Biomedical subjects

R Faccio

Publications and source records attributed to R Faccio.

5 recordsLinked to original sources

PubMed2006

Map kinase c-JUN N-terminal kinase mediates PMMA induction of osteoclasts.

Erosive osteolysis induced by implant-derived wear debris is mediated by recruitment and activation of osteoclasts in a pro-inflammatory microenvironment that is enriched with osteoclastogenic and pro-inflammatory cytokines such as RANKL and tumor necrosis factor alpha (TNF-alpha). These cytokines activate the transcription factor NF-kappaB and MAP kinases, including c-Jun, Erks, and p38, all known to be essential for the development of osteoclasts. We have recently documented that TNF and RANKL play a pivotal role in the development of inflammatory osteolysis. We have also found that polymethyl methacrylate (PMMA) particles stimulate osteoclastogenesis, at least in part, by induction of RANKL, TNF, and by activation of NF-kappaB and MAP kinases. More importantly, our data indicate that inhibitors of NF-kappaB and the MAP kinases p38 and ERK abrogate particle-induced osteoclastogenesis. In the current study, we investigated if inhibition of c-Jun N-Terminal kinase (JNK) pathway alters PMMA-induced osteoclastogenesis. Our findings point out that PMMA particles activate the JNK pathway in wild-type and TLR4-null (endotoxin-resistant) osteoclast precursors. This activation was selectively blocked in a dose-dependent fashion by the JNK inhibitor SP600125. Most importantly, we provide evidence that SP600125 inhibits osteoclast formation in a reversible manner. Collectively, our findings demonstrate that activation of the JNK pathway is essential for basal and PMMA-stimulated osteoclastogenesis, and buttress the potential significance of targeting the JNK pathway to inhibit osteolysis.

Animals↗

PubMed2001

A Glanzmann's mutation in beta 3 integrin specifically impairs osteoclast function.

Osteoclastic bone resorption requires cell-matrix contact, an event mediated by the alpha v beta 3 integrin. The structural components of the integrin that mediate osteoclast function are, however, not in hand. To address this issue, we generated mice lacking the beta 3 integrin gene, which have dysfunctional osteoclasts. Here, we show the full rescue of beta 3(-/-) osteoclast function following expression of a full-length beta 3 integrin. In contrast, truncated beta 3, lacking a cytoplasmic domain (h beta 3c), is completely ineffective in restoring function to beta 3(-/-) osteoclasts. To identify the components of the beta 3 cytoplasmic domain regulating osteoclast function, we generated six point mutants known, in other circumstances, to mediate beta integrin signaling. Of the six, only the S(752)P substitution, which also characterizes a form of the human bleeding disorder Glanzmann's thrombasthenia, fails to rescue beta 3(-/-) osteoclasts or restore ligand-activated signaling in the form of c-src activation. Interestingly, the double mutation Y(747)F/Y(759)F, which disrupts platelet function, does not affect the osteoclast. Thus similarities and distinctions exist in the mechanisms by which the beta 3 integrin regulates platelets and osteoclasts.

Amino Acid Sequence↗

PubMed1998

Activation of alphav beta3 integrin on human osteoclast-like cells stimulates adhesion and migration in response to osteopontin.

Integrins mediate cell adhesion and can induce different cellular responses, including changes in intracellular pH, changes and oscillation in intracellular free calcium, and protein phosphorylation on tyrosine. During bone resorption, the integrin alphav beta3 regulates adhesion of osteoclasts to bone extracellular matrix proteins, such us osteopontin (Opn). Adhesion via alphav beta3 is followed by osteoclast polarization onto the bone surface and by the onset of bone resorption. To characterize these events at the molecular level, we investigated the state of activation of alphav beta3 on the human osteoclast-like cell line GCT23 using the monoclonal antibody AP5 which binds to and can induce, under low calcium conditions, activated alphav beta3. By flow cytometry, approximately 50% of alphav beta3 on the surface of the osteoclast-like cell line GCT23 was reactive with AP5 and was therefore in the activated state. Incubation with AP5 in the presence of low calcium concentrations increased activated alphav beta3 to 90-100%. Activation of alphav beta3 increased the efficiency of GCT23 adhesion to Opn by 2-fold. Furthermore, haptotactic migration on Opn was also enhanced about 40% compared to control. We propose that changes in the activation state of alphav beta3 may be a regulation point for osteoclasts during bone resorption.

Antibodies, Monoclonal↗

PubMed1996

Human osteoclast-like cells selectively recognize laminin isoforms, an event that induces migration and activates Ca2+ mediated signals.

Osteoclast precursors are chemotactically attracted to sites of bone resorption via migration pathways that include transendothelial crossing in blood capillaries. Transendothelial migration involves poorly understood interactions with basal lamina molecules, including laminins. To investigate osteoclast-laminin interactions, we used human osteoclast-like cell lines obtained from giant cell tumors of bone (GCT 23 and GCT 24). These cell lines are a well-characterized model for osteoclast functions, such as bone resorption and the behaviour of osteoclast precursors. Both GCT cell lines adhered to laminin-2 (merosin) coated wells in standard adhesion assays, but failed to adhere to laminin-1 (EHS-laminin). By light microscopy, GCT cells on laminin-2 were partially spread, with a motile morphology. None of the anti-integrin antibodies tested inhibited GCT cells adhesion to laminin-2. Peptides containing the integrin adhesion site RGD or the laminin adhesion sequence IKVAV did not inhibit GCT cell adhesion to laminin-2. By immunofluorescence, beta 1 integrins were organized in focal adhesions. However, in the presence of monensin this reorganization of beta 1 integrins was abolished, indicating that it was probably due to secretion of fibronectin by GCT cells subsequent to adhesion to laminin-2. GCT cells transmigrated through membranes coated with laminin-2, much more efficiently than through membranes coated with collagen. Migration was induced by osteocalcin, as a chemoattractant, in a dose-dependent manner. At low osteocalcin concentrations, transmigration was detectable on laminin-2 but not collagen. In cells loaded with fura-2, a sharp increase in intracellular Ca2+ was detected upon addition of soluble laminin-2, but not laminin-1, due to release from thapsigargin-dependent intracellular stores. In summary, osteoclasts may recognize laminin isoforms differentially. Initial adhesion to laminin-2 appears to be due to integrin-independent mechanisms. Such adhesion, though, may trigger secretion of fibronectin that could then support spreading and efficient chemotactic migration. These mechanisms may play an important role in facilitating chemotactic migration of osteoclast precursors toward the bone surface.

Calcium↗

PubMed1994

Extracellular Ca2+ sensing is modulated by pH in human osteoclast-like cells in vitro.

Osteoclasts are polarized cells with a basolateral and an apical membrane exposed to different extracellular Ca2+ ([Ca2+]o) and H+ (pHe) concentrations. Osteoclast bone resorption is inhibited in vitro by increases of [Ca2+]o slightly above physiological levels, detected by a [Ca2+]o sensing causing elevations of the intracellular signal, [Ca2+]i. Nevertheless, during bone resorption the apical membrane is exposed to [Ca2+]o severalfold higher than physiological without apparent inhibition of osteoclast functions. Because pHe facing the apical membrane is acidic, in this single-cell [Ca2+]i and intracellular pH study we addressed the question of whether the responses of human osteoclast-like cells from a giant cell tumor of bone to elevated [Ca2+]o are altered by reducing pHe. We first observed that low pHe stimulated Ca2+ efflux and cell acidification. We then demonstrated that the amplitude of the [Ca2+]o-dependent [Ca2+]i "spikes" is downregulated by low pHe, with approximately 70-fold higher [Ca2+]o required to induce significant responses at pHe 6.0 compared with pHe 7.4. Similar downregulation was observed in authentic freshly isolated rat osteoclasts. Finally, we observed that occupancy of the [Ca2+]o sensing by Ca2+ prompted rapid and transient cell acidification partially counteracted by a Na(+)-dependent amiloride derivative-sensitive H+ transport. These results demonstrate that the cascade of events triggered by activation of the [Ca2+]o sensing is affected by environmental pH and in turn influences cellular H+ transport. Such pH-related features of the [Ca2+] o sensing mechanism might be relevant for the regulation of osteoclast-like function.

Calcium↗