Osteocalcin induces chemotaxis, secretion of matrix proteins, and calcium-mediated intracellular signaling in human osteoclast-like cells.

Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of beta 1 and beta 3 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins.

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. We measured [Ca2+]i in single fura 2-loaded single cells and found that activation of PKC by phorbol esters doubled the [Ca2+]o-induced [Ca2+]i elevation, whereas inhibition of the enzyme by H7, staurosporine or sphingosine, completely blocked the ability of the cell to respond to elevated [Ca2+]i. By contrast, a control inactive agent, 4Aphorbol, failed to modify the cellular response to elevated [Ca2+]o. We conclude that PKC plays a synergistic role in the regulation of osteoclast Ca(2+)-sensing. Since we have previously demonstrated that activation of PKA up-regulates the Ca(2+)-sensing as well, we hypothesize that such mechanism is positively fed-back by both PKA and PKC-dependent threonine/serine phosphorylations.

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. We conclude that in these cultures mature osteoclasts and their mononuclear precursors are present.

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. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

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. The released radioactivity was, in fact, 48% and 52% respectively compared to the untreated cultures. These data demonstrated that cytosolic calcium increase is one of the messengers of the pathway that inhibits, in this condition, bone resorption. Furthermore, as in parathyroid cells, extracellular calcium acts with a negative direct feedback mechanism that controls osteoclast activity.

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. This increase was dose-dependent. Its half maximal effect was achieved at 30 mM KCl. Voltage sensitive calcium channels in osteoclasts were inhibited by specific antagonists. Nicardipine, a dihydropyridine derivative, was the most effective, inducing complete block of the channels at 10(-6) M. Verapamil (phenylalkylamine) and diltiazem (benzodiazepine) were less effective. These results are consistent with the presence, on the osteoclast membrane, of L-type voltage-sensitive calcium channels.

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. Results showed that increased extracellular calcium concentration induces the inhibition of podosome expression indicating that these close-contact areas are capable of calcium-mediated regulation.