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O1. Selective inactivators of cysteine proteinases inhibit bone resorption
Bone, Vol. 15, No. 4, pp. 449-462,
1994 Copyright 8 1994 Elsevier Science Ltd F’rintedin the USA. All rights reserved 8756-32W94 $6.00 + .OO
Pergamon 8756-3282(94) EOOOS-M
ABSTRACTS FROM THE BONE AND TOOTH SOCIETY WINTER MEETING, DECEMBER 6, 1993 Venue: Royal College of Obstetricians and Gynaecologists, London. LocalOrganizer.Dr.
Tim Ame& Department of Anatomy and Developmental Biology, University College London.
Adhesive interactions are crucial to the activity of these differentiated cells both in terms of effects on extracellular matrices and as a mechanism for regulating the actions of other cells. We have examined the function of 1,25D3 as a regulator of cell adhesion molecules using monoblastic U937 cells and DH39, a stable transfectant variant of U937 expressing high levels of vitamin D receptors (VDR). DH39 were morphologically and phenotypically identical to U937 (as judged by a panel of myeloid markers), but expressed 6-10 fold higher levels of VDR and were much more sensitive to 1,25D3. Using fluorescence activated cell sorting (FACS) both U937 and DH39 (in the presence or absence of 1,25D3) were screened with panels of antibojies to cell adhesion molecules, including various intercellular adhesion molecules (ICAMs) and integrins.
01. Selective inactivalom of cysteine prdeinases inhibit bone resorption PA Hill*, D Buttle, JJ Reynolds, MC Meikle* Strangeways Research Laboratory, Cambridge CBl 4RN and *Department of Orthadontics and Paediatric Dentistry, UMDS (Guys), London Inactivators of cysteine proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vim The following four CP inactivators were tested: Ep475, a compound with low membrane permeability which inhibits cathepsins B, L, S, H and calpain; Ep453 the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectively inactivates cathepsin B; and CAO74Me, the membrane-permeant prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mouse calvarial explants and (2) assessing the extent of bone resorption in an isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475 and CA074Me inhibited both stimulated and basal bone resorption in vitro while CA074 was without effect; the inhibition was reversible and dosedependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of @-glucuronidase, N-acetyl-p-glucosaminidase or the spontaneous release of lactate dehydrogenase. Ep453, Ep475 and CA074Me dose-dependently inhibited the resorptive activity of isolated avian osteoclasts cultured on bone slices with a maximal effect at 5OpM. The number of resorption pits and their mean volume was reduced whilst the mean surface area remained unaffected. Again, CA074 was without effect. Ep453, Ep475 and CA074Me, but not CA074, when administered subcutaneously at a dose of 60 rg/g body weight inhibited bone resorption in vim measured by an in uiuolin vitro assay, by about 20 per cent. This study demonstrates that cathepsins B, L and/or S are involved in bone resorption in vitro and in vim Whilst cathepsin L and/or S act extracellularly, and possibly intracellularly, cathepsin B mediates its effect intracellularly possibly through the activation of other proteinases involved in subosteoclastic collagen degradation. Supported by the Medi.cal Research Council.
Two striking features were noted. Firstly, the high VDR levels present in DH39 were associated with decreased expression of ICAM 3, a newly identified receptor molecule for the integrin LFA-1. Secondly, in the presence of 1,25D3 (lo-EM) the VDR transfectants formed extensive homotypic clusters similar to those seen in granulomata of sarcoidosis. This 1,25D3-induced cell-cell adhesion was associated with increased expression of ~2 integrin and its associated a subunits CDlla,b, and c, as well as 2 novel integrins. The data indicate that 1,25D3 and its receptor are involved in the regulation of cell adhesion processes.
03. Nitric oxide production in cultures of human osteoblast-like cells DH Todd, PS Grabowski, M Helfrich, N Benjamin, SH Ralston Department of Medicine and Therapeutics, Aberdeen Royal Infirmary, Foresterhill, Aberdeen We have looked for evidence of production of the intercellular messenger nitric oxide (NO) in cultures of human osteoblast-like (hOB) cells. Without stimulation and when inflammatory cytokines (interleukin 18 (lOU/ml), tumour necrosis factor a (25ng/ml), and interferon 7 (lOOU/ml)) were added individually, NO production was not detected, and only a slight effect was observed when pairs of these cytokines were added. When hOB cells were stimulated by all three cytokines, however, NO production (reflected by nitrite) showed a ten-fold elevation over basal levels. Cytokine stimulated NO production was reduced hy 70-90% on addition of the L-arginine analogue L-NC monomethyl arginine (L-NMMA) which inhibits synthesis of NO by the enzyme NO synthase (NOS). Generation of NO was blocked by cycloheximide and actinomycin D; evidence that NO production in hOB cells is under transcriptional control. Dexamethasone reduced NO production, a characteristic of the inducible NOS type II enzyme. Cytokine induced NO production
02. Regulation of cell adhesion molecules by vitamin D h4 Hewison, L Faulkner*, S Vadher, E Hewson’, DR Katz* JLH O’Riordan Departments of Medicine and *immunology, University College London Medical School, London WIN 8AA The active form of vitamin D, 1,25_dihydroxyvitamin D3 (1,25D3) plays an important rclle in stimulating the differentiation of bone marrow-derived (cells such as monocytes and osteoclasts. 449
1994 Copyright 8 1994 Elsevier Science Ltd F’rintedin the USA. All rights reserved 8756-32W94 $6.00 + .OO
Pergamon 8756-3282(94) EOOOS-M
ABSTRACTS FROM THE BONE AND TOOTH SOCIETY WINTER MEETING, DECEMBER 6, 1993 Venue: Royal College of Obstetricians and Gynaecologists, London. LocalOrganizer.Dr.
Tim Ame& Department of Anatomy and Developmental Biology, University College London.
Adhesive interactions are crucial to the activity of these differentiated cells both in terms of effects on extracellular matrices and as a mechanism for regulating the actions of other cells. We have examined the function of 1,25D3 as a regulator of cell adhesion molecules using monoblastic U937 cells and DH39, a stable transfectant variant of U937 expressing high levels of vitamin D receptors (VDR). DH39 were morphologically and phenotypically identical to U937 (as judged by a panel of myeloid markers), but expressed 6-10 fold higher levels of VDR and were much more sensitive to 1,25D3. Using fluorescence activated cell sorting (FACS) both U937 and DH39 (in the presence or absence of 1,25D3) were screened with panels of antibojies to cell adhesion molecules, including various intercellular adhesion molecules (ICAMs) and integrins.
01. Selective inactivalom of cysteine prdeinases inhibit bone resorption PA Hill*, D Buttle, JJ Reynolds, MC Meikle* Strangeways Research Laboratory, Cambridge CBl 4RN and *Department of Orthadontics and Paediatric Dentistry, UMDS (Guys), London Inactivators of cysteine proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vim The following four CP inactivators were tested: Ep475, a compound with low membrane permeability which inhibits cathepsins B, L, S, H and calpain; Ep453 the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectively inactivates cathepsin B; and CAO74Me, the membrane-permeant prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mouse calvarial explants and (2) assessing the extent of bone resorption in an isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475 and CA074Me inhibited both stimulated and basal bone resorption in vitro while CA074 was without effect; the inhibition was reversible and dosedependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of @-glucuronidase, N-acetyl-p-glucosaminidase or the spontaneous release of lactate dehydrogenase. Ep453, Ep475 and CA074Me dose-dependently inhibited the resorptive activity of isolated avian osteoclasts cultured on bone slices with a maximal effect at 5OpM. The number of resorption pits and their mean volume was reduced whilst the mean surface area remained unaffected. Again, CA074 was without effect. Ep453, Ep475 and CA074Me, but not CA074, when administered subcutaneously at a dose of 60 rg/g body weight inhibited bone resorption in vim measured by an in uiuolin vitro assay, by about 20 per cent. This study demonstrates that cathepsins B, L and/or S are involved in bone resorption in vitro and in vim Whilst cathepsin L and/or S act extracellularly, and possibly intracellularly, cathepsin B mediates its effect intracellularly possibly through the activation of other proteinases involved in subosteoclastic collagen degradation. Supported by the Medi.cal Research Council.
Two striking features were noted. Firstly, the high VDR levels present in DH39 were associated with decreased expression of ICAM 3, a newly identified receptor molecule for the integrin LFA-1. Secondly, in the presence of 1,25D3 (lo-EM) the VDR transfectants formed extensive homotypic clusters similar to those seen in granulomata of sarcoidosis. This 1,25D3-induced cell-cell adhesion was associated with increased expression of ~2 integrin and its associated a subunits CDlla,b, and c, as well as 2 novel integrins. The data indicate that 1,25D3 and its receptor are involved in the regulation of cell adhesion processes.
03. Nitric oxide production in cultures of human osteoblast-like cells DH Todd, PS Grabowski, M Helfrich, N Benjamin, SH Ralston Department of Medicine and Therapeutics, Aberdeen Royal Infirmary, Foresterhill, Aberdeen We have looked for evidence of production of the intercellular messenger nitric oxide (NO) in cultures of human osteoblast-like (hOB) cells. Without stimulation and when inflammatory cytokines (interleukin 18 (lOU/ml), tumour necrosis factor a (25ng/ml), and interferon 7 (lOOU/ml)) were added individually, NO production was not detected, and only a slight effect was observed when pairs of these cytokines were added. When hOB cells were stimulated by all three cytokines, however, NO production (reflected by nitrite) showed a ten-fold elevation over basal levels. Cytokine stimulated NO production was reduced hy 70-90% on addition of the L-arginine analogue L-NC monomethyl arginine (L-NMMA) which inhibits synthesis of NO by the enzyme NO synthase (NOS). Generation of NO was blocked by cycloheximide and actinomycin D; evidence that NO production in hOB cells is under transcriptional control. Dexamethasone reduced NO production, a characteristic of the inducible NOS type II enzyme. Cytokine induced NO production
02. Regulation of cell adhesion molecules by vitamin D h4 Hewison, L Faulkner*, S Vadher, E Hewson’, DR Katz* JLH O’Riordan Departments of Medicine and *immunology, University College London Medical School, London WIN 8AA The active form of vitamin D, 1,25_dihydroxyvitamin D3 (1,25D3) plays an important rclle in stimulating the differentiation of bone marrow-derived (cells such as monocytes and osteoclasts. 449