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Time-dependent Changes in Protein Kinase C Distribution and Disappearance in Phorbol Ester-treated Human Osteosarcoma Cells¹

Laboratory of Taxicology, Harvard School of Public Health, and Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

To test directly whether protein kinase C activation is one of the required events leading to stimulation of prostaglandin production by bone cells, protein kinase C activity and prostaglandin E₂ release were measured in monolayer cultures of the clonal human osteosarcoma cell lines G-292 and SaOS-2 after exposure to phorbol myristate acetate (PMA). Both cell lines have specific receptors for PMA but only G-292 cells respond with increased prostaglandin E₂ production (M. A. Shupnik and A. H. Tashjian, Jr., J. Biol. Chem., 257: 12161–12164, 1982). The subcellular distribution of protein kinase C in both unstimulated osteosarcoma cell lines was similar; in an EDTA- and leupeptin-containing homogenization buffer, between 70 and 80% of the total enzyme activity was cytosolic. Short (<60 min) incubations with PMA induced marked decreases in cytosolic enzyme activity and parallel increases in particulate protein kinase C; thereafter, total measured cellular protein kinase C activity declined, mediated by decreases in both cytosolic and particulate protein kinase C specific activities. By 24 h cytosolic, particulate, and total protein kinase C activities were less than 10% of basal. Because the protein kinase C responses in both cell types were essentially the same, but only G-292 cells give a prostaglandin response to PMA, we conclude that protein kinase C activation by PMA is itself insufficient to stimulate prostaglandin E₂ production and that the lack of a prostaglandin response in SaOS-2 cells cannot be explained by lack of protein kinase C activation.

¹ This investigation was supported in part by Center Grant ES 00002 from the National Institute of Environmental Health Sciences, a cooperative agreement with the United States Environmental Protection Agency (OR 807809), and Research Grants AM 10206 and AM 11011 from the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases.

² On leave of absence from the Centre National de la Recherche Scientifique, Paris, France. Supported by La Fondation pour la Recherche Medicale and L'Association pour la Recherche sur le Cancer, France.

³ To whom requests for reprints should be addressed, at Laboratory of Toxicology, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.

Received 4/30/86. Revised 8/21/86. Revised 12/31/86. Accepted 1/27/87.

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