RESUMEN
Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues during development, physiological remodeling, and pathological bone loss. Osteoclasts have the ability to resorb substrate while concurrently migrating. However, the subcellular processes underlying migration are not well understood. It has been proposed that, in other cell types, cytosolic free Ca(2+) concentration ([Ca(2+) ]i ) regulates cell protrusion as well as retraction. Integration of these distinct events would require precise spatiotemporal patterning of subcellular Ca(2+) . The large size of osteoclasts offers a unique opportunity to monitor patterns of Ca(2+) during cell migration. We used ratiometric imaging to map [Ca(2+) ]i within rat and mouse osteoclasts. Migration was characterized by lamellipodial outgrowth at the leading edge, along with intermittent retraction of the uropod. Migrating osteoclasts displayed elevation of [Ca(2+) ]i in the uropod, that began prior to retraction. Dissipation of this [Ca(2+) ]i gradient by loading osteoclasts with the Ca(2+) chelator BAPTA abolished uropod retraction, on both glass and mineralized substrates. In contrast, elevation of [Ca(2+) ]i using ionomycin initiated prompt uropod retraction. To investigate downstream effectors, we treated cells with calpain inhibitor-1, which impaired uropod retraction. In contrast, lamellipodial outgrowth at the leading edge of osteoclasts was unaffected by any of these interventions, indicating that the signals regulating outgrowth are distinct from those triggering retraction. The large size of mature, multinucleated osteoclasts allowed us to discern a novel spatiotemporal pattern of Ca(2+) involved in cell migration. Whereas localized elevation of Ca(2+) is necessary for uropod retraction, lamellipod outgrowth is independent of Ca(2+) -a heretofore unrecognized degree of specificity underlying the regulation of osteoclast migration.
Asunto(s)
Calcio/metabolismo , Osteoclastos/citología , Fracciones Subcelulares/metabolismo , Animales , Movimiento Celular , Citosol/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Osteoclastos/metabolismo , Ratas , Ratas WistarRESUMEN
Osteopontin (OPN), an integrin-binding extracellular matrix glycoprotein, enhances osteoclast activity; however, its mechanisms of action are elusive. The Ca(2+)-dependent transcription factor NFATc1 is essential for osteoclast differentiation. We assessed the effects of OPN on NFATc1, which translocates to nuclei upon activation. Osteoclasts from neonatal rabbits and rats were plated on coverslips, uncoated or coated with OPN or bovine albumin. OPN enhanced the proportion of osteoclasts exhibiting nuclear NFATc1. An RGD-containing, integrin-blocking peptide prevented the translocation of NFATc1 induced by OPN. Moreover, mutant OPN lacking RGD failed to induce translocation of NFATc1. Thus, activation of NFATc1 is dependent on integrin binding through RGD. Using fluorescence imaging, OPN was found to increase the proportion of osteoclasts exhibiting transient elevations in cytosolic Ca(2+) (oscillations). OPN also enhanced osteoclast survival. The intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) suppressed Ca(2+) oscillations and inhibited increases in NFATc1 translocation and survival induced by OPN. Furthermore, a specific, cell-permeable peptide inhibitor of NFAT activation blocked the effects of OPN on NFATc1 translocation and osteoclast survival. This is the first demonstration that OPN activates NFATc1 and enhances osteoclast survival through a Ca(2+)-NFAT-dependent pathway. Increased NFATc1 activity and enhanced osteoclast survival may account for the stimulatory effects of OPN on osteoclast function in vivo.