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Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro

Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro Hindawi Publishing Corporation International Journal of Peptides Volume 2011, Article ID 605193, 7 pages doi:10.1155/2011/605193 Research Article Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro Jessica L. Costa, Dorit Naot, Jian-Ming Lin, Maureen Watson, Karen E. Callon, Ian R. Reid, Andrew B. Grey, and Jillian Cornish Department of Medicine, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Correspondence should be addressed to Jessica L. Costa, j.costa@auckland.ac.nz Received 15 May 2011; Accepted 7 July 2011 Academic Editor: Hubert Vaudry Copyright © 2011 Jessica L. Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ghrelin is released in response to fasting, such that circulating levels are highest immediately prior to meals. Bone turnover is acutely responsive to the fed state, with increased bone resorption during fasting and suppression during feeding. The current study investigated the hypothesis that ghrelin regulates the activity of bone cells. Ghrelin increased the bone-resorbing activity of rat osteoclasts, but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures. Ghrelin showed mitogenic activity in osteoblasts, with a strong effect in human cells and a weaker effect in rat osteoblasts. The expression of the human ghrelin receptor, GHSR, varied among individuals and was detectable in 25–30% of bone marrow and osteoblast samples. However, the rodent Ghsr expression was undetectable in bone cells and cell lines from rat and mouse. These data suggest that elevated levels of ghrelin may contribute to the higher levels of bone turnover that occurs in the fasted state. 1. Introduction [5, 12] as well as by directly acting on the pituitary [5]. The major form of circulating ghrelin is des-acyl ghrelin, which Bone is constantly remodeled through the actions of bone- does not signal through GHSR1a and was considered to be forming osteoblasts and bone-resorbing osteoclasts. Bone inactive, although recent studies suggest that des-acyl ghrelin turnover is acutely responsive to food intake, with bone re- has some biological activity [13, 14]. The ghrelin receptor, sorption increased during fasting and suppressed during GHSR, is a seven transmembrane-domain G-protein cou- feeding [1, 2]. Numerous factors, such as nutritional content pled receptor [15]. Two transcripts are produced from the or size of meals, may play a role in this relationship and there human GHSR gene by alternative splicing: GHSR1a encodes is evidence that some gut hormones, such as glucagon-like the functional receptor, and GHSR1b, which is truncated peptide-2 [3, 4], also affect the normal postprandial reduc- after transmembrane domain 5, appears to be unable to tion of bone resorption. stimulate intracellular signaling pathways [15]. Ghrelin is a 28-amino-acid peptide hormone primarily The activity of ghrelin to stimulate feeding produced a synthesized by the stomach and released in response to great interest in this peptide as a potential drug target. Re- fasting, such that circulating levels, which range from 100– ceptor antagonists or inverse agonists have been developed 120 pmol/L in rats [5] or 90–240 pmol/L in humans [6], are for potential treatments of obesity by the induction of highest immediately prior to meals and fall upon feeding satiety [16, 17] and ghrelin and receptor agonists have been [7, 8]. Two forms of ghrelin are found in the circulation: n- considered for treatment of cancer-related cachexia [18, 19]. octanoyl ghrelin, which is acylated with an n-octanoyl group In addition, ghrelin has been investigated for a therapeutic on serine residue 3, and des-acyl ghrelin [5]. The n-octan- oylated form of ghrelin binds the GH secretagogue receptor role in cardiovascular disease [20, 21] because of the (GHSR1a), increases food intake [9–11], and induces release expression of the ghrelin receptor in vascular smooth muscle of growth hormone from the pituitary via the hypothalamus cells of the cardiovascular system [22]. 2 International Journal of Peptides A number of studies investigated the effects of ghrelin in Thereweresix wellsineachgroup,and each experiment was skeletal tissue. Expression of the ghrelin receptor, GHSR1a, repeated three or four times. Mineralization and apoptosis has been demonstrated by reverse transcription-PCR, im- assays were carried out on this cell type as previously munohistochemistry, and western blot analysis in several described [31]. Mineralization was determined by von Kossa osteoblasts and osteoblastic cell lines [23, 24]and astudy of stain, and apoptosis by terminal deoxynucleotidyl transfer- the expression of GHSR1a in human tissues demonstrated a ase-mediated deoxyuridine triphosphate nick end labeling weak positive signal in bone marrow [25]. In vitro studies (TUNEL). showed effects of ghrelin in primary osteoblast-like cells and Cultures of primary human osteoblasts were prepared cell lines, with highly variable responses in proliferation, dif- using normal human trabecular bone. Osteoblasts were ferentiation, and survival assays [23, 24, 26–28]. Osteoclast grown from enzyme-treated bone chips, using a modified response to ghrelin has never been investigated previously. methodofRobey andTermine [32] and treated with ex- However, as a growth hormone secretagogue, ghrelin is likely perimental compounds 24 or 48 hours prior to end of the to have an indirect effect on osteoclasts, as growth hormone incubation. The experiments were carried out three times increases formation and function of osteoclasts isolated from with similar results. mouse or rabbit bone marrow or spleen [29]. The aim of the current study was to further investigate 2.4. Bone Marrow Culture. Bone marrow was obtained from the activity of ghrelin in bone cells, and in particular its pos- the long bones of normal Swiss CD-1 male mice aged 4– sible role in the acute stimulation of bone resorption that 6 weeks, as previously described [30, 31]. Briefly, marrow occurs during fasting. We used assays of both osteoclast for- cells were grown in 48-well plates and on days 0, 2, and mation and mature osteoclast function to investigate ghre- −8 5, 1,25-dihydroxyvitamin D3 (10 M) was added to all lin’s role in bone resorption, in addition to testing the effects wells. On days 2 and 4, cultures were fed with 0.5 mL fresh of ghrelin on osteoblasts, and investigating the expression of medium containing test substances. Recombinant human os- the ghrelin receptor in bone cells. teoprotegerin (10 ng/mL) was included as a positive control for inhibition of osteoclastogenesis. After 7 days of culture, TRAP-positive multinucleated cells (containing three or 2. Methods more nuclei) were counted in all wells. There were at least 2.1. Materials. Acylated ghrelin with n-octanoyl at serine eight wells for each group, and the experiment was carried residue 3 was purchased from American Peptide Company out four times with similar results. (Sunnyvale, CA) and this form was used exclusively in all studies. BSA was purchased from ICPbio International, Ltd 2.5. Mature Isolated Osteoclast Culture. Rat osteoclasts were (Auckland, New Zealand). Gibco brand FBS and media were isolated from the long bones of 1-2-day-old rats, as pre- used (Invitrogen, Auckland, New Zealand). Tritiated thymi- viously described [30, 31] and incubated on bovine bone dine was purchased from NZ Scientific, Ltd (Auckland, New slices with test substances or vehicle for 24 hours. After Zealand). Tartrate-resistant acid phosphatase (TRAP) stain- incubation, the bone slices were fixed and stained for TRAP. ing kit, 1,25-dihydroxyvitamin D3, osteoprotegerin (OPG), The potent inhibitor of osteoclast activity, salmon calcitonin and salmon calcitonin were purchased from Sigma (NSW, −10 (10 M), was used as a positive control. The number of Australia). TRAP-positive multinucleated cells containing three or more nuclei on each bone slice was counted, the cells were removed 2.2. Cell and Tissue Samples from Human Subjects and from by gentle scrubbing, and then the bone slices were stained Animals. Animals were purchased from the Vernon Jensen for 30 seconds with toluidine blue. After several washes in Unit of the University of Auckland. All animal procedures water, the bone slices were dried and assessed for the pits were approved by the Animal Ethics Committee of our insti- excavated by the osteoclasts, using reflected light microscopy tution. Human bone samples were collected from consenting with metallurgic lenses. The results for each bone slice subjects undergoing hip and knee arthroplasty for arthritis. were expressed as the ratio of the number of pits to the The study had the approval of the local institutional review number of osteoclasts. There were 6–8 bone slices in each board. group and the experiment was carried out more than three times. 2.3. Primary Osteoblast Proliferation, Differentiation and Apoptosis Assays. Rat osteoblasts were isolated from 20-day 2.6. Bone Organ Culture. Hemicalvariae labeled in vivo fetal rat calvariae, as previously described [30, 31]. For with Ca were collected as described previously [31]and proliferation assays, cells were seeded into 24-well plates in incubated with experimental substances or vehicle for 48 5% FBS/MEM/5 μg/mL ascorbic-acid-2-phosphate (AA2P) hours. Calcium release was assessed and used to calculate the 45 45 for 24 h. Cells were growth-arrested in 0.1% BSA/MEM/ percent of Ca released over total Ca injected per animal 5 μg/mL AA2P for 24 hours. Fresh media and experimental (3 μCi), and H-thymidine incorporation was measured compounds were then added for a further 24 hours. Cells as an indicator of cell proliferation. Parathyroid hormone 3 −8 were pulsed with H-thymidine 6 hours before the end of the (PTH) (10 M) was used as a positive control. There were experimental incubation. The experiment was terminated five to seven hemicalvariae in each group, and the experiment and cell counts or thymidine incorporation determined. was carried out more than three times. International Journal of Peptides 3 1.5 1 200 0.5 −9 −8 −10 −10 −9 −8 010 M10 M10 M 0 10 M10 M10 M 10 ng/mL Ghrelin sCT Ghrelin OPG Figure 1: Ghrelin increases bone resorption by mature rat Figure 2: Osteoclast differentiation in mouse bone marrow cultures osteoclasts. Osteoclasts were incubated on bovine bone slices for is not affected by ghrelin. Bone marrow cells were cultured for 7 24 hours, and the number of TRAP-positive multinucleated cells days with experimental substances or vehicle. Cells were fixed and on each bone slice was counted. Cells were then removed, the stained and the number of TRAP positive cells with three nuclei bone slices were stained with toluidine blue, and the number of or more was determined in each well. MNC: multinucleated cells, excavated pits was determined by reflected light microscopy. The OPG: osteoprotegerin (positive control). Data are mean ± SEM; results are expressed as the ratio of the number of pits to the ∗ indicates P< 0.05 compared to untreated control. number of osteoclasts on a bone slice, with 6–12 bone slices in each group. MNC: multinucleated cells. sCT: salmon calcitonin (positive control). Data are mean ± SEM; indicates P< 0.05 compared to untreated control. increase in the number of pits excavated by osteoclasts, whereas at 10 nM there was only a 15% increase which was not significantly different from the control (Figure 1). 2.7. RNA Isolation, Reverse Transcription, and Quantitative Osteoclast differentiation was investigated using a murine PCR . RNA was collected from flash frozen mouse hypotha- bone marrow assay, where the mixed population of cells lamic tissue or cultured cells with Qiagen RNeasy minikits, from the bone marrow is cultured in the presence of 1,25- and genomic DNA was removed using the RNase-free DNase dihydroxyvitamin D3 that promotes osteoclast formation. set (Qiagen) according to manufacturer’s instructions. RNA The addition of ghrelin at concentrations of 0.1–10 nM samples collected from cell cultures grown from human during the seven-day incubation period had no effect on the osteoblast and bone marrow have been previously described number of osteoclasts as compared to control (Figure 2). The [33]. cDNA was synthesized with Superscript III (Invitrogen) activity of ghrelin was also tested in an ex vivo organ culture and used for multiplex real-time PCR in ABI PRISM 7900HT of mouse calvariae. This experimental system is investigating Sequence Detection Systems (Applied Biosystems). Primers osteoclast activity rather than development as there is only and probe sets were purchased as TaqMan Gene Expression a minimal number of preosteoclasts in the calvariae. The Assays (Applied Biosystems) for mouse Ghsr,rat Ghsr, and ability of osteoclasts to resorb bone, as measured by % Ca human GHSR. All probes used to detect target genes were release, was unaffected by ghrelin (Figure 3). Measurement labeled with FAM and 18 S rRNA endogenous control probe of H thymidine incorporation in the same cultures showed was VIC labeled. The ΔΔCt method was used to calculate the no difference in cell proliferation in the presence of ghrelin relative levels of expression [34]. (data not shown). 2.8. Statistics. Data were analyzed using ANOVA with post 3.2. The Effects of Ghrelin on Osteoblasts. Human primary hoc Dunnett’s tests or Student’s t-test as indicated for osteoblasts incubated for 24 or 48 hours in the presence experiments with 2 or more treatments. Student’s t-test was of 0.1 nM ghrelin showed a 2-fold increase in proliferation, used where a single treatment was analyzed versus vehicle. A as measured by thymidine incorporation (Figure 4). Ghrelin 5% significance level (P< 0.05) is used throughout. Data are weakly induced cell proliferation in primary rat osteoblasts, presented as means ± SEM, unless indicated otherwise. with a 1.1-1.2-fold stimulation of thymidine incorporation (Figure 5(a)) and a similar, modest increase in cell numbers (Figure 5(b)). 3. Results We also investigated ghrelin’s effects on osteoblast 3.1. The Effects of Ghrelin on Osteoclast Differentiation and differentiation. Primary rat osteoblasts were cultured for Activity. We used a number of in vitro experimental systems 21 days in media supplemented with L-ascorbic acid-2- to study the effects of ghrelin on osteoclast differentiation phosphate and β-glycerophosphate, and mineralized bone and activity. Mature osteoclasts were isolated from long nodule formation was measured by Von Kossa stain. Ghrelin bones of 1-2-day-old rats and cultured on slices of bovine had no effect on osteoblast differentiation in this assay (data bone. Ghrelin at a concentration of 1 nM induced a 30% not shown). The effect of ghrelin on osteoblast survival was Ratio of pits: TRAP positive MNC per bone slice Number of TRAP positive MNC cells/well 4 International Journal of Peptides 60 Table 1: Ghrelin receptor expression in bone. This study Method Result Primary tissue Quantitative 3/10 Human osteoblasts (n = 10) PCR positive 4/16 Human bone marrow (n = 16) positive Mouse bone marrow Negative Rat osteoblastic cells Negative Mouse hypothalamus Positive Cell lines Mouse osteoblastic MC3T3-E1 Negative Mouse macrophage RAW Negative 264.7 Kim et al. [23] −9 −8 −8 10 M10 M10 M Primary Tissue Ghrelin PTH Rat osteoblastic cells Figure 3: Osteoclast activity in mouse calvarial culture is not Western blot Positive Mouse osteoblastic cells affected by ghrelin. Calvariae labeled in vivo with Ca were Rat brain collected and incubated ex vivo with experimental substances or Cell lines vehicle for 48 hours. Values are expressed as percent released Mouse osteoblastic MC3T3-E1 45 45 Ca detected over total Ca injected (3 μCi). PTH: parathyroid ∗ Mouse preadipocytic 3T3-L1 RT-PCR, hormone (positive control). Data are mean ± SEM; indicates P< Positive Western blot Rat ROS17/2.8 osteogenic sarcoma 0.05 compared to untreated control. Rat UMR-106 osteogenic sarcoma Maccarinelli et al. [24] Primary Tissue RT-PCR, Rat osteoblastic cells Positive ∗ Western blot Ueberberg et al. [25] Primary Tissue Quantitative Human bone marrow Positive PCR, IHC Delhanty et al. [27] Primary Tissue Quantitative Human bone marrow (n = 2) Positive 0 PCR Ghrelin − + − + −10 Fukushima et al. [28] 10 M 24 hr 48 hr Primary Tissue RT-PCR, Figure 4: Ghrelin is mitogenic to primary human osteoblasts. Rat osteoblastic cells Positive Osteoblasts cultured from human trabecular bone were growth- IHC arrested in 0.1% BSA for 24 hours. Ghrelin was added for 24 or Rat femur IHC Positive 48 hours, and cells were pulsed with H-thymidine for the last 24 Cell lines hours of the incubation. Data are mean ± SEM; indicates P< 0.05 Rat UMR-106 osteogenic sarcoma RT-PCR Positive compared to untreated control by t-test. Guan et al. [35] Primary Tissue investigated using a TUNEL assay. Apoptosis was induced Rat bone marrow RPA Negative in primary rat osteoblast by serum withdrawal, and the RT-PCR: reverse transcription PCR. IHC: immunohistochemistry. RPA, cells were incubated for a further 24 hours in the presence ribonuclease protection assay. of ghrelin or controls. There was no significant change in the number of apoptotic bodies between the treatment and control wells, indicating that ghrelin had no effect on cell used to determine the expression of GHSR in bone cells. The survival in this experimental system (data not shown). GHSR TaqMan probe we used is designed to hybridize to a common sequence which is included in both of the alterna- 3.3. The Expression of Ghrelin Receptor, GHSR, in Bone Cells tively splices products of the gene: GHSR1a and GHSR1b. from Human, Rat, and Mouse Origin. Quantitative PCR was We tested the expression of GHSR in RNA extracted from Thymidine incorporation (treatment/control) Ca release (%) International Journal of Peptides 5 1.5 1.5 ∗∗ 1 1 0.5 0.5 −11 −10 −9 −11 −10 −9 0 0 10 M10 M 10 M 10 M10 M 10 M Ghrelin Ghrelin (a) (b) Figure 5: Ghrelin is weakly mitogenic to primary rat osteoblasts. Osteoblasts cultured from rat calvariae were growth-arrested in 0.1% BSA for 24 hours. Fresh media and experimental compounds were then added for a further 24 hours. (a) Thymidine incorporation: cells were pulsed with H-thymidine for the last 6 hours of incubation (ANOVA P = 0.018). (b) Cell counts (ANOVA P = 0.076). Data are mean ± SEM; indicates P< 0.05 compared to untreated control by t-test. human primary osteoblasts and bone marrow samples and though there is no change in their bone mineral content or found that the expression varies among individuals, with 3 density [37]. The lack of changes in these null models sug- out of 10 osteoblast samples and 4 out of 16 bone marrow gests a redundancy in the ghrelin-growth hormone system. samples showing a positive amplification signal. Rat primary Overexpression of ghrelin from the brain and stomach of osteoblast and mouse bone marrow cells showed no expres- transgenic mice does not induce any changes in body size, sion of Ghsr. We also tested the mouse osteoblastic cell line though these animals have increased food intake, while the MC3T3-E1, collecting RNA samples on different days during impact on bone is unknown [38]. the differentiation of the cells into mature osteoblasts in vitro Our study shows stimulation of osteoblast proliferation and found no expression of Ghsr. RNA samples collected in primary cells derived from rat calvariae and from human from the murine macrophage cell line RAW ,which bone. Stimulation of proliferation in similar cultures from 264.7 differentiate in vitro into osteoclasts, were also negative for rat and human origin has been demonstrated before [24, Ghsr. RNA extracted from mouse hypothalamic tissue which 27, 28]. However, the finding that ghrelin had no effect on was used as positive control showed high levels of Ghsr osteoblast differentiation and apoptosis varies from what expression (Table 1). has been shown by some of the other groups: increased osteoblast differentiation was determined in rat primary osteoblasts and in MC3T3-E1 cells [23, 24, 28], whereas 4. Discussion in the human osteoblast cell line SV-HFO ghrelin had no effect on differentiation [27], similar to what we found in the The gastric hormone ghrelin is primarily secreted from the current study. Kim et al. [23] showed that ghrelin inhibits stomach in response to hunger or fasting and it induces apoptosis in MC3T3-E1 cells, in contrast to our observations growth hormone secretion and food intake, most likely via that ghrelin had no effect on apoptosis in primary rat os- activation of type 1a receptors in the arcuate nucleus of teoblasts. A study of the effect of ghrelin on apoptosis in the hypothalamus. Here we investigated the direct impact of rat osteoblasts and various other primary cells of rat and ghrelin on bone cells to expand on previous in vitro studies human, as well as in human aldosteronoma and human adre- of ghrelin and show the first data regarding ghrelin’s impact nocortical carcinoma-derived cell lines, showed that ghrelin on osteoclasts in vitro. The finding that ghrelin stimulates did not affect apoptotic rate of normal cells, but significantly mature osteoclast activity to resorb bone suggests a possible enhanced apoptosis in tumor-derived cells [26]. Thus, the direct link between increased levels of circulating ghrelin and effect of ghrelin on apoptosis appears to depend on the cells stimulation of bone resorption induced by fasting. tested, with reports of inhibition, activation, and no effect on Ghrelin does not appear to be a primary requirement for apoptosis in different cells. bone formation and remodeling. In mouse models lacking Ghrelin receptors are widely distributed and have pre- either ghrelin or the ghrelin receptor, there are no known viously been shown to be present in bone cells and tissue changes to the skeleton. Mice lacking ghrelin [36]are normal by reverse-transcription PCR, western blots and immuno- in size and weight compared to their littermate controls, histochemistry [23–25, 28]. We used real-time PCR to test showing neither dwarfism nor obesity and no change in bone the expression of Ghsr in primary rat osteoblasts and mouse mineral content or density. Mice lacking the ghrelin receptor have normal body size with slightly reduced body weight bone marrow cells, and in the mouse cell lines MC3T3-E1 Thymidine incorporation (treatment/control) Cell numbers/well (treatment/control) 6 International Journal of Peptides and RAW , but failed to find any expression of the ghrel- [2] A. Schlemmer and C. 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Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro

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Copyright © 2011 Jessica L. Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2011/605193
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Hindawi Publishing Corporation International Journal of Peptides Volume 2011, Article ID 605193, 7 pages doi:10.1155/2011/605193 Research Article Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro Jessica L. Costa, Dorit Naot, Jian-Ming Lin, Maureen Watson, Karen E. Callon, Ian R. Reid, Andrew B. Grey, and Jillian Cornish Department of Medicine, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Correspondence should be addressed to Jessica L. Costa, j.costa@auckland.ac.nz Received 15 May 2011; Accepted 7 July 2011 Academic Editor: Hubert Vaudry Copyright © 2011 Jessica L. Costa et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ghrelin is released in response to fasting, such that circulating levels are highest immediately prior to meals. Bone turnover is acutely responsive to the fed state, with increased bone resorption during fasting and suppression during feeding. The current study investigated the hypothesis that ghrelin regulates the activity of bone cells. Ghrelin increased the bone-resorbing activity of rat osteoclasts, but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures. Ghrelin showed mitogenic activity in osteoblasts, with a strong effect in human cells and a weaker effect in rat osteoblasts. The expression of the human ghrelin receptor, GHSR, varied among individuals and was detectable in 25–30% of bone marrow and osteoblast samples. However, the rodent Ghsr expression was undetectable in bone cells and cell lines from rat and mouse. These data suggest that elevated levels of ghrelin may contribute to the higher levels of bone turnover that occurs in the fasted state. 1. Introduction [5, 12] as well as by directly acting on the pituitary [5]. The major form of circulating ghrelin is des-acyl ghrelin, which Bone is constantly remodeled through the actions of bone- does not signal through GHSR1a and was considered to be forming osteoblasts and bone-resorbing osteoclasts. Bone inactive, although recent studies suggest that des-acyl ghrelin turnover is acutely responsive to food intake, with bone re- has some biological activity [13, 14]. The ghrelin receptor, sorption increased during fasting and suppressed during GHSR, is a seven transmembrane-domain G-protein cou- feeding [1, 2]. Numerous factors, such as nutritional content pled receptor [15]. Two transcripts are produced from the or size of meals, may play a role in this relationship and there human GHSR gene by alternative splicing: GHSR1a encodes is evidence that some gut hormones, such as glucagon-like the functional receptor, and GHSR1b, which is truncated peptide-2 [3, 4], also affect the normal postprandial reduc- after transmembrane domain 5, appears to be unable to tion of bone resorption. stimulate intracellular signaling pathways [15]. Ghrelin is a 28-amino-acid peptide hormone primarily The activity of ghrelin to stimulate feeding produced a synthesized by the stomach and released in response to great interest in this peptide as a potential drug target. Re- fasting, such that circulating levels, which range from 100– ceptor antagonists or inverse agonists have been developed 120 pmol/L in rats [5] or 90–240 pmol/L in humans [6], are for potential treatments of obesity by the induction of highest immediately prior to meals and fall upon feeding satiety [16, 17] and ghrelin and receptor agonists have been [7, 8]. Two forms of ghrelin are found in the circulation: n- considered for treatment of cancer-related cachexia [18, 19]. octanoyl ghrelin, which is acylated with an n-octanoyl group In addition, ghrelin has been investigated for a therapeutic on serine residue 3, and des-acyl ghrelin [5]. The n-octan- oylated form of ghrelin binds the GH secretagogue receptor role in cardiovascular disease [20, 21] because of the (GHSR1a), increases food intake [9–11], and induces release expression of the ghrelin receptor in vascular smooth muscle of growth hormone from the pituitary via the hypothalamus cells of the cardiovascular system [22]. 2 International Journal of Peptides A number of studies investigated the effects of ghrelin in Thereweresix wellsineachgroup,and each experiment was skeletal tissue. Expression of the ghrelin receptor, GHSR1a, repeated three or four times. Mineralization and apoptosis has been demonstrated by reverse transcription-PCR, im- assays were carried out on this cell type as previously munohistochemistry, and western blot analysis in several described [31]. Mineralization was determined by von Kossa osteoblasts and osteoblastic cell lines [23, 24]and astudy of stain, and apoptosis by terminal deoxynucleotidyl transfer- the expression of GHSR1a in human tissues demonstrated a ase-mediated deoxyuridine triphosphate nick end labeling weak positive signal in bone marrow [25]. In vitro studies (TUNEL). showed effects of ghrelin in primary osteoblast-like cells and Cultures of primary human osteoblasts were prepared cell lines, with highly variable responses in proliferation, dif- using normal human trabecular bone. Osteoblasts were ferentiation, and survival assays [23, 24, 26–28]. Osteoclast grown from enzyme-treated bone chips, using a modified response to ghrelin has never been investigated previously. methodofRobey andTermine [32] and treated with ex- However, as a growth hormone secretagogue, ghrelin is likely perimental compounds 24 or 48 hours prior to end of the to have an indirect effect on osteoclasts, as growth hormone incubation. The experiments were carried out three times increases formation and function of osteoclasts isolated from with similar results. mouse or rabbit bone marrow or spleen [29]. The aim of the current study was to further investigate 2.4. Bone Marrow Culture. Bone marrow was obtained from the activity of ghrelin in bone cells, and in particular its pos- the long bones of normal Swiss CD-1 male mice aged 4– sible role in the acute stimulation of bone resorption that 6 weeks, as previously described [30, 31]. Briefly, marrow occurs during fasting. We used assays of both osteoclast for- cells were grown in 48-well plates and on days 0, 2, and mation and mature osteoclast function to investigate ghre- −8 5, 1,25-dihydroxyvitamin D3 (10 M) was added to all lin’s role in bone resorption, in addition to testing the effects wells. On days 2 and 4, cultures were fed with 0.5 mL fresh of ghrelin on osteoblasts, and investigating the expression of medium containing test substances. Recombinant human os- the ghrelin receptor in bone cells. teoprotegerin (10 ng/mL) was included as a positive control for inhibition of osteoclastogenesis. After 7 days of culture, TRAP-positive multinucleated cells (containing three or 2. Methods more nuclei) were counted in all wells. There were at least 2.1. Materials. Acylated ghrelin with n-octanoyl at serine eight wells for each group, and the experiment was carried residue 3 was purchased from American Peptide Company out four times with similar results. (Sunnyvale, CA) and this form was used exclusively in all studies. BSA was purchased from ICPbio International, Ltd 2.5. Mature Isolated Osteoclast Culture. Rat osteoclasts were (Auckland, New Zealand). Gibco brand FBS and media were isolated from the long bones of 1-2-day-old rats, as pre- used (Invitrogen, Auckland, New Zealand). Tritiated thymi- viously described [30, 31] and incubated on bovine bone dine was purchased from NZ Scientific, Ltd (Auckland, New slices with test substances or vehicle for 24 hours. After Zealand). Tartrate-resistant acid phosphatase (TRAP) stain- incubation, the bone slices were fixed and stained for TRAP. ing kit, 1,25-dihydroxyvitamin D3, osteoprotegerin (OPG), The potent inhibitor of osteoclast activity, salmon calcitonin and salmon calcitonin were purchased from Sigma (NSW, −10 (10 M), was used as a positive control. The number of Australia). TRAP-positive multinucleated cells containing three or more nuclei on each bone slice was counted, the cells were removed 2.2. Cell and Tissue Samples from Human Subjects and from by gentle scrubbing, and then the bone slices were stained Animals. Animals were purchased from the Vernon Jensen for 30 seconds with toluidine blue. After several washes in Unit of the University of Auckland. All animal procedures water, the bone slices were dried and assessed for the pits were approved by the Animal Ethics Committee of our insti- excavated by the osteoclasts, using reflected light microscopy tution. Human bone samples were collected from consenting with metallurgic lenses. The results for each bone slice subjects undergoing hip and knee arthroplasty for arthritis. were expressed as the ratio of the number of pits to the The study had the approval of the local institutional review number of osteoclasts. There were 6–8 bone slices in each board. group and the experiment was carried out more than three times. 2.3. Primary Osteoblast Proliferation, Differentiation and Apoptosis Assays. Rat osteoblasts were isolated from 20-day 2.6. Bone Organ Culture. Hemicalvariae labeled in vivo fetal rat calvariae, as previously described [30, 31]. For with Ca were collected as described previously [31]and proliferation assays, cells were seeded into 24-well plates in incubated with experimental substances or vehicle for 48 5% FBS/MEM/5 μg/mL ascorbic-acid-2-phosphate (AA2P) hours. Calcium release was assessed and used to calculate the 45 45 for 24 h. Cells were growth-arrested in 0.1% BSA/MEM/ percent of Ca released over total Ca injected per animal 5 μg/mL AA2P for 24 hours. Fresh media and experimental (3 μCi), and H-thymidine incorporation was measured compounds were then added for a further 24 hours. Cells as an indicator of cell proliferation. Parathyroid hormone 3 −8 were pulsed with H-thymidine 6 hours before the end of the (PTH) (10 M) was used as a positive control. There were experimental incubation. The experiment was terminated five to seven hemicalvariae in each group, and the experiment and cell counts or thymidine incorporation determined. was carried out more than three times. International Journal of Peptides 3 1.5 1 200 0.5 −9 −8 −10 −10 −9 −8 010 M10 M10 M 0 10 M10 M10 M 10 ng/mL Ghrelin sCT Ghrelin OPG Figure 1: Ghrelin increases bone resorption by mature rat Figure 2: Osteoclast differentiation in mouse bone marrow cultures osteoclasts. Osteoclasts were incubated on bovine bone slices for is not affected by ghrelin. Bone marrow cells were cultured for 7 24 hours, and the number of TRAP-positive multinucleated cells days with experimental substances or vehicle. Cells were fixed and on each bone slice was counted. Cells were then removed, the stained and the number of TRAP positive cells with three nuclei bone slices were stained with toluidine blue, and the number of or more was determined in each well. MNC: multinucleated cells, excavated pits was determined by reflected light microscopy. The OPG: osteoprotegerin (positive control). Data are mean ± SEM; results are expressed as the ratio of the number of pits to the ∗ indicates P< 0.05 compared to untreated control. number of osteoclasts on a bone slice, with 6–12 bone slices in each group. MNC: multinucleated cells. sCT: salmon calcitonin (positive control). Data are mean ± SEM; indicates P< 0.05 compared to untreated control. increase in the number of pits excavated by osteoclasts, whereas at 10 nM there was only a 15% increase which was not significantly different from the control (Figure 1). 2.7. RNA Isolation, Reverse Transcription, and Quantitative Osteoclast differentiation was investigated using a murine PCR . RNA was collected from flash frozen mouse hypotha- bone marrow assay, where the mixed population of cells lamic tissue or cultured cells with Qiagen RNeasy minikits, from the bone marrow is cultured in the presence of 1,25- and genomic DNA was removed using the RNase-free DNase dihydroxyvitamin D3 that promotes osteoclast formation. set (Qiagen) according to manufacturer’s instructions. RNA The addition of ghrelin at concentrations of 0.1–10 nM samples collected from cell cultures grown from human during the seven-day incubation period had no effect on the osteoblast and bone marrow have been previously described number of osteoclasts as compared to control (Figure 2). The [33]. cDNA was synthesized with Superscript III (Invitrogen) activity of ghrelin was also tested in an ex vivo organ culture and used for multiplex real-time PCR in ABI PRISM 7900HT of mouse calvariae. This experimental system is investigating Sequence Detection Systems (Applied Biosystems). Primers osteoclast activity rather than development as there is only and probe sets were purchased as TaqMan Gene Expression a minimal number of preosteoclasts in the calvariae. The Assays (Applied Biosystems) for mouse Ghsr,rat Ghsr, and ability of osteoclasts to resorb bone, as measured by % Ca human GHSR. All probes used to detect target genes were release, was unaffected by ghrelin (Figure 3). Measurement labeled with FAM and 18 S rRNA endogenous control probe of H thymidine incorporation in the same cultures showed was VIC labeled. The ΔΔCt method was used to calculate the no difference in cell proliferation in the presence of ghrelin relative levels of expression [34]. (data not shown). 2.8. Statistics. Data were analyzed using ANOVA with post 3.2. The Effects of Ghrelin on Osteoblasts. Human primary hoc Dunnett’s tests or Student’s t-test as indicated for osteoblasts incubated for 24 or 48 hours in the presence experiments with 2 or more treatments. Student’s t-test was of 0.1 nM ghrelin showed a 2-fold increase in proliferation, used where a single treatment was analyzed versus vehicle. A as measured by thymidine incorporation (Figure 4). Ghrelin 5% significance level (P< 0.05) is used throughout. Data are weakly induced cell proliferation in primary rat osteoblasts, presented as means ± SEM, unless indicated otherwise. with a 1.1-1.2-fold stimulation of thymidine incorporation (Figure 5(a)) and a similar, modest increase in cell numbers (Figure 5(b)). 3. Results We also investigated ghrelin’s effects on osteoblast 3.1. The Effects of Ghrelin on Osteoclast Differentiation and differentiation. Primary rat osteoblasts were cultured for Activity. We used a number of in vitro experimental systems 21 days in media supplemented with L-ascorbic acid-2- to study the effects of ghrelin on osteoclast differentiation phosphate and β-glycerophosphate, and mineralized bone and activity. Mature osteoclasts were isolated from long nodule formation was measured by Von Kossa stain. Ghrelin bones of 1-2-day-old rats and cultured on slices of bovine had no effect on osteoblast differentiation in this assay (data bone. Ghrelin at a concentration of 1 nM induced a 30% not shown). The effect of ghrelin on osteoblast survival was Ratio of pits: TRAP positive MNC per bone slice Number of TRAP positive MNC cells/well 4 International Journal of Peptides 60 Table 1: Ghrelin receptor expression in bone. This study Method Result Primary tissue Quantitative 3/10 Human osteoblasts (n = 10) PCR positive 4/16 Human bone marrow (n = 16) positive Mouse bone marrow Negative Rat osteoblastic cells Negative Mouse hypothalamus Positive Cell lines Mouse osteoblastic MC3T3-E1 Negative Mouse macrophage RAW Negative 264.7 Kim et al. [23] −9 −8 −8 10 M10 M10 M Primary Tissue Ghrelin PTH Rat osteoblastic cells Figure 3: Osteoclast activity in mouse calvarial culture is not Western blot Positive Mouse osteoblastic cells affected by ghrelin. Calvariae labeled in vivo with Ca were Rat brain collected and incubated ex vivo with experimental substances or Cell lines vehicle for 48 hours. Values are expressed as percent released Mouse osteoblastic MC3T3-E1 45 45 Ca detected over total Ca injected (3 μCi). PTH: parathyroid ∗ Mouse preadipocytic 3T3-L1 RT-PCR, hormone (positive control). Data are mean ± SEM; indicates P< Positive Western blot Rat ROS17/2.8 osteogenic sarcoma 0.05 compared to untreated control. Rat UMR-106 osteogenic sarcoma Maccarinelli et al. [24] Primary Tissue RT-PCR, Rat osteoblastic cells Positive ∗ Western blot Ueberberg et al. [25] Primary Tissue Quantitative Human bone marrow Positive PCR, IHC Delhanty et al. [27] Primary Tissue Quantitative Human bone marrow (n = 2) Positive 0 PCR Ghrelin − + − + −10 Fukushima et al. [28] 10 M 24 hr 48 hr Primary Tissue RT-PCR, Figure 4: Ghrelin is mitogenic to primary human osteoblasts. Rat osteoblastic cells Positive Osteoblasts cultured from human trabecular bone were growth- IHC arrested in 0.1% BSA for 24 hours. Ghrelin was added for 24 or Rat femur IHC Positive 48 hours, and cells were pulsed with H-thymidine for the last 24 Cell lines hours of the incubation. Data are mean ± SEM; indicates P< 0.05 Rat UMR-106 osteogenic sarcoma RT-PCR Positive compared to untreated control by t-test. Guan et al. [35] Primary Tissue investigated using a TUNEL assay. Apoptosis was induced Rat bone marrow RPA Negative in primary rat osteoblast by serum withdrawal, and the RT-PCR: reverse transcription PCR. IHC: immunohistochemistry. RPA, cells were incubated for a further 24 hours in the presence ribonuclease protection assay. of ghrelin or controls. There was no significant change in the number of apoptotic bodies between the treatment and control wells, indicating that ghrelin had no effect on cell used to determine the expression of GHSR in bone cells. The survival in this experimental system (data not shown). GHSR TaqMan probe we used is designed to hybridize to a common sequence which is included in both of the alterna- 3.3. The Expression of Ghrelin Receptor, GHSR, in Bone Cells tively splices products of the gene: GHSR1a and GHSR1b. from Human, Rat, and Mouse Origin. Quantitative PCR was We tested the expression of GHSR in RNA extracted from Thymidine incorporation (treatment/control) Ca release (%) International Journal of Peptides 5 1.5 1.5 ∗∗ 1 1 0.5 0.5 −11 −10 −9 −11 −10 −9 0 0 10 M10 M 10 M 10 M10 M 10 M Ghrelin Ghrelin (a) (b) Figure 5: Ghrelin is weakly mitogenic to primary rat osteoblasts. Osteoblasts cultured from rat calvariae were growth-arrested in 0.1% BSA for 24 hours. Fresh media and experimental compounds were then added for a further 24 hours. (a) Thymidine incorporation: cells were pulsed with H-thymidine for the last 6 hours of incubation (ANOVA P = 0.018). (b) Cell counts (ANOVA P = 0.076). Data are mean ± SEM; indicates P< 0.05 compared to untreated control by t-test. human primary osteoblasts and bone marrow samples and though there is no change in their bone mineral content or found that the expression varies among individuals, with 3 density [37]. The lack of changes in these null models sug- out of 10 osteoblast samples and 4 out of 16 bone marrow gests a redundancy in the ghrelin-growth hormone system. samples showing a positive amplification signal. Rat primary Overexpression of ghrelin from the brain and stomach of osteoblast and mouse bone marrow cells showed no expres- transgenic mice does not induce any changes in body size, sion of Ghsr. We also tested the mouse osteoblastic cell line though these animals have increased food intake, while the MC3T3-E1, collecting RNA samples on different days during impact on bone is unknown [38]. the differentiation of the cells into mature osteoblasts in vitro Our study shows stimulation of osteoblast proliferation and found no expression of Ghsr. RNA samples collected in primary cells derived from rat calvariae and from human from the murine macrophage cell line RAW ,which bone. Stimulation of proliferation in similar cultures from 264.7 differentiate in vitro into osteoclasts, were also negative for rat and human origin has been demonstrated before [24, Ghsr. RNA extracted from mouse hypothalamic tissue which 27, 28]. However, the finding that ghrelin had no effect on was used as positive control showed high levels of Ghsr osteoblast differentiation and apoptosis varies from what expression (Table 1). has been shown by some of the other groups: increased osteoblast differentiation was determined in rat primary osteoblasts and in MC3T3-E1 cells [23, 24, 28], whereas 4. Discussion in the human osteoblast cell line SV-HFO ghrelin had no effect on differentiation [27], similar to what we found in the The gastric hormone ghrelin is primarily secreted from the current study. Kim et al. [23] showed that ghrelin inhibits stomach in response to hunger or fasting and it induces apoptosis in MC3T3-E1 cells, in contrast to our observations growth hormone secretion and food intake, most likely via that ghrelin had no effect on apoptosis in primary rat os- activation of type 1a receptors in the arcuate nucleus of teoblasts. A study of the effect of ghrelin on apoptosis in the hypothalamus. Here we investigated the direct impact of rat osteoblasts and various other primary cells of rat and ghrelin on bone cells to expand on previous in vitro studies human, as well as in human aldosteronoma and human adre- of ghrelin and show the first data regarding ghrelin’s impact nocortical carcinoma-derived cell lines, showed that ghrelin on osteoclasts in vitro. The finding that ghrelin stimulates did not affect apoptotic rate of normal cells, but significantly mature osteoclast activity to resorb bone suggests a possible enhanced apoptosis in tumor-derived cells [26]. Thus, the direct link between increased levels of circulating ghrelin and effect of ghrelin on apoptosis appears to depend on the cells stimulation of bone resorption induced by fasting. tested, with reports of inhibition, activation, and no effect on Ghrelin does not appear to be a primary requirement for apoptosis in different cells. bone formation and remodeling. In mouse models lacking Ghrelin receptors are widely distributed and have pre- either ghrelin or the ghrelin receptor, there are no known viously been shown to be present in bone cells and tissue changes to the skeleton. Mice lacking ghrelin [36]are normal by reverse-transcription PCR, western blots and immuno- in size and weight compared to their littermate controls, histochemistry [23–25, 28]. We used real-time PCR to test showing neither dwarfism nor obesity and no change in bone the expression of Ghsr in primary rat osteoblasts and mouse mineral content or density. Mice lacking the ghrelin receptor have normal body size with slightly reduced body weight bone marrow cells, and in the mouse cell lines MC3T3-E1 Thymidine incorporation (treatment/control) Cell numbers/well (treatment/control) 6 International Journal of Peptides and RAW , but failed to find any expression of the ghrel- [2] A. Schlemmer and C. Hassager, “Acute fasting diminishes the 264.7 circadian rhythm of biochemical markers of bone resorption,” in receptor. This was not due to technical problems, as European Journal of Endocrinology, vol. 140, no. 4, pp. 332– expression was robust in hypothalamic positive controls. In 337, 1999. contrast to the lack of expression in bone cells of rodents, [3] K. V. Haderslev, P. B. Jeppesen, B. Hartmann et al., “Short- we found that bone cells from humans expressed the ghrel- term administration of glucagon-like peptide-2. Effects on in receptor, although we could only detect low levels of bone mineral density and markers of bone turnover in expression in less than half of the osteoblast and bone mar- short-bowel patients with no colon,” Scandinavian Journal of row samples tested. The expression of GHSR in cells from Gastroenterology, vol. 37, no. 4, pp. 392–398, 2002. 26 different patients showed individual variability that [4] D.B.Henriksen,P.Alexandersen, N. H. 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