Bone Cell Activity Research Articles

The effect of alendronate doped calcium phosphates on bone cells activity

This study demonstrates that octacalcium phosphate (OCP) is a suitable substrate for alendronate local action towards bone cells. The results of the structural, spectroscopic, and microscopic investigation show that soaking OCP into alendronate solutions provoked the deposition of long crystalline rod-shaped formations, most likely a calcium alendronate complex, onto the calcium phosphate. The amount of alendronate loaded onto OCP increased as a function of the bisphosphonate concentration in solution. Osteoblast and osteoclast response was tested in single and in co-cultures on OCP containing 6.4wt.% AL (OCP-AL), and for comparison on hydroxyapatite (HA) containing a similar amount (5.9wt.%) of AL (HA-AL), as well as on pure OCP and HA as reference materials. Alendronate loaded materials displayed a beneficial effect on osteoblast activity and differentiation, whereas they inhibited osteoclast proliferation and differentiation. Crosstalking between osteoblast-like MG63 cells and human osteoclasts enhanced their response to alendronate. Moreover, OCP displayed a greater stimulating effect than HA on osteoblast differentiation, and AL promotion of osteoblast differentiation and mineralization was enhanced in OCP-AL with respect to HA-AL.

The alignment of MC3T3-E1 osteoblasts on steps of slip traces introduced by dislocation motion

Bone tissue shows a highly anisotropic microstructure comprising biological apatite and collagen fibrils produced by the mutual activities of bone cells, which dominates its mechanical function. Accordingly, directional control of osteoblasts is crucial for forming anisotropic bone tissue. A new approach was proposed for controlling cell directionality by using crystallographic slip traces caused by dislocation glide. Dislocations were introduced into α-titanium single crystals by plastic deformation of (011¯0)[21¯1¯0] slip system, inducing a step-like structure with acute angles between the surface normal and the slip plane. Topographical properties of step patterning, including step interval and step height, could be controlled by varying the compressive plastic strain. The step geometry introduced by plastic deformation strongly influenced osteoblast elongation, and it aligned preferentially along slip traces. Ti substrates under 10% plastic strain with step height of approximately 300 nm and step interval of 10 μm induced osteoblast alignment most successfully. Actin stress fibers elongated parallel to slip traces, with polarized vinculin accumulation between steps.

Clopidogrel (Plavix), a P2Y12 receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo

Clopidogrel (Plavix), a selective P2Y(12) receptor antagonist, is widely prescribed to reduce the risk of heart attack and stroke and acts via the inhibition of platelet aggregation. Accumulating evidence now suggests that extracellular nucleotides, signaling through P2 receptors, play a significant role in bone, modulating both osteoblast and osteoclast function. In this study, we investigated the effects of clopidogrel treatment on (1) bone cell formation, differentiation, and activity in vitro; and (2) trabecular and cortical bone parameters in vivo. P2Y(12) receptor expression by osteoblasts and osteoclasts was confirmed using qPCR and Western blotting. Clopidogrel at 10 µM and 25 µM inhibited mineralized bone nodule formation by 50% and >85%, respectively. Clopidogrel slowed osteoblast proliferation with dose-dependent decreases in cell number (25% to 40%) evident in differentiating osteoblasts (day 7). A single dose of 10 to 25 µM clopidogrel to mature osteoblasts also reduced cell viability. At 14 days, ≥10 µM clopidogrel decreased alkaline phosphatase (ALP) activity by ≤70% and collagen formation by 40%, while increasing adipocyte formation. In osteoclasts, ≥1 µM clopidogrel inhibited formation, viability and resorptive activity. Twenty-week-old mice (n = 10-12) were ovariectomized or sham treated and dosed orally with clopidogrel (1 mg/kg) or vehicle (NaCl) daily for 4 weeks. Dual-energy X-ray absorptiometry (DXA) analysis showed clopidogrel-treated animals had decreases of 2% and 4% in whole-body and femoral bone mineral density (BMD), respectively. Detailed analysis of trabecular and cortical bone using micro-computed tomography (microCT) showed decreased trabecular bone volume in the tibia (24%) and femur (18%) of clopidogrel-treated mice. Trabecular number was reduced 20%, while trabecular separation was increased up to 15%. Trabecular thickness and cortical bone parameters were unaffected. Combined, these findings indicate that long-term exposure of bone cells to clopidogrel in vivo could negatively impact bone health.

Genes associate with abnormal bone cell activity in bone metastasis

Bone is one of the most frequent sites of metastasis in patients with malignancies. Up to 90% of patients with multiple myeloma, and 60% to 75% patients with prostate cancer and breast cancer develop bone metastasis at the later stages of their diseases. Bone metastases are responsible for tremendous morbidity in patients with cancer, including severe bone pain, pathologic fractures, spinal cord and nerve compression syndromes, life-threatening hypercalcemia, and increased mortality. Multiple factors produced by tumor cells or produced by the bone marrow microenvironment in response to tumor cells play important roles in activation of osteoclastic bone resorption and modulation of osteoblastic activity in patients with bone metastasis. In this chapter, we will review the genes that play important roles in bone destruction, tumor growth, and osteoblast activity in bone metastasis and discuss the potential therapies targeting the products of these genes to block both bone destruction and tumor growth.

Metabolic acidosis increases fibroblast growth factor 23 in neonatal mouse bone

Fibroblast growth factor 23 (FGF23) significantly increases with declining renal function, leading to reduced renal tubular phosphate reabsorption, decreased 1,25-dihydroxyvitamin D, and increased left ventricular hypertrophy. Elevated FGF23 is associated with increased mortality. FGF23 is synthesized in osteoblasts and osteocytes; however, the mechanisms by which it is regulated are not clear. Patients with chronic kidney disease have decreased renal acid excretion leading to metabolic acidosis, which has a direct effect on bone cell activity. We hypothesized that metabolic acidosis would directly increase bone cell FGF23 production. Using cultured neonatal mouse calvariae, we found that metabolic acidosis increased medium FGF23 protein levels as well as FGF23 RNA expression at 24 h and 48 h compared with incubation in neutral pH medium. To exclude that the increased FGF23 was secondary to metabolic acidosis-induced release of bone mineral phosphate, we cultured primary calvarial osteoblasts. In these cells, metabolic acidosis increased FGF23 RNA expression at 6 h compared with incubation in neutral pH medium. Thus metabolic acidosis directly increases FGF23 mRNA and protein in mouse bone. If these results are confirmed in humans with chronic kidney disease, therapeutic interventions to mitigate acidosis, such as bicarbonate administration, may also lower levels of FGF23, decrease left ventricular hypertrophy, and perhaps even decrease mortality.

Potential Anti-Catabolic and Anabolic Properties of Strontium Ranelate

Osteoporosis is a major public health problem for adults above 55 years of age, which leads to an increase in bone fragility. Last decade has witnessed remarkable advances in molecular biology and genetics that led to detailed understanding of the bone remodeling cycle and new therapeutic targets for its treatment have emerged. Thus, besides classical approach (vitamin D and calcium administration, bisphosphonates, oestrogen, raloxifene), new therapeutic agents such as parathyroid hormone (PTH) compounds, anti-RANKL antibodies and strontium ranelate are or will be increasingly used in the treatment of osteoporosis. In this review, we have presented the importance and therapeutic potential of strontium ranelate as a dual agent in the current treatment of osteoporosis.

Vitamin D supplementation during pregnancy and pregnancy outcomes

Vitamin D supplementation during pregnancy and pregnancy outcomes

Abstract 5317: Role of TGF-beta superfamily ligand and mechanism of osteoblastic tumorigenesis of prostate cancer cells

Abstract Prostate cancer is the second leading cause of cancer related death in men in the US. The skeleton is the most common site for prostate cancer metastasis, which often results in osteoblastic lesions. Transforming growth factor-beta (TGFβ), which is a multifunctional cytokine, is important for the regulation of bone mass and matrix properties. Our group has recently shown that TGFβ signaling helps in promoting osteoblastic tumor growth by a novel human prostate cancer cell line, PacMetUT1. However, the mechanism by which prostate cancer cells metastasize to bone and how they induce formation of osteoblastic lesions remains to be intensively investigated. Bone cells such as osteoblasts express aromatase, which is the enzyme required for estrogen biosynthesis in humans. Recent data further implicates an aberrant aromatase expression and estrogen signaling in the development of prostate malignancy. Hence we hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would in turn lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ superfamily ligands on aromatase expression in pre-osteoblastic cells. Treatment with TGFβ1 and BMP-4/7 resulted in an increase in aromatase expression in both mouse pre-osteoblasts and human bone marrow-derived mesenchymal stem cells (MSC's). This increase in aromatase expression was abrogated after transient knockdown of Smad's in MSC's. Treatment with letrozole (aromatase inhibitor) as well as an anti-estrogen (ICI 182, 780) resulted in a decrease in osteoblastic differentiation of mesenchymal stem cells as measured by alkaline phosphatase activity assay and expression of osteogenic markers by quantitative real-time PCR analysis. Furthermore, PacMetUT1 cells were found to be responsive to 17-β estradiol treatment. 17-β estradiol treatment of PacMetUT1 cells resulted in an enhanced anchorage- dependent and independent cell growth in vitro. 17-β estradiol also induced a change in cell morphology of PacMetUT1 cells with a reduction in E-cadherin expression and an increase in Vimentin expression at both protein and mRNA level. This affect was abrogated after stable estrogen receptor alpha (ERα) knockdown in PacMetUT1 cells. Our results demonstrate an aromatase-mediated induction of osteogenesis by TGFβ superfamily ligands in the pre-osteoblastic cells and a stimulatory effect of estrogen signaling on PacMetUT1 cell tumorigencity in vitro. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis of prostate cancer cells in vivo and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy to alleviate bone metastasis in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5317. doi:1538-7445.AM2012-5317

Abstract 5651: p53-mediated apoptotic signaling overcomes the resistance to oncolytic adenovirus in human osteosarcoma cells

Abstract Despite major advances in the treatment of bone and soft tissue sarcomas, about one fourth of the patients show a poor response to conventional therapy, resulting in subsequent recurrence and leading to a poor prognosis. Therefore, the development of a novel therapeutic strategy is required to cure patients with bone and soft tissue sarcomas. We previously developed an oncolytic adenovirus, OBP-301, in which human telomerase reverse transcriptase (hTERT) gene promoter drives viral E1 gene for replication. OBP-301 induces tumor-specific oncolytic cell death in a telomerase-dependent manner. Recently, we revealed that OBP-301 shows cytopathic activity in human bone and soft tissue sarcoma cells. However, some human osteosarcoma cells were less sensitive to cytopathic activity of OBP-301. To enhance the cytopathic activity of OBP-301, we recently developed a novel telomerase-specific oncolytic adenovirus, OBP-702, which expresses tumor suppressor p53 gene. In this study, we investigated the antitumor effect of OBP-702 in OBP-301-sensitive (OST, HOS and U2OS) and OBP-301-resistant (MNNG/HOS and SaOS-2) human osteosarcoma cells. We compared the antitumor effects of OBP-702 and OBP-301 using XTT assay. The 50% inhibiting dose (ID50) value of OBP-702 and OBP-301 for each cell was calculated using cell viability data obtained on day 5 after virus infection. We further evaluated the expression of p53, p21 and cleaved poly (ADP-ribose) polymerase (PARP) proteins using western blot analysis in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells after infection with OBP-702 or Ad-p53, which is a p53-expressing replication-defective adenovirus. OBP-702 showed more cytopathic activity compared to OBP-301 in both OBP-301-sensitive and OBP-301-resistant osteosarcoma cells. The ID50 value of OBP-702 was lower than that of OBP-301 in all cell lines. OBP-702 induced not only oncolysis, but also apoptotic cell death with the cleavage of PARP. OBP-702 infection induced more profound p53 expression than Ad-p53. However, p53-downstream target p21 proteins were not activated by OBP-702. Taken together, we demonstrated that the p53-expressing oncolytic adenovirus OBP-702 has a much stronger antitumor effect than OBP-301 in human osteosarcoma cells. Oncolytic adenovirus-mediated p53 gene transduction would induce profound apoptosis through p53 upregulation without p21 activation, resulting in the enhancement of antitumor effect by OBP-301. OBP-702 would be a promising antitumor reagent for the treatment of OBP-301-resistant human osteosarcoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5651. doi:1538-7445.AM2012-5651

The NPY System and its Neural and Neuroendocrine Regulation of Bone

The past decade has seen a significant expansion of our understanding of the interaction between the neural system and bone. While innervation of bone was long appreciated, the discovery of central relays from the hypothalamus to the cells of bone has seen the identification of a number of efferent neural pathways to bone. The neuropeptide Y (NPY) system has proven to represent a major central pathway, regulating the activity of osteoblasts and osteoclasts, through signaling of central and peripheral ligands, through specific receptors within the hypothalamus and the osteoblast. Moreover, this pathway is now recognized as acting to coordinate both skeletal and energy homeostasis. This review examines the mechanism and actions of the NPY pathway to regulate bone mass and bone cell activity.

Clopidogrel and the risk of osteoporotic fractures: a nationwide cohort study

The P2Y(12) inhibitor clopidogrel inhibits platelet aggregation and is used in the treatment and prevention of coronary artery disease. It is widely used and, in combination with acetylsalicylic acid, is the standard of care for acute coronary syndrome and percutaneous coronary intervention. The mode of action of clopidogrel involves pathways that are important to the metabolic activity in bone cells, although to our knowledge whether P2Y(12) receptors are involved in the regulation of bone metabolism has not yet been investigated. Therefore, the objective of the present study was to investigate the association between clopidogrel use and risk of fractures. We investigated the association between clopidogrel use and fracture incidence in a nationwide cohort study within the Danish population of approximately 5.3 million individuals. All patients who were prescribed clopidogrel during the years 1996-2008 were included in the study (n = 77 503), and three nonusers were randomly selected, matched for age and gender (n = 232 510), for each clopidogrel-treated subject. Treatment with clopidogrel was associated with both increased overall fracture risk and increased risk of osteoporotic fractures, especially in subjects with a treatment duration of more than 1 year. However, individuals with low exposure to clopidogrel (<0.01 defined daily dose) had a lower risk of fracture than never users. Use of the P2Y(12) inhibitor clopidogrel is associated with risk of fractures. There seems to be a biphasic relation so that lower doses are associated with decreased fracture risk, whereas higher doses (recommended dose range) are associated with increased risk. More studies are warranted to determine the potential in vivo effect of platelet aggregation inhibitors on bone metabolism.

Monitoring Molecular, Functional and Morphologic Aspects of Bone Metastases Using Non-Invasive Imaging

Bone is among the most common locations of metastasis and therefore represents an important clinical target for diagnostic follow-up in cancer patients. In the pathogenesis of bone metastases, disseminated tumor cells proliferating in bone interact with the local microenvironment stimulating or inhibiting osteoclast and osteoblast activity. Non-invasive imaging methods monitor molecular, functional and morphologic changes in both compartments of these skeletal lesions - the bone and the soft tissue tumor compartment. In the bone compartment, morphologic information on skeletal destruction is assessed by computed tomography (CT) and radiography. Pathogenic processes of osteoclast and osteoblast activity, however, can be imaged using optical imaging, positron emission tomography (PET), single photon emission CT (SPECT) and skeletal scintigraphy. Accordingly, conventional magnetic resonance imaging (MRI) and CT as well as diffusion- weighted MRI and optical imaging are used to assess morphologic aspects on the macroscopic and cellular level of the soft tissue tumor compartment. Imaging methods such as PET, MR spectroscopy, dynamic contrast-enhanced techniques and vessel size imaging further elucidate on pathogenic processes in this compartment including information on metabolism and vascularization. By monitoring these aspects in bone lesions, new insights in the pathogenesis of skeletal metastases can be gained. In translation to the clinical situation, these novel methods for the monitoring of bone metastases might be applied in patients to improve follow-up of these lesions, in particular after therapeutic intervention. This review summarizes established and experimental imaging techniques for the monitoring of tumor and bone cell activity including molecular, functional and morphological aspects in bone metastases.

Impact of an obesogenic diet program on bone densitometry, micro architecture and metabolism in male rat

BackgroundThe relationships between fat mass and bone tissue are complex and not fully elucidated. A high-fat/high-sucrose diet has been shown to induce harmful effects on bone micro architecture and bone biomechanics of rat. When such diet leads to obesity, it may induce an improvement of biomechanical bone parameters in rodent.Here, we examined the impact of a high-fat/high-sucrose diet on the body composition and its resulting effects on bone density and structure in male rats. Forty three Wistar rats aged 7 months were split into 3 groups: 1 sacrificed before diet (BD, n = 14); 1 subjected to 16 weeks of high-fat/high-sucrose diet (HF/HS, n = 14); 1 subjected to standard diet (Control, n = 15). Abdominal circumference and insulin sensitivity were measured and visceral fat mass was weighed. The bone mineral density (BMD) was analyzed at the whole body and tibia by densitometry. Microcomputed tomography and histomorphometric analysis were performed at L2 vertebrae and tibia to study the trabecular and cortical bone structures and the bone cell activities. Osteocalcin and CTX levels were performed to assess the relative balance of the bone formation and resorption. Differences between groups have been tested with an ANOVA with subsequent Scheffe post-hoc test. An ANCOVA with global mass and global fat as covariates was used to determine the potential implication of the resulting mechanical loading on bone.ResultsThe HF/HS group had higher body mass, fat masses and abdominal circumference and developed an impaired glucose tolerance (p < 0.001). Whole body bone mass (p < 0.001) and BMD (p < 0.05) were higher in HF/HS group vs. Control group. The trabecular thickness at vertebrae and the cortical porosity of tibia were improved (p < 0.05) in HF/HS group. Bone formation was predominant in HF/HS group while an unbalance bone favoring bone resorption was observed in the controls. The HF/HS and Control groups had higher total and abdominal fat masses and altered bone parameters vs. BD group.ConclusionsThe HF/HS diet had induced obesity and impaired glucose tolerance. These changes resulted in an improvement of quantitative, qualitative and metabolic bone parameters. The fat mass increase partly explained these observations.

Role of cannabinoids in the regulation of bone remodeling.

The endocannabinoid system plays a key role in regulating a variety of physiological processes such as appetite control and energy balance, pain perception, and immune responses. Recent studies have implicated the endocannabinoid system in the regulation of bone cell activity and bone remodeling. These studies showed that endogenous cannabinoid ligands, cannabinoid receptors, and the enzymes responsible for ligand synthesis and breakdown all play important roles in bone mass and in the regulation of bone disease. These findings suggest that the endocannabinoid pathway could be of value as a therapeutic target for the prevention and treatment of bone diseases. Here, we review the role of the skeletal endocannabinoid system in the regulation of bone remodeling in health and disease.

Nitric oxide mediates low magnesium inhibition of osteoblast-like cell proliferation

An adequate intake of magnesium (Mg) is important for bone cell activity and contributes to the prevention of osteoporosis. Because (a) Mg is mitogenic for osteoblasts and (b) reduction of osteoblast proliferation is detected in osteoporosis, we investigated the influence of different concentrations of extracellular Mg on osteoblast-like SaOS-2 cell behavior. We found that low Mg inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the up-regulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor l-N6-(iminoethyl)-lysine-HCl and genetic silencing of iNOS by small interfering RNA restored the normal proliferation rate of the cells.Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining Mg homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.

Rheumatoid arthritis is associated with increased DKK1 expression and disturbances in the bone turnover regulating genes

Materials and methods RA patients submitted to hip replacement surgery were recruited. They were matched to a group of primary OP patients for bone mineral density and major clinical fracture risk factors (age, gender, BMI). Trabecular bone microarchitecture was assessed by micro-computed tomography and bone mechanical behavior by compression tests. Bone cell activity was analyzed by studying gene expression.

Osteocytic network is more responsive in calcium signaling than osteoblastic network under fluid flow

Osteocytes, regarded as the mechanical sensor in bone, respond to mechanical stimulation by activating biochemical pathways and mediating the cellular activities of other bone cells. Little is known about how osteocytic networks respond to physiological mechanical stimuli. In this study, we compared the mechanical sensitivity of osteocytic and osteoblastic networks under physiological-related fluid shear stress (0.5 to 4 Pa). The intracellular calcium ([Ca(2+)](i)) responses in micropatterned in vitro osteoblastic or osteocytic networks were recorded and analyzed. Osteocytes in the network showed highly repetitive spikelike [Ca(2+)](i) peaks under fluid flow stimulation, which are dramatically different from those in the osteoblastic network. The number of responsive osteocytes in the network remained at a constant high percentage (>95%) regardless of the magnitude of shear stress, whereas the number of responsive osteoblasts in the network significantly depends on the strength of fluid flow. All spatiotemporal parameters of calcium signaling demonstrated that osteocytic networks are more sensitive and dynamic than osteoblastic networks, especially under low-level mechanical stimulations. Furthermore, pathway studies were performed to identify the molecular mechanisms responsible for the differences in [Ca(2+)](i) signaling between osteoblastic and osteocytic networks. The results suggested that the T-type voltage-gated calcium channels (VGCC) expressed on osteocytes may play an essential role in the unique kinetics of [Ca(2+)](i) signaling in osteocytic networks, whereas the L-type VGCC is critical for both types of cells to release multiple [Ca(2+)](i) peaks. The extracellular calcium source and intracellular calcium store in ER-, ATP-, PGE₂-, NO-, and caffeine-related pathways are found to play similar roles in the [Ca(2+)](i) signaling for both osteoblasts and osteocytes. The findings in this study proved that osteocytic networks possess unique characteristics in sensing and processing mechanical signals.

Abstract B106: Role of TGF-beta superfamily ligand and estrogen signaling in mediating osteoblastic tumorigenesis of prostate cancer cells.

Abstract Prostate cancer metastasizes to bone in 80% of patients suffering from advanced disease. The nature of bone lesions is predominantly osteoblastic resulting in severe bone pain, hypocalcemia and nerve compression syndromes. Transforming growth factor-beta (TGFβ), which is a multifunctional cytokine, is a critical regulator of osteogenesis. Our group has recently shown that TGFβ signaling helps in promoting osteoblastic metastasis by a novel human prostate cancer cell line, PacMetUT1. TGFβ has been suggested to be one of the physiological upregulatory factors of bone aromatase in human osteoblast-like cells. Recent data further implicates an aberrant aromatase expression and estrogen signaling in the development of prostate malignancy. However, the effect of TGFβ superfamily ligands on aromatase expression and estrogen production and the mechanism by which osteoblastic lesions are formed have not been intensively investigated in prostate cancer cells. We hypothesize that TGFβ and other growth factors released from tumor cells can induce aromatase gene expression and activity in bone cells leading to an enhanced estradiol synthesis which would in turn lead to an increase in bone formation as estrogen is a known osteogenic factor. We examined the effect of TGFβ and BMP-4/7 on aromatase expression and osteogenic differentiation of pre-osteoblastic cells in vitro. Treatment with TGFβ 1 and BMP-4/7 resulted in an increase in aromatase expression in both mouse pre-osteoblasts and human bone marrow-derived mesenchymal stem cells. Treatment with letrozole (aromatase inhibitor) as well as an anti-estrogen (ICI 182, 780) resulted in a decrease in osteoblastic differentiation of mesenchymal stem cells as measured by alkaline phosphatase activity assay and expression of osteogenic markers by quantitative real-time PCR analysis. Furthermore, 17-β estradiol treatment of PacMetUT1 cells resulted in an enhanced anchorage-dependent and independent cell growth in vitro. This effect was very significantly inhibited by tamoxifen treatment, which is a selective estrogen receptor modulator. 17-β estradiol also induced a change in cell morphology of PacMetUT1 cells with a reduction in E-cadherin expression. Our results demonstrate an aromatase-mediated induction in osteogenesis by TGFβ superfamily ligands in the pre-osteoblastic cells and a stimulatory effect of estrogen signaling on PacMetUT1 cell tumorigencity in vitro. Future studies will delineate whether the induction of aromatase mediates osteoblastic bone metastasis of prostate cancer cells in vivo and if combination treatment of TGFβ and aromatase inhibitors can be used as a novel therapeutic strategy, to alleviate bone metastasis in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B106.

Structure and quantification of microvascularisation within mouse long bones: What and how should we measure?

Bone marrow vascularisation is involved in both remodeling and hematopoïesis. Challenged mouse models often require imaging and quantitative assessment of blood vessels and bone cell activities for a better understanding of the role of the vascular system. In this study we compared images of mouse hind limb long bone vascularisation after infusion of either barium sulfate or lead chromate-loaded silicon. The images were then analyzed through histology as well as low-resolution and synchrotron-radiation microtomography. We show that barium sulfate infusion provides the best vessel images and furthermore, that it is compatible with staining procedures used in bone histomorphometry and CD31 immunohistochemistry. Bone marrow vascularisation displays large structural and spatial distribution heterogeneity, including large lobular clusters of sinusoids and an unexpectedly substantial amount of capillaries in the adipocytes-rich distal third of the tibia. For an unbiased assessment of bone vascular development/changes, these features must be taken into account. We describe the conditions under which the quantification of microvascularisation on histological sections of barium-infused long bones is reproducible, as applied to seven-month-old male C57/Bl6J and mixed CD1/129Sv/J mice, and we propose a nomenclature for the histological parameters measured. Finally, we validate our technique by studying the effect of ovariectomy on mouse tibial vascular density.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFβ-dependent expression of the Wnt antagonist, sclerostin

IntroductionBreast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.MethodsThe aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.ResultsWe show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.ConclusionsThis study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.