Abstract
Cyclooxygenase-2 (Cox-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being up-regulated by diverse stimuli. Significantly Cox-2 mRNA is absent from rat pheochromocytoma (PC12) cells, both basally and following stimulation with a range of agonists. Using PC12 cells engineered to stably express isopropyl-1-thio-beta-D-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis. Cox-2 bioactivity had no effect on nerve growth factor-induced differentiation, epidermal growth factor-induced proliferation, or aromatic L-amino acid decarboxylase expression. However, trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining. The specificity of this effect was confirmed using two pharmacologically distinct nonsteroidal anti-inflammatory drugs, indomethacin and NS398. Investigations showed that the activity of the pro-apoptotic protease caspase-3 was reduced in PCXII cells. This study demonstrates that Cox-2-derived prostaglandins exert cytoprotective effects in trophic factor withdrawal apoptosis and provides evidence that this is, at least in part, due to suppression of caspase-3 activity.
Highlights
From the ‡Department of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and ʈMedizinische Poliklinik, University of Bonn, D-53111, Bonn, Germany
Using PC12 cells engineered to stably express isopropyl-1-thio--D-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis
Because PC12 cells are used as a cell culture model of nerve growth factor (NGF)-induced neuronal differentiation and have facilitated studies into the signaling pathways leading to the differentiated phenotype [11], this cell line represents an ideal tool for investigations into the effects of Cox-2 overexpression on cellular differentiation
Summary
From the ‡Department of Medicine and Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 and ʈMedizinische Poliklinik, University of Bonn, D-53111, Bonn, Germany. Trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining.
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