in T 4 levels from WT mice [35,36]. This euthyroid status probably arises as a conse- quence of the high free T 4 serum pool in the TTR KO mice [34]. Such a euthyroid status is essential for normal skeletal development and maintenance, and therefore it is hard to see how the bone phenotype of TTR KO mice could be related to thyroid hormones. It is additionally possible that in TTR KO mice, as a consequence of PAM overexpression, increased levels of other amidated neuropeptides may produce some complexity. In this respect, although contradictory results have been reported for the action in bone of some amidated neuropeptides, such as substance P, others, such as pancreatic polypeptide and calcitonin gene-related peptide, have been described as stimulat- ing the differentiation of MC3T3-E1 cells [37] or increasing the number of bone colonies formed from bone marrow stromal cells (MSC) in vitro [38], simi- larly to what is reported here in the absence of TTR. However, although not discarding the possible influ- ence of the putative increases in the levels of other amidated neuropeptides in this model, which should be NPY is expressed in osteoblasts A. F. Nunes et al. 270 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS addressed in future experiments, TTR KO mice not only show increased NPY levels when compared with other NPY overexpression models, but also present an accompanying NPY overexpression phenotype. This phenotype includes decreased energy expenditure, decreased depressive-like behavior, and increased car- bohydrate consumption and preference, and most of these features are not commonly observed in other NPY overexpression models [19]. It is noteworthy that the increased NPY levels in TTR KO mice are unre- lated to increased NPY mRNA expression, and result from increased processing and amidation by PAM, which is upregulated in TTR KO animals. In fact, although TTR is not expressed in BMSCs, PAM expression is increased in TTR KO BMSCs, suggesting that TTR KO osteoblasts have intrinsically augmented PAM expression in relation to WT cells, as a conse- quence of their physiological TTR-free environment. A similar finding was reported for TTR KO neurons (like BMSCs, neurons lack TTR expression), as these cells were also shown to display intrinsically decreased neurite outgrowth, as a consequence of their physio- logical TTR-free environment [39]. NPY control of bone mass is still controversial. On the one hand, there are two different mouse models with increased NPY expression that show high cancel- lous bone mass, the Y2 receptor KO mice [5] and mice lacking leptin (ob ⁄ ob mice) [7,16]. Although sharing a similar high cancellous bone phenotype, both models differ in cortical bone regulation, with increased corti- cal bone mass in Y2 receptor KO mice and decreased cortical density in ob ⁄ ob mice [23]. On the other hand, no NPY signaling in the bone, as is the case in Y1 receptor KO mice, leads to high bone mass [6], and central NPY overexpression yields decreased osteoblast activity [18] and bone mass [7], with no alteration in cancellous bone volume [17,18]. With regard to this central NPY overexpression, the consequential increase in leptin levels [40,41] cannot be excluded as the cause of the effects observed. Furthermore, the apparent dis- crepancy between Y1 and Y2 receptor KO models regarding NPY signaling and bone phenotype was recently clarified by the hypothesis that the increased central NPY levels observed in the Y2 receptor-defi- cient mice lead to Y1 receptor downregulation on bone cells, which would explain their increased bone mass phenotype [15]. The fact that deletion of both Y1 and Y2 receptors did not produce additive effects on increased bone mass further supports this hypothesis, as it suggests a common pathway from the hypothala- mus to the bone involving both Y2 and Y1 signaling [6], with probable central Y2 and peripheral Y1 effects on bone tissue. The NPY KO mouse is not very help- ful in this matter, as its bone mass is normal [8]. Here we show that in TTR KO mice, an additional model showing increased NPY levels, an increased cancellous bone mass phenotype is observed, in agreement with the Y2 receptor KO and ob ⁄ ob mouse phenotypes, fur- ther suggesting that increased NPY content might be related to increased cancellous bone mass. Despite all the concerns discussed above regarding the use of TTR KO mice as a model of increased NPY levels, the main advantage of these animals over other NPY over- expression models is that, in addition to the increase in NPY levels, the leptin level is not altered [42], exclud- ing its interference in the bone phenotype observed. In summary, we provide evidence that NPY is expressed in bone cells, namely in osteoblasts. Further- more, we report that in a model of increased amidated neuropeptide levels, showing an NPY overexpression phenotype, an increased bone mass phenotype is pres- ent. Finally, on the basis of these findings, further work is needed to determine the localization of NPY and NPY receptors during bone injury, disease, and aging, and thereby elucidate the possible role of NPY in the bone regeneration process. Experimental procedures Animals Mice were handled according to the European Communi- ties Council Directive (86 ⁄ 609 ⁄ EEC) and national rules, and all studies performed were approved by the Portuguese General Veterinarian Board. Male WT and TTR KO [33] littermate offspring of heterozygous breeding pairs, in the 129 ⁄ Sv background, were maintained at 24 ± 1 °C under a 12 h light ⁄ dark cycle and fed regular chow and tap water ad libitum. Prior to all experimental procedures, animals were anesthetized with ketamine (1 mgÆg )1 body weight) ⁄ mede- tomidine (0.02 lgÆg )1 body weight). Animals were killed with an overdose of anesthetic. Immunohistochemistry Femurs from 3 month old male WT (n = 6) and TTR KO (n = 5) littermates were fixed in 4% paraformaldehyde in NaCl ⁄ P i , decalcified in TBD-1 commercial solution (Thermo Electron Corporation), and embedded in paraffin; serial 4 lm thick longitudinal sections were then cut. For studies during embryonic development, 16 day or 18 day WT pregnant females were killed by cervical dislocation, and the fetuses were collected by cesarian section. Sections were then deparaffinized, dehydrated in a modified alcohol series, and blocked for the endogenous peroxidase activity. NPY immunohistochemistry was performed with the MOM Kit (Vector, Peterborough, UK), following the manufacturer’s A. F. Nunes et al. NPY is expressed in osteoblasts FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS 271 instructions. Briefly, bone sections from WT and TTR KO mice, as well as sections from NPY KO mice (prepared simi- larly to WT and TTR KO mouse samples; a kind gift from H. Herzog, Garvan Institute, Australia) were incubated in the MOM kit blocking reagent for 1 h at room temperature, prior to incubation with the monoclonal NPY antibody NPY05 (generously provided by E. Grouzmann, University Hospital, Lausanne, Switzerland; diluted 1 : 2000 in MOM diluent) for 1 h at room temperature. NPY05 is specific for the amidated form of NPY [30]. Antigen visualization was performed with the MOM avidin–biotinylated peroxidase complex reagent (Vector), using 3-amino-9-ethyl carbazole (Sigma, Lisbon, Portugal) as substrate. On parallel control sections, the primary antibody was replaced by mouse IgG (Sigma). Immunohistochemical investigations for NF200 and PGP9.5, both markers of nerve fibers, osteocalcin (a positive control for osteoblast staining), PAM and OSCAR (a marker of preosteoclasts and mature osteoclasts) were also performed. Briefly, sections were incubated in blocking buffer (1% BSA and 4% bovine serum in NaCl ⁄ P i ) for 30 min at 37 °C in a moist chamber, and then incubated with primary antibodies at the appropriate dilution in blocking buffer, overnight at 4 °C. The dilutions used were 1 : 2500 for rabbit anti-NF200 IgG (Sigma), 1 : 4000 for rabbit anti- PGP9.5 IgG (Serotec, Kidlington, UK), 1 : 500 for goat anti- osteocalcin IgG (Biomedical Technologies Inc., Stoughton, MA, USA), 1 : 500 for rabbit anti-PAM IgG (a kind gift from R. Mains, University of Connecticut Health Center), and 1 : 100 for mouse anti-OSCAR IgG (Santa Cruz Bio- technology, Heidelberg, Germany). Antigen visualization was performed with the biotin–extravidin–peroxidase kit (Sigma), using 3-amino-9-ethylcarbazole (Sigma) as sub- strate. On parallel control sections, the primary antibody was replaced with blocking buffer. Immunohistochemical analy- sis was performed independently by two observers. For quantification of PAM immunohistochemistry, the number of labeled cellsÆmm )2 was scored in three nonoverlapping micrographs with a magnification of · 40. Bone histology Femurs were harvested from 3 month old male WT (n = 6) and TTR KO (n = 5) mice. After their length had been measured, bones were fixed in 4% paraformaldehyde in NaCl ⁄ P i , decalcified as described above, and embedded in paraffin. Serial 10 lm thick longitudinal sections were cut. Sections were then deparaffinized, dehydrated in a modified alcohol series, and stained for hematoxylin ⁄ eosin. MicroCT analysis Dissected hindlimbs (femur plus tibia from WT and TTR KO littermates, n = 9 and n = 10, respectively) were scanned with high resolution (5 lm pixel size) microCT (Skyscan 1172; Skyscan, Kontich, Belgium). The whole mouse femur and tibia were reconstructed, and the trabecu- lar bone in the proximal metaphysis, comprising a region starting 0.25 mm from the growth plate and extending 1.5 mm (or 300 tomograms) distally, was analyzed. Histo- morphometric analysis in two and three dimensions was performed with Skyscan software (ct-analyser v. 1.5.1.3, Skyscan). For analysis of trabecular bone, cortical bone including the trabecular compartment was excluded by operator-drawn regions of interest, and 3D algorithms were used to determine the bone volume percentage (bone volume ⁄ trabecular volume). BMD measurement by microCT Volumetric BMD values of the trabecular bone compart- ment within the femural and tibial metaphysis were mea- sured from the same regions of interest used to derive the microarchitectural parameters, using the manufacturer’s instructions. Briefly, two calibration phantoms (Skyscan) with densities of 0.25 and 0.75 gÆcm )3 and a sample of water were scanned and reconstructed using the same settings used for the femurs and tibiae. The gray scale density values were converted into Hounsfield units, which were then used to compute the mean volumetric BMD of each femur and tibia. Cell cultures MC3T3-E1 mouse osteoblastic cell line culture MC3T3-E1 cells, established as an osteoblastic cell line from normal mouse calvaria, were grown in alpha-MEM (Invitro- gen, Carlsbad, CA, USA) supplemented with 10% (v ⁄ v) fetal bovine serum (Invitrogen), 0.5% (v ⁄ v) gentamicin (Invitro- gen), 1% (v ⁄ v) fungizone (Invitrogen), 50 lgÆmL )1 vitamin C (Sigma) and 10 mm b-glycerophosphate (Sigma) in a humidi- fied 5% CO 2 incubator at 37 °C. The medium was changed twice weekly. At confluence, the cells were trypsinized and seeded in 24-well plates at a cell seeding density of 4 · 10 4 cells per well. BMSC culture Primary BMSCs were obtained according to the method developed by Maniatopoulos et al. [43]. Briefly, femurs and tibias from 1 month old male WT and TTR KO littermates were aseptically excised from the hindlimbs, the epiphyses were cut off, and the marrow was flushed with standard culture medium, which consisted of alpha-MEM supple- mented with 10% fetal bovine serum, 50 lgÆmL )1 gentami- cin sulfate, and 2.5 lgÆmL )1 amphotericin B (Invitrogen). Cells were seeded in 75 cm 2 plastic culture flasks, and incu- bated in a humidified incubator (37 °C and 5% CO 2 ). The medium was changed after the first 24 h to remove nonad- herent cells. Subsequently, the adherent cells were cultured for 10 days, the medium being renewed every 3 days. NPY is expressed in osteoblasts A. F. Nunes et al. 272 FEBS Journal 277 (2010) 263–275 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... and other tissues in transthyretin- null mice Am J Physiol 272, E485–E493 NPY is expressed in osteoblasts 36 Palha JA, Fernandes R, de Escobar GM, Episkopou V, Gottesman M & Saraiva MJ (2000) Transthyretin regulates thyroid hormone levels in the choroid plexus, but not in the brain parenchyma: study in a transthyretin- null mouse model Endocrinology 141, 326 7–3 272 37 Kingery WS, Offley SC, Guo TZ, Davies... leptin and y2 receptor pathways J Bone Miner Res 20, 185 1–1 857 Allison S, Baldock P, Enriquez R, Lin E, During M, Gardiner E, Eisman J, Sainsbury A & Herzog H (2008) Critical interplay between neuropeptide Y and sex steroid pathways in bone and adipose tissue homeostasis J Bone Miner Res 24, 29 4–3 04 Nunes AF, Saraiva MJ & Sousa MM (2006) Transthyretin knockouts are a new mouse model for increased neuropeptide. .. neuropeptide Y FASEB J 20, 16 6–1 68 Mains RE, Cullen EI, May V & Eipper BA (1987) The role of secretory granules in peptide biosynthesis Ann NY Acad Sci 493, 27 8–2 91 Prigge ST, Mains RE, Eipper BA & Amzel LM (2000) New insights into copper monooxygenases and peptide amidation: structure, mechanism and function Cell Mol Life Sci 57, 123 6–1 259 Eipper BA, Stoffers DA & Mains RE (1992) The biosynthesis of neuropeptides:... 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Neuropeptide Y expression and function during osteoblast differentiation – insights from transthyretin knockout mice Ana F. Nunes 1,2, *,. By immunohistochemistry, we demonstrated, both at embryonic stages and in the adult, that NPY is synthesized by osteoblasts, osteocytes, and chondrocytes.