Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis

Sialic acid, which is located at the end of the carbohydrate moiety of cell surface glycoconjugates, is involved in many biologic responses, such as intercellular reactions and virus-cell fusion, especially in hematopoietic cells. Here we provide experimental evidence that the sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation. Lectin histochemical study demonstrated the existence of both alpha (2,3)-linked-sialic acid and alpha (2,6)-linked-sialic acid in mouse bone marrow-derived macrophages and in the RAW264.7 macrophage cell line, which are osteoclast precursors. Flow cytometric analysis of surface lectin staining revealed the kinetics of these sialic acids during osteoclastogenesis: alpha (2,3)-linked-sialic acid was abundantly expressed throughout osteoclastogenesis, whereas alpha (2,6)-linked-sialic acid levels declined at the terminal stage of osteoclast differentiation. To investigate the role of sialic acid in osteoclast differentiation, we performed an osteoclastogenesis assay with or without exogenous sialidase treatment. Desialylated cells formed TRAP-positive mononuclear cells, but did not become multinuclear cells despite the normal expression of osteoclast markers such as cathepsin K, integrin beta3, and nuclear factor-ATc1. Flow cytometric analysis also demonstrated that exogenous sialidase effectively removed alpha (2,6)-linked-sialic acid, but only slightly changed the alpha (2,3)-linked-sialic acid content, suggesting that alpha (2,6)-linked-sialic acid might be involved in osteoclast differentiation. Findings from knockdown analysis using small interfering RNA oligonucleotides against alpha 2,6-sialyltransferase support this idea: alpha (2,6)-linked-sialic acid-deficient cells markedly inhibit the formation of multinuclear osteoclasts. Our findings suggest that alpha (2,6)-linked-sialic acid of cell surface glycoconjugates has a role in osteoclast differentiation, possibly via its role in the cell-cell fusion process.     

RANKL-induced expression of tetraspanin CD9 in lipid raft membrane microdomain is essential for cell fusion during osteoclastogenesis.

We showed that CD9, a member of tetraspanin superfamily proteins, is expressed in a specific membrane microdomain, called "lipid raft," and is crucial for cell fusion during osteoclastogenesis after activation of the RANK/RANKL system. INTRODUCTION: Osteoclasts are bone-resorbing multinuclear polykaryons that are essential for bone remodeling and are formed through cell fusion of mononuclear macrophage/monocyte lineage precursors. Although osteoclastogenesis has been shown to be critically regulated by the RANK/RANKL system, the mechanism how precursor cells fuse with each other remains unclear. We examined the function of CD9, a member of tetraspanin superfamily, which has previously been shown to form macromolecular membrane microdomains and to regulate cell-cell fusion in various cell types. MATERIALS AND METHODS: We used RAW264.7, a macrophage/monocyte lineage cell line, which can differentiate into osteoclast-like polykaryons on the application of RANKL. Expression and distribution of CD9 was assessed by Western blotting, fluorescence-assorted cell sorting (FACS) and immunohistochemistry with light and electron microscopy. A specific neutralizing antibody and RNA interference were used to inhibit the function of CD9, and green fluorescent protein (GFP)-CD9 was exogenously expressed to enhance the effect of CD9. The distribution of CD9 in lipid microdomain was examined by biochemical (sucrose density gradient) isolation and imaging technique. RESULTS: CD9 is expressed on cell surfaces of RAW264.7, which is enhanced by RANKL. Targeted inhibition of CD9 decreases the number of osteoclast-like cells. On the other hand, overexpression of CD9 promotes spontaneous cell fusion even in the absence of RANKL. CD9 is localized in detergent-insoluble "lipid raft" microdomain in RANKL stimulation, and disruption of lipid rafts markedly reduces the formation of osteoclast-like polykaryons. Immunohistochemical studies of bone tissues revealed the expression of CD9 in osteoclasts in vivo. CONCLUSIONS: These data suggest that function of tetraspanin CD9 and its expression in lipid rafts are crucial for cell fusion during osteoclastogenesis.     

TRAF2 is essential for TNF-alpha-induced osteoclastogenesis.

TRAF2-deficient mice show embryonic lethality, and we developed a new in vitro differentiation system to show the function of TRAF2 in osteoclastogenesis, in which osteoclast progenitors are derived from the fetal liver of TRAF2-deficient mice. Using this system, we showed that TRAF2 is required for TNF-alpha-induced osteoclastogenesis. INTRODUCTION: TNF receptor-associated factor 2 (TRAF2) is a signal transducer for RANK and for two TNF receptor isotypes, TNFR1 and TNFR2. Because TRAF2-deficient mice show embryonic lethality, it has remained unclear whether TRAF2 is crucial in RANKL- or TNF-alpha-induced osteoclastogenesis. MATERIALS AND METHODS: Osteoclast progenitors derived from fetal liver were cultured in the presence of monocyte macrophage colony-stimulating factor (M-CSF), and flow cytometry for characterization of surface markers on these cells was performed. To examine the involvement of TRAF2 in osteoclast differentiation, we cultured osteoclast progenitors from TRAF2-deficient and wildtype mice with soluble RANKL or TNF-alpha in the presence of M-CSF, and counted the number of TRACP(+) multinucleate cells formed. c-jun N-terminal kinase (JNK) and NF-kappaB activation in osteoclast progenitors was examined by Western blot analysis and electrophoretic mobility shift assay, respectively. Nuclear factor of activated T cells (NFATc1) expression and activation were analyzed by RT-PCR and immunofluorescence staining, respectively. To examine whether TRAF2 overexpression induced osteoclastogenesis, TRAF2 was overexpressed in osteoclast progenitors form wildtype bone marrow by retrovirus infection. RESULTS AND CONCLUSIONS: Osteoclast progenitors from normal fetal liver, which were cultured with M-CSF, expressed surface molecules c-fms, Mac-1, and RANK, and could differentiate into TRACP(+) multinucleate cells in the presence of soluble RANKL or TNF-alpha. RANKL-induced osteoclastogenesis gave a reduction of 20% in the progenitors from TRAF2-deficient mice compared with that of the cells from littermate wildtype mice, whereas TNF-alpha-induced osteoclastogenesis was severely impaired in the cells from the TRAF2-deficient mice. Only a few TRACP(+) multinucleate cells were formed, and TNF-alpha-mediated activation of JNK, NF-kappaB, and NFATc1 was defective. TRAF2 overexpression induced differentiation of osteoclast progenitors from wildtype mice into TRACP(+) multinucleate cells. These results suggest that TRAF2 plays an important role in TNF-alpha-induced osteoclastogenesis.     

Determination of chitinases family during osteoclastogenesis

Mammalian chitinases consisting of CHIA, CHIT1, CHI3L1, CHI3L2 and CHID1 exert important biological roles in the monocyte lineage and chronic inflammatory diseases. Pathological bone resorption is a cause of significant morbidity in diseases affecting the skeleton such as rheumatoid arthritis, osteoporosis, periodontitis and cancer metastasis. The biologic role of chitinases in bone resorption is poorly understood. In this study, we evaluated the expression of the chitinases family during osteoclast differentiation. The expression of CHIA, CHI3L2 and CHID1 resulted unchanged during osteoclast differentiation, whereas CHIT1 and CHI3L1 increased significantly. We also observed that CHIT1 and CHI3L1 are involved in osteoclast function. Indeed, silencing CHIT1 and CHI3L1 with siRNA resulted in a significant decrease in bone resorption activity. In addition, transfection with CHIT1 or CHI3L1 siRNA and co-transfection with both decreased the levels of the pro-differentiative marker MMP9. Overall, these discoveries reveal a novel and crucial role for both CHIT1 and CHI3L1 in promoting bone resorption and identifying new potential candidate markers for therapeutic targeting.     

E-cadherin expression associated with differentiation and prognosis in patients with non-small cell lung cancer

BACKGROUND: E-Cadherin plays a major role in maintaining the intercellular junctions in epithelial tissues. The reduction of E-cadherin expression in cancer cells may be associated with tumor differentiation, metastasis, and a poor prognosis. METHODS: Immunohistochemistry for E-cadherin expression was performed on 109 tumors from patients with non-small cell lung cancer who underwent operations. RESULTS: With respect to membranous immunostaining, 57 carcinomas were E-cadherin-positive, 39 carcinomas E-cadherin-reduced, and 13 carcinomas E-cadherin-negative. The percentage of poorly differentiated tumors in the impaired E-cadherin expression group was significantly higher than that in the E-cadherin-positive group (p = 0.005). Furthermore, the frequency of lymph node metastases in tumors with impaired E-cadherin expression was significantly higher than that in the E-cadherin-positive tumors (p = 0.011). A Cox regression analysis revealed that E-cadherin expression was a significant factor in the prediction of survival for patients with non-small cell lung cancer (p = 0.002). CONCLUSIONS: E-Cadherin expression was associated with tumor differentiation, lymph node metastasis, and prognosis in patients with non-small cell lung cancer.     

E-cadherin: a marker for differentiation and invasiveness in prostatic carcinoma

Summary Considerable controversy exists concerning the value of histomorphological data in the assessment of the malignant potential of prostatic carcinomas. We investigated the expression pattern of E-cadherin in human prostate at the translational level. E-cadherin is a specific epithelial cell-cell adhesion molecule which has previously been found to be expressed in well-differentiated non-invasive carcinoma cell lines but is lost in many poorly differentiated invasive cell lines. The E-cadherin expression pattern in the prostate samples was correlated with histopathological findings in the same specimens. We found strong E-cadherin expression in normal prostate and benign prostatic hyperplasia. A decrease in or loss of E-cadherin was seen in 13 of 14 locally advanced and in 8 of 9 poorly differentiated prostatic carcinomas. We conclude that downregulation of E-cadherin expression plays a role in prostate carcinogenesis and invasiveness.     

Roles of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis

We have isolated osteoclast precursors (OCPs) from cocultures of mouse calvarial cells and bone marrow cells without adding any osteotropic factors. OCPs expressed Mac-1, Mac-2, and Gr-1 antigens but not osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptors, and they differentiated into TRAP-positive cells within 48 h on a fixed calvarial cell layer pretreated with osteotropic factors such as 1α,25-dihydroxyvitamin D₃. In the present study, we investigated the regulatory mechanisms of OCP formation from hemopoietic cells and TRAP-positive cell formation from OCPs. Calvarial osteoblasts obtained from macrophage-colony stimulating factor (M-CSF) -deficient op/op mice failed to support OCP formation or the differentiation of OCPs into TRAP-positive cells. Both OCP formation and TRAP-positive cell formation supported by osteoblasts were completely inhibited by osteoclastogenesis inhibitory factor (OCIF, also called OPG), which is a decoy receptor of osteoclast differentiation factor (ODF; also called TRANCE, RANKL, and OPGL). When bone marrow cells were cultured for 4 days with soluble ODF (sODF/sRANKL) together with M-CSF, OCPs were formed even in the absence of osteoblasts. When OCPs were treated with sODF/sRANKL and M-CSF in the absence of osteoblasts, they differentiated into TRAP-positive cells within 48 h even in the presence of hydroxyurea. Northern blotting analysis revealed that osteoblasts constitutively expressed a certain level of ODF/RANKL mRNA. These results indicated that M-CSF and sODF/sRANKL produced by osteoblasts are two essential factors for both OCP formation and TRAP-positive osteoclast formation. Received: July 17, 1999 / Accepted: Nov. 12, 1999     

Mustn1 is expressed during chondrogenesis and is necessary for chondrocyte proliferation and differentiation in vitro

Mustn1 encodes a small nuclear protein expressed specifically in the musculoskeletal system that was originally identified as a strongly up-regulated gene during bone regeneration, especially in fracture callus proliferating chondrocytes. Further experiments were undertaken to investigate its expression and role during chondrogenesis. Initially, whole mount mouse in situ hybridization was carried out and revealed Mustn1 expression in areas of active chondrogenesis that included limb buds, branchial arches and tail bud. To elucidate its function, experiments were carried out to perturb Mustn1 by overexpression and silencing in the pre-chondrocytic RCJ3.1C5.18 (RCJ) cell line. In these cells, Mustn1 is normally differentially regulated, with a spike in expression 2 days after induction of differentiation. Further, Mustn1 was successfully overexpressed in multiple RCJ cell lines by ～ 2–6 fold, and reduced to ～ 32–52% in silenced cell lines as compared to parental Mustn1 levels. Overexpressing, silenced, control, and parental RCJ cell lines were assayed for proliferation and differentiation. No statistically significant changes were observed in either proliferation or proteoglycan production when Mustn1 overexpressing lines were compared to parental and control. By contrast, both proliferation rate and differentiation were significantly reduced in Mustn1 silenced cell lines. Specifically, RNAi silenced cell lines showed reductions in populations of ～ 55–75%, and also ～ 34–40% less matrix (proteoglycan) production as compared to parental and random control lines. Further, this reduction in matrix production was accompanied by significant downregulation of chondrogenic marker genes, such as Sox9, Collagen type II (Col II), and Collagen type X (Col X). Lastly, reintroduction of Mustn1 into a silenced cell line rescued this phenotype, returning proliferation rate, matrix production, and chondrogenic marker gene expression back to parental levels. Taken together these data suggest that Mustn1 is a necessary regulator of chondrocyte function.     

Why history is important for thoracic surgeons

There are numerous examples of lessons to be learned from acquaintance with surgical history. Notwithstanding these considerations--the admonition to read and think about history, the lessons learned from technical misadventures, and the need to add humanistic practices to our scientific endeavors--the real reward from our study of medical history lies in the pure job of being educated in one more way. This implies understanding our contemporary position in the unrolling course of medical history: from remote history through the enlightenment after the reawakening from the dark ages, to the surgical spurt in the latter half of the nineteenth century, and onward through the dramatic advances of our passing millennium.     

Function of OPG as a traffic regulator for RANKL is crucial for controlled osteoclastogenesis

The amount of the receptor activator of NF‐κB ligand (RANKL) on the osteoblastic cell surface is considered to determine the magnitude of the signal input to osteoclast precursors and the degree of osteoclastogenesis. Previously, we have shown that RANKL is localized predominantly in lysosomal organelles, but little is found on the osteoblastic cell surface, and consequently, the regulated subcellular trafficking of RANKL in osteoblastic cells is important for controlled osteoclastogenesis. Here we have examined the involvement of osteoprotegerin (OPG), which is currently recognized as a decoy receptor for RANKL, in the regulation of RANKL behavior. It was suggested that OPG already makes a complex with RANKL in the Golgi apparatus and that the complex formation is necessary for RANKL sorting to the secretory lysosomes. It was also shown that each structural domain of OPG is indispensable for exerting OPG function as a traffic regulator. In particular, the latter domains of OPG, whose physiologic functions have been unclear, were indicated to sort RANKL molecules to lysosomes from the Golgi apparatus. In addition, the overexpression of RANK‐OPG chimeric protein, which retained OPG function as a decoy receptor but lost the function as a traffic regulator, inhibited endogenous OPG function as a traffic regulator selectively in osteoblastic cells and resulted in the upregulation of osteoclastogenic ability despite the increased number of decoy receptor molecules. Conclusively, OPG function as a traffic regulator for RANKL is crucial for regulating osteoclastogenesis at least as well as that as a decoy receptor. © 2010 American Society for Bone and Mineral Research.     

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50–65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.     

T-cell leukemia translocation-associated gene (TCTA) protein is required for human osteoclastogenesis

Synovial tissues of patients with rheumatoid arthritis (RA) include factors regulating bone resorption, such as receptor activator NF-κB ligand (RANKL), TNFα, IL-6, IL-17 and IFNγ. However, in addition to these cytokines, other factors expressed in synovial tissues may play a role in resorbing bone. Here, our objective was to identify novel proteins expressed in synovial tissues of RA that regulate human osteoclastogenesis. Proteins were purified from synovial tissues of patients with RA, using gel filtration chromatography, ion-exchange chromatography, reverse-aspect HPLC, and mass spectrometry. We evaluated the effects of the purified fractions on human osteoclastogenesis induced by RANKL and M-CSF. We determined the amino acid sequences showing inhibitory activity on human osteoclastogenesis. In addition, we synthesized novel peptides from the molecule including the amino acid sequences. Then, we evaluated the effects of the peptides and antibodies against the molecule on human osteoclastogenesis from monocytes and mature osteoclasts, and on pit formation by mature osteoclasts using Osteologic^R discs. We examined the effect of the peptide on the expression of both mRNA and protein of NFATc1. We also examined the effect of RANKL on the expression of mRNA of the molecule on osteoclasts and macrophages. We identified a small peptide including Gly-Gln-Asn (GQN) with inhibitory activity on human osteoclastogenesis. We then found that GQN is included in the amino acid sequence of the extra-cellular domain of TCTA protein, which is expressed ubiquitously in normal human tissues, but whose function has not been clarified. We designed novel peptides, including GQN, from the sequence of TCTA protein. One of these peptides (29-mer), but not a scrambled peptide for the 29-mer peptide, potently inhibited RANKL-induced human osteoclastogenesis. The peptide also inhibited pit formation of mature human osteoclasts and suppressed the formation of large osteoclasts in the culture of mature osteoclasts. Furthermore, polyclonal antibodies against TCTA protein suppressed the formation of large osteoclasts in the cultures of both monocytes and mature osteoclasts, supporting our hypothesis. Peptide A did not significantly inhibit the expression of both mRNA and protein of NFATc1 in osteoclasts. Our novel peptide and polyclonal antibodies against the peptide inhibited human osteoclastogenesis and the function of mature osteoclasts, preventing cellular fusion by TCTA protein and a putative counterpart molecule.