Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERalpha), during osteoclast differentiation.

Alendronate reduces adhesion of human osteoclast-like cells to bone and bone protein-coated surfaces.

Bisphosphonates (BPs) are potent inhibitors of bone resorption and are therapeutically effective in disease of increased bone turnover, but their mechanism(s) of action remain to be elucidated. Using as experimental model human osteoclast-like cell lines derived from giant cell tumors of bone, extensively characterized for their osteoclast features, we investigated the adhesive properties of osteoclasts on bone slices and on different proteins of the extracellular matrix in the presence of BPs. Adhesion assays using bone slices pretreated with ALN, at the established active concentration, showed that, although the morphology of osteoclasts plated onto pretreated bone slices was not modified, the number of adherent cells was reduced by the treatment of about 50% vs.

Altered expression of basement membrane proteins and their integrin receptors in lichen planus: possible pathogenetic role of gelatinases A and B.

Lichenoid lesions of the skin are characterized by a band-like dermal inflammatory infiltrate and structural alterations of the basement membrane (BM). The etiopathogenesis of these lesions, of which lichen planus (LP) is perhaps the prototypic example, is unknown. Acute cases of LP are accompanied by the destruction of epidermal BM, degeneration of basal keratinocytes with loss of tonofilaments and hemidesmosomes, vesicular alterations, and even blister formation.

Evidence for a K+ channel requirement in spreading of rat basophilic leukemia cells on fibronectin-coated surfaces.

We investigated the ionic requirements for the early events of cell-extracellular matrix interactions leading to cell spreading. We found that potassium ions were required specifically in several cell types. Adhesion to fibronectin- (FN) coated surfaces was independent of K+ in the medium.

New model for bone resorption study in vitro: human osteoclast-like cells from giant cell tumors of bone.

Cells harvested from 12 human giant cell tumors of bone and kept in culture for several passages were characterized for bone-resorbing capability, total and tartrate-resistant acid phosphatase activity, response to the calciotropic hormone calcitonin, cell proliferation, multinucleation after passages, and presence of calcium sensing. Cells obtained from three tumors presented a complete panel of osteoclast characteristics and maintained their multinuclearity after several passages. Cells from four other tumors increased their cAMP levels after treatment with calcitonin, and the other five apparently consisted of cells of stromal origin.

Ipriflavone directly inhibits osteoclastic activity.

Ipriflavone, an isoflavone derivative, is a new drug used in an attempt to decrease bone loss in osteoporosis. Experimental studies have shown that this compound acts by inhibiting osteoclastic bone resorption both in vivo and in vitro, but the mechanism of its inhibitory action on resorbing cells remains unclear. Using bone resorption assays, video image analysis together with measurements of intracellular free calcium in isolated osteoclasts, we show here that IP directly inhibits osteoclastic activity by the modulation of intracellular free calcium.

Human osteoclast-like cells recognize laminin via an RGD independent mechanism.

Interactions between cells from human giant cell tumors of bone and the extracellular matrix protein laminin were studied. Cells were capable of recognizing this substratum via a RGD-independent mechanism. Recognition induces adhesion and spreading onto laminin.

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.

Protein kinase C affects microfilaments, bone resorption, and [Ca2+]o sensing in cultured osteoclasts.

The effects of protein kinase C (PKC) in the control of osteoclast activity are still unknown. We investigated the role of the enzyme in the control of microfilament organization, podosome assembly, bone resorption, and extracellular Ca2+ sensing in chicken and rabbit osteoclasts treated with agents known to affect PKC activity. Cells were treated for 20 min with a PKC activator [phorbol 12-myristate 13-acetate (PMA)], a PKC inhibitor (staurosporine), a protein kinase A (PKA) inhibitor (H-9), a guanosine 3',5'-cyclic monophosphate-dependent protein kinase-PKA-PKC inhibitor (H-7), or with the inactive phorbol, 4 alpha-phorbol, to examine microfilaments by decoration with rhodamine-phalloidin.

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o.

Integrin expression and adhesion property of osteoclast-like cells from giant cell tumours of bone.

Cells cultured from human giant cell tumours of bone were used to study interactions with different extracellular matrix proteins as Collagen, Fibronectin, Osteocalcin, Thrombospondin and Bone Sialoprotein II. Cells were capable of recognizing these substrata; beta 3 integrin subunit was distributed in focal adhesions, together with beta 1 on BSPII, FN, and in presence of serum, whereas and presented a diffuse organization onto the other substrate. beta 1 alone was expressed over collagen coated coverslips.

Functional and biochemical characterization of osteoclast-like cells derived from giant cell tumours of bone.

Cells harvested from human giant cell tumours of bone were characterized on the basis of morphological features, proliferative capacity, total(AP) and tartrate resistant acid phosphatase (TRAP) activity, and hormonal response. Culture were formed by mononucleated and multinucleated cells. Mononucleated cells showed fibroblastic morphology, whereas multinucleated cells showed osteoclastic phenotype.

Protein kinase C-dependent phosphorylation regulates osteoclast calcium-sensing.

Osteoclasts display a membrane Ca(2+)-sensing mechanism capable of detecting the extracellular calcium concentration ([Ca2+]o), and to induce increase of [Ca2+]i and inhibition of bone resorption. The ultimate result of the stimulation of such sensing is probably the activation of protein kinase C (PKC). To demonstrate whether PKC plays a role in the control of the osteoclast activity, we treated rabbit single osteoclasts with agents known to activate or to inhibit the enzyme.

Recognition of osteopontin and related peptides by an alpha v beta 3 integrin stimulates immediate cell signals in osteoclasts.

We have investigated the nature of immediate cell signals produced by occupancy of the chicken osteoclast alpha v beta 3 integrin. Synthetic osteopontin 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 a monoclonal antibody to the alpha v beta 3 integrin, LM609.

Effects of calcium-phosphate-based materials on proliferation and alkaline phosphatase activity of newborn rat periosteal cells in vitro.

The effects of dental materials, intended for bone substitution, on cell growth and alkaline phosphatase activity of newborn rat periosteal cells have been studied in vitro. Confluent periosteal cells were exposed to three apatite-based materials (400 micrograms/mL) with different physico-chemical properties. The materials were a beta-tricalcium phosphate with a microporous granular structure obtained by sinterization (Synthograft, Johnson & Johnson, East Windsor, NY), a 40-60-mesh microporous durapatite ceramic (Periograf, Sterling Drug, Inc.

Parathyroid hormone binding to cultured avian osteoclasts.

Parathyroid hormone (PTH) increases serum calcium concentration via a controversial cellular mechanism. We investigated whether PTH binds avian osteoclasts. Isolated hypocalcaemic hen osteoclasts were incubated with [125I]--bovine PTH (1-84).

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption.

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips.

The role of protein kinase C in the osteoclast activity.

Isolated chicken osteoclasts in culture have been treated with 100 nM PMA for 20 minutes, and processed for the decoration of the microfilaments with fluorescent phalloidin. Results demonstrated that this phorbol ester, which activates the protein kinase C, induces the assembly of microfilaments in stress-fibers, and enlarges the microfilamentous core of podosomes. This results indicate that the protein kinase C mediates specific arrangement of microfilaments in osteoclasts.

Osteoclast bone resorption is enhanced in the presence of osteoblasts.

Bone resorption activity by osteoclasts has been evaluated in a co-culture system in which osteoclasts have been plated in the presence of osteoblasts. The system prevents cell-cell contact but permits diffusion of molecules through the pores of a millipore membrane that separates the two compartments in which the two cell types have been plated. Results demonstrated that osteoblasts exert a stimulatory effect over osteoclast bone resorption due to soluble molecules capable of passing through the membrane pores.

Osteoblast-osteoclast interaction in bone resorption. Preliminary results.

Osteoclastic bone resorption has been evaluated in vitro by release of tritiated collagen fragments from 3H-proline prelabeled bone particles incubated for 48 hours in presence of avian isolated osteoclasts. Cells were co-incubated with periosteum-free chick calvarial fragments by interposition of 0.4 micron millipore membrane transwells, in presence or absence of 10(-8) M 1.

Cytosolic free calcium dependent regulation of osteoclast bone resorbing activity.

Osteoclasts are sensitive to KCl-induced depolarization and to increased extracellular calcium concentration, and respond to these treatments with cytosolic calcium increase. In this study we evaluated the possibility that these experimental conditions could affect osteoclast bone resorption. We found that, incubating osteoclasts with 3H-proline previously labeled bone particles the resorbing activity was inhibited by both depolarization and extracellular calcium concentration increase.

Voltage dependent calcium channel expression in isolated osteoclasts.

In this study the expression of voltage-dependent calcium channels on osteoclast plasma membrane has been investigated. We found that osteoclasts were sensitive to KCl-induced depolarization. In this circumstance a 4 fold transient cytosolic calcium concentration ([Ca2+]i) increase was observed.

Podosome expression in osteoclasts: influence of high extracellular calcium concentration.

In this study the effect of high extracellular calcium concentration has been evaluated, by immunofluorescence, on podosome expression in chicken osteoclasts. Cells were cultured in presence of 0.2 and 4 mM calcium for 90 minutes and microfilaments were detected, after fixation and permeabilization, by decoration with rodhamine conjugated phalloidin.

Immunolocalization of beta 3 subunit of integrins in osteoclast membrane.

Utilizing isolated and cultured osteoclasts it has been possible to establish that they adhere to the substrate through specialized close contact areas, the podosomes, that in fully spread osteoclasts in vitro or in vivo are located within the clear zone. The cytochemical organization of podosomes has further been investigated in order to elucidate their possible involvement in the control of substrate recognition, that precedes bone resorption. An immunofluorescence investigation, performed utilizing human osteoclasts, shows that the beta 2 integrin subunit that in human monocytes is expressed and located in podosomes is absent in human osteoclasts, while the beta 3 subunit of the vitronectin receptor is expressed by osteoclasts, but not by other monocyte-derived cells and colocalizes with vinculin around the actin core of the podosome.