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T J Chambers

Publications and source records attributed to T J Chambers.

At least 127 records · Page 7Linked to original sources
PubMed1991

Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures.

Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10(-6)-10(-9) M) and PGE1 (10(-6)-10(-7) M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption was increased (10(-7) M PGE2 and 10(-6) M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Alprostadil↗
PubMed1991

Effects of transforming growth factor beta 1 on the regulation of osteoclastic development and function.

Transforming growth factor (TGF) beta 1 is a multifunctional cytokine with powerful effects on osteoblastic cells. Its role in the regulation of osteoclast generation and function, however, is unclear. It has been reported both to stimulate and to inhibit resorption in organ culture and to inhibit multinuclear cell formation in bone marrow cultures. We tested the effects of TGF-beta 1 on bone resorption by osteoclasts isolated from neonatal rat long bones. We found potent stimulation of osteoclastic bone resorption, mediated by osteoblastic cells, with an EC50 of 10 pg/ml, considerably lower than that of well-documented osteotropic hormones. Stimulation was not mediated by Swiss mouse 3T3 cells, a nonosteoblastic cell line. TGF-beta 1 strongly inhibited the generation of calcitonin receptor (CTR)-positive cells in mouse bone marrow cultures, but as for isolated osteoclasts, bone resorption per CTR-positive cell was increased. The inhibition of CTR-positive cell formation was associated with suppression of maturation of other bone marrow derivatives and may be related more to the known ability of TGF-beta 1 to suppress the proliferation of primitive hematopoietic cells than to a specific role of TGF-beta 1 in osteoclast generation.

Animals↗
PubMed1991

The giant cells recruited by subcutaneous implants of mineralized bone particles and slices in rabbits are not osteoclasts.

We have compared structural and functional characteristics of native osteoclasts and the multinucleated giant cells (MNGC) recruited by subcutaneous implants of mineralized bone particles and slices in normal rabbits. Weekly evaluation of the implants for 5 weeks showed distinct differences between MNGC and osteoclasts in the host with respect to morphology and the ability to stain for tartrate-resistant acid phosphatase and acid ATPase. An osteoclast-specific monoclonal antibody bound strongly to osteoclasts but not MNGC. Ground bone slices similarly implanted were surrounded by MNGC but did not show resorption pits by scanning electron microscopy. These data show that the MNGC recruited to subcutaneous implants of mineralized bone particles and slices lack the enzymatic, cell surface, and functional features of osteoclasts.

Acid Phosphatase↗
PubMed1991

Heparin augments osteoclast resorption-stimulating activity in serum.

Increased numbers of mast cells are commonly seen at sites of increased bone resorption and in osteoporosis. Long-term administration of heparin, a major component of mast cell granules, causes osteoporosis. We therefore tested the effect of heparin on bone resorption by osteoclasts disaggregated from neonatal rat long bones. We found that, in the absence of serum, heparin was without effect on osteoclast function. However, in the presence of newborn calf serum, rat serum, or bovine platelet-poor plasma-derived serum, heparin, in the range 25-100 micrograms/ml, induced an increase in osteoclastic bone resorption. Heparin appeared to act through binding and enhancement of an osteoclast resorption-stimulating activity (ORSA) present in serum. A number of known factors that show an affinity for heparin, including transforming growth factor-beta, platelet-derived growth factors, insulin-like growth factors I or II, acidic or basic fibroblast growth factors, fibronectin, or laminin, could not substitute for ORSA, suggesting that the activity may represent a novel heparin-binding factor. The ability of glycosaminoglycans (GAGs) and related molecules to enhance resorption was dependent on the degree of sulfation and on their size: The high molecular weight GAG heparan sulfate and polysaccharides fucoidan or dextran sulfate showed a similar effect, while low molecular weight heparin, chondroitin-2-sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were without effect. We propose that mast cells or heparin therapy increases bone resorption through augmentation of the activity of a factor involved in the local and systemic regulation of osteoclastic bone resorption.

Animals↗
PubMed1991

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.

Acid Phosphatase↗
PubMed1991

Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonates, osteoclasts, and bone.

Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to the cells that resorb Cl2MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorption in vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl2MBP. The greater sensitivity of resorption to AHPrBP in vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormones in vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl2MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generation in vivo in AHPrBP-treated rats. These results suggest that the comparisons of potency in vitro do not include all the factors responsible for determining bisphosphonate potency in vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10(-3) M AHPrBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Production and characterization of new monoclonal antibodies to human osteoclasts.

Two monoclonal antibodies raised against human osteoclastoma were found to show antiosteoclastic activity on frozen sections of tumor. Immunoreactivity was localized on the membrane surface. These antibodies exhibited no activity against tissue macrophages and human visceral tissue except kidney, where they stained tubules but not glomeruli. In addition, no activity was observed against rabbit or rat osteoclasts, suggesting that they might react with unique epitopes on human osteoclasts.

Animals↗
PubMed1991

A comparison of the effects of inhibitors of carbonic anhydrase on osteoclastic bone resorption and purified carbonic anhydrase isozyme II.

We have assessed the effects of five sulfonamides with widely varying inhibitory activity for carbonic anhydrase (CA) in the bone slice assay using disaggregated rat osteoclasts (OCs), and in the Maren assay where the catalytic activity of purified CA isozyme II (CA II) was measured. There was an excellent correlation between the relative potencies of the compounds in the two assays: ethoxzolamide (ETH) greater than acetazolamide (AZ) greater than M&B 21659 greater than M&B 9811 greater than M&B 7973. In the bone slice assay, ETH and AZ were found to be the most potent inhibitors of OC bone resorption, with IC50 values of 0.09 and 0.8 microM, respectively (from plan surface area of bone resorbed). These results support previous observations showing that OCs use CA II to generate protons during bone resorption and that CA II activity is essential for OCs to be able to resorb bone.

Acetazolamide↗
PubMed1991

Processing of the yellow fever virus nonstructural polyprotein: a catalytically active NS3 proteinase domain and NS2B are required for cleavages at dibasic sites.

The vaccinia virus-T7 transient expression system was used to further examine the role of the NS3 proteinase in processing of the yellow fever (YF) virus nonstructural polyprotein in BHK cells. YF virus-specific polyproteins and cleavage products were identified by immunoprecipitation with region-specific antisera, by size, and by comparison with authentic YF virus polypeptides. A YF virus polyprotein initiating with a signal sequence derived from the E protein fused to the N terminus of NS2A and extending through the N-terminal 356 amino acids of NS5 exhibited processing at the 2A-2B, 2B-3, 3-4A, 4A-4B, and 4B-5 cleavage sites. Similar results were obtained with polyproteins whose N termini began within NS2A (position 110) or with NS2B. When the NS3 proteinase domain was inactivated by replacing the proposed catalytic Ser-138 with Ala, processing at all sites was abolished. The results suggest that an active NS3 proteinase domain is necessary for cleavage at the diabasic nonstructural cleavage sites and that cleavage at the proposed 4A-4B signalase site requires prior cleavage at the 4B-5 site. Cleavages were not observed with a polyprotein whose N terminus began with NS3, but cleavage at the 4B-5 site could be restored by supplying the the NS2B protein in trans. Several experimental results suggested that trans cleavage at the 4B-5 site requires association of NS2B and the NS3 proteinase domain. Coexpression of different proteinases and catalytically inactive polyprotein substrates revealed that trans cleavage at the 2B-3 and 4B-5 sites was relatively efficient when compared with trans cleavage at the 2A-2B and 3-4A sites.

Animals↗
PubMed1991

Identification of osteoclast precursors in multilineage hemopoietic colonies.

The osteoclast is known to be derived from the hemopoietic stem cell, but its lineage and the mechanisms by which its differentiation is regulated are largely unknown. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hemopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semi-solid medium from mouse spleen cells in the presence of erythropoietin with either Wehi 3B-conditioned medium or interleukin 3 (IL3). After 7 days, individual colonies were picked. Half of each colony was phenotyped by the morphology of cells in cytospin preparations; the second half of each was incubated for 7 days with a bone marrow-derived cell line (ts8) that induces osteoclastic differentiation from hemopoietic cells, on bone slices in the presence of 1,25-dihydroxyvitamin D3. After incubation, bone resorption was assessed by scanning electron microscopy. No resorption was induced in cells derived from single-lineage colonies, but resorptive cells differentiated in 17% of granulocyte-macrophage (GM) colonies and 38% of multilineage colonies. Since only a minority of GM colonies contained osteoclastic precursors, this suggests that the GM colonies that contained osteoclasts were not typical GM colonies but may have been a form of multilineage colony analagous to other multilineage colonies that contain granulocytes, macrophages, and a third cell type. No resorptive cells were formed when IL3-derived colonies were incubated on bone slices without ts8 cells. The results suggest that osteoclasts are derived from a multilineage precursor, upon which IL3 acts to generate cells capable of osteoclastic differentiation, which form resorptive cells upon incubation with bone marrow stromal cells in the presence of 1,25-dihydroxyvitamin D3.

Animals↗
PubMed1991

High concentrations of 17 beta-estradiol stimulate trabecular bone formation in adult female rats.

Although the effects of low concentrations of 17 beta-estradiol (E2) on bone formation and resorption are well described, little is known of the effects of E2 on bone at concentrations that circulate during pregnancy. We, therefore, investigated the effects of administration of high dose E2 to 3-month-old female Wistar rats on biochemical and histomorphometric indices of bone formation and resorption. Animals receiving exogenous E2 (4 mg/kg.day for 17 days; n = 9) showed a mean serum E2 concentration of 17.5 +/- 2.9 nM, compared with 0.6 +/- 0.2 nM in those receiving vehicle alone (n = 10). The bone formation rate, measured at the proximal tibial metaphysis after the administration of double fluorochrome labels, was greatly increased in the E2 group (13.6 +/- 2.0 x 10(-2) microns3/microns2.day) compared to controls (3.9 +/- 0.9), as was serum alkaline phosphatase (E2, 133.6 +/- 10.1 IU; controls, 87.5 +/- 5.5). This increase in the rate of bone formation was associated with a significant increase in trabecular bone volume. E2 treatment did not affect urinary hydroxyproline excretion or histomorphometric indices of bone resorption. These findings suggest that high concentrations of E2 strongly stimulate the formation of trabecular bone. This may represent an important mechanism by which calcium stores are accumulated during pregnancy in rats, in anticipation of the mineral requirements of lactation.

Alkaline Phosphatase↗
PubMed1991

The effect of sex hormones on bone resorption by rat osteoclasts.

Although sex hormones are important in the attainment and maintenance of bone mass, the mechanism by which they exert their effect is unknown. We therefore tested the effects of estradiol-17 beta, dihydrotestosterone, and progesterone on osteoclasts, the cells that resorb bone. Osteoclasts were disaggregated from neonatal rat long bones, and incubated with or without the addition of osteoblastic cells or osteoblast-like cell lines. Bone resorption was assessed by scanning electron microscopy as the extent of excavation of the bone surface after incubation. We found dihydrotestosterone (1-100 nmol/l) and progesterone (10-1000 nmol/l) to have no significant effect on bone resorption. By contrast, E2 (1 nmol/l) reduced bone resorption in osteoclast cultures to which osteoblasts had been added, by approximately 25%, although consistent inhibition with other concentrations (0.01, 0.1, 10 nmol/l) was not observed. To our surprise, E2 was also associated with a delayed, dose-responsive, stimulation of bone resorption, in the range 0.1-10 nmol/l, in osteoclast cultures free from added osteoblastic cells. Tamoxifen, which itself had no effect on bone resorption, appeared to antagonise these E2 effects. Although the physiological significance of the stimulatory effect is unclear, we hypothesize that its presence prevented us, and previous workers, from observing dose-responsive inhibition of bone resorption by E2 in vitro.

Animals↗
PubMed1990

Structure-activity relationship of human calcitonin-gene-related peptide.

The calcitonin-calcitonin-gene-related peptide (CGRP) gene complex encodes a small family of peptides: calcitonin, CGRP and katacalcin. Calcitonin is a circulating hormone that prevents skeletal breakdown by inhibiting the resorption of bone by osteoclasts. CGRP, a potent vasodilator, is involved in normal regulation of blood flow. The calcitonins structurally resemble the CGRP peptides, and both are known to cross-react at each others' receptors. The present study was undertaken to examine the structural prerequisites for biological activity of the intact CGRP molecule. We therefore prepared eight chymotryptic and tryptic fragments of CGRP and synthesized its acetylated and S-carboxyamidomethylcysteinyl analogues. The analogues were purified by h.p.l.c. and their structures were confirmed by fast-atom bombardment mass spectrometry. We have examined the effects of structurally modified analogues and fragments of human CGRP in a calcitonin-receptor-mediated assay, the osteoclast bone resorption assay, and in one or two CGRP-receptor-mediated assays, the rabbit skin blood flow assay and the oedema formation assay. The results showed that (1) in the osteoclast bone resorption assay, both CGRP peptides, alpha and beta, were equipotent, and were both at least 1000-fold were both approx. 1000-fold more potent than salmon calcitonin; human calcitonin had no effect; (3) the bis- and N-acetylated CGRP analogues retained reduced levels of biological activity in all assays, whereas S-carboxyamidomethylcysteinyl-human CGRP was without activity; and (4) all tryptic and chymotryptic fragments of CGRP were without biological activity, with the exception of hCGRP-(Ala1-Lys35): this fragment had much reduced activity compared with the intact peptide in inhibiting osteoclastic bone resorption and increasing blood flow in the rabbit skin. The results suggest that: (1) calcitonin and CGRP act at distinct receptors to mediate different physiological effects; (2) minor amino acid substitutions, as between the alpha and beta forms of CGRP (these two forms have 94% structural similarity) do not result in differences in biological activity; (3) the intact peptide is required for full biological activity of the CGRP molecule, and even the loss of two amino acids at the C-terminus of the molecule results in a marked decrease in activity; (4) the disulphide bridge appears to play an important role in the interaction of the intact CGRP molecule with its receptor; and (5) the C-terminal region is probably necessary for the peptide to assume the right conformation in the interaction with the receptor.

Amino Acid Sequence↗
PubMed1990

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue.

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.

Animals↗
PubMed1990

Na+/H+ antiporter is the primary proton transport system used by osteoclasts during bone resorption.

We have examined the effects of inhibitors of proton transport systems on osteoclastic bone resorption using an in vitro bone slice assay, where osteoclasts (OCs) are free from the influence of other bone cells. Amiloride (AM) and dimethylamiloride (DMA), inhibitors of the Na+/H+ antiporter, were potent inhibitors of bone resorption (IC50 approximately 9 and 0.7 microM for AM and DMA, respectively). Omeprazole (OM), a potent inhibitor of parietal cell K+/H+(-)ATPase, was a poor inhibitor of OC bone resorption (IC50 approximately 100 microM). These results strongly suggest that the Na+/H+ antiporter is the primary proton system used by OCs during bone resorption.

Adenosine Triphosphatases↗