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 -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-alpha 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.
Teti
A
A
Department of Experimental Medicine, School of Medicine, University of L'Aquila, Italy.
Huwiler
A
A
Paniccia
R
R
Sciortino
G
G
Pfeilschifter
J
J
D000818:Animals; D001859:Bone Neoplasms / Q000201:enzymology* / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion; D003124:Colorimetry; D005870:Giant Cell Tumors / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000096:biosynthesis* / Q000187:drug effects; D008856:Microscopy, Fluorescence; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000187:drug effects; D051381:Rats; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium