Bcl-2 protects macrophages from nitric oxide-induced apoptosis.
Endogenously generated or exogenously supplied nitric oxide (NO)-induced apoptotic cell death in the mouse macrophage cell line RAW 264.7. Apoptotic signaling caused an early accumulation of the tumor suppressor p53 prior to DNA fragmentation. Contrary to the notion of specific activating signals, inhibitory transduction mechanisms largely remain unknown. Therefore, RAW 264.7 macrophages were stably transfected with human Bcl-2, an anti-apoptotic protein. Bcl-2 transfectants showed substantial protection from cell death induced following the exposure to NO donors such as S-nitrosoglutathione (GSNO) and spermine-NO. In contrast, in RAW 264. 7 parent or in neomycin control-transformed cells, these NO donors induced internucleosomal DNA cleavage in a dose-dependent manner. Similarly, expression of the inducible NO synthase in response to lipopolysaccharide and interferon-gamma also caused apoptosis in RAW macrophages and neo controls within 24 h. In contrast, Bcl-2 transfectants appeared highly resistant, although inducible NO synthase levels increased along with concomitant nitrite production similar to control cells. The expression of p53 and Bax was also explored in controls and Bcl-2 transfectants after GSNO addition. GSNO induced p53 expression in Bcl-2 transfectants at levels comparable with nontransfected RAW macrophages. Moreover, GSNO induced increases in the steady-state levels of Bax protein in parental and Bcl-2-transfected cells. We conclude therefore, that Bcl-2 acts downstream of p53, presumably nullifying the NO-mediated increase in Bax protein in RAW 264.7 cells.
Messmer
U K
UK
University of Erlangen Nürnberg, Faculty of Medicine, Department of Medicine IV-Experimental Division, Loschgestrabetae 8, 91054 Erlangen, Federal Republic of Germany.
Reed
U K
UK
Brüne
B
B
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004790:Enzyme Induction; D006801:Humans; D008264:Macrophages / Q000502:physiology*; D051379:Mice; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C494827:Bax protein, mouse; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase