Cellular and molecular mechanisms of bone resorption.
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Animals↗
Biomedical subjects
S L Teitelbaum
Publications and source records attributed to S L Teitelbaum.
Isolation and reconstitution of a vacuolar-type proton pump of osteoclast membranes.
A vacuolar-type proton-translocating ATPase was extracted from ruffled membranes of chicken osteoclasts with 1% polyoxyethylene 9-lauryl ether (C12E9) and was purified 13-fold by glycerol gradient centrifugation. The isolated pump appears by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to have a subunit composition similar to that of the clathrin-coated vesicle proton pump, in that subunits of apparent molecular masses of 116, 71, 57, 40, 39, 33, and 17 kDa are present in the osteoclast pump preparation. In addition, the 116-, 71-, 57-, and 40-kDa components were shown to cross-react with specific antisera generated against the homologous subunits of the clathrin-coated vesicle proton pump. The isolated osteoclast H(+)-ATPase was reconstituted into liposomes prepared from purified lipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol) by a cholate-dilution, freeze-thaw method. Proton transport catalyzed by the reconstituted pump was inhibited by bafilomycin A1 (10 nM) and N-ethylmaleimide (1 mM) but was insensitive to vanadate. We propose that osteoclast-mediated bone resorption is effected by a vacuolar-type proton pump with functional and structural similarities to that isolated from clathrin-coated vesicles.
Animals↗
Interleukin-4 inhibits bone resorption and acutely increases cytosolic Ca2+ in murine osteoclasts.
Interleukin-4 (IL-4) is an immune cytokine recently shown to inhibit bone resorption. To determine whether IL-4 directly acts on osteoclasts, we have analyzed its effect on cytosolic calcium concentration [Ca2+]i and bone resorptive function of murine osteoclastic cells generated from bone marrow/stromal cell co-cultures. IL-4 exposure induced an immediate and sustained increase in [Ca2+]i that remained elevated for at least 10 min. This IL-4 effect was dose-dependent, with the maximal effect (209 +/- 15% of baseline, n = 16) at 200 units/ml and an apparent ED0.5 of 60 units/ml. The IL-4-induced [Ca2+]i rise required extracellular Ca2+ influx, since the response was prevented by LaCl3, and voltage-gated Ca2+ channel blockers, although the IL-4 effect was more sensitive to nicardipine and nifedipine than to diltiazem. Depolarization by high extracellular K+ concentration also raised [Ca2+]i, and, under these conditions, osteoclasts failed to respond to IL-4. On the other hand, when intracellular Ca2+ stores were depleted by thapsigargin, IL-4 still induced an increase in [Ca2+]i, although smaller in amplitude and transient. Calcitonin also produced [Ca2+]i increases in osteoclasts, yet it only slightly desensitized these cells to IL-4. Furthermore, IL-4 was much less effective on osteoclasts pretreated (5-10 min) with either forskolin or 8-bromo-cAMP. Both IL-4 and calcitonin were effective even when [Ca2+]i had been increased by exposure to high extracellular Ca2+. Finally, IL-4 dose dependently inhibited the bone-resorptive activity of mature osteoclasts. Therefore, IL-4 signal transduction in osteoclasts involves a rapid and sustained elevation of [Ca2+]i mediated by a voltage-dependent Ca2+ influx, in combination with Ca2+ release from intracellular stores. Modulation of osteoclast [Ca2+]i represents a potential mechanism by which IL-4 inhibits bone resorption.
Animals↗
Enrichment of generated murine osteoclasts.
Biochemical and molecular studies of osteoclasts generally require cells in a reasonable degree of purity. The chicken has been extremely useful in this regard, as abundant avian osteoclasts can be generated in vitro entirely from pure populations of marrow macrophage precursors. Propagation of murine osteoclasts is, in contrast, far less efficient, demanding the presence of stromal cells. The aims of this study were to develop a method by which murine osteoclasts generated in culture, can be effectively enriched while maintaining viability and, to explore the mechanisms by which stromal cells promote murine osteoclast generation and survival. We find that 10(6) fractionated murine marrow cells enriched, for marrow-residing colony-forming units (CFU-cs), yield 3000-4000 tartrate-resistant acid phosphatase (TRAP)-expressing multinucleated giant cells when cultured for 12 days with ST-2 stromal cells. These cells are osteoclasts as evidenced by their ability to "pit" bone slices, resorb radiolabeled bone particles, and generate cyclic AMP in response to calcitonin. Treatment of these generated osteoclast cultures with bacterial collagenase for 2 hours at 37 degrees selectively removes virtually all ST-2 cells, yielding a > 60% pure population of TRAP and calcitonin receptor-expressing cells, 90% of which are viable. These cells continue to respond to calcitonin and survive for 24 hours in the absence of ST-2 cells. We also found that murine osteoclast generation depends upon contact of osteoclast precursors with viable ST-2 cells. Furthermore, the stromal cells secrete macrophage colony-stimulating factor (CSF-1), and the anti-CSF-1 antibody 5A1 inhibits murine osteoclastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Acid Phosphatase↗
1,25-Dihydroxyvitamin D3 transcriptionally activates the beta 3-integrin subunit gene in avian osteoclast precursors.
Osteoclasts are polykaryons and the principal, if not exclusive, resorptive cell of bone. They are members of the monocyte/macrophage family whose precursors differentiate under the influence of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption is dependent on osteoclast-bone attachment, and we have shown that the integrin alpha v beta 3 is critical to the resorptive process. Thus, we asked whether 1,25-(OH)2D3 enhances the expression of alpha v beta 3 on the surface of osteoclast precursors and if the steroid modulates expression of the beta 3-integrin subunit. We found that 1,25-(OH)2D3 promotes the plasma membrane appearance of alpha v beta 3 on avian bone marrow-derived osteoclast precursors and does so at physiological concentrations (10(-11) M) of the steroid. The effect is time dependent, appearing within 1 day of treatment. A full-length avian cDNA was cloned to explore the molecular mechanisms of beta 3 expression. The deduced amino acid sequence of the cDNA is 81% identical and 89% similar to that of human beta 3. Northern analysis demonstrates that beta 3 mRNA levels in vitamin D-treated osteoclast precursors mirror protein expression. Nuclear run-on experiments document the transcriptional nature of the event.
Amino Acid Sequence↗
Mechanisms of tumor-induced osteolysis.
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Bone Neoplasms↗
Interleukin-4 enhances murine bone marrow macrophage M-CSF receptor expression by a posttranscriptional mechanism.
Activated T-lymphocytes secrete interleukin-4 (IL-4), which has been shown to modulate a variety of monocyte activities requiring monocyte/macrophage colony-stimulating factor (M-CSF). To account for this interaction, we postulated that IL-4 acts on target cells by altering the expression of the M-CSF receptor (M-CSFr). To test this hypothesis, murine bone marrow macrophages were cultured under conditions that down-regulate M-CSFr and the effect of IL-4 on the reexpression of the receptor measured by binding of 125I-labeled M-CSF to the cells. The data show that incubation with IL-4 results in a dose-dependent, 2-3 x increase in M-CSFr with no change in binding affinity and a maximal effect on binding at about 12 h. This increase in M-CSFr is dependent upon new, specific protein synthesis as shown by the inhibitory action of cycloheximide, and gel analysis of radiolabeled, specific protein, immunoprecipitated with anti-M-CSFr antibody. Treatment with IL-4 does not stimulate M-CSFr mRNA expression but, consistent with enhanced receptor levels, does result in a heightened proliferative response to M-CSF. Thus, IL-4 affects M-CSF treated monocytic cells, at least in part, by altering the expression of M-CSFr.
Animals↗
Cloning of the promoter for the avian integrin beta 3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3.
We have cloned the promoter of the avian beta 3 integrin gene. Using a probe comprising the 5'-untranslated region of an avian macrophage beta 3 cDNA, characterized by 5' rapid amplification of cDNA ends, several clones were isolated from an avian genomic library. One major and one minor transcriptional start site were identified at +1 and -47 base pairs, respectively, with the latter coinciding with a consensus sequence of an initiator. DNA sequence analysis of 800 base pairs 5' of the transcriptional start site fails to reveal either a TATA or CAAT box. In addition to an initiator, the first 200 base pairs contain consensus sequences for the binding of AP-1 and SP-1. A 3.5-kilobase fragment located immediately upstream of the transcriptional start site exhibits functional promoter activity, and deletion analysis reveals both suppressor and enhancer elements. In light of our observation that 1,25-dihydroxyvitamin D3 (D3) accelerates beta 3 transcription, we determined whether the avian beta 3 promoter contains a vitamin D response element (VDRE). Transfected reporter constructs containing the first 1.5 kilobases upstream of the major beta 3 transcriptional start site respond to D3 with enhanced luciferase activity. Analysis of this region reveals a classical VDRE consensus sequence, located at -756 to -770. The following observations support the hypothesis that this sequence represents a functional VDRE: 1) a 600-base pair genomic fragment or a 29-base pair oligomer, each containing the putative VDRE, respond to D3 when transfected into HD11 cells; 2) a 67-base pair DNA fragment derived from genomic DNA and containing the candidate beta 3 VDRE specifically binds the vitamin D receptor-retinoid X receptor beta complex; and 3) avian osteoclast precursor-derived nuclear extracts bind to a synthetic oligomer containing the beta 3 VDRE-like sequence and, in turn, are specifically displaced by unlabeled beta 3 VDRE and anti-vitamin D receptor antibody.
Amino Acid Sequence↗
Osteoporosis induced in mice by overproduction of interleukin 4.
Osteoporosis is a common disease in which loss of bone mass results in skeletal fragility. The development of therapies for this disorder has been hampered by the lack of a convenient animal model. Here we describe a disorder in bone homeostasis in transgenic mice that inappropriately express the cytokine interleukin 4 (IL-4) under the direction of the lymphocyte-specific proximal promoter for the lck gene. Bone disease in lck-IL-4 mice appeared to result from markedly decreased bone formation by osteoblasts, features strikingly similar to those observed in cases of severe low-turnover human involutional osteoporosis. By 2 months of age, female and male lck-IL-4 mice invariably developed severe osteoporosis of both cortical and trabecular bone. Osteoporosis was observed in two independently derived founder animals, indicating that this phenotype was directly mediated by the IL-4 transgene.
Actins↗
Regulation of mouse bone marrow macrophage mannose receptor expression and activation by prostaglandin E and IFN-gamma.
The macrophage mannose receptor mediates the clearance of microorganisms and glycoproteins containing terminal mannose oligosaccharides. Cell surface expression of this receptor progresses with macrophage differentiation, and thus may be critical to the scavenger function of tissue and circulating macrophages. Bone marrow macrophages, which were used in this study, differentiate in culture and express functional mannose receptors. The cytokine IFN-gamma triggered activation of these macrophages and down-regulated cell surface expression of the mannose receptor after 48 h. Macrophage activation, as assessed by the generation of superoxide radicals, was inversely correlated with mannose receptor expression. The number of surface receptors was diminished by exposure to IFN-gamma, whereas the binding affinity of the mannose receptor remained unchanged. Treatment with IFN-gamma reduced receptor biosynthesis yet did not alter receptor degradation. Mannose receptor biosynthesis is up-regulated by PG of the E series, and these anti-inflammatory agents reversed the effects of IFN-gamma on receptor expression. Down-regulation of the mannose receptor by IFN-gamma was fully reversible by PGE, indicating that receptor levels are dependent on the functional state of the cell rather than being linked to terminal cell differentiation. The regulation of the receptor by cytokines and anti-inflammatory reagents suggests that the mannose receptor plays a critical role in macrophage scavenger functions and potentially in modulating inflammatory reactions.
Animals↗
Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption.
We have investigated the mechanism by which osteoclasts adhere to and resorb bone. We show that these cells express beta 1 and beta 3 integrins which are involved in attachment to purified bone matrix proteins. Binding to osteopontin and bone sialoprotein is mediated by alpha v beta 3, while a beta 1 integrin is responsible for attachment to fibronectin. Both the rapid attachment by osteoclasts to intact bone particles and their subsequent resorption are blocked by a monoclonal antibody directed to the alpha v beta 3 complex but not by an antibody against beta 1 integrins. Attachment of osteoclasts to bone is also inhibited with soluble osteopontin, Arg-Gly-Asp-containing peptides derived from both osteopontin and bone sialoprotein, or a monospecific polyclonal antibody against osteopontin. We conclude that both osteoclast adherence to bone and subsequent resorption of its matrix are dependent on interactions between the bone matrix proteins osteopontin and/or bone sialoprotein and the integrin alpha v beta 3. Moreover, collagen, which constitutes 90% of its organic matrix, is minimally involved in binding of chicken osteoclasts to bone.
Amino Acid Sequence↗
Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization.
Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.
Amino Acid Sequence↗
Bone remodeling and the osteoclast.
Bone remodeling is a process initiated by the osteoclast, and thus, its understanding is prerequisite to regulation of bone turnover. The last decade has witnessed major advances in our understanding of osteoclast biology, specifically as relates to the ontogeny of the cell and the mechanisms by which it degrades bone. It is now possible to isolate and generate osteoclasts and maintain them in relative purity. Using these models, a number of laboratories have shown that ion transport by the osteoclast plays a major role in its ability to resorb bone. Furthermore, osteoclast-bone matrix attachment, mediated at least in part by integrins, is pivotal to the resorptive process. These discoveries are likely to lead to insight into control of the remodeling process.
Animals↗
1,25-Dihydroxyvitamin D3 and macrophage colony-stimulating factor-1 synergistically phosphorylate talin.
Macrophage colony stimulating factor (CSF-1) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage-like cells. In this regard, we utilized the CSF-1 dependent murine macrophage-like line BAC 1.25F5. CSF-1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF-1 receptor, and a 210 kDa moiety. 1,25(OH)2D3 exposure for 24 h prior to addition of CSF-1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)2D3 dose- and time-dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti-talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF-1 and 1,25(OH)2D3, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)2D3/CSF-1 induced phosphorylation of the putative CSF-1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)2D3 pretreated cells to phorbol 12-myristate 13-acetate (PMA) in place of CSF-1 also prompts talin phosphorylation. Finally, 1,25(OH)2D3 enhances 3[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF-1 and 1,25(OH)2D3 act synergistically via PKC to phosphorylate talin, a cytoskeletal-associated protein.
Alkaloids↗
Binding of osteopontin to the osteoclast integrin alpha v beta 3.
Occupancy of the chicken osteoclast alpha v beta 3 integrin stimulates immediate cell signals. Peptides from osteopontin containing Arg-Gly-Asp and peptides from the osteopontin and bone sialoprotein sequences containing Arg-Gly-Asp stimulated immediate reductions in osteoclast cytosolic Ca2+. The changes in cytosolic Ca2+ required the Arg-Gly-Asp sequence, and were blocked by LM609, a monoclonal antibody to the alpha v beta 3 integrin. Osteoclast stimulation by the proteins through the integrin did not require immobilization since soluble peptides produced changes in cytosolic Ca2+ and inhibited osteoclast binding to bone particles and bone resorption. The decrease in cytosolic Ca2+ stimulated by osteopontin and related peptides was due to activation of a plasma membrane Ca(2+)-ATPase. Thus, the data suggest that ligand binding to the osteoclast alpha v beta 3 integrin results in a reduction in cytosolic Ca2+ which participates in regulation of osteoclast function.
Animals↗
Systemic mastocytosis and osteoporosis.
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Biopsy↗
Acid and base effects on avian osteoclast activity.
Osteoclasts generate a massive acid flux to mobilize bone calcium. Local extracellular acidification by polarized vacuolar-type H(+)-ATPase, balanced by contralateral HCO3-(-)Cl- exchange to maintain physiological intracellular pH, is theorized to drive this process. It follows that extracellular pH, PCO2, or HCO3- concentration ([HCO3-]) should impact bone matrix dissolution. However, the effects on bone resorption of the concentrations of these ions or their transmembrane gradients are unknown. Furthermore, because bone management is a vital process, regulatory feedback may minimize such effects. Thus a complex relationship between bone resorption and pH, PCO2, and [HCO3-] is expected but requires experimental determination. We measured bone resorption by isolated avian osteoclasts while varying these parameters across the physiological range. Bone degradation increased 50% from pH 7.3 to 6.7, whether achieved by changing [HCO3-] (2.3-38 mM) at constant HCO3- or PCO2 (15-190 mmHg) at constant [HCO3-]. However, at constant pH, changing PCO2 and [HCO3-] within physiological limits did not affect bone resorption. In contrast, total HCO3- removal at pH 7.4 reduced bone degradation by rat or avian osteoclasts substantially, confirming that normal acid secretion requires HCO3-. These observations support a model coupling osteoclastic bone resorption to proton and HCO3- transport but indicate that [HCO3-] is not rate limiting under physiological conditions. Extracellular pH changes affect osteoclastic bone resorption measurably, but not dramatically, at physiological [HCO3-].
Acid-Base Equilibrium↗
Recent advances toward understanding osteoclast physiology.
Osteoclasts develop from precursor cells of the monocyte series. However, specialized differentiation for efficient bone degradation separates the osteoclast from the macrophage. The physical reasons for these differences are emerging from the study of osteoclastic physiology and biochemistry. Key osteoclast specializations are multinucleation, formation of a tightly sealed extracellular compartment on bone, and high-capacity secretion of HCl and acid proteases into this extracellular site. Multinucleation increases efficiency of extracellular attachment processes. The attachment process is mediated by cell membrane integrins, and is sensitive to changes in intracellular or extracellular calcium. Acid production exploits carbonic acid as the source of acid and conjugate base equivalents, reflected in abundant osteoclastic carbonic anhydrase type II expression. Secretion of acid involves extremely high expression of vacuolar-type H(+)-ATPase and a chloride channel in the cell's specialized acid secreting organelle, the ruffled membrane, which is polarized to the osteoclast's bone attachment. Acid secretion is balanced by chloride-bicarbonate exchange in the cell's nonbone attached membranes; this functionally resembles the band 3 chloride-bicarbonate exchanger of the red cell carbon dioxide transport system. Bone collagen is degraded by acid proteases secreted into the acid degradation site via the mannose-6-phosphate receptor system, which is targeted to lysosomes in other cells. Functional deficits, as in osteopetrosis, may affect any of the elements involved in osteoclast differentiation. Furthermore, new antiosteoclastic therapeutic agents may inhibit osteoclast biochemistry intentionally, such as for the control of hypercalcemia of malignancy.
Bicarbonates↗