Maureen Watson

University of Auckland, Окленд, Auckland, New Zealand

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Publications (24)84.36 Total impact

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    ABSTRACT: Osteoporosis is an (age-related) skeletal disorder leading to bone fragility and increased incidence of fractures. Majority of osteoporosis medications reduce the activity of osteoclasts and hence bone resorption. These, however, do not regenerate damaged bone tisse.2 Conversely, anabolic agents have the ability to increase bone mass and strength, potentially reversing structural damage. Therefore, the discovery of novel, cost-effective bone anabolic agents is a priority to treat those suffering from this disabling disease. The application of pancreatic peptide preptin and the cyclic hexapeptide dianthin G, obtained from Dianthus superbus -chinese medical plant-, as bone anabolic agents have been previously reported. Herein, we report a structure-activity study of these anabolics, designed to identify the functionally important amino acid residues of each peptide and then, using peptidomimetic techniques to incorporate modifications designed to enhance their metabolic stability and/or anabolic activity. An alanine scan of preptin (1-16), a truncated but active form of the native preptin, revealed that Ser-3, Asp-12, and Pro-14 residues are not essential for the proliferation activity. Using Fmoc-SPPS, preptin (1-16) was then modified by the introduction of non-proteinogenic amino acid residues. Furthermore, preptin (1-16) was conformationally constrained via solution phase cyclisation to produce head-to-tail and head-to-side chain cyclic analogues. An alanine scan of dianthin G revealed that Thr-3, Phe-5, and Gly-6 residues are not essential for the biological activity. Cyclic analogues were synthesized using Grubbs’ ring closing metathesis (stapling). The proliferative activity and in vitro metabolic stability of preptin (1-16) and dianthin G analogues are currently being investigated.
    No preview · Conference Paper · Jun 2015
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    ABSTRACT: The first synthesis and osteoblast proliferative activity of the naturally occurring cyclic peptides dianthins G and H is described. The greater potency of naturally occurring dianthin G over dianthin H at physiological concentrations mirrored the osteoblast proliferative activity observed for synthetic dianthins G and H. Six alanine-scan analogues of the more potent dianthin G were also synthesised and osteoblast assays revealed that four of the six residues can be further modified for improved activity. We also confirmed by variable temperature 1H NMR spectroscopic analysis that the sets of major and minor signals observed for dianthins G and H in DMSO-d6 are in fact due to cis–trans rotational isomers of the proline ring.
    No preview · Article · Oct 2014 · Tetrahedron
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    ABSTRACT: Osteoporosis is a skeletal disorder of aging leading to bone fragility and an increase in bone fractures. The majority of current therapies for osteoporosis are anti-resorptive which do not have the ability to re-build lost bone. Anabolic agents however, have the ability to increase bone mass and strength. Therefore, the discovery of low cost, bone anabolic agents is a priority to treat osteoporosis and improve the quality of life of osteoporotic patients. The 34-amino acid pancreatic peptide preptin has been shown to be anabolic to bone in vitro and in vivo possibly via phosphorylation of p42/44 MAP kinases. We have identified the smaller peptide fragment, preptin (1-16) and due to its smaller size and retained anabolic activity to bone formation, is a promising candidate for further therapeutic development. Alanine scanning has been commonly used as a useful and valuable tool to probe the activity of peptides. Substitution of a residue side chain by a methyl group provides a convenient means to assess which side chains are responsible for the biological activity. An alanine scan study on preptin (1-16) was therefore undertaken and the results showed that substituting Ser-3, Asp-12, or Pro-14 with Ala retained the proliferation activity of the native preptin in vitro. Preptin’s peptidic nature suggests a moderately rapid systemic clearance and susceptibility to proteolytic degradation. Therefore, additional modifications using natural and non-natural amino acids were introduced on the native sequence in order to improve enzymatic resistance and enhance dosage profile. In addition to that, we aim to explore the therapeutic potential of cyclic preptin (1-16) analogues. Because of their reduced conformational freedom relative to their linear precursors, cyclic peptides display improved metabolic stability and/or biological activity. We will therefore attempt different synthetic approaches for the chemical synthesis of three cyclic analogues of preptin (1-16), to evaluate for osteoblast proliferation activity and metabolic stability
    No preview · Conference Paper · Jul 2014
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    ABSTRACT: Preptin, a 34-amino acid residue peptide hormone is co-secreted with insulin from the β-pancreatic cells and is active in fuel metabolism. We have previously established that a shorter fragment of preptin, namely preptin-(1-16), stimulates bone growth by proliferation and increasing the survival rate of osteoblasts. This was demonstrated in both in vitro and in vivo models. These findings suggest that preptin-(1-16) could play an important role in the anabolic therapy of osteoporosis. However, due to the large size of the peptide it is not an ideal therapeutic agent. The aim of this study was to identify the shortest preptin analogue that retains or even increases the bone anabolic activity as compared to the parent preptin-(1-16) peptide. Truncations were made in a methodical manner from both the N-terminus and the C-terminus of the peptide, and the effect of these deletions on the resulting biological activity was assessed. In order to improve the enzymatic stability of the shortest yet active analogue identified, ruthenium-catalysed ring closing metathesis was used to generate a macrocyclic peptide using allylglycine residues as handles for ring formation. We have successfully identified a short 8-amino acid preptin (1-8) fragment that retains an anabolic effect on the proliferation of primary rat osteoblasts and enhances bone nodule formation. Preptin (1-8) is a useful lead compound for the development of orally active therapeutics for the treatment of osteoporosis.
    No preview · Article · Jul 2014 · Bioorganic & Medicinal Chemistry
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    ABSTRACT: Several adipokines are known to influence skeletal metabolism. Fasting-induced adipose factor (FIAF) is an adipokine that gives rise to 2 further peptides in vivo, the N-terminal coiled-coil domain (FIAFCCD) and C-terminal fibrinogen-like domain (FIAFFLD). The skeletal action of these peptides is still uncertain. Our results show that FIAFCCD is a potent inhibitor of osteoclastogenesis and function, as seen in mouse bone marrow and RAW264.7 cell cultures, and in a resorption assay using isolated primary mature osteoclasts. The inhibitory effects at 500 ng/mL were approximately 90%, 50% and 90%, respectively, in these assays. FIAFCCD also stimulated osteoblast mitogenesis by approximately 30% at this concentration. In comparison, FIAFFLD was only active in decreasing osteoblast mitogenesis, and intact FIAF had no effect in any of these assays. In murine bone marrow cultures, FIAFCCD reduced the expression of macrophage colony-stimulating factor (M-CSF), nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP), and to lesser extent suppressed the expression of connective tissue growth factor (CTGF). FIAFCCD also decreased expression of M-CSF and CTGF in stromal/osteoblastic ST2 cells. Its effect on receptor activator of nuclear factor κB (RANKL) and osteoprotegerin expression in bone marrow was not consistent with its inhibitory action on osteoclastogenesis, but it decreased RANKL expression in ST2 cells. In RAW264.7 cell cultures, FIAFCCD significantly reduced the expression of NFATc1 and DC-STAMP. In conclusion, FIAFCCD inhibits osteoclast differentiation and function in vitro and decreases expression of genes encoding key osteoclastogenic factors such as M-CSF, CTGF, NFATc1, and DC-STAMP. FIAFCCD's action on osteoclasts may be independent of the RANKL/osteoprotegerin pathway. These results suggest a novel mechanism by which adipose tissue may regulate bone resorption and skeletal health.
    No preview · Article · Oct 2013 · Endocrinology
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    ABSTRACT: The class-I PI 3-kinases mediate the effects of many hormones and growth factors on a wide range of cellular processes and activating mutations or gene amplifications of Class-I PI 3-kinase isoforms are known to contribute to oncogenic processes in a range of tumours. Consequently a number of small molecule PI 3-kinase inhibitors are under development and in clinical trial. The central signalling role of PI 3-kinase in many cellular processes suggests there will be on target side effects associated with the use of these agents. To gain insights into what these might be we have investigated the effect of extended daily dosing of 8 small molecule inhibitors of class-Ia PI 3-kinases. Animals were characterized in metabolic cages to analyse food intake, oxygen consumption and movement. Insulin tolerance and body composition were analysed at the end of the experiment, the latter using echo-MRI. Bone volume and strength was assessed by micro-CT and 3-point bending, respectively. Surprisingly, after sustained dosing with pan-PI 3-kinase inhibitors and selective inhibitors of the p110α isoform there was a resolution of the impairments in insulin tolerance observed in drug naïve animals treated with the same drugs. However, pan-PI 3-kinase inhibitors and selective inhibitors of the p110α have deleterious effects on animal growth, animal behaviour and bone volume and strength. Together these findings identify a range of on target effects of PI 3-kinase inhibitors and suggest use of these drugs in humans may have important adverse effects on metabolism, body composition, behaviour and skeletal health. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jul 2013 · FEBS Journal
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    ABSTRACT: This report describes the investigations into the cause and treatment of metabolic bone disease (MBD) in captive native New Zealand frogs (Leiopelma spp.) and the role of fluoride in the disease. MBD was diagnosed in Leiopelma archeyi and Leiopelma hochstetteri in 2008 at three institutions: Auckland Zoo, Hamilton Zoo, and the University of Otago. Most of these frogs had originally been held at the University of Canterbury for several years (2000-2004) but some were collected directly from the wild. Radiographs on archived and live frogs showed that MBD had been present at Canterbury, but at a lower rate (3%) than in the current institutions (38-67%). Microcomputed tomography showed that the femoral diaphyses of the captive frogs at Auckland Zoo had greater bone volume, bone surface, cross-sectional thickness, and mean total cross-sectional bone perimeter, which is consistent with osteofluorosis. On histology of the same femurs, there was hyperplasia, periosteal growth, and thickening of trabeculae, which are also consistent with skeletal fluorosis. An increase in fluoride levels in the water supply preceded the rise in the incidence of the above pathology, further supporting the diagnosis of osteofluorosis. Analysis of long-standing husbandry practices showed that ultraviolet B (UVB) exposure and the dietary calcium:phosphorus ratio were deficient when compared with wild conditions-likely causing chronic underlying MBD. To prevent multifactorial MBD in captive Leiopelma, the authors recommend increasing dietary calcium by incorporating into the captive diet inherently calcium-rich invertebrates; increasing exposure to natural or artificial (UVB) light; and using defluoridated water. Addressing these three factors at Auckland Zoo reduced morbidity, bone fractures, and mortality rates.
    Full-text · Article · Sep 2012 · Journal of Zoo and Wildlife Medicine
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    ABSTRACT: Osteoporotic fracture is a significant public health problem, resulting in fractures in >50% of women and in almost one third of men age 65 and older. Most of the existing therapies act by slowing bone loss, through inhibiting the action of bone resorbing cells. However, more substantial reductions of fracture numbers will only result from treatments that can rebuild bone. Our own animal studies demonstrated the anabolic potential of the small but unstable octapeptide fragment of amylin-(1-37), namely amylin-(1-8) containing one disulfide bridge (Cys/2 and Cys/7) [Am. J. Physiol. Endocrinol. Metab.2000, 279, E730]. Herein, we describe the synthesis of amylin-(1-8) octapeptide and seven analogues thereof wherein the disulfide bridge is modified either via insertion of different linkers or bridges of a different nature in order to improve the stability and/or bone anabolic activity of the parent peptide. The peptide analogues were screened for proliferative activity in primary foetal rat bone-forming cells or osteoblasts at physiological concentrations. One such analogue showed promising biological activity.
    No preview · Article · Feb 2012 · Bioorganic & medicinal chemistry
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    ABSTRACT: 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.
    Full-text · Article · Sep 2011 · International Journal of Peptides
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    ABSTRACT: Nilotinib is a tyrosine kinase inhibitor (TKI) developed to manage imatinib-resistance in patients with chronic myeloid leukemia (CML). It inhibits similar molecular targets to imatinib, but is a significantly more potent inhibitor of Bcr-Abl. Nilotinib exhibits off-target effects in other tissues, and of relevance to bone metabolism, hypophosphataemia has been reported in up to 30% of patients receiving nilotinib. We have assessed the effects of nilotinib on bone cells in vitro and on bone metabolism in patients receiving nilotinib for treatment of CML. We firstly investigated the effects of nilotinib on proliferating and differentiating osteoblastic cells, and on osteoclastogenesis in murine bone marrow cultures and RAW264.7 cells. Nilotinib potently inhibited osteoblast proliferation (0.01-1uM), through inhibition of the platelet-derived growth factor (PDGFR). There was a biphasic effect on osteoblast differentiation such that it was reduced by lower concentrations of nilotinib (0.1-0.5uM), with no effect at higher concentrations (1uM). Nilotinib also potently inhibited osteoclastogenesis, predominantly by stromal-cell dependent mechanisms. Thus, nilotinib decreased osteoclast development in murine bone marrow cultures, but did not affect osteoclastogenesis in RAW264.7 cells. Nilotinib treatment of osteoblastic cells increased expression and secretion of OPG and decreased expression of RANKL. In 10 patients receiving nilotinib, levels of bone turnover markers were in the low-normal range, despite secondary hyperparathyroidism, findings that are similar to those in patients treated with imatinib. Bone density tended to be higher than age and gender-matched normal values. These data suggest that nilotinib may have important effects on bone metabolism. Prospective studies should be conducted to determine the long-term effects of nilotinib on bone density and calcium metabolism.
    No preview · Article · Aug 2011 · Bone
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    ABSTRACT: Lactoferrin, an iron-binding glycoprotein present in milk and other exocrine secretions in mammals, is anabolic to bone at physiological concentrations. Lactoferrin stimulates the proliferation, differentiation and survival of osteoblasts, as well as potently inhibiting osteoclastogenesis in bone marrow cultures. In the current study we further investigated the mechanism of action of lactoferrin in osteoblasts. We used low-density arrays to measure the level of expression of 45 genes in MC3T3-E1 osteoblast-like cells treated with lactoferrin, and identified transient, dose-dependent increases in the transcription levels of interleukin-6, of the pro-inflammatory factor prostaglandin-endoperoxide synthase 2 (Ptgs2), and of the transcription factor nuclear factor of activated T cells (Nfatc1). We demonstrated similar changes in primary osteoblast cultures from human and rat. Levels of prostaglandin E2 were increased in conditioned media collected from osteoblasts treated with lactoferrin, indicating that the activity of the enzyme cyclooxygenase 2 (COX2), which is encoded by Ptgs2, was also up-regulated. Using a luciferase reporter construct we showed that lactoferrin induced transcription from the NFAT consensus sequence. We found that inhibiting either COX2 or NFATc1 activity blocked the mitogenic effect of lactoferrin in osteoblasts and that inhibition of NFATc1 activity partially blocked the transcriptional activation of Ptgs2. Our study has provided the first evidence that COX2 and NFATc1 activities are increased by lactoferrin, and demonstrated a role for each of these molecules as mediators of the mitogenic effects of lactoferrin in osteoblasts.
    No preview · Article · Aug 2011 · Bone
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    ABSTRACT: Leptin, a major hormonal product of the adipocyte, regulates appetite and reproductive function through its hypothalamic receptors. The leptin receptor is present in osteoblasts and chondrocytes, and previously we have shown leptin to be an anabolic bone factor in vitro, stimulating osteoblast proliferation and inhibiting osteoclastogenesis. Leptin increases bone mass and reduces bone fragility when administered peripherally but also can indirectly reduce bone mass when administered into the central nervous system. However, data from animal models deficient in either leptin (ob/ob) or its receptor (db/db) remain contradictory. We compared the bone phenotype of leptin receptor-deficient (db/db) and wild-type mice using micro-computed tomographic (µCT) analysis of the proximal tibias and vertebrae. In the tibia, db/db mice had reduced percent trabecular bone volume (13.0 ± 1.62% in wild-type versus 6.01 ± 0.601% in db/db mice, p = .002) and cortical bone volume (411 ± 21.5 µm(3) versus 316 ± 3.53 µm(3), p = .0014), trabecular thickness (48.4 ± 001.07 µm versus 45.1 ± 0.929 µm, p = .041) and trabecular number (2.68 ± 0.319 mm(-1) versus 1.34 ± 0.148 mm(-1), p = .0034). In the fifth lumbar vertebral body, the trabecular thickness and cortical thickness were decreased in the db/db versus wild-type mice (0.053 ± 0.0011 mm versus 0.047 ± 0.0013 mm, p = .0002 and 0.062 ± 0.00054 mm versus 0.056 ± 0.0009 mm, p = .0001), respectively, whereas the trabecular and cortical percent bone volume and trabecular number did not reach significance. The total (endosteal and periosteal) cortical perimeter (12.2 ± 0.19 mm versus 13.2 ± 0.30 mm, p = .01) was increased. The serum osteocalcin levels were reduced in the db/db mice, suggesting that bone formation rates are decreased. The material properties of db/db femurs were determined by three-point bending and nanoindentation, showing decreased bone strength (13.3 ± 0.280 N versus 7.99 ± 0.984 N, p = .0074) and material stiffness (28.5 ± 0.280 GPa versus 25.8 ± 0.281 GPa, p < .0001). These results demonstrate that bone mass and strength are reduced in the absence of leptin signaling, indicating that leptin acts in vivo as an anabolic bone factor. This concurs with results of in vitro studies and of peripheral leptin administration in vivo and suggests that leptin's direct effects on bone cells are likely to override its actions via the central nervous system.
    Preview · Article · Aug 2011 · Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
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    ABSTRACT: Indentation is a mature technique that has been widely used in materials science to investigate the mechanical properties of metals and thin films. The indentation technique provides accurate modulus and hardness values of materials over many length scales and can target specific microstructures within heterogeneous materials. A more traditional engineering approach for mechanical properties is three point bend testing which provides an indication of the general fracture performance of the material. The breaking force and toughness results determined are based on the materials overall structure and composition. However, for both techniques, the testing specimen requires certain degree of process. This study evaluated a new indentation technique, which is able to penetrate biological tissues, apply compressive loads on the bone surface and record the resulting displacement, using wild type rats fed with a standard diet. In this study, both femurs from the same animal were tested followed by the three point bending to reach structural failure. We found a correlation between the two techniques and the properties of the bone in the animal model.
    No preview · Article · Jul 2011
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    ABSTRACT: Bone erosion is a common manifestation of chronic tophaceous gout. To investigate the effects of monosodium urate monohydrate (MSU) crystals on osteoblast viability and function. The MTT assay and flow cytometry were used to assess osteoblast cell viability in the MC3T3-E1 and ST2 osteoblast-like cell lines, and primary rat and primary human osteoblasts cultured with MSU crystals. Quantitative real-time PCR and von Kossa stained mineralised bone formation assays were used to assess the effects of MSU crystals on osteoblast differentiation using MC3T3-E1 cells. The numbers of osteoblasts and bone lining cells were quantified in bone samples from patients with gout. MSU crystals rapidly reduced viability in all cell types in a dose-dependent manner. The inhibitory effect on cell viability was independent of crystal phagocytosis and was not influenced by differing crystal length or addition of serum. Long-term culture of MC3T3-E1 cells with MSU crystals showed a reduction in mineralisation and decreased mRNA expression of genes related to osteoblast differentiation such as Runx2, Sp7 (osterix), Ibsp (bone sialoprotein), and Bglap (osteocalcin). Fewer osteoblast and lining cells were present on bone directly adjacent to gouty tophus than bone unaffected by tophus in patients with gout. MSU crystals have profound inhibitory effects on osteoblast viability and differentiation. These data suggest that bone erosion in gout occurs at the tophus-bone interface through alteration of physiological bone turnover, with both excessive osteoclast formation, and reduced osteoblast differentiation from mesenchymal stem cells.
    No preview · Article · May 2011 · Annals of the rheumatic diseases
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    ABSTRACT: Fracture risk is increased in patients with schizophrenia, who often receive long-term therapy with anti-psychotic drugs. The mechanisms by which skeletal fragility is increased in patients with psychosis include increased risk of falling, but direct skeletal toxicity of anti-psychotic drugs is a possibility that has not been investigated. We examined the skeletal effects, in vivo and in vitro, of a typical anti-psychotic drug, haloperidol, which primarily inhibits dopaminergic signaling, and an atypical anti-psychotic drug, clozapine, which predominantly inhibits serotonergic signaling. In growing rats, 42 days of clozapine treatment reduced whole body bone mineral density by 15% (P<0.01 vs vehicle), and trabecular and cortical bone volume, as assessed by microcomputed tomography, by 29% and 15%, respectively (P<0.05 vs vehicle for each). Treatment with haloperidol did not affect bone density. Clozapine, but not haloperidol, transiently increased levels of serum corticosterone, and decreased levels of serum testosterone. In vitro, clozapine dose-dependently decreased osteoblast mitogenesis, osteoblast differentiation and osteoclastogenesis, while haloperidol did not affect any of these parameters. These data demonstrate that clozapine, but not haloperidol, exerts adverse skeletal effects in rodents, and that this effect may be attributable to direct actions to reduce osteoblast growth and function. Long-term administration of clozapine may therefore negatively affect bone health, and clinical studies to investigate this possibility are warranted.
    No preview · Article · Dec 2010 · Schizophrenia Research
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    ABSTRACT: Imatinib mesylate is a tyrosine kinase inhibitor used in the management of disorders in which activation of c-Abl, PDGFR, or c-Kit signaling plays a critical role. In vitro, imatinib stimulates osteoblast differentiation, inhibits osteoblast proliferation and survival, and decreases osteoclast development. Patients treated with imatinib exhibit altered bone and mineral metabolism, with stable or increased bone mass. However, recovery from the underlying disease and/or weight gain might contribute to these effects. We therefore investigated the skeletal effects of imatinib in healthy rats. We evaluated the effects of imatinib on bone volume, markers of bone turnover, and bone histomorphometry in mature female rats treated for 5 weeks with either vehicle, imatinib 40 mg/kg daily, or imatinib 70 mg/kg daily. Compared to vehicle, imatinib reduced trabecular bone volume/tissue volume (mean [SD]: vehicle 26.4% [5.4%], low-dose imatinib 24.8% [4.9%] [P = 0.5], high-dose imatinib 21.1% [5.7%] [P = 0.05]), reduced osteoblast surface (mean [SD]: vehicle 12.8% [5.8%], low-dose 6.8% [1.9%] [P < 0.01], high-dose 7.8 [3.1%] [P < 0.05]), and reduced serum osteocalcin (mean change from baseline [95% CI]: vehicle -8.2 [-26.6 to 10.2] ng/ml, low dose -79.7 [-97.5 to -61.9] ng/ml [P < 0.01 vs. vehicle], high-dose -66.0 [-82.0 to -50.0] ng/ml [P < 0.05 vs. vehicle]). Imatinib did not affect biochemical or histomorphometric indices of bone resorption. These results suggest that, in healthy animals, treatment with imatinib does not increase bone mass and that the improvements in bone density reported in patients receiving imatinib may not be a direct effect of the drug.
    No preview · Article · Oct 2010 · Calcified Tissue International
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    ABSTRACT: Signaling through phosphatidylinositol-3 kinases (PI3K) regulates fundamental cellular processes such as survival and growth, and these lipid kinases are currently being investigated as therapeutic targets in several contexts. In skeletal tissue, experiments using pan-specific PI3K inhibitors have suggested that PI3K signaling influences both osteoclast and osteoblast function, but the contributions of specific PI3K isoforms to these effects have not been examined. In the current work, we assessed the effects of pharmacological inhibitors of the class Ia PI3Ks, alpha, beta, and delta, on bone cell growth, differentiation and function in vitro. Each of the class Ia PI3K isoforms is expressed and functionally active in bone cells. No consistent effects of inhibitors of p110-beta or p110-delta on bone cells were observed. Inhibitors of p110-alpha decreased osteoclastogenesis by 60-80% (p<0.001 vs control) by direct actions on osteoclast precursors, and decreased the resorptive activity of mature osteoclasts by 60% (p<0.01 vs control). The p110-alpha inhibitors also decreased the growth of osteoblastic and stromal cells (p<0.001 vs control), and decreased differentiated osteoblast function by 30% (p<0.05 vs control). These data suggest that signaling through the p110-alpha isoform of class Ia PI3Ks positively regulates the development and function of both osteoblasts and osteoclasts. Therapeutic agents that target this enzyme have the potential to significantly affect bone homeostasis, and evaluation of skeletal endpoints in clinical trials of such agents is warranted.
    No preview · Article · Nov 2009 · Biochemical and Biophysical Research Communications
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    ABSTRACT: The fibroblast growth factors (FGFs) are a group of at least 25 structurally related peptides that are involved in many biological processes. Some FGFs are active in bone, including FGF-1, FGF-2, and FGF-18, and recent evidence indicates that FGF-8 is osteogenic, particularly in mesenchymal stem cells. In the current study, we found that FGF-8 was expressed in rat primary osteoblasts and in osteoblastic UMR-106 and MC3T3-E1 cells. Both FGF-8a and FGF-8b potently stimulated the proliferation of osteoblastic cells, whereas they inhibited the formation of mineralized bone nodules in long-term cultures of osteoblasts and reduced the levels of osteoblast differentiation markers, osteocalcin, and bone sialoprotein. FGF-8a induced the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in osteoblastic cells; however, its mitogenic actions were not blocked by either the MAPK kinase (MEK) inhibitor U-0126 or the PI 3-kinase (PI3K) inhibitor LY-294002. Interestingly, FGF-8a, unlike FGF-8b and other members of the family, inhibited osteoclastogenesis in mouse bone marrow cultures, and this was via a receptor activator of NF-kappaB ligand (RANKL)/osteoprotegerin (OPG)-independent manner. However, FGF-8a did not affect osteoclastogenesis in RAW 264.7 cells (a macrophage cell line devoid of stromal cells) exogenously stimulated by RANKL, nor did it affect mature osteoclast function as assessed in rat calvarial organ cultures and isolated mature osteoclasts. In summary, we have demonstrated that FGF-8 is active in bone cells, stimulating osteoblast proliferation in a MAPK-independent pathway and inhibiting osteoclastogenesis via a RANKL/OPG-independent mechanism. These data suggest that FGF-8 may have a physiological role in bone acting in an autocrine/paracrine manner.
    Full-text · Article · May 2009 · AJP Endocrinology and Metabolism
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    ABSTRACT: Fat mass impacts on both bone turnover and bone density and is a critical risk factor for osteoporotic fractures. Adipocyte-derived hormones may contribute to this relationship, and adiponectin is a principal circulating adipokine. However, its effects on bone remain unclear. We have, therefore, investigated the direct effects of adiponectin on primary cultures of osteoblastic and osteoclastic cells in vitro and determined its integrated effects in vivo by characterizing the bone phenotype of adiponectin-deficient mice. Adiponectin was dose-dependently mitogenic to primary rat and human osteoblasts ( approximately 50% increase at 10 microg/ml) and markedly inhibited osteoclastogenesis at concentrations of 1 microg/ml or greater. It had no effect on osteoclastogenesis in RAW-264.7 cells or on bone resorption in isolated mature osteoclasts. In adiponectin knockout (AdKO) male C57BL/6J mice, trabecular bone volume and trabecular number (assessed by microcomputed tomography) were increased at 14 wk of age by 30% (P = 0.02) and 38% (P = 0.0009), respectively. Similar, nonsignificant trends were observed at 8 and 22 wk of age. Biomechanical testing showed lower bone fragility and reduced cortical hardness at 14 wk. We conclude that adiponectin stimulates osteoblast growth but inhibits osteoclastogenesis, probably via an effect on stromal cells. However, the AdKO mouse has increased bone mass, suggesting that adiponectin also has indirect effects on bone, possibly through modulating growth factor action or insulin sensitivity. Because adiponectin does influence bone mass in vivo, it is likely to be a contributor to the fat-bone relationship.
    Preview · Article · Apr 2009 · Endocrinology
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    ABSTRACT: Clinical studies have shown that total body fat mass is related to both bone density and fracture risk and that fat ingestion reduces bone turnover. These effects are at least partially mediated by endocrine mechanisms, but it is possible that lipids might act directly on bone. We assessed the effects of broad fractions of milk lipids in osteoblasts, bone marrow, and neonatal mouse calvariae. Several milk fractions and their hydrolysates inhibited osteoclastogenesis in bone marrow cultures, so we assessed the effects of free fatty acids in this model. Saturated fatty acids (0.1-10 microg/ml) inhibited osteoclastogenesis in bone marrow cultures and RAW264.7 cells. This effect was maximal for C14:0 to C18:0 fatty acids. The introduction of greater than 1 double bond abrogated this effect; omega3 and omega6 fatty acids had comparable low activity. Osteoblast proliferation was modestly increased by the antiosteoclastogenic compounds, ruling out a nonspecific toxic effect. Active fatty acids did not consistently change expression of receptor activator of nuclear factor-kappaB ligand or osteoprotegerin in osteoblastic cells nor did they affect the activity of key enzymes in the mevalonate pathway. However, receptors known to bind fatty acids were found to be expressed in osteoblastic (GPR120) and osteoclastic (GPR40, 41, 43, 120) cells. A synthetic GPR 40/120 agonist mimicked the inhibitory effects of fatty acids on osteoclastogenesis. These findings provide a novel link between lipid and bone metabolism, which might contribute to the positive relationship between adiposity and bone density as well as provide novel targets for pharmaceutical and nutriceutical development.
    Preview · Article · Jul 2008 · Endocrinology