Monocyte Chemotactic Protein-1 (MCP1) Accumulation in Human Osteoclast Precursor Cultures.

In vitro osteoclast methods require constant treatment with macrophage colony stimulating factor (M-CSF) to support precursor survival and addition of the differentiation agent receptor activator of NF-κB ligand (RANKL). Constant exposure to granulocyte macrophage colony stimulating factor (GM-CSF) suppresses human osteoclast formation in vitro. Addition of the chemokine monocyte chemotactic protein-1 (MCP1) to such cultures dramatically increases osteoclast formation and overcomes GM-CSF mediated suppression. We investigated the effect of M-CSF, GM-CSF and the combination of M-CSF and GM-CSF treatment on the expression of chemokines in human CD14+ cells in culture. Of assayed chemokines, MCP1 was the most abundant in terms of mRNA transcript and protein in M-CSF treated cultures and was suppressed by GM-CSF. MCP1 protein accumulated up to 50 ng/mL in culture medium, greatly exceeding other assayed chemokines. C-C chemokine receptor-2 (CCR2) is the receptor for MCP1: the formation of osteoclast-like cells was inhibited by constant exposure to the CCR2 antagonist RS102895, in part by decreasing expression of RANK, the receptor for RANKL.

Intermittent Parathyroid Hormone Accelerates Stress Fracture Healing More Effectively Following Cessation of Bisphosphonate Treatment.

Parathyroid hormone (PTH) and bisphosphonates (BPs), including alendronate (ALN), have opposing effects on bone dynamics. The extent to which PTH remains effective in the treatment of stress fracture (SFx) in the presence of an ongoing BP treatment has not been tested. SFx was induced in 150 female Wistar rats, divided into five equal groups (n = 30). All rats were pretreated with ALN (1 µg/kg-1/day-1) for 14 days prior to SFx induction, followed by ALN cessation or continuation for the duration of the experiment; this was combined with daily PTH (8 µg/100 g-1/day-1) on SFx induction for 14 days, followed by cessation or continuation of ALN after SFx induction or an equivalent vehicle as a control. Ulnas were examined 2 weeks or 6 weeks following SFx. Two toluidine blue- and two tartrate-resistant acid phosphatase-stained sections were examined for histomorphometric analysis using Osteomeasure software. There was a significant interaction between the effects of time and treatment type on the woven bone width and apposition rate, as well as an improvement in the woven bone architecture. However, woven bone variables remained unaffected by the cessation or continuation of ALN. Cessation of ALN increased osteoclast number when compared with the ALN-PTH continuation group (p = 0.006), and vehicle (p = 0.024) after 2 weeks. There was a significant interaction between the effects of time and treatment type on the number of osteoclasts per unit BMU area and length. The number of osteoclasts per unit BMU area and length was significantly greater in ALN cessation groups. It was concluded that intermittent short-duration iPTH treatment effectively increased remodeling of SFx with a concurrent BP treatment, provided that BP was ceased at the time of SFx. Our results could help develop shorter iPTH treatment protocols for the clinical management of SFxs and guide clinical decision-making to cease BP treatment in cases of SFx. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) Drives Activation of Bone Remodelling and Skeletal Metastasis.

PURPOSE OF REVIEW: The purpose of this review is to explore the role of monocyte chemoattractant protein-1 (MCP-1 or CCL2) in the processes that underpin bone remodelling, particularly the action of osteoblasts and osteoclasts, and its role in the development and metastasis of cancers that target the bone. RECENT FINDINGS: MCP-1 is a key mediator of osteoclastogenesis, being the highest induced gene during intermittent treatment with parathyroid hormone (iPTH), but also regulates catabolic effects of continuous PTH on bone including monocyte and macrophage recruitment, osteoclast formation and bone resorption. In concert with PTH-related protein (PTHrP), MCP-1 mediates the interaction between tumour-derived factors and host-derived chemokines to promote skeletal metastasis. In breast and prostate cancers, an osteolytic cascade is driven by tumour cell-derived PTHrP that upregulates MCP-1 in osteoblastic cells. This relationship between PTHrP and osteoblastic expression of MCP-1 may drive the colonisation of disseminated breast cancer cells in the bone. There is mounting evidence to suggest a pivotal role of MCP-1 in many diseases and an important role in the establishment of comorbidities. Coupled with its role in bone remodelling and the regulation of bone turnover, there is the potential for pathological relationships between bone disorders and bone-related cancers driven by MCP-1. MCP-1's role in bone remodelling and bone-related cancers highlights its potential as a novel anti-resorptive and anti-metastatic target.

Single injection of PTH improves osteoclastic parameters of remodeling at a stress fracture site in rats.

Stress fractures (SFx) result from repetitive cyclical loading of bone. They are frequent athletic injuries and underlie atypical femoral fractures following long-term bisphosphonate (BP) therapy. We investigated the effect of a single PTH injection on the healing of SFx in the rat ulna. SFx was induced in 120 female Wistar rats (300 ± 15 g) during a single loading session. A single PTH (8 µg.100g-1 ) or vehicle (VEH) saline injection was administered 24 h after loading. Rats were divided into four groups (n = 15) and ulnae were examined 1, 2, 6, or 10 weeks following SFx. Two Toluidine Blue and TRAP-stained sections of the SFx were examined for histomorphometric analysis using Osteomeasure™ software. An increase in osteoclast number (N.Oc) and perimeter (Oc.Pm) was observed two weeks following PTH treatment (p < 0.01). At 6 weeks, bone formation was the main activity in BMUs. At 10 weeks, the proportion of healing along the SFx line remained 50% greater in PTH groups (p = 0.839), leading to a 43% reduction in the porosity area of BMU (p = 0.703). The main effect of time was a significant variable along the entire SFx remodeling cycle, with significant interactions between time and treatment type affecting (N.Oc) (p = 0.047) and (Oc.Pm) (p = 0.002). We conclude that a single PTH injection increases osteoclastogenesis by the second week of the remodeling cycle in a SFx in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

CCL2 and CCR2 are Essential for the Formation of Osteoclasts and Foreign Body Giant Cells.

Osteoclasts are multinucleated cells responsible for bone resorption. They are derived from the fusion of cells in the monocyte/macrophage lineage. Monocytes and macrophages can also fuse to form foreign body giant cells (FBGC). Foreign body giant cells are observed at the interface between a host and a foreign body such as implants during a foreign body reaction. Macrophages are attracted to the site of bone resorption and foreign body reactions by different cytokines. Chemokine (C-C) ligand-2 (CCL2) is an important chemotactic factor and binds to a receptor CCR2. In this study we investigated the importance of CCL2 and the receptor CCR2 in the formation of osteoclasts and FBGC. CCL2 mRNA was more highly expressed in giant cell culture than macrophages, being 9-fold and 16-fold more abundant in osteoclasts and FBGC respectively. Significantly fewer osteoclasts and FBGC were cultured from the bone marrow of CCL2 and CCR2 knockout mice, when compared to wild type. Not only were the number of giant cells reduced but there was a significant reduction in the number of nuclei and the size of these cells in the cultures of CCL2 and CCR2 knockout mice. Formation of osteoclasts and FBGC were recovered in cultures by addition of exogenous CCL2 to the media containing marrow cells from CCL2-/- mice. We conclude that CCL2 and its receptor CCR2 are important for the formation of osteoclasts and FBGC and absence of these genes causes inhibition of osteoclast and FBGC formation.

MCP-1 as a potential target to inhibit the bone invasion by oral squamous cell carcinoma.

Bone invasion is a common complication of oral squamous cell carcinoma (OSCC), and this study sought to explore whether suppressed expression of monocyte chemotactic protein-1 (MCP-1) can be used to inhibit the bone invasion by OSCC. Strong staining of MCP-1 protein was observed from 10 archival blocks of OSCC by immunohistochemistry (IHC). Real-time PCR showed MCP-1 mRNA was highly expressed by OSCC cell lines (SCC25, HN5, and Tca8113), and SCC25 cells had the highest expression. An expression construct of a dominant negative variant of MCP-1 with 7 amino acids truncated (7ND), in the vector pcDNA was used to transfect SCC25 cells, and resultant stabilized SCC25 cells (SCC25-7ND) were generated by antibiotic selection. 10% conditioned media (CM, supernatant) of SCC25-7ND cells efficiently inhibited the formation of human osteoclasts grown from CD14(+) monocyte subpopulation, comparing with 10% CM of SCC25 cells. Further, cells of SCC25 or SCC25-7ND were injected onto the surface of calvariae of nude mice to establish an animal model of bone invasion by OSCC. H&E staining showed well-differentiated OSCC was formed in both groups, tumour cells invading the bone while osteoclasts locating in typical resorption lacunae. TRAP staining indicated significantly fewer osteoclasts were found in calvariae with cells of SCC25-7ND in comparison to cells of SCC25. These data demonstrate the relevance of MCP-1 with research on bone invasion by OSCC, and suggest the potential value of MCP-1 as a target to inhibit this common complication.

Novel target genes of RUNX2 transcription factor and 1,25-dihydroxyvitamin D3.

The RUNX2 transcription factor is indispensable for skeletal development and controls bone formation by acting as a signaling hub and transcriptional regulator to coordinate target gene expression. A signaling partner of RUNX2 is the nuclear vitamin D receptor (VDR) that becomes active when bound by its ligand 1,25-dihydroxyvitamin D3 (VD3). RUNX2 and VDR unite to cooperatively regulate the expression of numerous genes. In this study, we overexpressed RUNX2 in NIH3T3 fibroblasts concomitantly treated with VD3 and show that RUNX2 alone, or in combination with VD3, failed to promote an osteoblastic phenotype in NIH3T3 cells. However, the expression of numerous osteoblast-related genes was up-regulated by RUNX2 and large-scale gene expression profiling using microarrays identified over 800 transcripts that displayed a twofold of greater change in expression in response to RUNX2 overexpression or VD3 treatment. Functional analysis using gene ontology (GO) revealed GO terms for ossification, cellular motility, biological adhesion, and chromosome organization were enriched in the pool of genes regulated by RUNX2. For the set of genes whose expression was modulated by VD3, the GO terms response to hormone stimulus, chemotaxis, and metalloendopeptidase activity where overrepresented. Our study provides a functional insight into the consequences of RUNX2 overexpression and VD3 treatment in NIH3T3 cells in addition to identifying candidate genes whose expression is controlled by either factor individually or through their functional cooperation.

Dominant negative MCP-1 blocks human osteoclast differentiation.

Human osteoclasts were differentiated using receptor activator of NFκB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) from colony forming unit-granulocyte macrophage (CFU-GM) precursors of the myeloid lineage grown from umbilical cord blood. Gene expression profiling using quantitative polymerase chain reaction (Q-PCR) showed more than 1,000-fold induction of chemokine MCP-1 within 24 h of RANKL treatment. MCP-1 mRNA content exceeds that of other assayed chemokines (CCL1, 3, 4, and 5) at all time points up to day 14 of treatment. MCP-1 induction preceded peak induction of calcium signaling activator calmodulin 1 (CALM1) and transcription factors JUN and FOS, which were at 3 days. Key osteoclast related transcription factors NFATc1 and NFATc2 showed peak induction at 7 days, while marker genes for osteoclast function cathepsin K and tartrate resistance acid phosphatase (TRAP) were maximally induced at 14 days, corresponding with mature osteoclast function. To test whether the early and substantial peak in MCP-1 expression is part of human osteoclast differentiation events, a dominant negative inhibitor of MCP-1 (7ND) was added simultaneously with RANKL and M-CSF, resulting in blockade of CALM1, JUN and NFATc2 induction and strong inhibition of human osteoclast differentiation. These data show that a cascade of gene expression leading to osteoclast differentiation depends on intact early MCP-1 induction and signaling in human osteoclasts.

Polyalanine repeat polymorphism in RUNX2 is associated with site-specific fracture in post-menopausal females.

Runt related transcription factor 2 (RUNX2) is a key regulator of osteoblast differentiation. Several variations within the RUNX2 gene have been found to be associated with significant changes in BMD, which is a major risk factor for fracture. In this study we report that an 18 bp deletion within the polyalanine tract (17A>11A) of RUNX2 is significantly associated with fracture. Carriers of the 11A allele were found to be nearly twice as likely to have sustained fracture. Within the fracture category, there was a significant tendency of 11A carriers to present with fractures of distal radius and bones of intramembranous origin compared to bones of endochondral origin (p = 0.0001). In a population of random subjects, the 11A allele was associated with decreased levels of serum collagen cross links (CTx, p = 0.01), suggesting decreased bone turnover. The transactivation function of the 11A allele showed a minor quantitative decrease. Interestingly, we found no effect of the 11A allele on BMD at multiple skeletal sites. These findings suggest that the 11A allele is a biologically relevant polymorphism that influences serum CTx and confers enhanced fracture risk in a site-selective manner related to intramembranous bone ossification.

Foreign body giant cells and osteoclasts are TRAP positive, have podosome-belts and both require OC-STAMP for cell fusion.

Macrophages have the ability to fuse and form multinucleated giant cells such as Osteoclast (OCs) and FBGCs. Osteoclast stimulatory transmembrane protein (OC-STAMP) is an important cell surface protein involved in the formation of OCs. This study sought to determine if OC-STAMP also regulates formation of FBGCs using expression analysis and subsequent inhibition studies. qPCR and Western blot analysis showed that OC-STAMP expression is significantly higher in FBGCs compared to control monocytes (P < 0.05). Four days following cell culture, OCs were positive for TRAP and F-actin ring formation, but FBGCs were not. In contrast, FBGCs were positive for TRAP and showed podosome belts comprised of F-actin on Day 8. FBGCs were subsequently plated onto dentine, but despite presenting some morphologic features of OCs (OC-STAMP expression, TRAP reactivity, and podosome belts) they failed to resorb bone. To evaluate a role for OC-STAMP in FBGCs, we inhibited this cell surface protein with anti-OC-STAMP antibody and observed that cell fusion and podosome belt formation was inhibited in both OCs and FBGCs. Our data support the hypothesis that OC-STAMP is a regulatory molecule for FBGCs; and that they are functionally distinct from OCs, despite similarities in gene expression profile, podosome belt formation, and TRAP expression.

Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (-0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

Interaction of Burkholderia pseudomallei and Burkholderia thailandensis with human monocyte-derived dendritic cells.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic in areas of South-East Asia and northern Australia, and is classed as a category B select agent by the Centers for Disease Control and Prevention (CDC). Factors that determine whether host infection is achieved or if disease is chronic or acute are unknown but the type of host immune response that is mounted is important. B. pseudomallei can replicate within macrophages, causing them to multinucleate. In light of the common lineage of macrophages with dendritic cells (DCs), and the role played by DCs in orchestration of the immune response, we investigated the interactions of a variety of B. pseudomallei and B. thailandensis strains with DCs. This study demonstrates that, in the majority of cases, infection of human monocyte-derived dendritic cells is dramatically decreased or cleared by 12 h post-infection, showing a lack of ability to replicate and survive within DCs. Additionally we have shown that B. pseudomallei activates DCs, as measured by cytokine secretion, and live bacteria are not required for activation.

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.

Beta3-adrenergic receptor gene, body mass index, bone mineral density and fracture risk in elderly men and women: the Dubbo Osteoporosis Epidemiology Study (DOES).

BACKGROUND: Recent studies have suggested that the Arg allele of beta3-adrenergic receptor (ADRB3) gene is associated with body mass index (BMI), which is an important predictor of bone mineral density (BMD) and fracture risk. However, whether the ADRB3 gene polymorphism is associated with fracture risk has not been investigated. The aim of study was to examine the inter-relationships between ADRB3 gene polymorphisms, BMI, BMD and fracture risk in elderly Caucasians. METHODS: Genotypes of the ADRB3 gene were determined in 265 men and 446 women aged 60+ in 1989 at entry into the study, whose BMD were measured by DXA (GE Lunar, WI USA) at baseline. During the follow-up period (between 1989 and 2004), fractures were ascertained by reviewing radiography reports and personal interviews. RESULTS: The allelic frequencies of the Trp and the Arg alleles were 0.925 and 0.075 respectively, and the relative frequencies of genotypes Trp/Trp, Trp/Arg and Arg/Arg 0.857, 0.138 and 0.006 respectively. There was no significant association between BMI and ADRB3 genotypes (p = 0.10 in women and p = 0.68 in men). There was also no significant association between ADRB3 genotypes and lumbar spine or femoral neck BMD in either men and women. Furthermore, there were no significant association between ADRB3 genotypes and fracture risk in both women and men, either before or after adjusting for and, BMD and BMI. CONCLUSION: The present data suggested that in Caucasian population the contribution of ADRB3 genotypes to the prediction of BMI, BMD and fracture risk is limited.

Association of functionally different RUNX2 P2 promoter alleles with BMD.

UNLABELLED: RUNX2 gene SNPs were genotyped in subjects from the upper and lower deciles of age- and weight-adjusted femoral neck BMD. Of 16 SNPs in RUNX2 and its two promoters (P1 and P2), only SNPs in the P2 promoter were significantly associated with BMD. These P2 promoter SNPs were functionally different in gel-shift and promoter activity assays. INTRODUCTION: Specific osteoblast genes are induced by Runx2, a cell-specific transcription factor that is a candidate gene for controlling BMD. We tested the hypothesis that RUNX2 genetic variation is associated with BMD. MATERIALS AND METHODS: From a population repository of normal subjects, the age- and weight-adjusted femoral neck BMD was ranked, and the upper and lower deciles (n = 132 each) were taken to represent the adjusted extremes of the population distribution. In these 264 subjects, we identified 16 allelic variations within the RUNX2 gene and promoters (P1 and P2) through DNA sequencing and denaturing high-performance liquid chromatography. Characterization of these alleles was performed through allele-specific cloning, transfection into ROS 17/2.8 cells, luciferase reporter analysis, and electrophoretic mobility shift assays. RESULTS: Within the P2 promoter were three polymorphic nucleotides for which the minor alleles were over-represented in the upper decile of BMD (0.117 and 0.064 in the upper and lower deciles, respectively). These alleles are in near complete linkage disequilibrium with each other and represent a haplotype block that is significantly associated with increased BMD. The common and rare P2 promoter alleles were cloned upstream of luciferase, and when transfected into osteoblast-like cells, the construct representing the rare haplotype showed significantly greater P2 promoter activity than the common haplotype. CONCLUSIONS: Because the high BMD allele had higher P2 promoter activity, the data suggest that greater RUNX2 P2 promoter activity is associated with higher BMD.

Induction of chemokines and chemokine receptors CCR2b and CCR4 in authentic human osteoclasts differentiated with RANKL and osteoclast like cells differentiated by MCP-1 and RANTES.

Chemokines MCP-1 and RANTES are induced when authentic bone resorbing human osteoclasts differentiate from monocyte precursors in vitro. In addition, MCP-1 and RANTES can stimulate the differentiation of cells with the visual appearance of osteoclasts, being multinuclear and positive for tartrate resistance acid phosphatase (TRAP +). We show here that MIP1alpha is also potently induced by RANKL during human osteoclast differentiation and that this chemokine also induces the formation of TRAP + multinucleated cells in the absence of RANKL. MIP1alpha was able to overcome the potent inhibition of GM-CSF on osteoclast differentiation, permitting the cells to pass through to TRAP + multinuclear cells, however these were unable to form resorption pits. Chemokine receptors CCR2b and CCR4 were potently induced by RANKL (12.6- and 49-fold, P = 4.0 x 10(-7) and 4.0 x 10(-8), respectively), while CCR1 and CCR5 were not regulated. Chemokine treatment in the absence of RANKL also induced MCP-1, RANTES and MIP1alpha. Unexpectedly, treatment with MCP-1 in the absence of RANKL resulted in 458-fold induction of CCR4 (P = 1.0 x 10(-10)), while RANTES treatment resulted in twofold repression (P = 1.0 x 10(-4)). Since CCR2b and CCR4 are MCP-1 receptors, these data support the existence of an MCP-1 autocrine loop in human osteoclasts differentiated using RANKL.

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.

Optimized transfection of diced siRNA into mature primary human osteoclasts: inhibition of cathepsin K mediated bone resorption by siRNA.

Osteoclasts are large multinucleated cells responsible for bone resorption. Bone resorption is dependent on the liberation of calcium by acid and protease destruction of the bone matrix by proteinases. The key proteinase produced by the osteoclast is cathepsin K. Targeted knock-down of cathepsin K was performed using small inhibitory RNA (siRNA). siRNA is a method that introduces short double-stranded RNA molecules that instruct the RNA-induced silencing complex (RISC) to degrade mRNA species complementary to the siRNA. Transfection of siRNA by lipid cations allows for short-term inhibition of expression of the targeted gene. We show that transfection of primary human osteoclasts with siRNA to cathepsin K reduces expression by > or = 60% and significantly inhibits bone resorption with a reduction of both resorption pit numbers (P = 0.018) and resorbed area (P = 0.013). We also show that FuGENE 6 is an effective lipid transfection reagent with which to transfect primary human osteoclasts, that does not produce off-target effects.