Role In Osteoclastogenesis Research Articles

THU0073 Loss of Tyrosine Kinases Leads to Enhanced Osteoclastogenesis in a Murine Model of Rheumatoid Arthritis

BackgroundTyrosine Kinases (TEC) family members like Btk and Tec play an important role in osteoclastogenesis. However, little is known about the role of Tec on osteoclast under auto-inflammatory conditions such...

AB0080 Serum interleukin-34 levels increase in rheumatoid arthritis but are not associated with joint damage

BackgroundInterleukin-34 (IL-34) plays a critical role in osteoclastogenesis via colony-stimulating factor 1 receptor and enhances monocyte viability and chemokines production [1,2]. Recently, Chemel et al. reported that the expression of...

Wear particle-mediated expressions of pro-inflammatory cytokines, NF-κB and RANK were impacted by lanthanum chloride in RAW264.7 cells

To explore the impact of different concentrations of lanthanum chloride (LaCl3) on critical components of wear particle-mediated signaling pathways in inflammation and osteoclastogenesis, RAW264.7 cells were naturally divided into eight groups and analyzed by CCK-8 assay, flow cytometry, ELISA, RT-PCR and western blot after treatments. The results showed that three concentrations of LaCl3 had no influence on viability of RAW264.7 cells and down-regulated receptor activator of nuclear factor κB (RANK) instead of macrophage colony-stimulating factor receptor (M-CSFR). Additionally, 2.5 and 10 μmol/L LaCl3 could significantly inhibit gene and protein levels of pro-inflammatory cytokines (tumor necrosis factor-α and interleukin-1β, i.e., TNF-α and IL-1β and NF-κB/p65, but 100 μmol/L LaCl3 did not exert an obvious inflammation-inhibiting effect, and even induced inflammation. In conclusion, these findings demonstrated that LaCl3 was able to suppress wear particle-induced inflammation and activation of NF-κB in a certain range of concentrations in vitro and mainly decrease the expression of RANK, but not M-CSFR, all of which were generally recognized to play a pivotal role in osteoclastogenesis.

Sargachromanol G inhibits osteoclastogenesis by suppressing the activation NF-κB and MAPKs in RANKL-induced RAW 264.7 cells

Inflammatory cytokines play a major role in osteoclastogenesis, leading to the bone resorption that is frequently associated with osteoporosis. Sargachromanol G (SG), isolated from the brown alga Sargassum siliquastrum, inhibits the production of inflammatory cytokines. In the present study, we determined the effect of SG on receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation. SG inhibited RANKL-induced osteoclast differentiation from RAW264.7 cells without signs of cytotoxicity. Additionally, the expression of osteoclastic marker genes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K (CTSK), matrix metalloproteinase 9 (MMP9), and calcitonin receptor (CTR), was strongly inhibited. SG inhibited RANKL-induced activation of NF-κB by suppressing RANKL-mediated IκB-α degradation. Furthermore, SG inhibited RANKL-induced phosphorylation of mitogen activated protein kinases (p38, JNK, and ERK). This study identified SG as an inhibitor for osteoclast formation and provided evidence that natural compounds, such as SG, are an alternative medicines for preventing and treating osteolysis.

CD109 Plays a Role in Osteoclastogenesis

Osteoclasts are large multinucleated cells that arise from the fusion of cells from the monocyte/macrophage lineage. Osteoclastogenesis is mediated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) and involves a complex multistep process that requires numerous other elements, many of which remain undefined. The primary aim of this project was to identify novel factors which regulate osteoclastogenesis. To carry out this investigation, microarray analysis was performed comparing two pre-osteoclast cell lines generated from RAW264.7 macrophages: one that has the capacity to fuse forming large multinucleated cells and one that does not fuse. It was found that CD109 was up-regulated by>17-fold in the osteoclast forming cell line when compared to the cell line that does not fuse, at day 2 of the differentiation process. Results obtained with microarray were confirmed by RT-qPCR and Western blot analyses in the two cell lines, in the parental RAW264.7 cell line, as well as primary murine monocytes from bone marrow. A significant increase of CD109 mRNA and protein expression during osteoclastogenesis occurred in all tested cell types. In order to characterize the role of CD109 in osteoclastogenesis, CD109 stable knockdown cell lines were established and fusion of osteoclast precursors into osteoclasts was assessed. It was found that CD109 knockdown cell lines were less capable of forming large multinucleated osteoclasts. It has been shown here that CD109 is expressed in monocytes undergoing RANKL-induced osteoclastogenesis. Moreover, when CD109 expression is suppressed in vitro, osteoclast formation decreases. This suggests that CD109 might be an important regulator of osteoclastogenesis. Further research is needed in order to characterize the role played by CD109 in regulation of osteoclast differentiation.

Vps35 loss promotes hyperresorptive osteoclastogenesis and osteoporosis via sustained RANKL signaling

Receptor activator of NF-κB (RANK) plays a critical role in osteoclastogenesis, an essential process for the initiation of bone remodeling to maintain healthy bone mass and structure. Although the signaling and function of RANK have been investigated extensively, much less is known about the negative regulatory mechanisms of its signaling. We demonstrate in this paper that RANK trafficking, signaling, and function are regulated by VPS35, a major component of the retromer essential for selective endosome to Golgi retrieval of membrane proteins. VPS35 loss of function altered RANK ligand (RANKL)-induced RANK distribution, enhanced RANKL sensitivity, sustained RANKL signaling, and increased hyperresorptive osteoclast (OC) formation. Hemizygous deletion of the Vps35 gene in mice promoted hyperresorptive osteoclastogenesis, decreased bone formation, and caused a subsequent osteoporotic deficit, including decreased trabecular bone volumes and reduced trabecular thickness and density in long bones. These results indicate that VPS35 critically deregulates RANK signaling, thus restraining increased formation of hyperresorptive OCs and preventing osteoporotic deficits.

IL-11 produced by breast cancer cells augments osteoclastogenesis by sustaining the pool of osteoclast progenitor cells

BackgroundInterleukin (IL)-11, a cytokine produced by breast cancer, has been implicated in breast cancer-induced osteolysis (bone destruction) but the mechanism(s) of action remain controversial. Some studies show that IL-11 is able to promote osteoclast formation independent of the receptor activator of NF-κB ligand (RANKL), while others demonstrate IL-11 can induce osteoclast formation by inducing osteoblasts to secrete RANKL. This work aims to further investigate the role of IL-11 in metastasis-induced osteolysis by addressing a new hypothesis that IL-11 exerts effects on osteoclast progenitor cells.MethodsTo address the precise role of breast cancer-derived IL-11 in osteoclastogenesis, we determined the effect of breast cancer conditioned media on osteoclast progenitor cells with or without an IL-11 neutralizing antibody. We next investigated whether recombinant IL-11 exerts effects on osteoclast progenitor cells and survival of mature osteoclasts. Finally, we examined the ability of IL-11 to mediate osteoclast formation in tissue culture dishes and on bone slices in the absence of RANKL, with suboptimal levels of RANKL, or from RANKL-pretreated murine bone marrow macrophages (BMMs).ResultsWe found that freshly isolated murine bone marrow cells cultured in the presence of breast cancer conditioned media for 6 days gave rise to a population of cells which were able to form osteoclasts upon treatment with RANKL and M-CSF. Moreover, a neutralizing anti-IL-11 antibody significantly inhibited the ability of breast cancer conditioned media to promote the development and/or survival of osteoclast progenitor cells. Similarly, recombinant IL-11 was able to sustain a population of osteoclast progenitor cells. However, IL-11 was unable to exert any effect on osteoclast survival, induce osteoclastogenesis independent of RANKL, or promote osteoclastogenesis in suboptimal RANKL conditions.ConclusionsOur data indicate that a) IL-11 plays an important role in osteoclastogenesis by stimulating the development and/or survival of osteoclast progenitor cells and b) breast cancer may promote osteolysis in part by increasing the pool of osteoclast progenitor cells via tumor cell-derived IL-11. However, given the heterogeneous nature of the bone marrow cells, the precise mechanism by which IL-11 treatment gives rise to a population of osteoclast progenitor cells warrants further investigation.

Salivary soluble receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio in periodontal disease and health

PurposeThe receptor activator of nuclear factor kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system plays a significant role in osteoclastogenesis, activation of osteoclasts, and regulation of bone resorption. This study aimed to evaluate the use of the salivary soluble RANKL (sRANKL)/OPG ratio as a diagnostic marker for periodontitis in nonsmokers.MethodsTwenty-five patients with chronic periodontitis and 25 individuals with a healthy periodontium were enrolled in this study. Samples containing 5 mL of unstimulated saliva were obtained from each subject. Salivary sRANKL and OPG concentrations were determined using a standard enzyme-linked immunosorbent assay. Statistical analysis was performed using SPSS ver. 18.0.ResultsThe levels of sRANKL and OPG were detectable in all of the samples. Positive relationships were found between the plaque index and clinical attachment level and both the salivary concentration of sRANKL and the salivary sRANKL/OPG ratio (P<0.05). The salivary concentration of sRANKL and the sRANKL/OPG ratio were significantly higher in the periodontitis group than in the healthy group (P=0.004 and P=0.001, respectively). In contrast, the OPG concentration showed no significant differences between the groups (P=0.455).ConclusionsThese findings suggest that the salivary sRANKL/OPG ratio may be helpful in the screening and diagnosis of periodontitis. However, longitudinal studies with larger populations are needed to confirm these results.

5-Lipoxygenase Mediates RANKL-Induced Osteoclast Formation via the Cysteinyl Leukotriene Receptor 1

5-Lipoxygenase (5-LO) catalyzes the formation of two major groups of leukotrienes, leukotriene B4 and cysteinyl leukotrienes (CysLTs), and it has been implicated as a promising drug target to treat various inflammatory diseases. However, its role in osteoclastogenesis has not been investigated. In this study, we used mouse bone marrow-derived macrophages (BMMs) to show that 5-LO inhibitor suppresses RANKL-induced osteoclast formation. Inhibition of 5-LO was associated with impaired activation of multiple signaling events downstream of RANK, including ERK and p38 phosphorylation, and IκB degradation, followed by a decrease in NFATc1 expression. Ectopic overexpression of a constitutively active form of NFATc1 partly rescued the antiosteoclastogenic effect of 5-LO inhibitor. The knockdown of 5-LO in BMMs also resulted in a significant reduction in RANKL-induced osteoclast formation, accompanied by decreased expression of NFATc1. Similar effects were shown with CysLT receptor (CysLTR)1/2 antagonist and small RNA for CysLTR1 in BMMs, indicating the involvement of CysLT and CysLTR1 in 5-LO-mediated osteoclastogenesis. Finally, 5-LO inhibitor suppressed LPS-induced osteoclast formation and bone loss in the in vivo mouse experiments, suggesting a potential therapeutic strategy for treating diseases involving bone destruction. Taken together, the results of this study demonstrate that 5-LO is a key mediator of RANKL-induced osteoclast formation and possibly a novel therapeutic target for bone-resorption diseases.

Analysis of RANKL gene polymorphism (rs9533156 and rs2277438) in Iranian patients with chronic periodontitis and periimplantitis

ObjectiveRANK/OPG/RANKL pathway plays a significant role in osteoclastogenesis, osteoclast activation, and regulation of bone resorption. The aim of this study was to investigate the association of RANKL gene polymorphisms (rs9533156 and rs2277438) with chronic periodontitis and peri-implantitis in an Iranian population. Design77 patients with chronic periodontitis, 40 patients with peri-implantitis and 89 periodontally healthy patients were enrolled in this study. 5cc of blood was obtained from the cephalic vein of subjects arms and transferred into tubes containing EDTA. Genomic DNA was extracted using Miller's Salting Out technique. The DNA was transferred into 96 division plates, transported to Kbioscience Institute in United Kingdom and analyzed using the Kbioscience Competitive Allele Specific PCR (KASP) technique. Differences in the frequencies of genotypes and alleles in the disease and control groups were analyzed using Chi-square and Fisher's exact statistical tests. ResultsComparison of frequency of alleles in SNP rs9533156 of RANKL gene between the chronic periodontitis group with the control and peri-implantitis groups revealed statistically significant differences (P=0.024 and P=0.027, respectively). Comparison of genotype expression of SNP rs9533156 on RANKL gene between the peri-implantitis group with chronic periodontitis and control groups revealed statistically significant differences (P=0.001); the prevalence of CT genotype was significantly higher amongst the chronic periodontitis group. Regarding SNP rs2277438 of RANKL gene, comparison of prevalence of genotypes and frequency of alleles did not reveal any significant differences (P=0.641/P=0.537, respectively). ConclusionsThe results of this study indicate that CT genotype of rs9533156 RANKL gene polymorphism was significantly associated with peri-implantitis, and may be considered as a genetic determinant for peri-implantitis.

RANKL downregulates cell surface CXCR6 expression through JAK2/STAT3 signaling pathway during osteoclastogenesis

The receptor activator of nuclear factor-κB ligand (RANKL), as a member of the tumor necrosis factor (TNF) family, plays an essential role in osteoclast differentiation and function. Chemokines and their receptors have recently been shown to play critical roles in osteoclastogenesis, however, whether CXCL16-CXCR6 plays role in RANKL-mediated osteoclastogenesis is unknown. In this study, we first reported that RANKL decreased CXCR6 in a dose-dependent manner, which may be through deactivation of Akt and STAT3 signaling induced by CXCL16. Interestingly, RANKL-mediated CXCR6 reduction may be associated to the activation of STAT3 by phosphorylation. When STAT3 activation was blocked by JAK2/STAT3 inhibitor AG490, RANKL failed to shut down CXCR6 expression during osteoclastogenesis. However, CXCL16 alone did not augment RANKL-mediated osteoclast differentiation and did not alter RANKL-receptor RANK mRNA expression. These results demonstrate that reduction of CXCL16-CXCR6 is critical in RANKL-mediated osteoclastogenesis, which is mainly through the activation of JAK2/STAT3 signaling. CXCL16-CXCR6 axis may become a novel target for the therapeutic intervention of bone resorbing diseases such as rheumatoid arthritis and osteoporosis.

Expression profiling of microRNAs in RAW264.7 cells treated with a combination of tumor necrosis factor alpha and RANKL during osteoclast differentiation

Tumor necrosis factor alpha (TNF-α), a cytokine involved in the pathogenesis of periodontal disease, induces osteoclast differentiation and indirectly promotes alveolar bone resorption. We investigated TNF-α-regulated osteoclast differentiation, focusing on microRNAs. MicroRNAs are small, noncoding RNAs that are involved in various biological processes, including cellular differentiation, proliferation and apoptosis. Aside from miR-21, miR-155 and miR-223, the identities of the microRNAs that play roles in osteoclast differentiation are unknown. Notably, no previous studies have reported the expression profiling of microRNAs during TNF-α-regulated osteoclast differentiation. We used microarrays to screen the levels of expression of mature microRNAs in RAW264.7 cells treated with a combination of TNF-α and RANKL, or RANKL alone for 0, 24 or 82 h during osteoclast formation. We validated the results of the microarray analyses through quantitative RT-PCR analyses of representative microRNAs in RAW264.7 cells and murine bone marrow macrophages. During osteoclast formation, the expression of 44 mature microRNAs differed by more than twofold between untreated cells and cells treated with a combination of TNF-α and RANKL, and the expression of 52 mature microRNAs differed upon RANKL treatment. According to quantitative RT-PCR analyses, miR-378 was upregulated and miR-223 was downregulated during osteoclast formation. Furthermore, miR-21, miR-29b, miR-146a, miR-155 and miR-210 were highly expressed during osteoclast differentiation in TNF-α/RANKL-treated cells compared with RANKL-treated cells. These results suggest that miR-223 and miR-378 may play important roles in osteoclastogenesis, and that miR-21, miR-29b, miR-146a, miR-155 and miR-210 are involved in TNF-α-regulated osteoclast differentiation.

Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma

AbstractBruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF–induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5nM) and MM patients. It decreased SDF-1–induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell–induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Overexpression of microRNA-223 in rheumatoid arthritis synovium controls osteoclast differentiation

MicroRNAs, a class of noncoding RNAs, play roles in human diseases. MicroRNA-223 (miR-223) is reported to play critical roles in osteoclastogenesis. The purpose of this study was to analyze the expression pattern of miR-223 in rheumatoid arthritis (RA) synovium and examine the suppression of osteoclastogenesis from human peripheral blood mononuclear cells (PBMC) by overexpression of miR-223. Expression of miR-223 in synovium from RA patients was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and section in situ hybridization. MiR-223 was overexpressed in an osteoclastogenesis coculture system with PBMC and RA synovial fibroblast. At 3 weeks after transfection of double-stranded miR-223, the formation of tartrate-resistant acid phosphatase (TRAP)-stained multinucleated cells was analyzed to evaluate the inhibitory effect of miR-223 on osteoclastogenesis. MiR-223 was more highly expressed in RA synovium than in osteoarthritis (OA) synovium due to the increased number of miR-223-positive cells in RA synovium. MiR-223 was expressed in the superficial and sublining layers, and macrophages, monocytes, and CD4 T cells also expressed miR-223. The number of TRAP-positive multinucleated cells was significantly decreased by overexpression of miR-223 in a dose-dependent manner. The expression of osteoclastogenesis marker genes was significantly down-regulated by miR-223 overexpression. MiR-223 is intensely expressed in RA synovium, and overexpression of miR-223 suppresses osteoclastogenesis in vitro. This study demonstrates the possibility of gene therapy with miR-223 to treat bone destruction in RA patients.

The R740S mutation in the V-ATPase a3 subunit increases lysosomal pH, impairs NFATc1 translocation, and decreases in vitro osteoclastogenesis

Vacuolar H(+) -ATPase (V-ATPase), a multisubunit enzyme located at the ruffled border and in lysosomes of osteoclasts, is necessary for bone resorption. We previously showed that heterozygous mice with an R740S mutation in the a3 subunit of V-ATPase (+/R740S) have mild osteopetrosis resulting from an ∼90% reduction in proton translocation across osteoclast membranes. Here we show that lysosomal pH is also higher in +/R740S compared with wild-type (+/+) osteoclasts. Both osteoclast number and size were decreased in cultures of +/R740S compared with +/+ bone marrow cells, with concomitant decreased expression of key osteoclast markers (TRAP, cathepsin K, OSCAR, DC-STAMP, and NFATc1), suggesting that low lysosomal pH plays an important role in osteoclastogenesis. To elucidate the molecular mechanism of this inhibition, NFATc1 activation was assessed. NFATc1 nuclear translocation was significantly reduced in +/R740S compared with +/+ cells; however, this was not because of impaired enzymatic activity of calcineurin, the phosphatase responsible for NFATc1 dephosphorylation. Protein and RNA expression levels of regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of NFATc1 activation and a protein degraded in lysosomes, were not significantly different between +/R740S and +/+ osteoclasts, but the RCAN1/NFATc1 ratio was significantly higher in +/R740S versus +/+ cells. The lysosomal inhibitor chloroquine significantly increased RCAN1 accumulation in +/+ cells, consistent with the hypothesis that higher lysosomal pH impairs RCAN1 degradation, leading to a higher RCAN1/NFATc1 ratio and consequently NFATc1 inhibition. Our data indicate that increased lysosomal pH in osteoclasts leads to decreased NFATc1 signaling and nuclear translocation, resulting in a cell autonomous impairment of osteoclastogenesis in vitro.

Osteoclastogenesis is negatively regulated by D‐serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.

Epigenetic regulation of osteoclastogenesis by histone modification

Osteoclasts are multinuclear fusion cells derived from monocyte and macropharge lineage cells, which are differentiated in the presence of M-CSF and RANKL. Some transcription factors, such as NFATc1, are known to be associated with osteoclastogenesis however, the epigenetic regulation is not uncovered. Recently it was revealed that the activation and the repression of gene in the ES cells were strictly regulated by the histone modification, such as H3K4/H3K27 trimethylation mark. In this section we review the epigenetic regulation by histone modification and the role in osteoclastogenesis.

Abstract 2934: Targeting Bruton's tyrosine kinase with PCI-32765 blocks growth and survival of multiple myeloma and Waldenström macroglobulinemia via potent inhibition of osteoclastogenesis, cytokines/chemokine secretion, and myeloma stem-like cells in the bone marrow microenvironment

Abstract Specific expression of Bruton's tyrosine kinase (Btk) in osteoclasts (OC), but not osteoblasts (OB), suggests its role in osteoclastogenesis. Since Btk has not been characterized in multiple myeloma (MM) and Waldenström Macroglobulinemia (WM), we investigate effects of PCI-32765, an oral, potent, and selective Btk inhibitor with promising clinical activity in B-cell malignancies, on OC differentiation and function within MM bone marrow (BM) milieu, as well as on MM and WM cancer cells. In CD14+ OC precursor cells, PCI-32765 abrogated RANKL/M-CSF-induced Btk activation and downstream PLCγ2, resulting in decreased number of multinucleated OC (&amp;gt;3 nuclei) by tartrate-resistant acid phosphatase (TRAP) staining and inhibition of TRAP5b (ED50 = 17 nM), a specific mature OC marker. It induced defective bone resorption activity, as evidenced by diminished pit formation on dentine slices. Lack of effect of Dexamethasone on osteoclastic activity was overcome by combination of Dexamethasone with PCI-32765. PCI-32765 potently downregulates cytokine and chemokine secretion from OC cultures, i.e., MIP1α, MIP1α, IL-8, TGFβ1, RANTES, APRIL, SDF-1, and activin A (ED50 = 0.1-0.48 nM). It significantly decreased IL-6, SDF-1, MIP1α, MIP1α, and M-CSF in 2-week cultures of CD138-negative cells from active MM patients, associated with decreased TRAP staining. In MM and WM cells, immunoblotting analysis showed Btk expression in higher percentage of CD138+ myeloma cells from patients (4 out of 5 samples) than MM cell lines (5 out of 9 cell lines), whereas microarray analysis demonstrated increased expression of Btk and its downstream signaling components in WM cells than in CD19+ normal BM cells (p&amp;lt;0.001). Importantly, PCI-32765 blocked SDF-1-induced adhesion and migration of MM cell lines and patient MM cells via blockade of Btk signaling cascade. Quantigene analysis further showed PCI-32765-inhibited MIP1α mRNA in MM cells and many NF-κB-targeted transcripts in OC-lineages. PCI-32765 mitigated MM cell growth and survival triggered by IL-6 and coculture with BM stromal cells (BMSCs) or OCs. It blocked WM cell proliferation and induced apoptosis. Furthermore, myeloma stem-like cells from MM patients expressed Btk and PCI-32765 (10-100 nM) specifically blocked their abilities to form colonies in methylcellulose (n=5). In contrast, no toxicity was observed in Btk-negative BMSCs and OB. Oral administration of PCI-32765 (12 mg/kg) in mice significantly suppressed MM cell growth (p&amp;lt; 0.03) and MM cell-induced osteolysis on implanted human bone chips in a humanized myeloma (SCID-hu) model. Together, these results strongly support clinical trials of targeting Btk by PCI-32765 in the BM microenvironment to improve patient outcome in MM and WM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2934. doi:1538-7445.AM2012-2934

Abstract SY15-01: Cellular context-specific tumor suppression by PTPN11

Cartilage tumors, accounting for 22% of skeletal system tumors, are characterized by the formation of exostoses, enchondroma(s) or both, and cause significant morbidity and mortality. Both benign and malignant cartilaginous tumors can develop at various ages, and can show autosomal dominant inheritance or occur sporadically. The molecular mechanism underlying the development and progression of these cartilaginous lesions remains incompletely understood. Shp2, encoded by the Ptpn11 gene, is one of two Src homology 2 domain-containing protein-tyrosine phosphatases, and is required for most, if not all, receptor tyrosine kinase (RTK), cytokine, and integrin signaling pathways. Global deletion of Shp2 in mice results in early embryonic lethality, whereas postnatal Shp2 deficiency in various tissues/cells has diverse effects on their development and function. Several human malignancies, most notably childhood myeloproliferative disorders, are associated with Ptpn11 gain-of-function (GOF) mutations. Several lines of evidence indicate that Shp2 plays an important role in skeletal development and homoeostasis; however, little is known about its role in vivo. Recently Ptpn11 truncated mutations (presumably protein null) are reported to cause human metachondromatosis, a benign cartilage tumor syndrome with malignant potentials. By taking a tissue-specific gene knockout approach, we report here that Shp2 loss-of-function mutation, in contrast to its GOF mutants in other tissues/cells, causes widespread and severe cartilaginous tumors, strongly suggesting that Shp2 has a tissue specific-tumor suppressor function. These studies also identify the target of cartilage malignancies caused by Shp2 deficiency as a novel cell population with stem/progenitor properties located within the groove of Ranvier. Materials and Methods: Mice carrying Ptpn11 floxed (fl) or cathepsin K-Cre (Ctsk-Cre) were described previously. To generate Ctsk-expressing cell specific Shp2 deficient mice and study the role of Shp2 in these cells to regulate skeletal development and homeostasis, Ptpn11 floxed mice were bred to Ctsk-Cre mice to generate Ptpn11fl/fl;Ctsk-Cre (KO) and Ptpn11fl/+;Ctsk-Cre (Control) animals. To track the fate of Ctsk + cartilaginous cells in vivo in the presence or absence of Shp2, Roza26lacZ (R26lacZ) and Roza26EYFP (R26YFP) reporter alleles were bred to Control and KO mice to generate Control/R26YFP, KO/R26YFP, Control/R26lacZ and KO/R26lacZ compound mice, respectively. YFP-positive cartilaginous cells were isolated by serial enzymatic digestions of epiphyseal cartilage with hyaluronidase/Trypsin/ collagenase D and FACS sorting. Cell surface marker expression was determined by FACS analysis after staining with fluorescence-labeled antibodies. For histological analysis, skeletal tissues were removed from mice after euthanasia and fixed in 4% paraformaldehyde; after decalcification, embedding, and sectioning, they were stained with HELysMCre mcie, revealed that the cartilage phenotype in KO mice was not osteoclast-autonomous. Therefore to identify the cellular origin of the cartilage lesions in KO mice, we conducted a reporter study by using Ctsk-Cre mediated R26lacZ or YFP expression. Surprisingly, we found that the Ctsk promoter is active not only in mature osteoclasts, but also in a subset of cells that live in perichondrial groove of Ranvier. Deficiency of Shp2 in these cells causes unrestrained proliferation, chondrogenic differentiation, and exostoses/enchondroma(s) formation. These cells were found to share several mesenchymal/chondroprogenitor markers, such as CD44, CD90, Stro1 and jagged1. Biochemical analysis shows that Shp2 deficiency impaired Erk activation and caused elevated expression of Indian hedgehog (Ihh) and PTHrP in cartilage lesions. In summary, we specifically ablated Shp2 in Ctsk-expressing cells (primarily in mature osteoclasts and subsets of cartilaginous cells) in mice and found that Shp2, in addition to having a role in osteoclastogenesis in vitro and in vivo, negatively regulates the proliferation and chondrogenic differentiation of a unique population of ctsk+ perichondrial cartilaginous cells. Loss of Shp2 in these cells causes cell proliferation, chondrogenic differentiation, and cartilage tumorigenesis at the metaphyses of tubular bones. This phenotype is strikingly similar to the human disease metachondromatosis. Mechanistically, we believe that this pathogenic process is triggered by ectopic and/or elevated Ihh expression due to impaired Erk activation in Shp2- deficient perichondrial cells. If confirmed, these data identify hedgehog pathway inhibitors as potential therapeutic agents in metachromatosis patients. Our results, in concert with previous studies, show that, depending on the cellular context, Shp2 can act as an oncogene or a tumor suppressor gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY15-01. doi:1538-7445.AM2012-SY15-01

MiR-133a in Human Circulating Monocytes: A Potential Biomarker Associated with Postmenopausal Osteoporosis

BackgroundOsteoporosis mainly occurs in postmenopausal women, which is characterized by low bone mineral density (BMD) due to unbalanced bone resorption by osteoclasts and formation by osteoblasts. Circulating monocytes play important roles in osteoclastogenesis by acting as osteoclast precursors and secreting osteoclastogenic factors, such as IL-1, IL-6 and TNF-α. MicroRNAs (miRNAs) have been implicated as important biomarkers in various diseases. The present study aimed to find significant miRNA biomarkers in human circulating monocytes underlying postmenopausal osteoporosis.Methodology/Principal FindingsWe used ABI TaqMan® miRNA array followed by qRT-PCR validation in circulating monocytes to identify miRNA biomarkers in 10 high and 10 low BMD postmenopausal Caucasian women. MiR-133a was upregulated (P=0.007) in the low compared with the high BMD groups in the array analyses, which was also validated by qRT-PCR (P=0.044). We performed bioinformatic target gene analysis and found three potential osteoclast-related target genes, CXCL11, CXCR3 and SLC39A1. In addition, we performed Pearson correlation analyses between the expression levels of miR-133a and the three potential target genes in the 20 postmenopausal women. We did find negative correlations between miR-133a and all the three genes though not significant.Conclusions/SignificanceThis is the first in vivo miRNA expression analysis in human circulating monocytes to identify novel miRNA biomarkers underlying postmenopausal osteoporosis. Our results suggest that miR-133a in circulating monocytes is a potential biomarker for postmenopausal osteoporosis.