Platelets and osteoblasts: secretome connections.

Megakaryocyte hyperplasia associated with myeloproliferative neoplasms commonly leads to abnormal bone tissue deposition in the bone marrow, known as osteosclerosis. In this study, we aimed to synthesize the known proteomics literature describing factors released by megakaryocytes and platelets and to examine if any of the secreted factors have a known ability to stimulate the bone-forming cells, osteoblasts. Using a systematic search of Medline, we identified 77 articles reporting on factors secreted by platelets and megakaryocytes. After a full-text screening and analysis of the studies, we selected seven papers that reported proteomics data for factors secreted by platelets from healthy individuals. From 60 proteins reported in at least two studies, we focused on 23 that contained a putative signal peptide, which we searched for a potential osteoblast-stimulatory function. From nine proteins with a positive effect on osteoblast formation and function, two extracellular matrix (ECM) proteins, secreted protein acidic and rich in cysteine (SPARC) and tissue inhibitor of metalloproteinase-1 (TIMP1), and three cellular proteins with known extracellular function, the 70-kDa heat shock protein (HSP70), thymosin-ß4 (TB4), and super dismutase (SOD), were identified as hypothetical candidate molecules to be examined as potential mediators in mouse models of osteomyelofibrosis. Thus, careful analysis of prior literature can be beneficial in assisting the planning of future experimental studies.

Male Marfan mice are predisposed to high-fat diet-induced obesity, diabetes, and fatty liver.

Gene mutations in the extracellular matrix protein fibrillin-1 cause connective tissue disorders including Marfan syndrome (MFS) with clinical symptoms in the cardiovascular, skeletal, and ocular systems. Patients with MFS also exhibit alterations in adipose tissues, which in some individuals leads to lipodystrophy, whereas in others to obesity. We have recently demonstrated that fibrillin-1 regulates adipose tissue homeostasis. Here, we examined how fibrillin-1 abnormality affects metabolic adaptation to different diets. We used two MFS mouse models: hypomorph Fbn1mgR/mgR mice and Fbn1C1041G/+ mice with a fibrillin-1 missense mutation. When Fbn1mgR/mgR mice were fed with high-fat diet (HFD) for 12 wk, male mice were heavier than littermate controls (LCs), whereas female mice gained less weight compared with LCs. Female Fbn1C1041G/+ mice on an HFD for 24 wk were similarly protected from weight gain. Male Fbn1C1041G/+ mice on an HFD demonstrated higher insulin levels, insulin intolerance, circulating levels of cholesterol, and high-density lipoproteins. Moreover, male HFD-fed Fbn1C1041G/+ mice showed a higher liver weight and a fatty liver phenotype, which was reduced to LC levels after orchiectomy. Phosphorylation of protein kinase-like endoplasmic reticulum kinase (PERK) and the expression of sterol regulatory element-binding protein 1 (Srebp1) in livers of HFD-fed male Fbn1C1041G/+ mice were elevated. In conclusion, the data demonstrate that male mice of both the MFS models are susceptible to HFD-induced obesity and diabetes. Moreover, male Fbn1C1041G/+ mice develop a fatty liver phenotype, likely mediated by a baseline increased endoplasmic reticulum stress. In contrast, female MFS mice were protected from the consequence of HFD.

Fibrillin-1 regulates white adipose tissue development, homeostasis, and function.

Fibrillin-1 is an extracellular glycoprotein present throughout the body. Mutations in fibrillin-1 cause a wide spectrum of type I fibrillinopathies, including Marfan syndrome characterized by clinical manifestations in adipose tissues, among others. This study addresses the hypothesis that fibrillin-1 regulates adipocyte development and plays a vital role in adipose tissue homeostasis. We employed two mouse models - Fbn1mgR/mgR (20-25% of normal fibrillin-1) and Fbn1C1041G/+ (missense mutation in fibrillin-1) to examine the role of fibrillin-1 in adipose tissue development and homeostasis. Fibrillin-1 was detected around mature adipocytes in both mouse and human white adipose tissues. As expected, Fbn1mgR/mgR mice displayed a significant reduction of fibrillin-1 in white adipose tissue, and no change was observed for Fbn1C1041G/+ mice, each compared to their respective littermates. Male Fbn1mgR/mgR mice had more white and brown adipose tissues, whereas female Fbn1mgR/mgR and both male and female Fbn1C1041G/+ showed no difference compared to their respective wild-type littermates. Consistent with this data, male Fbn1mgR/mgR mice displayed hyperinsulinemia and an insulin resistance phenotype with higher levels of cholesterol and high-density lipoproteins in the serum. Fibrillin-1 deficiency in male Fbn1mgR/mgR mice also promoted adipogenic gene expression and led to hypertrophic expansion of mature adipocytes. To further elucidate the fibrillin-1-dependent adipogenic mechanisms in cell culture, we used primary bone marrow derived mesenchymal stem/stromal cells (MSCs) from Fbn1mgR/mgR, Fbn1C1041G/+ and wild-type mice. Increased lipid content, adipogenic differentiation and pAKT levels were observed when MSCs from both male and female Fbn1mgR/mgR mice were differentiated. Furthermore, a recombinant fragment spanning the C-terminal half of fibrillin-1 significantly reduced adipocyte differentiation i) by binding to MSCs and inhibiting adipogenic commitment, and ii) by sequestering insulin, together suppressing the AKT signaling pathway. This fibrillin-1 fragment also rescued enhanced adipogenic differentiation of MSCs derived from Fbn1mgR/mgR mice. Overall, this study shows that altered adipose tissue homeostasis observed in fibrillin-1 deficient mice depends on the type of fibrillin-1 deficiency and the biological sex, and it shows that fibrillin-1 is a negative regulator of adipogenesis.

Fibrillin-1-regulated miR-122 has a critical role in thoracic aortic aneurysm formation.

Thoracic aortic aneurysms (TAA) in Marfan syndrome, caused by fibrillin-1 mutations, are characterized by elevated cytokines and fragmentated elastic laminae in the aortic wall. This study explored whether and how specific fibrillin-1-regulated miRNAs mediate inflammatory cytokine expression and elastic laminae degradation in TAA. miRNA expression profiling at early and late TAA stages using a severe Marfan mouse model (Fbn1mgR/mgR) revealed a spectrum of differentially regulated miRNAs. Bioinformatic analyses predicted the involvement of these miRNAs in inflammatory and extracellular matrix-related pathways. We demonstrate that upregulation of pro-inflammatory cytokines and matrix metalloproteinases is a common characteristic of mouse and human TAA tissues. miR-122, the most downregulated miRNA in the aortae of 10-week-old Fbn1mgR/mgR mice, post-transcriptionally upregulated CCL2, IL-1ß and MMP12. Similar data were obtained at 70 weeks of age using Fbn1C1041G/+ mice. Deficient fibrillin-1-smooth muscle cell interaction suppressed miR-122 levels. The marker for tissue hypoxia HIF-1α was upregulated in the aortic wall of Fbn1mgR/mgR mice, and miR-122 was reduced under hypoxic conditions in cell and organ cultures. Reduced miR-122 was partially rescued by HIF-1α inhibitors, digoxin and 2-methoxyestradiol in aortic smooth muscle cells. Digoxin-treated Fbn1mgR/mgR mice demonstrated elevated miR-122 and suppressed CCL2 and MMP12 levels in the ascending aortae, with reduced elastin fragmentation and aortic dilation. In summary, this study demonstrates that miR-122 in the aortic wall inhibits inflammatory responses and matrix remodeling, which is suppressed by deficient fibrillin-1-cell interaction and hypoxia in TAA.

Active hematopoiesis triggers exosomal release of PRDX2 that promotes osteoclast formation.

Hematopoietic disorders, particularly hemolytic anemias, commonly lead to bone loss. We have previously reported that actively proliferating cancer cells stimulate osteoclastogenesis from late precursors in a RANKL-independent manner. We theorized that cancer cells exploit the physiological role of bone resorption to support expanding hematopoietic bone marrow and examined if hematopoietic cells can trigger osteoclastogenesis. Using phlebotomy-induced acute anemia in mice, we found strong correlation between augmented erythropoiesis and increased osteoclastogenesis. Conditioned medium (CM) from K562 erythroleukemia cells and primary mouse erythroblasts stimulated osteoclastogenesis when added to RANKL-primed precursors from mouse bone marrow or RAW264.7 cells. Using immunoblotting and mass spectrometry, PRDX2 was identified as a factor produced by erythroid cells in vitro and in vivo. PRDX2 was detected in K562-derived exosomes, and inhibiting exosomal release significantly decreased the osteoclastogenic capacity of K562 CM. Recombinant PRDX2 induced osteoclast formation from RANKL-primed primary or RAW 264.7 precursors to levels comparable to achieved with continuous RANKL treatment. Thus, increased bone marrow erythropoiesis secondary to anemia leads to upregulation of PRDX2, which is released in the exosomes and acts to induce osteoclast formation. Increased bone resorption by the osteoclasts expands bone marrow cavity, which likely plays a supporting role to increase blood cell production.

Role of Altered Metabolic Microenvironment in Osteolytic Metastasis.

Metastatic bone disease is generally incurable and leads to pathological fractures, pain, hypercalcemia, spinal cord compression and decreased mobility. The skeleton is the major site of bone metastases from solid cancers, including breast and prostate carcinoma. Bone metastasis is facilitated by activation of bone-resorbing osteoclasts, terminally differentiated multinucleated cells formed by fusion from monocytic precursors. Cancer cells are known to produce specific factors that stimulate osteoclast differentiation and function. Of interest, cancer cells are also known to alter their own bioenergetics increasing the use of glycolysis for their survival and function. Such change in energy utilization by cancer cells would result in altered levels of cell-permeable metabolites, including glucose, lactate, and pyruvate. Osteoclast resorption is energy-expensive, and we have previously demonstrated that during differentiation osteoclasts actively adapt to their bioenergetics microenvironment. We hypothesize that altered bioenergetics state of cancer cells will also modify the bioenergetics substrate availability for the tissue-resident bone cells, potentially creating a favorable milieu for pathological osteolysis. The goals of this review are to analyze how metastasizing cancer cells change the availability of energy substrates in bone microenvironment; and to assess how the altered bioenergetics may affect osteoclast differentiation and activity.

Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis.

Bone metastasis from breast and prostate carcinomas is facilitated by activation of bone-resorbing osteoclasts. Using proteomics approaches, we have identified peroxiredoxin-4 (PRDX4) as a cancer-secreted mediator of osteoclastogenesis. We now report characterization of L-plastin in the conditioned media (CM) of MDA-MB-231 human breast cancer cells using immunoblotting and mass spectrometry. The osteoclastogenic potential of MDA-MB-231 CM with siRNA-silenced L-plastin was significantly reduced. L-plastin was detected in cancer-derived exosomes, and inhibition of exosomal release significantly decreased the osteoclastogenic capacity of MDA-MB-231 CM. When added to osteoclast precursors primed with RANKL for 2 days, recombinant L-plastin induced calcium/NFATc1-mediated osteoclastogenesis to the levels similar to continuous treatment with RANKL. Using shRNA, we generated MDA-MB-231 cells lacking L-plastin, PRDX4, or both and injected these cell populations intratibially in CD-1 immunodeficient mice. Micro-CT and histomorphometric analysis demonstrated a complete loss of osteolysis when MDA-MB-231 cells lacking both L-plastin and PRDX4 were injected. A meta-analysis established an increase in L-plastin and PRDX4 mRNA expression in numerous human cancers, including breast and prostate carcinomas. This study demonstrates that secreted L-plastin and PRDX4 mediate osteoclast activation by human breast cancer cells.

Regulation of Osteoclast Growth and Fusion by mTOR/raptor and mTOR/rictor/Akt.

Osteoclasts are giant bone cells formed by fusion from monocytes and uniquely capable of a complete destruction of mineralized tissues. Previously, we have demonstrated that in energy-rich environment not only osteoclast fusion index (the number of nuclei each osteoclast contains), but also cytoplasm volume per single nucleus was increased. The goal of this study was to investigate the regulation of metabolic sensor mTOR during osteoclast differentiation in energy-rich environment simulated by addition of pyruvate. We have found that in the presence of pyruvate, the proportion of mTOR associated with raptor increased, while mTOR-rictor-mediated Akt phosphorylation decreased. Inhibition of mTOR with rapamycin (10 nM) significantly interfered with all aspects of osteoclastogenesis. However, rapamycin at 1 nM, which preferentially targets mTOR-raptor complex, was only effective in control cultures, while in the presence of pyruvate osteoclast fusion index was successfully increased. Inhibition of Akt drastically reduced osteoclast fusion, however in energy-rich environment, osteoclasts of comparable size were formed through increased cytoplasm growth. These data suggest that mTOR-rictor mediated Akt signaling regulates osteoclast fusion, while mTOR-raptor regulation of protein translation contributes to fusion-independent cytoplasm growth. We demonstrate that depending on the bioenergetics microenvironment osteoclastogenesis can adjust to occur through preferential multinucleation or through cell growth, implying that attaining large cell size is part of the osteoclast differentiation program.

Fibulin-5 Regulates Angiopoietin-1/Tie-2 Receptor Signaling in Endothelial Cells.

BACKGROUND: Fibulin-5 is an extracellular matrix glycoprotein that plays critical roles in vasculogenesis and embryonic development. Deletion of Fibulin-5 in mice results in enhanced skin vascularization and upregulation of the angiogenesis factor angiopoietin-1 (Ang-1), suggesting that Fibulin-5 functions as an angiogenesis inhibitor. In this study, we investigate the inhibitory effects of Fibulin-5 on Ang-1/TIE-2 receptor pathway signaling and cell survival in human endothelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant wild-type and RGE-mutant Fibulin-5 proteins were generated through stable transfection of HEK293 and CHO cells, respectively. In vitro solid phase binding assays using pure proteins revealed that wild-type Fibulin-5 does not bind to Ang-1 or TIE-2 proteins but strongly binds to heparin. Binding assays using human umbilical vein endothelial cells (HUVECs) indicated that wild-type Fibulin-5 strongly binds to cells but RGE-mutant Fibulin-5, which is incapable of binding to integrins, does not. Pre-incubation of HUVECs for 1 hr with Fibulin-5 significantly increased caspase 3/7 activity, ERK1/2 phosphorylation, and expressions of the transcription factor early growth response 1 (EGR1) and the dual-specificity phosphatase 5 (DUSP5). Fibulin-5 also strongly attenuated Ang-1-induced TIE-2 and AKT phosphorylation, decreased Ang-1-induced expressions of the transcription factors Inhibitor of DNA Binding 1 (ID1) and Kruppel-like Factor 2 (KLF2), and reversed the inhibitory effect of Ang-1 on serum deprivation-induced cytotoxicity and caspase 3/7 activity. CONCLUSION/SIGNIFICANCE: We conclude that Fibulin-5 strongly binds to the endothelial cell surface through heparin-sulfate proteoglycans and possibly integrins and that it exerts strong anti-angiogenic effects by reducing endothelial cell viability and interfering with the signaling pathways of the Ang-1/TIE-2 receptor axis.

Peroxiredoxin 4: a novel secreted mediator of cancer induced osteoclastogenesis.

Bone is a common site of metastasis from breast and prostate carcinoma, where activation of bone resorbing osteoclasts is important for cancer progression. A large body of evidence indicates that soluble factors produced by the cancer cells act to promote osteoclast formation. Using mass spectrometry, we identified peroxiredoxin (PRDX) as a secreted mediator of cancer-induced osteoclastogenesis. Both breast (MCF7 and MDA-MB-231) and prostate (PC3 and LNCaP) carcinoma cells secreted PRDX4. PRDX4 knockdown using shRNA (shPRDX4) diminished PRDX4 secretion from MDA-MB-231 and PC3 cells and significantly decreased the ability of cancer-derived factors to induce osteoclast formation from late precursors in vitro. Tibial injection of shPRDX4 PC3 cells led to the development of significantly smaller osteolytic lesions characterized by significantly reduced osteoclast numbers compared to control PC3 cells. A meta-analysis demonstrated an increase in PRDX4 mRNA expression in carcinoma and metastatic breast and prostate tissues. Moreover, high expression of PRDX4 in the primary breast tumor was consistently associated with metastasis at 5 years. These data identify a novel function of secreted PRDX4 in mediating osteoclast activation by cancer cells.

Effects of low frequency cyclic mechanical stretching on osteoclastogenesis.

Bone cells are continuously exposed to mechanical deformations originating from movement. Mechanical stimulation at fundamental frequencies associated with most frequent normal locomotion (0.167-10Hz) has been reported to suppress differentiation of osteoclasts. However, the effects of very low frequency (0.01Hz) stimulation (which could be a frequency component of normal movement and also relevant to locomotion of movement-impaired individuals) on osteoclasts are poorly understood. We examined differentiation of osteoclasts from mouse bone marrow precursors and RAW 264.7 monocytes cultured on an extendable silicone surface that was dynamically stretched at 0.01Hz. Three stimulation regimes were applied: (i) continuously during 4 days of differentiation, (ii) non-continuously, 8h/day for 4 days, and (iii) post-differentiation, when stimulation was applied for 24h after osteoclasts were noted. Low frequency mechanical stimulation did not inhibit osteoclastogenesis. Moreover, the expression of osteoclast marker genes was upregulated in mechanically stimulated cells compared to static control. Conditioned medium collected from osteoclast cultures stimulated non-continuously or post-differentiation induced differentiation of osteoclast precursors plated in standard tissue culture plates. Extracellular signal-regulated kinase (ERK) phosphorylation was increased in mechanically-stimulated cultures compared to static control. Thus, low frequency mechanical stimulation has qualitatively different effects on osteoclast formation compared to stimulation associated with the fundamental frequencies of normal movement.

Autocrine signaling is a key regulatory element during osteoclastogenesis.

Osteoclasts are responsible for bone destruction in degenerative, inflammatory and metastatic bone disorders. Although osteoclastogenesis has been well-characterized in mouse models, many questions remain regarding the regulation of osteoclast formation in human diseases. We examined the regulation of human precursors induced to differentiate and fuse into multinucleated osteoclasts by receptor activator of nuclear factor kappa-B ligand (RANKL). High-content single cell microscopy enabled the time-resolved quantification of both the population of monocytic precursors and the emerging osteoclasts. We observed that prior to induction of osteoclast fusion, RANKL stimulated precursor proliferation, acting in part through an autocrine mediator. Cytokines secreted during osteoclastogenesis were resolved using multiplexed quantification combined with a Partial Least Squares Regression model to identify the relative importance of specific cytokines for the osteoclastogenesis outcome. Interleukin 8 (IL-8) was identified as one of RANKL-induced cytokines and validated for its role in osteoclast formation using inhibitors of the IL-8 cognate receptors CXCR1 and CXCR2 or an IL-8 blocking antibody. These insights demonstrate that autocrine signaling induced by RANKL represents a key regulatory component of human osteoclastogenesis.

A new class of bioactive glasses: calcium-magnesium sulfophosphates.

Low-melting ionic sulfophosphate glasses from the system P2O5-SO4-MO-Na2O (M = Zn(2+), Ca(2+) or Mg(2+)) have been previously shown by us to allow tuneable aqueous dissolution and also enable processing temperatures well below 400°C. Sulfate ions are extremely safe for use in the body as decades of use of calcium sulfate bone grafts testifies and there is no known limit on their adult oral toxicity. This glass system therefore offers great potential for use as biomaterials, especially in organic-inorganic hybrid systems such as glass-polymer composites for tissue engineering or drug encapsulation and delivery applications. A compositional region was identified where stable sulfophosphates of the type P2O5-SO4-(Ca, Mg, Zn)O-Na2O can be fabricated. For these glasses, the viscosity-temperature-dependence, glass transformation temperatures (Tg ) and the onset of crystallization were evaluated as the primary processing parameters. As a first step in exploring their potential as a biomaterial, in this study we examine the bioactivity of several compositions of these glasses using fibroblast, monocyte, and osteoclast cell culture models to determine cellular responses in terms of attachment, proliferation, differentiation, and toxicity.

Fibrillin-1 directly regulates osteoclast formation and function by a dual mechanism.

Mutations in the fibrillin-1 gene give rise to a number of heritable disorders, which are all characterized by various malformations of bone as well as manifestations in other tissues. However, the role of fibrillin-1 in the development and homeostasis of bone is not well understood. Here, we examined the role of fibrillin-1 in regulating osteoclast differentiation from primary bone-marrow-derived precursors and monocytic RAW 264.7 cells. The soluble N-terminal half of fibrillin-1 (rFBN1-N) strongly inhibited osteoclastogenesis, whereas the C-terminal half (rFBN1-C) did not. By contrast, when rFBN1-N was immobilized on calcium phosphate, it did not affect osteoclastogenesis but modulated osteoclast resorptive activity, which was evident by a larger number of smaller resorption pits. Using a panel of recombinant sub-fragments spanning rFBN1-N, we localized an osteoclast inhibitory activity to the 63 kDa subfragment rF23 comprising the N-terminal region of fibrillin-1. Osteoclastic resorption led to the generation of small fibrillin-1 fragments that were similar to those identified in human vertebral bone extracts. rF23, but not rFBN1-N, was found to inhibit the expression of cathepsin K, matrix metalloproteinase 9 and Dcstamp in differentiating osteoclasts. rFBN1-N, but not rF23, exhibited interaction with RANKL. Excess RANKL rescued the inhibition of osteoclastogenesis by rFBN1-N. By contrast, rF23 disrupted RANKL-induced Ca(2+) signaling and activation of transcription factor NFATc1. These studies highlight a direct dual inhibitory role of N-terminal fibrillin-1 fragments in osteoclastogenesis, the sequestration of RANKL and the inhibition of NFATc1 signaling, demonstrating that osteoclastic degradation of fibrillin-1 provides a potent negative feedback that limits osteoclast formation and function.

Moderate excess of pyruvate augments osteoclastogenesis.

Cell differentiation leads to adaptive changes in energy metabolism. Conversely, hyperglycemia induces malfunction of many body systems, including bone, suggesting that energy metabolism reciprocally affects cell differentiation. We investigated how the differentiation of bone-resorbing osteoclasts, large polykaryons formed through fusion and growth of cells of monocytic origin, is affected by excess of energy substrate pyruvate and how energy metabolism changes during osteoclast differentiation. Surprisingly, small increases in pyruvate (1-2 mM above basal levels) augmented osteoclastogenesis in vitro and in vivo, while larger increases were not effective in vitro. Osteoclast differentiation increased cell mitochondrial activity and ATP levels, which were further augmented in energy-rich conditions. Conversely, the inhibition of respiration significantly reduced osteoclast number and size. AMP-activated protein kinase (AMPK) acts as a metabolic sensor, which is inhibited in energy-rich conditions. We found that osteoclast differentiation was associated with an increase in AMPK levels and a change in AMPK isoform composition. Increased osteoclast size induced by pyruvate (1 mM above basal levels) was prevented in the presence of AMPK activator 5-amino-4-imidazole carboxamide ribonucleotide (AICAR). In keeping, inhibition of AMPK using dorsomorphin or siRNA to AMPKγ increased osteoclast size in control cultures to the level observed in the presence of pyruvate. Thus, we have found that a moderate excess of pyruvate enhances osteoclastogenesis, and that AMPK acts to tailor osteoclastogenesis to a cell's bioenergetics capacity.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone.

INTRODUCTION: Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. METHODS: To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors. RESULTS: ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5. CONCLUSIONS: Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

Monocyte proliferation and differentiation to osteoclasts is affected by density of collagen covalently bound to a poly(dimethyl siloxane) culture surface.

Osteoclast differentiation is affected by substrate characteristics and environmental conditions; these parameters are therefore of interest for understanding bone remodeling. As a step toward osteoclast mechanotransduction experiments, we aimed to optimize conditions for osteoclast differentiation on extendable poly(dimethylsiloxane) (PDMS) substrates. Because cells attach poorly on PDMS alone, chemical modification by covalent attachment of collagen type I was performed. Effects of collagen surface concentrations on monocyte fusion and osteoclast differentiation were examined. Osteoclasts differentiated on modified PDMS were fewer in number (by ∼50%) than controls on polystyrene physically modified by nonspecific attachment of collagen, and exhibited somewhat different morphologies. Nevertheless, for certain choices of the chemical modification procedures, appropriate differentiation on PDMS was still evident by qRT-PCR analysis for tartrate-resistant acid phosphate (TRAP) and cathepsin K (CTSK) gene expression, positive TRAP staining, fluorescent phalloidin staining showing actin ring formation and bone resorption assays. At relatively high collagen surface densities, monocyte clumps appeared on PDMS suggesting substrate-induced alterations to monocyte fusion. Covalently bound collagen can therefore be used to promote osteoclast differentiation on extendable PDMS substrates. Under appropriate conditions osteoclasts retain similar functionality as on polystyrene, which will enable future studies of osteoclast interactions with microstructured surfaces and mechanostimulation.

Rapamycin inhibits osteolysis and improves survival in a model of experimental bone metastases.

Breast cancer metastasis to bone results in pain, pathological fractures and hypercalcemia. Activation of osteoclasts is critical for the formation of osteolytic lesions by metastasizing tumors. Although the potent drugs, zoledronic acid and Denosumab were introduced, the presence of resistant or intolerant cases necessitated the continued search of osteoclast-targeting treatments. Rapamycin acts through the mTOR pathway, which is important for osteoclast formation. Mouse mammary carcinoma 4T1 cells were injected into the tibia of balb/c mice. Rapamycin treatment significantly decreased the osteoclast population and osteolysis associated with experimental metastases. Our data indicate the benefit of rapamycin in treating metastases-associated osteolytic disease.

CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone.

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Breast cancer cells inhibit spontaneous and bisphosphonate-induced osteoclast apoptosis.

Breast cancer metastasizes to bone where it stimulates formation of bone-resorbing osteoclasts. Bisphosphonates constitute an important treatment for osteolytic metastases. The goal of this study was to assess the effects of soluble factors produced by breast cancer cells on osteoclast survival and responsiveness to bisphosphonates. Osteoclasts derived from the murine monocytic cell line RAW264.7 or from primary mouse bone marrow were cultured for 24-48 h untreated, with 10% conditioned media (CM) from human (MDA-MB-231) or mouse (4T1) metastatic breast carcinoma cells, or with a pro-survival factor RANKL. Cancer-derived factors maintained osteoclast survival at the levels comparable to those observed with RANKL. Alendronate (10⁻4M) or pamidronate (10⁻7M) induced osteoclast apoptosis in untreated and, to a smaller extent, in RANKL-treated cultures, resulting in a significant decrease in osteoclast number and size, induction of caspase-3 cleavage and up-regulation of BIM. In the presence of cancer-derived factors, bisphosphonates were ineffective in inducing osteoclast apoptosis, resulting in only modest decrease in osteoclast numbers and not in size. MDA-MB-231 CM prevented bisphosphonate-induced cleavage of caspase-3 and up-regulation of BIM. MCSF-neutralizing antibody attenuated the effect of MDA-MB-231 CM by ~50%, but could not fully restore osteoclast responsiveness to alendronate. Inhibition of phospholipase C (PLC)-γ interfered with MDA-MB-231-induced down-regulation of BIM and prevented anti-apoptotic action of cancer-derived factors on osteoclasts. Our data suggest that factors produced by the metastatic breast cancer cells promote osteoclast survival and block the apoptotic effect of bisphosphonates in MCSF and PLC-dependent manner, potentially compromising bisphosphonate effectiveness in the bone metastasis setting.