100 results on '"Bar-Shavit Z"'
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2. Enhancement of Phagocytosis by Neurotensin, a Newly Found Biological Activity of the Neuropeptide
- Author
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Goldman, R., Bar-Shavit, Z., Shezen, E., Terry, S., Blumberg, S., Normann, Sigurd J., editor, and Sorkin, Ernst, editor
- Published
- 1982
- Full Text
- View/download PDF
3. Functional Similarity and Diversity in Peritoneal Macrophage Populations Induced in Vivo by Various Stimuli
- Author
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Goldman, R., Bar-Shavit, Z., Raz, A., Escobar, Mario R., editor, and Friedman, Herman, editor
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- 1980
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- View/download PDF
4. 1,25-Dihydroxyvitamin D3 and the regulation of macrophage function
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Bar-Shavit, Z., Noff, D., Edelstein, S., Meyer, M., Shibolet, S., and Goldman, R.
- Published
- 1981
- Full Text
- View/download PDF
5. Enhancement of Phagocytosis by Neurotensin, a Newly Found Biological Activity of the Neuropeptide
- Author
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Goldman, R., primary, Bar-Shavit, Z., additional, Shezen, E., additional, Terry, S., additional, and Blumberg, S., additional
- Published
- 1982
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- View/download PDF
6. TNF-? expression is transcriptionally regulated by RANK ligand
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Zou, W., primary, Amcheslavsky, A., additional, Takeshita, S., additional, Drissi, H., additional, and Bar-Shavit, Z., additional
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- 2004
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7. Developmental regulation of proenkephalin gene expression in osteoblasts.
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Rosen, H, primary, Krichevsky, A, additional, Polakiewicz, R D, additional, Benzakine, S, additional, and Bar-Shavit, Z, additional
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- 1995
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8. Proenkephalin A in bone-derived cells.
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Rosen, H., primary, Polakiewicz, R. D., additional, Benzakine, S., additional, and Bar-Shavit, Z., additional
- Published
- 1991
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9. 25-Hydroxyvitamin D3 metabolism in a human leukemia cell line.
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Bar-Shavit, Zvi, Horst, Ronald, Chappel, Jean, Ross, F., Gray, Richard, Teitelbaum, Steven, Bar-Shavit, Z, Horst, R L, Chappel, J C, Ross, F P, Gray, R W, and Teitelbaum, S L
- Subjects
VITAMIN D metabolism ,CELL differentiation ,CELL lines ,COMPARATIVE studies ,RESEARCH methodology ,MEDICAL cooperation ,MONOCYTES ,RESEARCH ,VITAMIN D ,EVALUATION research ,ACUTE myeloid leukemia - Abstract
1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a potent inducer of monocytic differentiation of the human promyelocytic leukemia cell line, HL-60. We have noted that 25-hydroxyvitamin D3 (25(OH)D3) in high doses is also capable of promoting monocytic differentiation of this cell line. To test the possibility that the latter activity is due to conversion of 25OHD3 to 1,25(OH)2D3 by HL-60, we exposed HL-60 cells to 25OHD3 and analyzed the products by HPLC and radioreceptor assay. When chromatographed in the traditional solvent system (isopropanol-hexane), a new peak appears which migrates with authentic 1,25(OH)2D3. However, in a solvent system containing dichloromethane, 90% of the peak migrates with another metabolite, 19-Nor-10-Keto-25OHD3 (19-Nor-25OHD3). Production of this metabolite is enhanced by living cells and is synthesized by both virgin HL-60 and those which have undergone differentiation. We next determined if authentic 19-Nor-25OHD3 also promotes differentiation of this cell. As assessed by appearance of the monocyte-specific surface antigen (63D3) and macrophage-specific esterase activity, we find that this metabolite does, in fact, induce monocytic differentiation of HL-60 with a potency of approximately 1/200 that of 1,25(OH)2D3 and similar to that of 25OHD3. In agreement with the effect upon cell maturation, 19-Nor-25OHD3 displaces 3H-1,25(OH)2D3 from its HL-60 receptor with an efficiency comparable to 25OHD3. Hence, HL-60 cells convert 25OHD3 to 19-Nor-25OHD3, and 19-Nor-25OHD3 induces monocytic differentiation of HL-60 with comparable efficiency to its precursor, 25OHD3. [ABSTRACT FROM AUTHOR]
- Published
- 1986
- Full Text
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10. Natural cytotoxicity on tumour cells of human macrophages obtained from diverse anatomical sites
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Alberto Mantovani, Bar Shavit, Z., Peri, G., Polentarutti, N., Bordignon, C., Sessa, C., Mangioni, C., Mantovani, A., Bar Shavit, Z., Peri, G., Polentarutti, N., Bordignon, Claudio, Sessa, C., and C., Mangioni
- Subjects
Adult ,Cytotoxicity, Immunologic ,Kinetics ,Phagocytes ,Macrophages ,Humans ,Female ,Interferons ,Neoplasms, Experimental ,Cells, Cultured ,Monocytes ,Research Article - Abstract
Human mononuclear phagocytes were isolated from peripheral blood, peritoneal exudate and early lactation milk by adherence on microexudate-coated plastic and exposure to ethylene diamine tetracetic acid. Their cytolytic activity was measured as 3H-thymidine release from prelabelled target cells over 48-72 hr and cytostasis was evaluated in a spectrophotometric 72-hr assay. The murine SV40-transformed mKSA-TU5 line and the human E cell line, derived from an ovarian carcinoma, were employed as targets. Peripheral blood monocytes, in vitro-matured monocyte-derived macrophages, peritoneal macrophages and milk macrophages were all significantly cytolytic and cytostatic on these target cells at attacker to target cell ratios ranging from 5:1 to 40:1. When monocytes were cultivated in vitro, no loss of cytocidal capacity occurred over the first 10 days of culture, whereas later on, when epithelioid and giant cells predominate in the cultures, mononuclear phagocytes had little cytotoxic activity. Adherent cells obtained from cord blood or from the peripheral blood of old donors had natural cytotoxicity similar to monocytes obtained from young adult volunteers. Peripheral blood monocytes and peritoneal macrophages showed enhanced cytolytic activity after exposure to partially purified human fibroblast interferon. These experiments suggest that in the human mononuclear phagocyte series cytotoxicity on tumour cells is not restricted to circulating monocytes but is also expressed by macrophages obtained from diverse anatomical sites.
- Published
- 1980
11. Modulation of vitamin D increased H2O2 production and MAC-2 expression in the bone marrow-derived macrophages by estrogen
- Author
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Abu-Amer, Y. and Bar-Shavit, Z.
- Abstract
The calcium-regulating hormone 1,25-dihydroxyvitamin D
3 (1,25(OH)2 D3 ) is recognized as an immunomodulator. Members of the macrophage-monocyte lineage are targets for 1,25(OH)2 D3 action. The hormone enhances the ability of bone marrow-derived macrophages (BMDMs) to produce H2 O2 , increases the expression of the macrophage specific surface antigen MAC-2, increases the release of tumor necrosis factor-a (TNF-a), and inhibits BMDM proliferation. In the present study we examine the possibility that estrogen modulates 1,25(OH)2 D3 effects on BMDMs. The active form, 17ß-estradiol, failed to affect any of the BMDM functions by itself. On the other hand, 17ß-estradiol increased the effects of 1,25(OH)2 D3 production by BMDMs and on MAC-2 expression on these cells. The inactive estrogen analog 17a-estradiol was unable to elicit these effects. Moreover, 17ß-estradiol did not affect the lipopolysaccharide (LPS)-induced increase in H2 O2 production by BMDMs. Modulation of BMDM proliferation and TNF-a release from these cells by 1,25(OH)2 D3 were not affected by the estrogen. The experiments were performed with BMDMs harvested from vitamin D-depleted and repleted mice, and always under similar conditions, the various functions were more pronounced in the cells derived from the repleted mice. Our data are consistent with the hypothesis that 17ß-estradiol modulates the interactions of 1,25(OH)2 D3 with BMDMs and consequently is able to affect biological responses to 1,25(OH)2 D3 in these cells. We propose that this cell system is a convenient, nontransformed model for studying cellular activities of 1,25(OH)2 D3 .- Published
- 1994
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12. Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line.
- Author
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Hruska, K A, Bar-Shavit, Z, Malone, J D, and Teitelbaum, S
- Abstract
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [(Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.
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- 1988
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13. Differentiation of a human leukemia cell line and expression of collagenase inhibitor.
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Bar-Shavit, Z, Teitelbaum, S L, Stricklin, G P, Eisen, A Z, Kahn, A J, and Welgus, H G
- Abstract
A human collagenase inhibitor (CI) of Mr 28,500 has been extensively characterized in skin fibroblasts and identified in a variety of connective tissues. Because human alveolar macrophages synthesize and secrete both a collagenase and CI that are immunologically and functionally identical to their counterparts in fibroblasts, we studied the production of such proteins by an immature human cell line (HL60) that can be induced to differentiate along monocytic or granulocytic pathways. The cells failed to synthesize collagenase under any culture condition tested. However, upon exposure to 1,25-dihydroxyvitamin D3 or phorbol esters (PMA), both of which promote monocytic differentiation of HL60, these cells synthesized and released CI in a dose-dependent manner. Furthermore, the extent of CI expression was paralleled by the acquisition by such cells of the monocytic marker 63D3, indicating that inhibitor production and differentiation are closely correlated. This CI was immunologically and functionally identical to that produced by human macrophages and human skin fibroblasts. The quantity of CI synthesized by PMA-stimulated cells was 3- to 5-fold greater than produced by human alveolar macrophages, approximately equal to 1 microgram per 10(6) cells per day. In contrast, undifferentiated HL60 cells produced little or no detectable CI (less than or equal to 10-20 ng per 10(6) cells per day). Interestingly, when HL60 cells were stimulated to undergo granulocytic differentiation by dimethyl sulfoxide or retinoic acid, they also produced the "monocytic" CI.
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- 1985
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14. 1,25-Dihydroxyvitamin D3 and the regulation of macrophage function
- Author
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Bar-Shavit, Z., Noff, D., Edelstein, S., Meyer, M., Shibolet, S., and Goldman, R.
- Abstract
Summary: Vitamin D
3 deficient (D− ) mice show a depressed inflammatory response and both inflammatory peritoneal macrophages and bone marrow polymorphonuclear leukocytes of D− mice exhibit a decreased spontaneous migration under agarose. The impaired phagocytic response of peritoneal macrophages from D− mice can be corrected by incubation with 1,25-dihydroxyvitamin D3 and is not affected by interaction with other vitamin D3 metabolites. Transfer of mice from the D− to the D+ state results in correction of both the inflammatory and the phagocytic response. Intactness of phagocyte function is thus directly dependent on vitamin D3 metabolism.- Published
- 1981
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15. 1,25-Dihydroxyvitamin D3 modulates bone marrow macrophage precursor proliferation and differentiation. Up-regulation of the mannose receptor.
- Author
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Clohisy, D R, Bar-Shavit, Z, Chappel, J C, and Teitelbaum, S L
- Abstract
1,25-Dihydroxyvitamin D3 (1,25−(OH)2D3), the biologically active form of vitamin D3, has been shown to inhibit proliferation and promote monocytic differentiation of leukemic cell lines. In the present communication, we extend these observations to normal bone marrow macrophage precursors, and 1) identify the stage of monocytic maturation wherein the steroid exerts its antiproliferative effect, and 2) demonstrate that 1,25−(OH)2D3 promotes bone marrow macrophage differentiation as manifest by specific up-regulation of the lineage-specific membrane protein, the mannose-fucose receptor. In these experiments, the 1,25−(OH)2D3-mediated inhibitory effect on colony formation was shown to be independent of attendant levels of colony stimulating factor-1 and targeted through the adherent bone marrow macrophage precursor. Examination of this steroid-sensitive adherent precursor population demonstrates that its specific binding of 125I-mannose bovine serum albumin spontaneously and progressively increases with time in culture. Whereas adherent bone marrow macrophages cultured for 2 days express 3 × 10(4) mannose receptors/cell, the number of binding sites increases to 7 × 10(4)/cell by day 4. When bone marrow macrophage precursors are exposed to 1,25-(OH)2D3, an additional stepwise enhancement of 125I-mannose bovine serum albumin obtains with time. Four days of culture with the steroid results in 1.6 × 10(5) mannose receptors/cell, a 100% increase as compared to control cells. Neither duration of culture nor exposure to 1,25-(OH)2D3 alters the KD of 125I-mannose bovine serum albumin which approximates 3-5 × 10(-9) ml-1. Finally, the “specificity” of vitamin D-mediated up-regulation of the mannose receptor was established by demonstrating that the steroid does not alter binding of 125I-alpha-thrombin by bone marrow-derived macrophage precursors.
- Published
- 1987
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16. Induction of monocytic differentiation and bone resorption by 1,25-dihydroxyvitamin D3.
- Author
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Bar-Shavit, Z, Teitelbaum, S L, Reitsma, P, Hall, A, Pegg, L E, Trial, J, and Kahn, A J
- Abstract
1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] stimulates bone resorption in man and other vertebrates, in part, by increasing the number of osteoclasts, the principal resorbing cells of bone. Because osteoclasts are very likely derived from a member(s) of the mononuclear phagocyte family, we determined if 1,25(OH)2D3 promotes maturation of these cells by studying its effects on the human promyelocytic leukemia cell line HL-60. Of the vitamin D3 metabolites tested, only 1,25(OH)2D3, at 10(-10) to 10(-7) M, induces the differentiation of HL60 into mono- and multinucleated macrophage-like cells. Phenotypic change is evident within 24 hr and reaches a plateau between 72 and 96 hr of incubation. The changes are metabolite-specific and include (i) adherence to substrate, (ii) acquisition of the morphological features of mature monocytes, (iii) a 4- to 6-fold enhancement in lysozyme synthesis and secretion, (iv) increase in the fraction of alpha-naphthyl acetate esterase-positive cells from approximately 2% to 100% of the population, and (v) the acquisition of several monocyte-associated cell surface antigens. More importantly, treated HL-60 cells acquire the capacity to bind and degrade bone matrix, two of the essential, functional characteristics of osteoclasts and related bone-resorbing cells. These results, considered together with the reported action of 1,25(OH)2D3 on nontransformed mononuclear cells, are consistent with the view that vitamin D3 enhances bone resorption and osteoclastogenesis in vivo by promoting the differentiation of precursor cells.
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- 1983
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17. Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line
- Author
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Ka, Hruska, Bar-Shavit Z, Jd, Malone, and Steven Teitelbaum
- Subjects
N-Formylmethionine Leucyl-Phenylalanine ,Kinetics ,Leukemia, Myeloid, Acute ,Calcitriol ,Humans ,Biological Transport ,Calcium ,Cell Differentiation ,Hydrogen-Ion Concentration ,Monocytes ,Cell Line - Abstract
1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.
18. Glucocorticoids and bone resorption
- Author
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Steven Teitelbaum, Bar-Shavit Z, Md, Fallon, Imbimbo C, Jd, Malone, and Aj, Kahn
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Hydrocortisone ,Leucine ,Macrophages ,Animals ,Humans ,Osteoclasts ,Cell Differentiation ,Bone Resorption ,Glucocorticoids ,Uridine ,Cells, Cultured
19. Enhancement of phagocytosis — A newly found activity of Substance P residing in its N-terminal tetrapeptide sequence
- Author
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Bar-Shavit, Z., primary, Goldman, R., additional, Stabinsky, Y., additional, Gottlieb, P., additional, Fridkin, M., additional, Teichberg, V.I., additional, and Blumberg, S., additional
- Published
- 1980
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20. Concanavalin A-mediated attachment and ingestion of yeast cells by macrophages
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Bar-Shavit, Z., primary and Goldman, R., additional
- Published
- 1976
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21. Neurotensin — macrophage interaction: Specific binding and augmentation of phagocytosis
- Author
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Bar-Shavit, Z., primary, Terry, S., additional, Blumberg, S., additional, and Goldman, R., additional
- Published
- 1982
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- View/download PDF
22. Human Alveolar Macrophages Produce a Fibroblast-like Collagenase and Collagenase Inhibitor
- Author
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Welgus, H.G., primary, Campbell, E.J., additional, Bar-Shavit, Z., additional, Senior, R.M., additional, and Teitelbaum, S.L., additional
- Published
- 1986
- Full Text
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23. Defective binding of macrophages to bone in rodent osteomalacia and vitamin D deficiency. In vitro evidence for a cellular defect and altered saccharides in the bone matrix.
- Author
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Bar-Shavit, Z, primary, Kahn, A J, additional, and Teitelbaum, S L, additional
- Published
- 1983
- Full Text
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24. Impaired macrophage activation in vitamin D3 deficiency: differential in vitro effects of 1,25-dihydroxyvitamin D3 on mouse peritoneal macrophage functions.
- Author
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Gavison, R, primary and Bar-Shavit, Z, additional
- Published
- 1989
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25. Human alveolar macrophages produce a fibroblast-like collagenase and collagenase inhibitor.
- Author
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Welgus, H G, primary, Campbell, E J, additional, Bar-Shavit, Z, additional, Senior, R M, additional, and Teitelbaum, S L, additional
- Published
- 1985
- Full Text
- View/download PDF
26. Regulation of myc gene expression in HL-60 leukaemia cells by a vitamin D metabolite
- Author
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Reitsma, P. H., primary, Rothberg, P. G., additional, Astrin, S. M., additional, Trial, J., additional, Bar-Shavit, Z., additional, Hall, A., additional, Teitelbaum, S. L., additional, and Kahn, A. J., additional
- Published
- 1983
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27. Glucocorticoids modulate macrophage surface oligosaccharides and their bone binding activity.
- Author
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Bar-Shavit, Z, primary, Kahn, A J, additional, Pegg, L E, additional, Stone, K R, additional, and Teitelbaum, S L, additional
- Published
- 1984
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28. Saccharides mediate the attachment of rat macrophages to bone in vitro.
- Author
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Bar-Shavit, Z, primary, Teitelbaum, S L, additional, and Kahn, A J, additional
- Published
- 1983
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29. Regulation of osteoclastogenesis by integrated signals from toll-like receptors.
- Author
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Krisher T and Bar-Shavit Z
- Subjects
- Animals, Cells, Cultured, Gene Expression, Interleukin-12 metabolism, Interleukin-6 metabolism, Lipopolysaccharides pharmacology, Mice, Inbred BALB C, Oligodeoxyribonucleotides pharmacology, Poly I-C pharmacology, RANK Ligand physiology, Receptor, Macrophage Colony-Stimulating Factor metabolism, Tumor Necrosis Factor-alpha metabolism, Cell Differentiation, Osteoclasts physiology, Toll-Like Receptors physiology
- Abstract
A variety of pathogen-derived molecules have been shown to cause bone loss by enhancing osteoclast differentiation through activation of toll-like receptors (TLRs). The pathogen-derived molecules (TLR-ligands) modulate osteoclastogenesis in a complex manner: inhibition of the osteoclast differentiation factor RANKL in early precursors and osteoclastogenesis stimulation in RANKL-primed cells. Since organisms may be challenged by several TLR ligands at a time, we investigated osteoclastogenesis modulation by simultaneous challenge with different TLR ligands. As an example we used ligands for TLR3 (Synthetic double stranded RNA [dsRNA], polyinosinic-polycytidylic acid [poly(I:C)] mimicking viral dsRNA), TLR4 (lipopolysaccharide [LPS], found in the outer membrane of Gram-negative bacteria) and TLR9 (Synthetic oligodeoxynucleotide mimicking bacterial DNA [CpG-ODN]). In osteoclastogenesis-inhibition, synergy between LPS and CpG-ODN or LPS and poly(I:C) while in stimulation, synergy between LPS and CpG-ODN or CpG-ODN and poly(I:C) were observed. Modulation of molecules involved in osteoclastogenesis (c-Fos, M-CSF receptors [M-CSFR], TNF-α, IL-6, and IL-12 and the three TLRs tested) was examined. The results indicate that M-CSFR plays a role only in the inhibitory effect while c-Fos plays a role in the two effects. TLR3 and TLR9 levels were increased by the TLRs ligands, suggesting that this may be part of the mechanism leading to the synergy. While TLRs activation in RANKL-primed cells, increasing osteoclastogenesis, explains pathogen-induced bone loss, activation of TLRs in early cells inhibiting osteoclastogenesis could attenuate excessive resorption, and promote differentiation of common precursor cells into inflammatory cells. The synergism between TLR ligands enables the individual to initiate response at a lower level of pathogen., (© 2014 Wiley Periodicals, Inc.)
- Published
- 2014
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30. An SNX10 mutation causes malignant osteopetrosis of infancy.
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Aker M, Rouvinski A, Hashavia S, Ta-Shma A, Shaag A, Zenvirt S, Israel S, Weintraub M, Taraboulos A, Bar-Shavit Z, and Elpeleg O
- Subjects
- Base Sequence, Consanguinity, Female, Genotype, Humans, Infant, Infant, Newborn, Male, Osteoclasts metabolism, Osteoclasts pathology, Osteopetrosis pathology, Pedigree, Polymorphism, Single Nucleotide, Mutation, Osteopetrosis genetics, Sorting Nexins genetics
- Abstract
Background: Osteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving blindness and deafness, and bone marrow failure. In ∼70% of the patients there is a molecularly defined failure to maintain an acid pH at the osteoclast-bone interface (the ruffled border) which is necessary for the bone resorptive activity., Methods and Results: In eight patients with infantile osteopetrosis which could be cured by bone marrow transplantation, the study identified by homozygosity mapping in distantly related consanguineous pedigrees a missense mutation in a highly conserved residue in the SNX10 gene. The mutation segregated with the disease in the families and was carried by one of 211 anonymous individuals of the same ethnicity. In the patients' osteoclasts, the mutant SNX10 protein was abnormally abundant and its distribution altered. The patients' osteoclasts were fewer and smaller than control cells, their resorptive capacity was markedly deranged, and the endosomal pathway was perturbed as evidenced by the distribution of internalised dextran., Conclusions: SNX10 was recently shown to interact with vacuolar type H(+)-ATPase (V-ATPase) which pumps protons at the osteoclast-bone interface. Mutations in TCIRG1, the gene encoding a subunit of the V-ATPase complex, account for the majority of cases of osteopetrosis. It is speculated that SNX10 is responsible for the vesicular sorting of V-ATPase from Golgi or for its targeting to the ruffled border. A mutation in SNX10 may therefore result in 'secondary V-ATPase deficiency' with a failure to acidify the resorption lacuna. Determination of the sequence of the SNX10 gene is warranted in molecularly undefined patients with recessive 'pure' osteopetrosis of infancy.
- Published
- 2012
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31. Taking a toll on the bones: regulation of bone metabolism by innate immune regulators.
- Author
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Bar-Shavit Z
- Subjects
- Animals, Bone Diseases metabolism, Bone Diseases microbiology, Bone Remodeling immunology, Bone and Bones metabolism, Cytokines metabolism, Humans, Immunity, Innate, Osteoblasts metabolism, Osteoclasts metabolism, Toll-Like Receptors metabolism, Bone Diseases immunology, Bone and Bones immunology, Osteoblasts immunology, Osteoclasts immunology, Toll-Like Receptors immunology
- Abstract
The interplay between the immune system and bone metabolism has been recognized as important for both of these systems. Various factors produced and released during immune responses markedly affect bone cells and bone metabolism. Meanwhile, niches for lymphocytes in bone also play an important role in the biology of these cells. Osteoimmunology, a new area of research focusing on associations between the immune and bone systems, is based on the concept that deeper investigation of the relationships between these systems will enhance our understanding of their biology and contribute to the discovery of novel therapeutic approaches for diseases of the two systems. Toll-like receptors (TLRs), the focus of this review, sense pathogen-derived molecules and initiate the inflammatory reactions of innate immune cells. TLRs are also expressed in bone cells, and their activation affects osteoclast differentiation and activity in a complex manner: TLR activation in early osteoclast precursors blocks the differentiation of those cells, while in cells that have already started their osteoclastic differentiation, it stimulates this process and increases the survival rates of mature osteoclasts (OCs). Activation of TLRs in osteoblasts (OBs) induces the production of osteoclastogenic cytokines, such as RANKL and TNF-alpha, thereby contributing to TLR ligand-induced osteoclastogenesis. These processes are the reason for the bone loss observed in variety of infectious diseases. The inhibition of osteoclastogenesis by TLR activation in early precursor cells may play a role in reducing the excessive bone loss caused by pathogenic infection and shifting the balance between the bone and immune systems during infection to recruit immune cells.
- Published
- 2008
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32. The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell.
- Author
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Bar-Shavit Z
- Subjects
- Animals, Cell Differentiation, Cell Nucleus metabolism, Humans, Models, Biological, Osteoclasts cytology, Bone Resorption, Hematopoietic System cytology, Osteoclasts immunology, Osteoclasts physiology
- Abstract
Osteoclasts are multinucleated cells that derive from hematopoietic progenitors in the bone marrow which also give rise to monocytes in peripheral blood, and to the various types of tissue macrophages. Osteoclasts are formed by the fusion of precursor cells. They function in bone resorption and are therefore critical for normal skeletal development (growth and modeling), for the maintenance of its integrity throughout life, and for calcium metabolism (remodeling). To resorb bone, the osteoclasts attach to the bone matrix, their cytoskeleton reorganizes, and they assume polarized morphology and form ruffled borders to secrete acid and collagenolytic enzymes and a sealing zone to isolate the resorption site. Identification of the osteoclastogenesis inducer, the receptor activator of nuclear factor-kappaB ligand (RANKL), its cognate receptor RANK, and its decoy receptor osteoprotegerin (OPG), has contributed enormously to the dramatic advance in our understanding of the molecular mechanisms involved in osteoclast differentiation and activity. This explosion in osteoclast biology is reflected by the large number of reviews which appeared during the last decade. Here I will summarize the "classical" issues (origin, differentiation, and activity) in a general manner, and will discuss an untouched issue (multinucleation) and a relatively novel aspect of osteoclast biology (osteoimmunology)., ((c) 2007 Wiley-Liss, Inc.)
- Published
- 2007
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33. Toll-like receptor 9 ligand blocks osteoclast differentiation through induction of phosphatase.
- Author
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Amcheslavsky A and Bar-Shavit Z
- Subjects
- Animals, Enzyme Induction, Extracellular Signal-Regulated MAP Kinases metabolism, Ligands, Male, Mice, Mice, Inbred BALB C, NF-kappa B metabolism, Oligodeoxyribonucleotides pharmacology, Osteoclasts drug effects, Phosphoric Monoester Hydrolases genetics, Phosphorylation, Proto-Oncogene Proteins c-fos genetics, Proto-Oncogene Proteins c-fos metabolism, RANK Ligand antagonists & inhibitors, RANK Ligand pharmacology, Cell Differentiation, Osteoclasts cytology, Osteoclasts metabolism, Phosphoric Monoester Hydrolases metabolism, RANK Ligand metabolism, Toll-Like Receptor 9 metabolism
- Abstract
Unlabelled: CpG-ODN, in addition to stimulation of osteoclastogenic signals in early osteoclast precursors, also induces phosphatase, shifting the pattern of ERK phosphorylation from sustained to transient. This shift results in the degradation of c-fos, an essential molecule for osteoclast differentiation. Therefore, CpG-ODN blocks osteoclast differentiation., Introduction: Activation of either Toll-like receptor 9 (TLR9) or RANK induces similar responses in osteoclast precursors. Paradoxically, activation of TLR9 results in inhibition of RANKL-induced osteoclastogenesis., Materials and Methods: We used bone marrow-derived osteoclast precursors. Analyses of signaling molecules phosphorylation were performed using Western blotting. Different levels of gene expression analyses were performed using RT-PCR, Northern, and run-on analyses (for RNA), and EMSA, Western, and pulse-chase experiments (for protein). Phosphatase activity was measured spectrophotometrically., Results: We found that RANKL and TLR9 ligand, oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN), induce sustained and transient extracellular signal-regulated kinase (ERK) phosphorylation, respectively. Furthermore, together they induce a transient phosphorylation of ERK. The duration of ERK phosphorylation is a key factor in determining induction of c-fos, a protein critical for osteoclastogenesis. Indeed, we found that CpG-ODN does not induce c-fos and inhibits its induction by RANKL by enhancing c-fos mRNA and protein degradation. Our observation that CpG-ODN, but not RANKL, induces the expression of the phosphatase PP2A suggests that CpG-ODN exerts its inhibitory activity by induction of ERK dephosphorylation. Moreover, together with the phosphatase inhibitor okadaic acid, CpG-ODN induces sustained ERK phosphorylation and c-fos expression., Conclusions: Our findings suggest that the increased rate of c-fos degradation by the TLR9 ligand mediates the inhibition of RANKL-induced osteoclast differentiation. The TLR9 ligand, through induction of dephosphorylation, prevents the sustained ERK phosphorylation needed for maintaining high c-fos levels that are essential for osteoclast differentiation.
- Published
- 2007
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34. Involvement of aquaporin 9 in osteoclast differentiation.
- Author
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Aharon R and Bar-Shavit Z
- Subjects
- Animals, Aquaporins metabolism, Bone Marrow Cells cytology, Carrier Proteins metabolism, Cell Differentiation, Cell Line, Cells, Cultured, Macrophages metabolism, Membrane Glycoproteins metabolism, Mice, Mice, Inbred BALB C, Osteoclasts metabolism, Phagocytes metabolism, Phloretin pharmacology, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Aquaporins physiology
- Abstract
Aquaporins (water channels) selectively enhance water permeability of membranes. Since osteoclast differentiation includes a dramatic increase in cell volume, we hypothesize that aquaporin(s) is/are critical for the formation of the multinucleated osteoclast from its mononuclear precursor. Our studies employ two cell models, bone marrow macrophages (BMMs) and the murine macrophage-like cell line, RAW264.7, as osteoclast precursors. Receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL) and macrophage-colony-stimulating factor or RANKL alone were used to induce osteoclast differentiation in BMMs or RAW264.7 cells, respectively. We first used qualitative reverse transcription (RT)-PCR to examine which of the aquaporins are expressed in osteoclasts and in their precursor cells. Out of the 10 aquaporins examined, only aquaporin 9 (AQP9) was expressed in osteoclast-lineage cells. AQP9 has unique aqueous pore properties mediating the passage of a wide variety of non-charged solutes in addition to water. Western analyses using specific antibodies revealed a higher AQP9 level in RANKL-treated than in untreated cells. Quantitative real-time RT-PCR analyses also demonstrated higher AQP9 mRNA levels in RANKL-treated cells. Finally, we examined the effect of phloretin, an AQP9 inhibitor, on RANKL-induced osteoclast differentiation. Cells were incubated with RANKL for 5 days, and phloretin was added for the last 2 days, when most fusion occurs. A dramatic reduction in osteoclast size and in the number of nuclei per osteoclast was observed in cultures containing phloretin. The inhibitor did not have a significant effect on the number and size of mononuclear phagocytes in cultures not treated with RANKL. Our results suggest a role for AQP9 in osteoclast differentiation, specifically in the fusion process.
- Published
- 2006
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35. Interleukin (IL)-12 mediates the anti-osteoclastogenic activity of CpG-oligodeoxynucleotides.
- Author
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Amcheslavsky A and Bar-Shavit Z
- Subjects
- Acid Phosphatase genetics, Acid Phosphatase metabolism, Animals, Antibodies pharmacology, Carrier Proteins pharmacology, Cell Differentiation genetics, Cell Line, Cells, Cultured, Dose-Response Relationship, Drug, Gene Expression drug effects, Gene Expression genetics, Interleukin-12 genetics, Interleukin-12 immunology, Interleukin-12 metabolism, Interleukin-12 physiology, Interleukin-12 Subunit p40, Isoenzymes genetics, Isoenzymes metabolism, Macrophages cytology, Macrophages drug effects, Macrophages metabolism, Male, Membrane Glycoproteins pharmacology, Mice, Mice, Inbred BALB C, NFATC Transcription Factors genetics, NFATC Transcription Factors metabolism, Osteoclasts cytology, Osteoclasts metabolism, Protein Subunits genetics, Protein Subunits metabolism, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Tartrate-Resistant Acid Phosphatase, Cell Differentiation drug effects, Interleukin-12 pharmacology, Oligodeoxyribonucleotides pharmacology, Osteoclasts drug effects
- Abstract
Bacterial DNA activates the innate immune system via interactions with Toll-like receptor 9 (TLR9). This receptor recognizes CpG-oligodeoxynucleotides (CpG-ODNs) mimicking the CpG dinucleotides in certain sequence contexts characterizing this DNA. Most studies have shown increased osteoclast differentiation by TLR ligands. We found that activation of TLRs (specifically TLR4 and TLR9) in early osteoclast precursors results in inhibition of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclast differentiation. Our objective is to identify the mechanism leading to this inhibitory effect of a TLR ligand. Since both RANKL-RANK and CpG-ODN-TLR9 interactions result in NF-kappaB activation, p38 and ERK phosphorylation, and TNF-alpha synthesis (all implicated in osteoclastogenesis), we hypothesized that CpG-ODN (but not RANKL) in addition induces the synthesis of an anti-osteoclastogenic factor. Control osteoclast precursors, and cells treated with RANKL, CpG-ODN, or their combination were studied using DNA arrays (GEArray Q Series Mouse NF-kappaB Signaling Pathway Gene Array, MM-016, SuperArray). We found a marked increase in the mRNA levels of the osteoclastogenesis inhibitor interleukin-12 (IL-12) in osteoclast precursors treated with CpG-ODN and CpG-ODN + RANKL. Northern and Western analyses, together with ELISA, confirmed the DNA array studies. In correlation with these findings, IL-12 inhibited RANKL-induced osteoclast differentiation and specific anti-IL-12-antibodies inhibited the anti-osteoclastogenic effect of CpG-ODN. In conclusion, activation of TLR9 by its ligand, CpG-ODN, results in synthesis and release of IL-12 opposing RANKL-induced osteoclast differentiation.
- Published
- 2006
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36. Organization of transcriptional regulatory machinery in osteoclast nuclei: compartmentalization of Runx1.
- Author
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Saltman LH, Javed A, Ribadeneyra J, Hussain S, Young DW, Osdoby P, Amcheslavsky A, van Wijnen AJ, Stein JL, Stein GS, Lian JB, and Bar-Shavit Z
- Subjects
- Animals, Carrier Proteins pharmacology, Cell Differentiation drug effects, Cell Lineage, Cell Nucleus drug effects, Cells, Cultured, Core Binding Factor Alpha 2 Subunit, Down-Regulation, Male, Membrane Glycoproteins pharmacology, Mice, Mice, Inbred BALB C, NF-kappa B metabolism, Nuclear Matrix metabolism, Organ Specificity, Osteoclasts drug effects, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Osteoclasts cytology, Osteoclasts metabolism, Proto-Oncogene Proteins metabolism, Transcription Factors metabolism, Transcription, Genetic
- Abstract
The osteoclast is a highly polarized multinucleated cell that resorbs bone. Using high resolution immunofluorescence microscopy, we demonstrated that all nuclei of an osteoclast are transcriptionally active. Each nucleus within the osteoclast contains punctately organized microenvironments where regulatory complexes that support transcriptional and post-transcriptional control reside. Functional equivalency of osteoclast nuclei is reflected by similar representation of regulatory proteins that support ribosomal RNA synthesis (nucleolin), mRNA transcription (RNA polymerase II, bromouridine triphosphate), processing of gene transcripts (SC35), signal transduction (NF-kappaB), and phenotypic gene expression (Runx1). Our results establish that gene regulatory machinery is architecturally associated and compartmentalized within intranuclear microenvironments of the multiple nuclei of osteoclasts to support physiologically responsive modifications in cellular structural and functional properties., (Copyright 2005 Wiley-Liss, Inc.)
- Published
- 2005
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37. Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.
- Author
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Amcheslavsky A, Hemmi H, Akira S, and Bar-Shavit Z
- Subjects
- Animals, Blotting, Northern, Blotting, Western, Carrier Proteins biosynthesis, Cell Differentiation, Cell Lineage, Coculture Techniques, CpG Islands, Culture Media, Serum-Free pharmacology, Dose-Response Relationship, Drug, Macrophages cytology, Membrane Glycoproteins biosynthesis, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Osteoblasts metabolism, Phosphorylation, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Time Factors, Toll-Like Receptor 9 genetics, Tumor Necrosis Factor-alpha biosynthesis, p38 Mitogen-Activated Protein Kinases metabolism, Bone Marrow Cells cytology, Gene Expression Regulation, Oligodeoxyribonucleotides pharmacology, Osteoblasts cytology, Osteoclasts cytology, Toll-Like Receptor 9 physiology
- Abstract
Unlabelled: CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity., Introduction: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL., Materials and Methods: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9-/-) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression., Results: As expected CpG-ODN did not exert any activity in cells derived from TLR9-/-mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9-/- and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis., Conclusions: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9.
- Published
- 2005
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38. TNF-alpha expression is transcriptionally regulated by RANK ligand.
- Author
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Zou W, Amcheslavsky A, Takeshita S, Drissi H, and Bar-Shavit Z
- Subjects
- Animals, Bone Marrow, Carrier Proteins pharmacology, Cell Line, Macrophages metabolism, Male, Membrane Glycoproteins pharmacology, Mice, Mice, Inbred BALB C, NF-kappa B metabolism, Osteoclasts metabolism, Promoter Regions, Genetic drug effects, RANK Ligand, RNA, Messenger metabolism, Receptor Activator of Nuclear Factor-kappa B, Stem Cells metabolism, Transcription, Genetic drug effects, Carrier Proteins physiology, Gene Expression Regulation physiology, Membrane Glycoproteins physiology, Transcription, Genetic physiology, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism
- Abstract
Tumor necrosis factor (TNF)-alpha is known for its osteoclastogenic and resorptive activities. Induction of osteoclastogenesis by receptor activator of NF-kappaB ligand (RANKL) is accompanied by increased TNF-alpha expression. In the present study we investigated the mechanism by which RANKL induces expression of TNF-alpha in osteoclast precursors. The macrophage-like cell-line, RAW 264.7 was used as a model for osteoclast precursors. To examine if RANKL-mediated increase in TNF-alpha expression involves increased stability of its transcript, RAW264.7 cells were treated with or without RANKL, and then a transcription inhibitor was added. At different time points, TNF-alpha and L32 mRNA levels were examined. TNF-alpha mRNA stability was not altered by RANKL. We next measured directly the transcription rate of TNF-alpha by a run-on assay and found that RANKL increases TNF-alpha transcription rate by 2.9-fold in RAW264.7 cells. We further characterized this transcriptional induction of TNF-alpha by RANKL. Gel shift assays using nuclear extracts derived from RANKL-treated RAW264.7 cells show increased specific NF-kappaB binding activity on the murine TNF-alpha promoter. Gliotoxin, known for its ability to inhibit NF-kappaB activation blocked RANKL-induced TNF-alpha expression. We finally used 1,260 bp of the murine TNF-alpha promoter fused to luciferase, as well as four mutants of this promoter carrying mutations in each of the four NF-kappaB sites to stably transfect RAW 264.7 cells. Reporter activity was increased in response to RANKL in wild type promoter transfected cells, whereas treatment of the mutants' transfected cells did not elicit reporter activity. In conclusion, RANKL induces TNF-alpha expression via a transcriptional mechanism, depending on the NF-kappaB sites in the TNF promoter., (2004 Wiley-Liss, Inc.)
- Published
- 2005
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39. Toll-like receptor 9 regulates tumor necrosis factor-alpha expression by different mechanisms. Implications for osteoclastogenesis.
- Author
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Amcheslavsky A, Zou W, and Bar-Shavit Z
- Subjects
- Animals, Blotting, Northern, Carrier Proteins administration & dosage, Carrier Proteins physiology, Cell Differentiation drug effects, Male, Membrane Glycoproteins administration & dosage, Membrane Glycoproteins physiology, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, NF-kappa B physiology, Oligodeoxyribonucleotides pharmacology, Osteoclasts drug effects, Polymorphism, Single Nucleotide, Promoter Regions, Genetic genetics, RANK Ligand, RNA, Messenger analysis, Receptor Activator of Nuclear Factor-kappa B, Species Specificity, Stem Cells physiology, Toll-Like Receptor 9, Transcription, Genetic, DNA-Binding Proteins physiology, Gene Expression Regulation physiology, Osteoclasts physiology, Receptors, Cell Surface physiology, Tumor Necrosis Factor-alpha genetics
- Abstract
CpG oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA, stimulate osteoclastogenesis via Toll-like receptor 9 (TLR9) in receptor activator of NF-kappa B ligand (RANKL)-primed osteoclast precursors. This activity is mediated via tumor necrosis factor (TNF)-alpha induction by CpG-ODN. To further reveal the role of the cytokine in TLR9-mediated osteoclastogenesis, we compared the ability of CpG-ODN to induce osteoclastogenesis in two murine strains, BALB/c and C57BL/6, expressing different TNF-alpha alleles. The induction of osteoclastogenesis and TNF-alpha release by CpG-ODN was by far more noticeable in BALB/c-derived than in C57BL/6-derived osteoclast precursors. Unexpectedly, as revealed by Northern analysis, CpG-ODN induction of TNF-alpha mRNA increase was more efficient in C57BL/6-derived cells. The cytokine transcript abundance was increased due to both increased message stability and rate of transcription. The difference between the two cell types was the result of a higher transcription rate in CpG-ODN-induced C57BL/6-derived cells caused by a single nucleotide polymorphism in kappa B2a site within the TNF-alpha promoter sequence. CpG-ODN enhanced the rate of the cytokine translation in BALB/c-derived cells. Thus, CpG-ODN modulated both transcription and translation of TNF-alpha. The induction of transcription was more evident in C57BL/6-derived cells, while the induction of translation took place only in BALB/c-derived osteoclast precursors. Altogether the cytokine was induced to a larger extent in BALB/c-derived osteoclast precursors, consistent with the increased CpG-ODN osteoclastogenic effect in these cells.
- Published
- 2004
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40. CpG oligodeoxynucleotides modulate the osteoclastogenic activity of osteoblasts via Toll-like receptor 9.
- Author
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Zou W, Amcheslavsky A, and Bar-Shavit Z
- Subjects
- Animals, Cells, Cultured, CpG Islands physiology, Male, Mice, Mice, Inbred BALB C, Oligodeoxyribonucleotides pharmacology, Osteoblasts cytology, Toll-Like Receptor 9, Cell Differentiation physiology, DNA-Binding Proteins physiology, Oligodeoxyribonucleotides metabolism, Osteoblasts physiology, Osteoclasts physiology, Receptors, Cell Surface physiology
- Abstract
Regulation of osteoclastogenesis by lipopolysaccharide (LPS) is mediated via its interactions with toll-like receptor 4 (TLR4) on both osteoclast- and osteoblast-lineage cells. We have recently demonstrated that CpG oligodeoxynucleotides (CpG ODNs), known to mimic bacterial DNA, modulate osteoclastogenesis via interactions with osteoclast precursors. In the present study we characterize the interactions of CpG ODNs with osteoblasts, in comparison with LPS. We find that, similar to LPS, CpG ODNs modulate osteoclastogenesis in bone marrow cell/osteoblast co-cultures, although in a somewhat different pattern. Osteoblasts express receptors for both LPS and CpG ODN (TLR4 and TLR9, respectively). The osteoblastic TLR9 transmits signals into the cell as demonstrated by NFkappaB activation as well as by extracellular-regulated kinase (ERK) and p38 phosphorylation. Similar to LPS, CpG ODN increases in osteoblasts the expression of tumor necrosis factor (TNF)-alpha and macrophage-colony stimulating factor (M-CSF). The two TLR ligands do not affect osteoprotegerin expression in osteoblasts. CpG ODN does not significantly affect receptor activator of NFkappaB ligand (RANKL) expression, in contrast to LPS, which induces the expression of this molecule. In the co-cultures CpG ODN induces RANKL expression in osteoblasts as a result of the more efficient TNF-alpha induction. CpG ODN activity (modulation of osteoclastogenesis, gene expression, ERK and p38 phosphorylation, and nuclear translocation of NFkappaB) is specific, because the control oligodeoxynucleotide, not containing CpG, is inactive. Furthermore, these effects (unlike the LPS effects) are inhibited by chloroquine, suggesting a requirement for endosomal maturation/acidification, the classic CpG ODN mode of action. We conclude that CpG ODN, upon TLR9 ligation, induces osteoblasts osteoclastogenic activity.
- Published
- 2003
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41. Modulation of TNF-alpha expression in bone marrow macrophages: involvement of vitamin D response element.
- Author
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Hakim I and Bar-Shavit Z
- Subjects
- Animals, Bone Marrow metabolism, Drug Synergism, Gene Expression Regulation, Lipopolysaccharide Receptors analysis, Lipopolysaccharide Receptors biosynthesis, Lipopolysaccharides pharmacology, Macrophages metabolism, Male, Mice, Mice, Inbred BALB C, RNA, Messenger analysis, RNA, Messenger biosynthesis, Transcription, Genetic, Tretinoin pharmacology, Tumor Necrosis Factor-alpha analysis, Tumor Necrosis Factor-alpha genetics, Bone Marrow drug effects, Calcitriol pharmacology, Macrophages drug effects, Tumor Necrosis Factor-alpha metabolism, Vitamin D Response Element physiology
- Abstract
The calcium-regulating hormone, 1,25(OH)(2)D(3), induces tumor necrosis factor-alpha (TNF-alpha) synthesis and release from bone marrow macrophages (BMMs). To investigate the mechanism of this regulation, we have examined the effects of 1,25(OH)(2)D(3) on the cytokine message. 1,25(OH)(2)D(3) increased TNF-alpha mRNA abundance in a dose- and time-dependent manner. The combined treatment of BMMs with LPS and 1,25(OH)(2)D(3) resulted in a synergistic increase of TNF-alpha. The steroid also increased the expression of CD14 (LPS receptor). Vitamin D receptors (VDRs) mediate 1,25(OH)(2)D(3) genomic effects by forming homodimers or heterodimers with retinoic acid receptors (RARs) or retinoic X receptors (RXRs). The RXR ligand, 9-cis retinoic acid (9cRA), reduced TNF-alpha mRNA abundance in BMMs, but increased CD14 mRNA levels. 1,25(OH)(2)D(3) or LPS did not affect TNF-alpha transcript stability. 9cRA, however, caused TNF-alpha mRNA destabilization. Next, we searched for potential vitamin D response elements (VDREs) in the promoter region (1.2 kb) of the TNF-alpha gene, and identified six such sequences. Using electrophoresis mobility shift assay (EMSA) we identified one of those sequences (-1008 to -994) as a likely candidate to be a VDRE (tnfVDRE). The binding of tnfVDRE to BMM-derived nuclear extract was increased following cell treatment with 1,25(OH)(2)D(3). No induction was observed with 9cRA treatment, but the retinoid enhanced the activity of 1,25(OH)(2)D(3) when added together. Previously characterized VDREs (mouse osteopontin and rat osteocalcin) competed effectively with tnfVDRE, demonstrating the nature of the TNF-alpha-derived sequence as a VDRE. We observed super-shift and block-shift of the complex in the presence of either anti-VDR or anti-RXR antibodies. Our data suggest that 1,25(OH)(2)D(3) increases TNF-alpha transcript abundance in BMMs via a transcriptional mechanism; 9cRA decreases TNF-alpha mRNA by destabilizing the transcript, and possibly also by forming transcriptionally inactive complex with 1,25(OH)(2)D(3) on the tnfVDRE. The receptor complex interacting with tnfVDRE found in the promoter of the cytokine gene is probably composed of VDR-RXR heterodimer., (Copyright 2003 Wiley-Liss, Inc.)
- Published
- 2003
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42. Dual modulation of osteoclast differentiation by lipopolysaccharide.
- Author
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Zou W and Bar-Shavit Z
- Subjects
- Animals, Carrier Proteins pharmacology, Cell Differentiation, Gene Expression radiation effects, Glycoproteins genetics, Interleukin-1 genetics, Macrophage Colony-Stimulating Factor pharmacology, Male, Membrane Glycoproteins pharmacology, Mice, Mice, Inbred BALB C, Osteoclasts cytology, Osteoclasts metabolism, Osteoprotegerin, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Tumor Necrosis Factor, Tumor Necrosis Factor-alpha genetics, Lipopolysaccharides pharmacology, Osteoclasts drug effects
- Abstract
Lipopolysaccharide (LPS) modulates bone resorption by augmentation of osteoclastogenesis. It increases in osteoblasts the production of RANKL, interleukin (IL)-1, prostaglandin E2 (PGE2), and TNF-alpha, each known to induce osteoclast activity, viability, and differentiation. We examined the role of direct interactions of LPS with osteoclast precursors in promoting their differentiation. To this end, we have used bone marrow mononuclear cell preparations in the absence of osteoblasts or stromal cells. We found that LPS does not induce osteoclast differentiation in these cells. Moreover, the inclusion of LPS blocked the osteoclastogenic activity of RANKL. However, LPS is a potent inducer of osteoclastogenesis in RANKL-pretreated cells, even if present in the absence of exogenous RANKL. Osteoprotegerin (OPG), does not affect the stimulatory phase of LPS modulation of osteoclastogenesis, ruling out involvement of endogenous RANKL. LPS induces the expression of TNF-a and IL-1beta in osteoclast precursors, regardless if they were or were not pretreated with RANKL. These two cytokines induced osteoclast differentiation in RANKL-pretreated cells. To examine if these cytokines mediate LPS effect in an autocrine mechanism, we measured the effect of their neutralization on LPS osteoclastogenic activity. Although neutralization of IL-1beta did not affect LPS activity, a marked inhibition was observed when TNF-alpha was neutralized. However, TNF-a expression was increased also in conditions in which LPS inhibited RANKL osteoclastogenic activity. We found that LPS reduces the expression of RANK and macrophage colony-stimulating factor (M-CSF) receptor. In summary, LPS impacts on osteoclastogenesis also via its interactions with the precursor cells. LPS inhibits RANKL activity by reducing the expression of RANK and M-CSF receptor and stimulates osteoclastogenesis in RANKL-pretreated cells via TNF-alpha.
- Published
- 2002
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43. CpG oligonucleotides: novel regulators of osteoclast differentiation.
- Author
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Zou W, Schwartz H, Endres S, Hartmann G, and Bar-Shavit Z
- Subjects
- Acid Phosphatase analysis, Animals, Antibodies pharmacology, Carrier Proteins antagonists & inhibitors, Carrier Proteins pharmacology, Cell Differentiation drug effects, Cells, Cultured, Cytokines biosynthesis, Cytokines genetics, Dose-Response Relationship, Drug, Isoenzymes analysis, Kinetics, Macrophages cytology, Macrophages drug effects, Macrophages physiology, Male, Membrane Glycoproteins antagonists & inhibitors, Membrane Glycoproteins pharmacology, Mice, Mice, Inbred BALB C, Osteoclasts cytology, Osteoclasts drug effects, RANK Ligand, RNA, Messenger biosynthesis, Receptor Activator of Nuclear Factor-kappa B, Receptor, Macrophage Colony-Stimulating Factor biosynthesis, Receptor, Macrophage Colony-Stimulating Factor genetics, Stem Cells cytology, Stem Cells drug effects, Stem Cells physiology, Tartrate-Resistant Acid Phosphatase, Tumor Necrosis Factor-alpha antagonists & inhibitors, Tumor Necrosis Factor-alpha immunology, Tumor Necrosis Factor-alpha physiology, Adjuvants, Immunologic pharmacology, Oligodeoxyribonucleotides pharmacology, Osteoclasts metabolism
- Abstract
The macrophage capability to recognize bacterial DNA is mimicked by oligodeoxynucleotides containing unmethylated CG dinucleotides ('CpG' motifs) in specific sequence contexts (CpG ODN). CpG ODN stimulates NF-kappaB activation in murine macrophages. In light of the pivotal role played by NF-kappaB in osteoclast differentiation, we examined the ability of CpG ODN to modulate osteoclastogenesis. CpG ODN alone induced TRAP-positive cells in bone marrow macrophage (BMM) cultures, but not multinucleation or calcitonin receptor expression. CpG ODN inhibited RANKL-induced osteoclastogenesis when present from the beginning of BMM culture, but strongly increased RANKL-induced osteoclastogenesis in RANKL-pretreated BMMs. CpG ODN enhanced the expression of interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). Antibodies to TNF-alpha and the TNF type 1 receptor, but not the addition of IL-1 receptor antagonist, blocked CpG ODN-induced osteoclastogenesis in RANKL-pretreated cultures. On the other hand, CpG ODN reduced expression of the M-CSF receptor, which is critical during the initiation of osteoclast differentiation. These results suggest that CpG ODN, via the induction of TNF-alpha, support osteoclastogenesis in cells that are committed to the osteoclast differentiation pathway but, due to down-modulation of M-CSF receptor, inhibit early steps of osteoclast differentiation. Thus, CpG ODN represents a potential therapeutic tool for treating bone diseases.
- Published
- 2002
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44. Tumor necrosis factor-alpha mediates RANK ligand stimulation of osteoclast differentiation by an autocrine mechanism.
- Author
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Zou W, Hakim I, Tschoep K, Endres S, and Bar-Shavit Z
- Subjects
- Acid Phosphatase metabolism, Animals, Animals, Newborn, Antibodies immunology, Antibodies pharmacology, Blotting, Western, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Cell Nucleus metabolism, Cells, Cultured, Dose-Response Relationship, Drug, Male, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, RANK Ligand, RNA, Messenger genetics, RNA, Messenger metabolism, Receptor Activator of Nuclear Factor-kappa B, Receptors, Tumor Necrosis Factor antagonists & inhibitors, Receptors, Tumor Necrosis Factor immunology, Receptors, Tumor Necrosis Factor metabolism, Stem Cells cytology, Stem Cells drug effects, Stem Cells metabolism, Time Factors, Tumor Necrosis Factor-alpha antagonists & inhibitors, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha immunology, Autocrine Communication drug effects, Carrier Proteins metabolism, Cell Differentiation drug effects, Membrane Glycoproteins metabolism, Osteoclasts cytology, Osteoclasts drug effects, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Osteoblasts or bone marrow stromal cells are required as supporting cells for the in vitro differentiation of osteoclasts from their progenitor cells. Soluble receptor activator of nuclear factor-kappaB ligand (RANKL) in the presence of macrophage colony-stimulating factor (M-CSF) is capable of replacing the supporting cells in promoting osteoclastogenesis. In the present study, using Balb/c-derived cultures, osteoclast formation in both systems-osteoblast/bone-marrow cell co-cultures and in RANKL-induced osteoclastogenesis-was inhibited by antibody to tumor necrosis factor-alpha (TNF-alpha), and was enhanced by the addition of this cytokine. TNF-alpha itself promoted osteoclastogenesis in the presence of M-CSF. However, even at high concentrations of TNF-alpha the efficiency of this activity was much lower than the osteoclastogenic activity of RANKL. RANKL increased the level of TNF-alpha mRNA and induced TNF-alpha release from osteoclast progenitors. Furthermore, antibody to p55 TNF-alpha receptors (TNF receptors-1) (but not to p75 TNF-alpha receptors (TNF receptors-2) inhibited effectively RANKL- (and TNF-alpha() induced osteoclastogenesis. Anti-TNF receptors-1 antibody failed to inhibit osteoclastogenesis in C57BL/6-derived cultures. Taken together, our data support the hypothesis that in Balb/c, but not in C57BL/6 (strains known to differ in inflammatory responses and cytokine modulation), TNF-alpha is an autocrine factor in osteoclasts, promoting their differentiation, and mediates, at least in part, RANKL's induction of osteoclastogenesis., (Copyright 2001 Wiley-Liss, Inc.)
- Published
- 2001
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45. The enkephalinergic osteoblast.
- Author
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Rosen H, Krichevsky A, and Bar-Shavit Z
- Subjects
- Animals, Bone and Bones innervation, Humans, Narcotics metabolism, Neuropeptides physiology, Receptors, Opioid physiology, Enkephalins physiology, Osteoblasts physiology
- Published
- 1998
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46. Regulation of TNF-alpha release from bone marrow-derived macrophages by vitamin D.
- Author
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Abu-Amer Y and Bar-Shavit Z
- Subjects
- Animals, Bone Marrow pathology, Cells, Cultured, Culture Media, Conditioned, Enzyme-Linked Immunosorbent Assay, Interferon-gamma pharmacology, Lipopolysaccharides pharmacology, Macrophages drug effects, Male, Mice, Mice, Inbred BALB C, Molecular Weight, Reference Values, Sensitivity and Specificity, Tumor Necrosis Factor-alpha isolation & purification, Bone Marrow Cells, Calcitriol pharmacology, Macrophages metabolism, Tumor Necrosis Factor-alpha biosynthesis, Vitamin D Deficiency metabolism
- Abstract
The calcium-regulating hormone 1,25-dihydroxyvitamin D3[1,25(OH)2D3] is recognized as an immunomodulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D-deficient mice (-D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells (+D MPs), and that bone marrow-derived macrophage (BMDM) differentiation is modulated by 1,25(OH)2D3. The release of tumor necrosis factor-alpha (TNF-alpha) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1,25(OH)2D3 in TNF-alpha synthesis and release. BMDMs were harvested from +D and -D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF-alpha. BMDMs did not release measurable amounts of TNF-alpha without stimulation. Addition of endotoxin (LPS) to the cultures, resulted in a marked stimulation of TNF-alpha release. 1,25(OH)2D3 increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon-gamma (IFN-gamma), yielded essentially similar results. +D and -D mice were injected with LPS and TNF-alpha levels in the serum were measured. A marked reduction (approximately fourfold) in the TNF-alpha levels was observed in the serum of -D mice as compared with +D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1,25(OH)2D3 is on TNF-alpha synthesis. Our findings suggest that 1,25(OH)2D3 plays a role in the regulation of TNF-alpha secretion by mononuclear phagocytes.
- Published
- 1994
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47. Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues.
- Author
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Rosen H and Bar-Shavit Z
- Subjects
- Amino Acid Sequence, Animals, Bone Development physiology, Bone and Bones cytology, Humans, Molecular Sequence Data, Opioid Peptides physiology, Pain physiopathology, Bone and Bones physiology, Enkephalins chemistry, Osteoblasts physiology, Peptides physiology, Protein Precursors chemistry
- Abstract
Proenkephalin encode a group of small peptides with opiate-like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone-derived cells, its modulation by bone active hormones, and the effects of enkephalin-derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin-derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin-derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain.
- Published
- 1994
- Full Text
- View/download PDF
48. Impaired bone marrow-derived macrophage differentiation in vitamin D deficiency.
- Author
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Abu-Amer Y and Bar-Shavit Z
- Subjects
- Animals, Antigens, Surface biosynthesis, Cell Differentiation physiology, Cell Division physiology, Cells, Cultured, Hydrogen Peroxide metabolism, Lysosomes enzymology, Macrophages physiology, Mice, Mice, Inbred BALB C, Vitamin D Deficiency immunology, Bone Marrow Cells, Cholecalciferol physiology, Macrophages cytology
- Abstract
The calcium-regulating hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is also recognized as an immunomodulator. In vitro addition of the hormone to bone marrow-derived macrophages (BMDMs) results in a decreased proliferation and an increased differentiation. In the present work we compare the in vitro differentiation of BMDMs derived from vitamin D-depleted and -repleted mice (-D and +D BMDMs, respectively). -D BMDMs proliferate in vitro slower than +D BMDMs. Addition of the hormone to BMDM cultures inhibited the rate of their proliferation, which was more pronounced in low-density cultures. The ability of mononuclear phagocytes to produce reactive oxygen metabolites is an important element in the microbicidal functions of these cells. We found that -D BMDMs produce less H2O2 than +D BMDMs, which was corrected by the in vitro addition of 1,25(OH)2D3. Analyses of various macrophage-specific surface antigens revealed a reduction in their expression on -D BMDMs. In vitro addition of 1,25(OH)2D3 to BMDM cultures increased the expression of these antigens. The activity of the lysosomal enzyme acid phosphatase was similarly affected by vitamin D deficiency and by the in vitro addition of the hormone. Thus, vitamin D deficiency is associated with impaired maturation of BMDMs suggesting that the hormone is a natural modulator of macrophage maturation.
- Published
- 1993
- Full Text
- View/download PDF
49. Osteoblast-like cell line maintains in vitro rat peritoneal mast cell viability and functional activity.
- Author
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Levi-Schaffer F and Bar-Shavit Z
- Subjects
- Animals, Cell Adhesion physiology, Cell Line, Cell Survival physiology, Mast Cells cytology, Osteoblasts cytology, Peritoneal Cavity cytology, Rats, Cell Communication physiology, Mast Cells physiology, Osteoblasts physiology
- Abstract
We studied the ability of the rat osteosarcoma derived cell-line with osteoblastic properties ROS-17/2.8 (ROS) to maintain in vitro rat peritoneal mast cells (MC) in a functional state. Highly purified (greater than 95%) MC were seeded on confluent ROS cells. The MC adhered tightly to the monolayers within a few hours and remained viable for at least 2 weeks, but did not proliferate. The MC retained their typical appearance, exhibiting highly granulated resting morphology when stained with alcian blue followed by safranin or with acidic toluidine blue. Furthermore, after 2 weeks, the MC were fully responsive to activation with compound 48/80 (3 micrograms/ml), releasing 75% of their histamine content, as compared to 3% in the absence of the secretagogue. Utilizing metabolically inactive ROS cells and prevention of contact between ROS and MC, we found that both release of factor(s) and cell-cell contact were required by ROS to exhibit their MC supporting activity. Various other cells and cell lines were unable to support MC viability. On the other hand, as demonstrated before, 3T3 fibroblasts were capable of promoting MC viability. Thus, MC viability and functional activity are specifically maintained by fibroblastic and osteoblastic cells. The abundance of MC in bone, and their participation in bone remodelling raise the possibility of physiological and pathological significance to interactions between MC and osteoblasts.
- Published
- 1990
50. Complement and Fc receptor-mediated phagocytosis of normal and stimulated mouse peritoneal macrophages.
- Author
-
Bar-Shavit Z, Raz A, and Goldman R
- Subjects
- Animals, Ascitic Fluid immunology, Complement C3b, Erythrocytes immunology, In Vitro Techniques, Lysosomes enzymology, Macrophages enzymology, Male, Mice, Mice, Inbred BALB C, Saccharomyces cerevisiae immunology, Complement System Proteins, Immunoglobulin Fc Fragments, Macrophages immunology, Phagocytosis
- Published
- 1979
- Full Text
- View/download PDF
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