Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts.

Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

Cardiovascular risk factor mediation of the effects of education and Genetic Risk Score on cardiovascular disease: a prospective observational cohort study of the Framingham Heart Study.

OBJECTIVES: Level of education and genetic risk are key predictors of cardiovascular disease (CVD). While several studies have explored the causal mechanisms of education effects, it remains uncertain to what extent genetic risk is mediated by established CVD risk factors. This study sought to investigate this and explored the mediation of education and genetic effects on CVD by established cardiovascular risk factors in the Framingham Heart Study (FHS). DESIGN: Prospective observational cohort study. PARTICIPANTS: 7017 participants from the FHS. SETTING: Community-based cohort of adults in Framingham, Massachusetts, USA. PRIMARY OUTCOME MEASURE: Incident CVD. The total effects of education and genetic predisposition using a 63-variant genetic risk score (GRS) on CVD, as well as those mediated by established CVD risk factors, were assessed via mediation analysis based on the counterfactual framework using Cox proportional hazards regression models. RESULTS: Over a median follow-up time of 12.0 years, 1091 participants experienced a CVD event. Education and GRS displayed significant associations with CVD after adjustment for age and sex and the established risk factors smoking, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), body mass index, systolic blood pressure (SBP) and diabetes. For education effects, smoking, HDL-C and SBP were estimated to mediate 18.8% (95% CI 9.5% to 43%), 11.5% (95% CI 5.7% to 29.0%) and 4.5% (95% CI 1.6% to 13.3%) of the total effect of graduate degree, respectively, with the collective of all risk factors combined mediating 38.5% (95% 24.1% to 64.9%). A much smaller proportion of the effects of GRS were mediated by established risk factors combined (17.6%, 95% CI 2.4% to 35.7%), with HDL-C and TC mediating 11.5% (95% CI 6.2% to 21.5%) and 3.1% (95% CI 0.2% to 8.3%), respectively. CONCLUSIONS: Unlike education inequalities, established risk factors mediated only a fraction of GRS effects on CVD. Further research is required to elucidate the underlying causal mechanisms of genetic contributions to CVD.

Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency.

Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation.

Microtubule dynamic instability controls podosome patterning in osteoclasts through EB1, cortactin, and Src.

In osteoclasts (OCs) podosomes are organized in a belt, a feature critical for bone resorption. Although microtubules (MTs) promote the formation and stability of the belt, the MT and/or podosome molecules that mediate the interaction of the two systems are not identified. Because the growing "plus" ends of MTs point toward the podosome belt, plus-end tracking proteins (+TIPs) might regulate podosome patterning. Among the +TIPs, EB1 increased as OCs matured and was enriched in the podosome belt, and EB1-positive MTs targeted podosomes. Suppression of MT dynamic instability, displacement of EB1 from MT ends, or EB1 depletion resulted in the loss of the podosome belt. We identified cortactin as an Src-dependent interacting partner of EB1. Cortactin-deficient OCs presented a defective MT targeting to, and patterning of, podosomes and reduced bone resorption. Suppression of MT dynamic instability or EB1 depletion increased cortactin phosphorylation, decreasing its acetylation and affecting its interaction with EB1. Thus, dynamic MTs and podosomes interact to control bone resorption.

Primordium of an artificial Bruch's membrane made of nanofibers for engineering of retinal pigment epithelium cell monolayers.

Transplanted retinal pigment epithelium (RPE) cells hold promise for treatment of age-related macular degeneration (AMD) and Stargardt disease (SD), but it is conceivable that the degenerated host Bruch's membrane (BM) as a natural substrate for RPE might not optimally support transplanted cell survival with correct cellular organization. We fabricated novel ultrathin three-dimensional (3-D) nanofibrous membranes from collagen type I and poly(lactic-co-glycolic acid) (PLGA) by an advanced clinical-grade needle-free electrospinning process. The nanofibrillar 3-D networks closely mimicked the fibrillar architecture of the native inner collagenous layer of human BM. Human RPE cells grown on our nanofibrous membranes bore a striking resemblance to native human RPE. They exhibited a correctly orientated monolayer with a polygonal cell shape and abundant sheet-like microvilli on their apical surfaces. RPE cells built tight junctions and expressed RPE65 protein. Flat 2-D PLGA film and cover glass as controls delivered inferior RPE layers. Our nanofibrous membranes may imitate the natural BM to such extent that they allow for the engineering of an in vivo-like human RPE monolayer that maintains the natural biofunctional characteristics. Such ultrathin membranes may provide a promising vehicle for a functional RPE cell monolayer implantation in the subretinal space in patients with AMD or SD.

Antimicrobial peptide coating of dental implants: biocompatibility assessment of recombinant human beta defensin-2 for human cells.

PURPOSE: Artificial materials such as dental implants are at risk of bacterial contamination in the oral cavity. Human beta defensins (HBDs), small cationic antimicrobial peptides that exert a broad-spectrum antibacterial function at epithelial surfaces and within some mesenchymal tissues, could probably help to reduce such contamination. HBDs also have protective immunomodulatory effects and have been reported to promote bone remodeling. The aim of this study, therefore, was to investigate the influence of recombinant HBD-2 on the proliferation and survival of cells in culture. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs), human osteoblasts, human keratinocytes (control), and the HeLa cancer cell line (control) were incubated with recombinant HBD-2 (1, 5, 10, or 20 µg/mL). Cell proliferation and cytotoxicity were evaluated via a water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase assays, respectively. RESULTS: HBD-2 was not toxic in any tested concentration to hMSCs, osteoblasts, keratinocytes, or HeLa cells. Furthermore, proliferation of hMSCs and osteoblasts increased after treatment with HBD-2 at all tested concentrations, and keratinocyte proliferation increased when treated at 20 µg/mL. In contrast, HeLa cancer cells were not affected by HBD-2 as tested. CONCLUSIONS: HBD-2 is not only biocompatible but also promotes proliferation of hMSCs, osteoblasts, and keratinocytes in culture. Further investigation of HBD-2 functional surface coating of artificial materials is recommended.

SLC4A2-mediated Cl-/HCO3- exchange activity is essential for calpain-dependent regulation of the actin cytoskeleton in osteoclasts.

Bone remodeling requires osteoclasts to generate and maintain an acidified resorption compartment between the apical membrane and the bone surface to solubilize hydroxyapatite crystals within the bone matrix. This acidification process requires (i) apical proton secretion by a vacuolar H(+)-ATPase, (ii) actin cytoskeleton reorganization into a podosome belt that forms a gasket to restrict lacunar acid leakage, and (iii) basolateral chloride uptake and bicarbonate extrusion by an anion exchanger to provide Cl(-) permissive for apical acid secretion while preventing cytoplasmic alkalinization. Here we show that osteoclast-targeted deletion in mice of solute carrier family 4 anion exchanger member 2 (Slc4a2) results in osteopetrosis. We further demonstrate a previously unrecognized consequence of SLC4A2 loss of function in the osteoclast: dysregulation of calpain-dependent podosome disassembly, leading to abnormal actin belt formation, cell spreading, and migration. Rescue of SLC4A2-deficient osteoclasts with functionally defined mutants of SLC4A2 indicates regulation of actin cytoskeletal reorganization by anion-exchange activity and intracellular pH, independent of SLC4A2's long N-terminal cytoplasmic domain. These data suggest that maintenance of intracellular pH in osteoclasts through anion exchange regulates the actin superstructures required for bone resorption.

A clinically-feasible protocol for using human platelet lysate and mesenchymal stem cells in regenerative therapies.

The transplantation of human stem cells seeded on biomaterials holds promise for many clinical applications in cranio-maxillo-facial tissue engineering and regenerative medicine. However, stem cell propagation necessary to produce sufficient cell numbers currently utilizes fetal calf serum (FCS) as a growth supplement which may subsequently transmit animal pathogens. Human platelet lysate (HPL) could potentially be utilized to produce clinical-grade stem cell-loaded biomaterials as an appropriate FCS substitute that is in line with clinically-applicable practice. The goal of this study was to investigate whether HPL can be successfully used to propagate human mesenchymal stem cells (HMSCs) seeded on clinically-approved collagen materials under clinically-applicable conditions using FCS as a control. HMSCs were isolated from bone marrow and cultured in the presence of 10% FCS or 10% HPL. Characterization of HMSCs was performed by flow cytometry and through osteogenic and adipogenic differentiation assays. Proliferative capacity of HMSCs on both matrices was investigated by mitochondrial dehydrogenase assays (WST) and tissue coverage scanning electron microscopy (SEM). The isolated HMSC differentiated into osteogenic and adipogenic cells authenticating the multipotentiality of the HMSCs. WST tests and the SEM images demonstrated that HPL was generally superior to FCS in promoting growth of seeded HMSCs. For all other tests HPL supported HMSCs at least equal to FCS. In conclusion, HPL is an effective growth factor to allow expansion of clinical-grade HMSCs on clinically-approved biomaterials for maxillofacial and oral implantology applications.

Nanospiderwebs: artificial 3D extracellular matrix from nanofibers by novel clinical grade electrospinning for stem cell delivery.

Novel clinical grade electrospinning methods could provide three-dimensional (3D) nanostructured biomaterials comprising of synthetic or natural biopolymer nanofibers. Such advanced materials could potentially mimic the natural extracellular matrix (ECM) accurately and may provide superior niche-like spaces on the subcellular scale for optimal stem-cell attachment and individual cell homing in regenerative therapies. The goal of this study was to design several novel "nanofibrous extracellular matrices" (NF-ECMs) with a natural mesh-like 3D architecture through a unique needle-free multi-jet electrospinning method in highly controlled manner to comply with good manufacturing practices (GMP) for the production of advanced healthcare materials for regenerative medicine, and to test cellular behavior of human mesenchymal stem cells (HMSCs) on these. Biopolymers manufactured as 3D NF-ECM meshes under clinical grade GMP-like conditions show higher intrinsic cytobiocompatibility with superior cell integration and proliferation if compared to their 2D counterparts or a clinically-approved collagen membrane.

Mass production of nanofibrous extracellular matrix with controlled 3D morphology for large-scale soft tissue regeneration.

AIM: Biomaterials that mimic the nanofibrous architecture of the natural extracellular matrix (ECM) are in the focus for stem cell hosting or delivery in tissue engineering of multilayered soft tissues such as skin, mucosa, or retina. Synthetic nanofibers for such ECM are usually produced by single-syringe electrospinning with only one needle-jet at very low production rates of 0.005-0.008 g·min⁻¹. The aim of this study was to utilize a novel industrial needle-free multijet electrospinning device with the potential for mass production of nanofibrous ECM (NF-ECM) exhibiting a controlled three-dimensional (3D) morphology for large-scale applications such as large area skin regeneration in patients with burns. METHODS: The novel NanoSpider™ NS200, an industrial apparatus originally designed for electrospinning of nanofibrous textile meshes, was used to fabricate 3D NF-ECMs of the following synthetic and natural biopolymers: collagen, gelatin, poly(caprolactone) (PCL), and poly(L-lactide-co-glycolide) (PLGA). Different concentrations of Gelatin polymer solution were electrospun under varying processing conditions, namely speed of spinning electrode rotation (u) and electric field intensity (E) by altering applied voltage (v) or the distance between electrodes (h) to achieve homogeneous desirable 3D morphology. Nanofiber diameters were assessed by scanning electron microscopy (SEM). Biocompatibility was tested by WST-1 (water-soluble tetrazolium salt) proliferation assay of seeded human mesenchymal stem cells (HMSCs). Biological performance of HMSCs on 3D PLGA NF-ECM was compared to two-dimensional (2D) PLGA film controls via SEM and confocal microscopy. Western blotting addressed the expression of surface adhesion proteins; focal adhesion kinase (FAK), phosphorylated FAK (pY397), α-tubulin, paxillin, vinculin. and integrin subunits; α5, αv, and ß1 proteins. RESULTS: Large-scale mass production of NF-ECM membranes with a highly homogenous nanofiber morphology and 3D architecture could be produced with an extremely high production rate of 0.394±0.013 g·min⁻¹·m⁻¹ when compared to standard procedures. This was achieved by electrospinning a 20% (wt)/v gelatin solution, in an electric field intensity of 0.381 kV·mm⁻¹. The nanofibers possessed diameters of around 180±40 nm with 28% deviation. HSMCs proliferation was significantly improved on NF-ECMs derived from collagen, gelatin, and PLGA when compared to PCL or flat coverglass controls (p<0.01). PLGA NF-ECM in 3D nanofibrous architecture possessed significantly superior biocompatibility when compared to flat 2D PLGA film (p<0.05). Furthermore, on 3D PLGA NF-ECMs, HSMCs expressed a higher amount of α-tubulin and paxillin compared to the HMSCs cultured on a 2D PLGA film (p<0.05). HMSCs exhibited a complex multifaceted morphology on all NF-ECMs, where cells appeared to be integrated into the 3D NF-ECMs niches with complex cell filopodia extending into to all directions. In contrast, HMSCs on flat 2D films of the same materials or on coverglass displayed a simple flattened, monolayered structure. CONCLUSION: Needle-free multijet electrospinning can be used to mass produce artificial ECMs with intrinsic biocompatibility and desirable integration of stem cells for large-scale applications.

Internal control genes for quantitative RT-PCR expression analysis in mouse osteoblasts, osteoclasts and macrophages.

BACKGROUND: Real-time quantitative RT-PCR (qPCR) is a powerful technique capable of accurately quantitating mRNA expression levels over a large dynamic range. This makes qPCR the most widely used method for studying quantitative gene expression. An important aspect of qPCR is selecting appropriate controls or normalization factors to account for any differences in starting cDNA quantities between samples during expression studies. Here, we report on the selection of a concise set of housekeeper genes for the accurate normalization of quantitative gene expression data in differentiating osteoblasts, osteoclasts and macrophages. We implemented the use of geNorm, an algorithm that determines the suitability of genes to function as housekeepers by assessing expression stabilities. We evaluated the expression stabilities of 18S, ACTB, B2M, GAPDH, HMBS and HPRT1 genes. FINDINGS: Our analyses revealed that 18S and GAPDH were regulated during osteoblast differentiation and are not suitable for use as reference genes. The most stably expressed genes in osteoblasts were ACTB, HMBS and HPRT1 and their geometric average constitutes a suitable normalization factor upon which gene expression data can be normalized. In macrophages, 18S and GAPDH were the most variable genes while HMBS and B2M were the most stably expressed genes. The geometric average of HMBS and B2M expression levels forms a suitable normalization factor to account for potential differences in starting cDNA quantities during gene expression analysis in macrophages. The expression stabilities of the six candidate reference genes in osteoclasts were, on average, more variable than that observed in macrophages but slightly less variable than those seen in osteoblasts. The two most stably expressed genes in osteoclasts were HMBS and B2M and the genes displaying the greatest levels of variability were 18S and GAPDH. Notably, 18S and GAPDH were the two most variably expressed control genes in all three cell types. The geometric average of HMBS, B2M and ACTB creates an appropriate normalization factor for gene expression studies in osteoclasts. CONCLUSION: We have identified concise sets of genes suitable to use as normalization factors for quantitative real-time RT-PCR gene expression studies in osteoblasts, osteoclasts and macrophages.

Myocyte enhancer factor 2c, an osteoblast transcription factor identified by dimethyl sulfoxide (DMSO)-enhanced mineralization.

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem cell-mediated therapies for fracture and other orthopedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of stimulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase activity and extracellular matrix mineralization. Furthermore, similar DMSO-mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1, we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among the numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical, and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased alkaline phosphatase activity, and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. A flow on knockdown of bone-specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c, suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

The bacterial gene lfpA influences the potent induction of calcitonin receptor and osteoclast-related genes in Burkholderia pseudomallei-induced TRAP-positive multinucleated giant cells.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1gamma), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensis-infected cells. The expression by B. pseudomallei-infected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis. Analysis of dentine resorption by B. pseudomallei-induced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.

Expression profile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis: identification of essential genes in osteoclasts.

UNLABELLED: RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology. INTRODUCTION: During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis. MATERIALS AND METHODS: The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest. RESULTS: Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts. CONCLUSIONS: We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.

MCP-1-induced human osteoclast-like cells are tartrate-resistant acid phosphatase, NFATc1, and calcitonin receptor-positive but require receptor activator of NFkappaB ligand for bone resorption.

MCP-1 (monocyte chemotactic protein-1) is a CC chemokine that is induced by receptor activator of NFkappaB ligand (RANKL) in human osteoclasts. In the absence of RANKL, treatment of human peripheral blood mononuclear cells with macrophage colony-stimulating factor and MCP-1 resulted in tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells that are positive for calcitonin receptor (CTR) and a number of other osteoclast markers, including nuclear factor of activated t cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). Although NFATc1 was strongly induced by MCP-1 and was observed in the nucleus, MCP-1 did not permit the formation of bone-resorbing osteoclasts, although these cells had the typical TRAP(+)/CTR(+) multinuclear phenotype of osteoclasts. Despite a similar appearance to osteoclasts, RANKL treatment was required in order for TRAP(+)/CTR(+) multinuclear cells to develop bone resorption activity. The lack of bone resorption was correlated with a deficiency in expression of certain genes related to bone resorption, such as cathepsin K and MMP9. Furthermore, calcitonin blocked the MCP-1-induced formation of TRAP(+)/CTR(+) multinuclear cells as well as blocking osteoclast bone resorption activity, indicating that calcitonin acts at two stages of osteoclast differentiation. Ablation of NFATc1 in mature osteoclasts did not prevent bone resorption activity, suggesting NFATc1 is involved in cell fusion events and not bone resorption. We propose that the MCP-1-induced TRAP(+)/CTR(+) multinuclear cells represent an arrested stage in osteoclast differentiation, after NFATc1 induction and cellular fusion but prior to the development of bone resorption activity.

Gene array identification of osteoclast genes: differential inhibition of osteoclastogenesis by cyclosporin A and granulocyte macrophage colony stimulating factor.

Treatment of adherent peripheral blood mononuclear cells (PBMCs) with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) stimulates the formation of multinucleate osteoclast-like cells. Treatment with M-CSF alone results in the formation of macrophage-like cells. Through the use of Atlas human cDNA expression arrays, genes regulated by RANKL were identified. Genes include numerous cytokines and cytokine receptors (RANTES and CSF2R proportional, variant ), transcription factors (nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and GA binding protein transcription factor alpha (GABPalpha)), and ribosomal proteins (60S L17 and 40S S20). Real-time PCR analysis showed significant correlation (R2 of 0.98 P < 0.01) with array data for all genes tested. Time courses showed differential activation patterns of transcription factors with early induction of FUSE binding protein 1 (FBP) and c-Jun, and later steady upregulation of NFATc1 and GABP by RANKL. Treatment with cyclosporin A, a known NFATc1 inhibitor, resulted in a blockade of osteoclast formation. The mononuclear cells resulting from high cyclosporin treatment (1,000 ng/ml) were cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) positive but expression of calcitonin receptor (CTR) was downregulated by more than 30-fold. Constant exposure of M-CSF- and RANKL-treated cells to GM-CSF resulted in inhibition of osteoclast formation and the downregulation of CTSK and TRAP implicating the upregulation of CSF2R in a possible feedback inhibition of osteoclastogenesis.