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 -alpha, -delta, and -epsilon, PKC isoforms, but not the -beta isoenzyme, are expressed by GCT23 cells, Immunofluorescence studies demonstrated that PKC-alpha, -delta, and -epsilon 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+](o)) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+](i)). An increase of [Ca2+](o) stimulated the translocation of PKC-cy from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+](o) sensing. By contrast, PKC-delta was not altered by exposure to elevated [Ca2+](o), whereas PKC-epsilon 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+](o)-induced [Ca2+](i) increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.

TRANSLOCATION OF PROTEIN-KINASE-C ISOENZYMES BY ELEVATED EXTRACELLULAR CA2+ CONCENTRATION IN CELLS FROM A HUMAN GIANT-CELL TUMOR OF BONE

TETI, ANNA MARIA;
1995-01-01

Abstract

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 -alpha, -delta, and -epsilon, PKC isoforms, but not the -beta isoenzyme, are expressed by GCT23 cells, Immunofluorescence studies demonstrated that PKC-alpha, -delta, and -epsilon 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+](o)) with a dose-dependent elevation of the cytosolic free Ca2+ concentration ([Ca2+](i)). An increase of [Ca2+](o) stimulated the translocation of PKC-cy from the cytosolic to the particulate fraction, suggesting the involvement of this isoenzyme in the signal transduction mechanism prompted by stimulation of the [Ca2+](o) sensing. By contrast, PKC-delta was not altered by exposure to elevated [Ca2+](o), whereas PKC-epsilon 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+](o)-induced [Ca2+](i) increases. The latter effect was specific, since it was reversed by treatment with the PKC inhibitors staurosporine and chelerythrine.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/5924
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