Translocation of protein kinase C isoenzymes by elevated extracellular Ca2+ concentration in cells from a human giant cell tumor of bone

In this study we investigated the protein kinase C isoenzymes expressed by human osteoclast-like cells harvested from a giant cell tumor of bone (GCT23 cells), and by freshly isolated rat osteoclasts. Immunoblotting analysis revealed that the -α, -δ, and -ε, PKC isoforms, but not the -β isoenzyme, are expressed by GCT23 cells. Immunofluorescence studies demonstrated that PKC-α, -δ, and -ε are homogeneously expressed by both mononuclear and multinucleated GCT23 cells, as well as by rat osteoclasts. Similar to authentic osteoclasts, GCT23 cells responded to an increase of extracellular Ca2+ concentration ([Ca2+]0) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). An increase of [Ca2+]0 stimulated the translocation of PKC-α from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+]0 sensing. By contrast, PKC-δ was not altered by exposure to elevated [Ca2+]0, whereas PKC-ε underwent reciprocal translocation, disappearing from the insoluble fraction and increasing in the cytosol. The effects of PKC on GCT23 cell functions were investigated by treatment with phorbol 12-myristate, 13-acetate (PMA). We observed that activation of PKC by PMA failed to affect adhesion onto the substrate, but down-regulated the [Ca2+]0-induced [Ca2+]i increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.