Tartrate-resistant Acid Phosphatase Expression Research Articles

Vav1 inhibits RANKL-induced osteoclast differentiation and bone resorption

Vav1 is a Rho/Rac guanine nucleotide exchange factor primarily expressed in hematopoietic cells. In this study, we investigated the potential role of Vav1 in osteoclast (OC) differentiation by comparing the ability of bone marrow mononuclear cells (BMMCs) obtained from Vav1-deficient (Vav1−/−) and wild-type (WT) mice to differentiate into mature OCs upon stimulation with macrophage colony stimulating factor and receptor activator of nuclear kappa B ligand in vitro. Our results suggested that Vav1 deficiency promoted the differentiation of BMMCs into OCs, as indicated by the increased expression of tartrate-resistant acid phosphatase, cathepsin K, and calcitonin receptor. Therefore, Vav1 may play a negative role in OC differentiation. This hypothesis was supported by the observation of more OCs in the femurs of Vav1−/− mice than in WT mice. Furthermore, the bone status of Vav1−/− mice was analyzed in situ and the femurs of Vav1−/− mice appeared abnormal, with poor bone density and fewer number of trabeculae. In addition, Vav1-deficient OCs showed stronger adhesion to vitronectin, an αvβ3 integrin ligand important in bone resorption. Thus, Vav1 may inhibit OC differentiation and protect against bone resorption.

BMSCs-Derived Exosomes Ameliorate Pain Via Abrogation of Aberrant Nerve Invasion in Subchondral Bone in Lumbar Facet Joint Osteoarthritis.

Lumbar facet joint osteoarthritis (LFJ OA) is regarded as one of the common causes of low back pain (LBP). The pathogenesis and underlying mechanism of this disease are largely unknown, there is still no effective disease-modifying therapy. This study aims to investigate the efficacy of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the pathogenesis and behavioral signs of LBP in the LFJ OA mouse model. The pathogenetic change in cartilage and aberrant nerve invasion in the subchondral bone of LFJ in a mouse model after treatment with BMSC-exosomes was evaluated.BMSC-exosomes could relieve pain via abrogation of aberrant CGRP-positive nerve and abnormal H-type vessel formation in the subchondral bone of LFJ. Moreover, BMSC-exosomes attenuated cartilage degeneration and inhibited tartrate-resistant acid phosphatase expression and RANKL-RANK-TRAF6 signaling activation to facilitate subchondral bone remodeling. These results indicated that BMSC-exosomes could relive behavioral signs of LBP and pathological processes in LFJ OA. BMSC-exosomes have a prominent protective effect and might be a potential therapeutic option for the treatment of LFJ OA causing LBP. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:670-679, 2020.

FMNL1 down-regulation suppresses bone metastasis through reducing TGF-β1 expression in non-small cell lung cancer (NSCLC)

Approximately 40% of patients with non-small cell lung cancer (NSCLC) develop bone metastasis. The formin protein formin-like 1 (FMNL1) plays a key role in the pathogenic processes of hematopoietic malignancies, and has been reported to be associated with the progression of multiple types of cancer. In the study, we found that FMNL1 expression was markedly up-regulated in primary NSCLC samples, and stronger expression of FNML1 was detected in bone metastasis. Reducing FMNL1 expression significantly suppressed cell proliferation in NSCLC cells. We also investigated the functional effects of FMNL1 knockdown on the inhibition of migration and invasion by meditating the expression of epithelial to mesenchymal transition (EMT)-associated signals in NSCLC cells. The transforming growth factor-β1 (TGF-β1)/SMADs signaling pathway was repressed in FMNL1-knockdown NSCLC cells. Further studies indicated that additional treatment with TGF-β1 could markedly abrogate FMNL1 knockdown-induced suppression of migration and invasion in NSCLC cells. In addition, NSCLC cell-induced osteoclastogenesis was also inhibited by FMNL1 deletion, as evidenced by the down-regulated expression of tartrate-resistant acid phosphatase (TRAP) and NFATc1. In vivo studies confirmed the results that FMNL1 knockdown markedly limited tumor growth. Importantly, decreasing FMNL1 reduced bone metastasis ability in vivo. Therefore, our results demonstrated that suppressing FMNL1 expression could inhibit bone metastasis in NSCLC through blocking TGF-β1 signaling, and FMNL1 might be a novel target for developing effective therapeutic strategy to limit the bone metastasis of NSCLC.

Anti-osteoporosis effects of osteoking via reducing reactive oxygen species

Ethnopharmacological relevanceOsteoking is a Traditional Chinese Medicine consisting of seven types of medicinal herbs originated from Yi nationality and has been used in clinic to treat bone diseases for thousands of years in China. Osteoking shows excellent clinical therapeutic effects on osteoporosis, but it is not clear whether Osteoking could exhibit beneficial effects against osteoporosis via reducing reactive oxygen species (ROS).Aim of the study: To explore whether the protective effects of Osteoking on osteoporosis related to ROS, we investigated the effects of Osteoking on osteogenesis differentiation under oxidative stress. Materials and methodsThe ovariectomized (OVX) osteoporosis model was established by ovarian surgery, and Osteoking was orally administrated for 84 days. Then the pathogenesis changes of femur were analyzed by Hematoxylin and eosin (H&E) and Masson's trichrome staining. The levels of ROS, malondialdehyde (MDA)and superoxide dismutase (SOD) from rats' serum were further measured. In vitro, mouse pre-osteoblastic MC3T3-E1 cells pre-treated with or without 0.25 mM tert-butyl hydroperoxide (t-BHP) for 2 h were cultured and treated with different dilutions of Osteoking or 20 μM N-Acetyl-L-cysteine for another 24 h, respectively. The intracellular ROS production and markers of oxidative damage of the MC3T3-E1 cells were determined using corresponding kits, respectively. The expressions of alkaline phosphatase (ALP), collagen type I, osteoprotegerin (OPG), TGF-β1, β-catenin, receptor activator of nuclear factor-κB ligand (RANKL) and interleukin-6 (IL-6) were further analyzed by qRT-PCR and western blotting upon treatment. ResultsOur results showed that Osteoking significantly improving trabecular microstructure by promoting collagen fiber repair and new bone or cartilage regeneration was demonstrated in OVX osteoporosis rat models by micro-CT analysis and histological staining results. Osteoking supplementation reduced the levels of ROS and MDA in OVX rat serum and increased SOD activities. In addition, Osteoking could also up-regulate the proteins expression levels of Runx2, osteocalcin (BGP) and osteoprotegerin (OPG) but reducing the expression of tartrate-resistant acid phosphatase (TRAP). In vitro, Osteoking could effectively inhibit the t-BHP-induced intracellular excessive ROS production and protect cells from oxidative stress in mouse pre-osteoblastic MC3T3-E1 cells. Meanwhile, the mRNA expressions of ALP, collagen type I, OPG, TGF-β1 and β-catenin were also up-regulated whereas the RANKL and IL-6 were down-regulated in Osteoking-treated MC3T3-E1 cells. ConclusionsA novel therapeutic mechanism of Osteoking on osteoporosis reveals by present investigation. Clinic effects of Osteoking to treat osteoporosis are closely related to its ability to reduce oxidative stress.

Association between Increased Inducible Costimulator/Inducible Costimulator Ligand Expression with Bone Destruction in Apical Periodontitis

IntroductionThe aim was to assess the association of inducible costimulator (ICOS) and ICOS ligand with bone destruction in apical periodontitis (AP). MethodsSpecimens from patients presenting with AP were obtained during apicoectomy and subjected to histopathologic analysis and molecular assessment of ICOS/ICOS ligand. In addition, the experimental AP was induced by exposing the pulp of first mandibular molars of rats. Histologic and radiographic examinations were performed to validate the periapical lesions. The immunolocalization and messenger RNA expression of ICOS/ICOS ligand were evaluated by immunofluorescence staining and quantitative real-time polymerase chain reaction. The osteoclastic activities in periapical lesions, including the lesion size and the expression of tartrate-resistant acid phosphatase and the receptor activator of nuclear factor kappa B ligand, were recorded and followed by correlation analysis with ICOS/ICOS ligand expression. ResultsIn excisional specimens from AP patients, a significantly increased expression of ICOS/ICOS ligand was found compared with the healthy control. In the experimental AP samples, the expression of ICOS/ICOS ligand, tartrate-resistant acid phosphatase, and receptor activator of nuclear factor kappa B ligand was significantly elevated in inflamed periapical tissues (AP group) when compared with the healthy control. The number of ICOS+/ICOS ligand+ cells was highly correlated with the periapical lesion size (r = 0.892, P < .01 and r = 0.930, P < .01, respectively). ConclusionsThe increased expression of ICOS/ICOS ligand in periapical lesions was associated with the inflammatory infiltration and alveolar bone destruction of AP.

Prognostic significance of tartrate-resistant acid phosphatase expression in breast cancer.

e12594 Background: Tartrate-resistant acid phosphatase (TRAP) is a metalloproteinase-like protein that is expressed in several primary and metastatic tumors, and its expression is positively correlated with the oncogenic process. Tartrate-resistant acid phosphatase is also a novel product of activated macrophage. We have previously demonstrated the clinical significance of TRAP expression in tumor-infiltrating macrophages and serum TRAP in patients with metastatic breast cancer (BC). Therefore, TRAP protein can potentially be a predictive and prognostic marker to evaluate disease progression and therapeutic response in breast cancer patients with bone metastasis. We aim to investigate the role of TRAP expression in breast cancer metastasis and survival. Methods: RNA-seq expression data were obtained from The Cancer Genome Atlas (TCGA) database. Estrogen receptor (ER)-positive, human epidermal growth factor receptor-2 (HER2)-negative, and TNBC subtypes were included in the analyses. The TRAP-overexpressed and -silenced breast cancer cells (MCF7, 4T1, MDA-MB-231) were used for validation. Survival data was also retrieved from the TCGA database to verify the prognostic biomarker. Results: Through TCGA database analysis, we found that TRAP expression correlated to the Ki-67 expression indicating the cancer cell proliferating activity. Additionally, TRAP expression positively correlated with mesenchymal markers (SNAIL, CDH1, MMP9, Fibronectin), and negatively correlated with epithelial markers (SMAD2, SOX10), implying that the TRAP expression is related to the breast cancer Epithelial-Mesenchymal-Transition process. This phenomenon was validated in TRAP-altered cell and confirmed inferior survival with TRAP-expressed breast cancer patients in TCGA database. Conclusions: Combining clinical TCGA data and cell-based analyses showed that TRAP expression was significantly associated with breast cancer proliferating activity, metastatic potential, and inferior survival. TRAP serves as a breast cancer prognostic biomarker and can be considered as a therapeutic target. Further investigation is warranted.

The PI3K inhibitor buparlisib suppresses osteoclast formation and tumour cell growth in bone metastasis of lung cancer, as evidenced by multimodality molecular imaging.

Non-small cell lung cancer (NSCLC) metastasis commonly occurs in bone, which often results in pathological fractures. Sustained phosphoinositide-3-kinase (PI3K) signalling promotes the growth of PI3K-dependent NSCLC and elevates osteoclastogenic potential. The present study investigated the effects of a PI3K inhibitor on NSCLC growth in bone and osteoclast formation, and aimed to determine whether it could control symptoms associated with bone metastasis. A bone metastasis xenograft model was established by implanting NCI-H460-luc2 lung cancer cells, which contain a phosphatidylinositol- 4,5-bisphosphate 3-kinase catalytic subunit α mutation, into the right tibiae of mice. After 1 week, the tumours were challenged with a PI3K inhibitor (buparlisib) or blank control for 3 weeks. Tumour growth and burden were longitudinally assessed in vivo via reporter gene bioluminescence imaging (BLI), small animal positron emission tomography/computed tomography (CT) [18F-fluorodeoxyglucose (18F-FDG)] and single-photon emission computed tomography/CT [99mTc-methylene diphosphonate (99mTc-MDP)] imaging. Tibia sections of intraosseous NCI-H460 tumours were analysed by immunohistochemistry (IHC), western blotting and flow cytometry. Dynamic weight bearing (DWB) tests were further performed to examine the improvement of symptoms associated with bone metastasis during the entire study. Administration of buparlisib significantly inhibited the progression of bone metastasis of NSCLC, as evidenced by significantly reduced uptake of 18F-FDG, 99mTc-MDP and BLI signals in the treated lesions. In addition, buparlisib appeared to inhibit the expression of tartrate-resistant acid phosphatase and receptor activator of nuclear factor-κB ligand, as determined by IHC. Buparlisib also resulted in increased cell apoptosis, as determined by a higher percentage of Annexin V staining and increased caspase 3 expression. Furthermore, buparlisib significantly increased weight-bearing capacity, as revealed by DWB tests. The PI3K inhibitor, buparlisib, suppressed osteoclast formation in vivo, and exhibited antitumour activity, thus leading to increased weight-bearing ability in mice with bone metastasis of lung cancer. Therefore, targeting the PI3K pathway may be a potential therapeutic strategy that prevents the structural skeletal damage associated with bone metastasis of lung cancer.

Hyperglycemia Induces Osteoclastogenesis and Bone Destruction Through the Activation of Ca2+/Calmodulin-Dependent Protein Kinase II.

Hyperglycemia induces osteoclastogenesis and bone resorption through complicated, undefined mechanisms. Ca2+/calmodulin-dependent protein kinase II (CaMKII) promotes osteoclastogenesis, and could be activated by hyperglycemia. Here, we investigated whether CaMKII is involved in hyperglycemia-induced osteoclastogenesis and subsequent bone resorption. Osteoclast formation, bone resorption, CaMKII expression and phosphorylation were measured under high glucose in vitro and in streptozotocin-induced hyperglycemia rats with or without CaMKII inhibitor KN93. The results showed that 25mmol/L high glucose in vitro promoted cathepsin K and tartrate-resistant acid phosphatase expression (p < 0.05) and osteoclast formation (p < 0.01) associated with enhancing β isoform expression (p < 0.05) and CaMKII phosphorylation (p < 0.001). Hyperglycemia promoted the formation of osteoclasts and resorption of trabecular and alveolar bone, and inhibited sizes of femur and mandible associated with enhanced CaMKII phosphorylation (p < 0.001) in rats. All these changes could be alleviated by KN93. These findings imply that CaMKII participates not only in hyperglycemia-induced osteoclastogenesis and subsequent bone resorption, but also in the hyperglycemia-induced developmental inhibition of bone.

Combined treatment with cinnamaldehyde and PTH enhances the therapeutic effect on glucocorticoid-induced osteoporosis through inhibiting osteoclastogenesis and promoting osteoblastogenesis

The study was to investigate the effect of combining treatment with cinnamaldehyde and parathyroid hormone (1−34) (PTH) on glucocorticoid-induced osteoporosis (GIO) and compare with monotherapy. Forty Sprague-Dawley male rats with GIO were divided into four groups randomly: control group (CON group, N = 10); group that intragastric administration with cinnamaldehyde (CIN group, N = 10); group that subcutaneous injection with PTH, three times per week(PTH group, N = 10); both administration with cinnamaldehyde and PTH (CIN + PTH group, N = 10). Distal femurs were harvested for hematoxylin and eosin (H&E) staining, micro-CT scanning and immunohistochemical analysis. Murine mesenchymal stem cells were cultured and dealt with the presence of dexamethasone(DEX group), DEX + cinnamaldehyde(DEX + CIN group), DEX + PTH(DEX + PTH group) or DEX + cinnamaldehyde + PTH(DEX + CIN + PTH group). Alkaline phosphatase (ALP) staining was performed subsequently. The results showed that bone formation in CIN + PTH group was notably promoted compared with other groups. And the expression of tartrate-resistant acid phosphatase (trap) and runt-related transcription factor 2 (runx2) in CIN + PTH group were down-regulated and up-regulated respectively compared with PTH group. In vitro study revealed that ALP-positive cell number in DEX + CIN + PTH group was obviously enhanced compared with other groups. The study revealed that combined treatment with cinnamaldehyde and PTH enhances the therapeutic effect on GIO through inhibiting osteoclastogenesis and promoting osteoblastogenesis.

RANKL-mediated osteoclastogenic differentiation of macrophages in the abdominal aorta of angiotensin II-infused apolipoprotein E knockout mice

ObjectiveOsteoclastogenic activation of macrophages (OCG) occurs in human abdominal aortic aneurysms (AAAs) and in calcium chloride-induced degenerative AAAs in mice, which have increased matrix metalloproteinase activity. As the activity of OCG in dissecting aneurysms is not clear, we tested the hypothesis that OCG contributes to angiotensin II (Ang II)-induced dissecting aneurysm (Ang II-induced AAA) in apolipoprotein E knockout mice. MethodsAAAs were produced in apolipoprotein E knockout mice via the administration of Ang II. Additionally, receptor activator of nuclear factor kB ligand (RANKL)-neutralizing antibody (5 mg/kg) was administered to one group of mice 7 days prior to Ang II infusion. Aneurysmal sections were probed for presence of RANKL and tartrate-resistant acid phosphatase via immunohistochemistry and immunofluorescence staining. Mouse aortas were also examined for RANKL and matrix metalloproteinase 9 expression via Western blot. In vitro murine vascular smooth muscle cells (MOVAS) and murine macrophages (RAW 264.7) were analyzed for the expression of osteogenic factors via Western blot, qPCR, and flow cytometry in response to Ang II or RANKL stimulation. The signaling pathway that mediates Ang II-induced RANKL expression in MOVAS cells was also investigated via application of TG101348, a Janus kinase 2 (JAK2) inhibitor, and Western blot analysis. ResultsImmunohistochemical staining of Ang II-induced AAA sections revealed OCG as evidenced by increased RANKL and tartrate-resistant acid phosphatase expression compared with control mice. Immunofluorescence staining of AAA sections revealed co-localization of vascular smooth muscle cells and RANKL, revealing vascular smooth muscle cells as one potential source of RANKL. Systemic administration of RANKL-neutralizing antibody suppressed Ang II-induced AAA, with significant reduction of the maximum diameter of the abdominal aorta compared with vehicle controls (1.5 ± 0.4 mm vs 2.2 ± 0.2 mm). Ang II (1 μM) treatment induced a significant increase in RANKL messenger RNA expression levels in MOVAS cells compared with the vehicle control (1.0 ± 0.2 vs 2.8 ± 0.2). The activities of JAK2 and signal transducer and activator of transcription 5 (STAT5) were also significantly increased by Ang II treatment. Inhibition of JAK2/STAT5 suppressed Ang II-induced RANKL expression, suggesting the involvement of the JAK2/STAT5 signaling pathway. ConclusionsOCG with increased RANKL expression was present in Ang II-induced AAA, and neutralization of RANKL suppressed AAA formation. As neutralization of RANKL has been used clinically to treat osteoporosis and other osteoclast-related diseases, additional study of the effectiveness of RANKL neutralization in AAA is warranted.

Effect of caspases and RANKL induced by heavy force in orthodontic root resorption.

ObjectiveOrthodontic root resorption (ORR) due to orthodontic tooth movement is a difficult treatment-related adverse event. Caspases are important effector molecules for apoptosis. At present, little is known about the mechanisms underlying ORR and apoptosis in the cementum. The aim of the present in vivo study was to investigate the expression of tartrate-resistant acid phosphatase (TRAP), caspase 3, caspase 8, and receptor activator of nuclear factor kappa-B ligand (RANKL) in the cementum in response to a heavy or an optimum orthodontic force.MethodsThe maxillary molars of male Wistar rats were subjected to an orthodontic force of 10 g or 50 g using a closed coil spring. The rats were sacrificed each experimental period on days 1, 3, 5, and 7 after orthodontic force application. And the rats were subjected to histopathological and immunohistochemical analyses.ResultsOn day 7 for the 50-g group, hematoxylin and eosin staining revealed numerous root resorption lacunae with odontoclasts on the root, while immunohistochemistry showed increased TRAP- and RANKL-positive cells. Caspase 3- and caspase 8-positive cells were increased on the cementum surfaces in the 50-g group on days 3 and 5. Moreover, the number of caspase 3- and caspase 8-positive cells and RANKL-positive cells was significantly higher in the 50-g group than in the 10-g group.ConclusionsIn our rat model, ORR occurred after apoptosis was induced in the cementum by a heavy orthodontic force. These findings suggest that apoptosis of cementoblasts is involved in ORR.

The role of TGFβ receptor 1-smad3 signaling in regulating the osteoclastic mode affected by fluoride

Studies that have focused on the role TGFβ signaling plays in osteoclast activity are gradually increasing; however, literature is rare in terms of fluorosis. The aim of this study is to observe the role the TβR1/Smad3 pathway plays in fluoride regulating cellsosteoclast-like cells that are under the treatment of TGFβ receptor 1 kinase. The RANKL-mediated osteoclast-like cells from RAW264.7 cells were used as osteoclast precursor model. The profile of miRNA expression in fluoride-treated osteoclast-like cells exhibited 303 upregulated miRNAs, 61 downregulated miRNAs, and further drew 37 signaling pathway maps by KEGG and Biocarta pathway enrichment analysis. TGFβ and its downstream effectors were included among them. Osteoclast viability, formation and function were detected via MTT method, bone resorption pit and tartrate-resistant acid phosphatase (TRACP) staining, respectively. Results demonstrated that different doses of fluoride exhibited a biphasic effect on osteoclast cell viability, differentiation, formation and function. It indicated that a low dose of fluoride treatment stimulated them, but high dose inhibited them. SB431542 acted as TβR1 kinase inhibitor and blocked viability, formation and function of osteoclast-like cells regulated by fluoride. The expression of the osteoclast marker, RANK, and TβR1/Smad3 at gene and protein level was analyzed under fluoride with and without SB431542 treatment. Fluoride treatment indicated little effect on the RANK protein expression; however it significantly influenced TRACP expression in osteoclast-like cells. The stimulation of fluoride on the expression of Smad3 gene and phosphorylated Smad3 protein exhibited dose-dependent manner. SB431542 significantly impeded phosphorylation of Smad3 protein and TRACP expression in osteoclast-like cells that were exposed to fluoride. Our work demonstrated that TGFβ signaling played a key role in fluoride regulating osteoclast differentiation, formation and function. It elucidated that TβR1/Smad3 pathway participated in the mechanism of biphasic modulation of osteoclast mode regulated by fluoride.

Biological Effects of Orthodontic Tooth Movement Into the Grafted Alveolar Cleft

Functional stimulus during orthodontic tooth movement into the grafted bone can lead to better alveolar bone grafting outcomes. The aim of this study was to analyze the biological effects of orthodontic tooth movement into the grafted alveolar cleft area with histologic staining, fluorescence staining, and real-time polymerase chain reaction (PCR). An animal model of orthodontic tooth movement into the grafted alveolar cleft area was established in 8-week-old Sprague-Dawley rats. The animals were divided into the experimental group and the control group. Four checkpoints were observed: before orthodontic stimuli, day 1 after orthodontic stimuli, day 3 after orthodontic stimuli, and day 5 after orthodontic stimuli. The cleft bone formation conditions, including the collagen fibers and the activities of the osteoclasts and osteoblasts, were evaluated by histologic staining. The expression of tartrate-resistant acid phosphatase (TRAP), receptor activator nuclear factor κB ligand, and Runt-related transcription factor 2 was detected by real-time PCR in both groups. Hematoxylin-eosin staining showed that the remodeling process of iliac autografts was completed when the orthodontic stress was applied, whereas the bone tissues first showed osteoclastogenesis and then osteogenesis. On the basis of TRAP staining, the osteoclasts increased to the maximal amount on day 3 and decreased thereafter. Evidence from tetracycline fluorescence staining indicated that no obvious changes in osteoblast activity were detected at the early stage; however, it gradually increased, especially in the region close to the root surface. According to real-time PCR, the expression of TRAP increased in both the early and middle stages, that of receptor activator nuclear factor κB ligand increased in the early stage, and that of Runt-related transcription factor 2 increased in the late stage. Moreover, the results showed significant differences between the experimental and control groups. Orthodontic tooth movement into the alveolar cleft bone graft area promoted bone remodeling of embedded bone, thus inducing bone resorption and subsequent deposition.

Abstract 2710: Molecular and clinical characteristics of IGHV4-34 expressing classic and variant HCL

Abstract Classic hairy cell leukemia (HCLc) is a B-cell malignancy with distinctive immunophenotype, typically expressing CD20, CD22, CD25, CD11c, CD103, CD123, annexin A1, tartrate-resistant acid phosphatase (TRAP), and BRAF V600E mutation. Purine analog therapy is highly effective with complete remission rates of approximately 85% in first line. HCL variant (HCLv) is recognized as a distinct entity, lacking CD25 and usually lacking annexin A1, tartrate-resistant acid phosphatase expression, CD123, and also lacks the BRAF V600E mutation. Patients respond poorly to purine analogs, with partial response in less than 50% and relatively poor overall survival from diagnosis. HCLc expressing B-cell receptor (BCR) with the IGHV4-34 immunoglobulin rearrangement has a poor prognosis like HCLv, whether immunophenotypically consistent with HCLv or HCLc, and also lacks BRAF V600E. We previously reported a series of 17 patients expressing IGHV4-34, immunophenotypically resembling HCLc in 7 and HCLv in 10 cases. Using routine deep sequencing of patient samples for immunoglobulin rearrangements, we have been able to accumulate 42 IGHV4-34 expressing cases including 23 classic HCL and 19 HCLv. Six (26%) of 23 HCLc vs 12 (63%) of 19 HCLv were truly unmutated, with VH gene germline identity (GI) 100%. Eleven (48%) of 23 HCLc and 7 (37%) of 19 HCLv were borderline-mutated (97≤GI&amp;lt;100), and 6 (26%) of 23 HCLc were significantly mutated (97&amp;lt;GI). There was no significant difference between the 5 groups with respect to either IGHD or IGHJ genes usage, or CDR3 length. However, we found a significant restriction of IGHD genes between 12 HCLv truly unmutated and 6 HCLc truly unmutated cases, in that there was no overlap of any IGHD genes between these 2 groups (p&amp;lt;0.0001). Of 28 cases test for BRAF V600E, only 5 were positive, all HCLc with GI 93.18-97.49%. The 23 BRAF wild-type (WT) cases included one with only one with GI=93.64%, but GI was &amp;gt;98.7% for the remaining 22 cases (p&amp;lt;0.0001). Of the 23 BRAF WT cases, 9 patients died of disease, 9 have active disease, and only 2 are in remission. Patients commonly had highly aggressive courses, with infiltration of HCLc/HCLv into spinal cord and cranial nerves, cardiac muscle, lungs, and cervical lymph nodes. Complete remission was achieved only through either recombinant immunotoxin therapy of combination purine analog and rituximab, but not by purine analogs alone. Additional clinical and genetic studies are ongoing to better characterize this poor-prognosis syndrome and determine if therapy can be optimized. Citation Format: Evgeny Arons, Katherine Potocka, Maryalice Stetler-Stevenson, Hong Zhou, Mark Raffeld, Mark Sokolsky, Sarah Davies, Robert J. Kreitman. Molecular and clinical characteristics of IGHV4-34 expressing classic and variant HCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2710. doi:10.1158/1538-7445.AM2017-2710

Inhibition of lipopolysaccharide-induced osteoclast formation and bone resorption in vitro and in vivo by cysteine proteinase inhibitors.

Inflammation-induced bone destruction is a major treatment target in many inflammatory skeletal diseases. The aim of this study was to investigate if the cysteine proteinase inhibitors cystatin C, fungal cysteine proteinase inhibitor (E-64), and N-benzyloxycarbonyl-arginyl-leucyl-valyl-glycyl-diazomethane acetate (Z-RLVG-CHN2) can inhibit LPS-induced osteoclast formation. Mouse bone marrow macrophages (BMMs) were isolated and primed with receptor activator of NF-κB ligand (RANKL) for 24 h, followed by stimulation with LPS, with and without inhibitors. Adult mice were injected locally with LPS and then treated with E-64 and osteoclast formation assessed by the number of cathepsin K+ multinucleated cells. Cystatin C inhibited LPS-induced osteoclast formation time and concentration dependently (IC50 = 0.3 μM). The effect was associated with decreased mRNA and protein expression of tartrate-resistant acid phosphatase (TRAP) and cathepsin K and of the osteoclastogenic transcription factors c-Fos and NFATc1. LPS-induced osteoclast formation on bone slices was also inhibited by cystatin C, resulting in decreased pit formation and release of bone matrix proteins. Similar data were obtained with E-64 and Z-RLVG-CHN2 Cystatin C was internalized in BMMs stimulated by LPS but not in unstimulated BMMs. Osteoclast formation induced by LPS was dependent on TNF-α, and the 3 inhibitors abolished LPS-induced TNF superfamily 2 (gene encoding TNF-α; Tnfsf2) mRNA expression without affecting Il1b, Il6, or oncostatin M (Osm) expression. Formation of osteoclasts in the skull bones after local LPS stimulation was inhibited by E-64. It is concluded that cysteine proteinase inhibitors effectively inhibit LPS-induced osteoclast formation in vivo and in vitro by inhibition of TNF-α expression. The targeting of cysteine proteinases might represent a novel treatment modality for prevention of inflammatory bone loss.

Abstract P4-03-17: Tartrate-resistant acid phosphatase (TRACP) modulates breast cancer pre-metastatic niche

Abstract Background: Tartrate-resistant acid phosphatase (TRACP or ACP5) is a metalloprotein enzyme that belongs to the acid phosphatase family and is mainly expressed by osteoclasts. It is a classic marker for bone resorption and osteoclast differentiation. TRACP expression is a useful serum marker for extensive bone metastasis. Recent studies have reported that TRACP promotes the invasion and distant metastasis of melanoma and breast cancer cells through the modulation of focal adhesion kinase (FAK) phosphorylation and epithelial cell migration. Therefore, TRACP has received considerable attention as a newly discovered proinvasion metastasis driver associated with different malignancies. Additionally, TRACP overexpression is indicative of the poor prognoses cancer patients and suggests that TRACP may have an important role in promoting tumor metastasis. Its detail mechanism in modulating cancer metastasis remains unknown. We have previously demonstrated the serum TRCAP 5a is a valuable marker in assessing disease activity, severity or systemic macrophage (inflammatory) burden. It has recently been found that up-regulation of TRACP promoted cancer cell invasion and lung metastasis, whereas TRACP knockdown inhibited these processes. Aims: No studies have previously addressed the question of extracellular effects of TRAP. The aim of this study was to evaluate if TRACP binds to and is endocytosed by cancer cells. We also aim to determine if extracellular TRACP affect oncogenesis or cancer progression, alter the cancer cell expressions of angiogenesis or adhesion molecules, or direct the treatment resistance or cancer prognosis. Results: Knockdown of TRACP significantly attenuated FoxM1-enhanced invasion and lung metastasis. Extracellular TRACP in culture media influenced the tumor invasion upon endocytosis. Significant increased invasion/migration was observed upon higher concentrations of TRACP. Tumor growth was significantly enhanced when the tumor xenografting previously primed with polarized M2Ø or TRACP protein, by enhanced metalloproteinase (MMP-9) and angiogenic factor (VEGF-A) expression Conclusions: TRACP or M2Ø-secreted TRACP could promote cancer growth and modulated the tissue and microenvironment and create pre-metastatic condition by cell adhesion/angiogenesis signaling alteration. TRACP-targeting strategy is plausible in breast cancer metastasis treatment. Citation Format: Dai M-S, Wu C-C, Chen L-C, Chen C-M, Chao T-Y. Tartrate-resistant acid phosphatase (TRACP) modulates breast cancer pre-metastatic niche [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-03-17.

Hepatic Osteodystrophy: The Mechanism of Bone Loss in Hepatocellular Disease and the Effects of Pamidronate Treatment

OBJECTIVES:The present study was designed to evaluate the bone phenotypes and mechanisms involved in bone disorders associated with hepatic osteodystrophy. Hepatocellular disease was induced by carbon tetrachloride (CCl4). In addition, the effects of disodium pamidronate on bone tissue were evaluated.METHODS:The study included 4 groups of 15 mice: a) C = mice subjected to vehicle injections; b) C+P = mice subjected to vehicle and pamidronate injections; c) CCl4+V = mice subjected to CCl4 and vehicle injections; and d) CCl4+P = mice subjected to CCl4 and pamidronate injections. CCl4 or vehicle was administered for 8 weeks, while pamidronate or vehicle was injected at the end of the fourth week. Bone histomorphometry and biomechanical analysis were performed in tibiae, while femora were used for micro-computed tomography and gene expression.RESULTS:CCl4 mice exhibited decreased bone volume/trabecular volume and trabecular numbers, as well as increased trabecular separation, as determined by bone histomorphometry and micro-computed tomography, but these changes were not detected in the group treated with pamidronate. CCl4 mice showed increased numbers of osteoclasts and resorption surface. High serum levels of receptor activator of nuclear factor-κB ligand and the increased expression of tartrate-resistant acid phosphatase in the bones of CCl4 mice supported the enhancement of bone resorption in these mice.CONCLUSION:Taken together, these results suggest that bone resorption is the main mechanism of bone loss in chronic hepatocellular disease in mice.

ICOS-Ligand Triggering Impairs Osteoclast Differentiation and Function In Vitro and In Vivo.

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14- cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS-B7h system.

Androgens have antiresorptive effects on trabecular disuse osteopenia independent from muscle atrophy

Aging hypogonadal men are at increased risk of osteoporosis and sarcopenia. Testosterone is a potentially appealing strategy to prevent simultaneous bone and muscle loss. The androgen receptor (AR) mediates antiresorptive effects on trabecular bone via osteoblast-lineage cells, as well as muscle-anabolic actions. Sex steroids also modify the skeletal response to mechanical loading. However, it is unclear whether the effects of androgens on bone remain effective independent of mechanical stimulation or rather require indirect androgen effects via muscle. This study aims to characterize the effects and underlying mechanisms of androgens on disuse osteosarcopenia. Adult male mice received a unilateral botulinum toxin (BTx) injection, and underwent sham surgery or orchidectomy (ORX) without or with testosterone (ORX+T) or dihydrotestosterone (ORX+DHT) replacement. Compared to the contralateral internal control hindlimb, acute trabecular number and bone volume loss was increased by ORX and partially prevented DHT. T was more efficient and increased BV/TV in both hindlimbs over sham values, although it did not reduce the detrimental effect of BTx. Both androgens and BTx regulated trabecular osteoclast surface as well as tartrate-resistant acid phosphatase expression. Androgens also prevented BTx-induced body weight loss but did not significantly influence paralysis or muscle atrophy. BTx and ORX both reduced cortical thickness via endosteal expansion, which was prevented by T but not DHT. In long-term follow-up, the residual trabecular bone volume deficit in sham-BTx hindlimbs was prevented by DHT but T restored it more efficiently to pre-treatment levels. Conditional AR deletion in late osteoblasts and osteocytes or in the satellite cell lineage increased age-related trabecular bone loss in both hindlimbs without influencing the effect of BTx on trabecular osteopenia. We conclude that androgens have antiresorptive effects on trabecular disuse osteopenia which do not require AR actions on bone via muscle or via osteocytes.

Inhibition of SDF-1α/CXCR4 Signalling in Subchondral Bone Attenuates Post-Traumatic Osteoarthritis.

Previous studies showed that SDF-1α is a catabolic factor that can infiltrate cartilage, decrease proteoglycan content, and increase MMP-13 activity. Inhibiting the SDF-1α/CXCR4 signalling pathway can attenuate the pathogenesis of osteoarthritis (OA). Recent studies have also shown that SDF-1α enhances chondrocyte proliferation and maturation. These results appear to be contradictory. In the current study, we used a destabilisation OA animal model to investigate the effects of SDF-1α/CXCR4 signalling in the tibial subchondral bone and the OA pathological process. Post-traumatic osteoarthritis (PTOA) mice models were prepared by transecting the anterior cruciate ligament (ACLT), or a sham surgery was performed, in a total of 30 mice. Mice were treated with phosphate buffer saline (PBS) or AMD3100 (an inhibitor of CXCR4) and sacrificed at 30 days post ACLT or sham surgery. Tibial subchondral bone status was quantified by micro-computed tomography (μCT). Knee-joint histology was analysed to examine the articular cartilage and joint degeneration. The levels of SDF-1α and collagen type I c-telopeptidefragments (CTX-I) were quantified by ELISA. Bone marrow mononuclear cells (BMMCs) were used to clarify the effects of SDF-1α on osteoclast formation and activity in vivo. μCT analysis revealed significant loss of trabecular bone from tibial subchondral bone post-ACLT, which was effectively prevented by AMD3100. AMD3100 could partially prevent bone loss and articular cartilage degeneration. Serum biomarkers revealed an increase in SDF-1α and bone resorption, which were also reduced by AMD3100. SDF-1α can promote osteoclast formation and the expression oftartrate resistant acid phosphatase (TRAP), cathepsin K (CK), and matrix metalloproteinase (MMP)-9 in osteoclasts by activating the MAPK pathway, including ERK and p38, but not JNK. In conclusion, inhibition of SDF-1α/CXCR4signalling was able to prevent trabecular bone loss and attenuated cartilage degeneration in PTOA mice.