Bcl-2 Protects Macrophages from Nitric Oxide-induced Apoptosis

doi:10.1074/jbc.271.33.20192

Bcl-2 Protects Macrophages from Nitric Oxide-induced Apoptosis*

From the University of Erlangen Nürnberg, Faculty of Medicine, Department of Medicine IV-Experimental Division, Loschgestraβe 8, 91054 Erlangen, Federal Republic of Germany and the
^‡ The Burnham Institute, Cancer Research Center, La Jolla, California 92037

^§To whom correspondence should be addressed:
University of Erlangen-Nürnberg, Faculty of Medicine, Dept. of Medicine IV, Loschgestraβe 8, 91054 Erlangen, Germany.
Tel.: 49-9131-856311; Fax: 49-9131-859202.

Abstract

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-γ 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.

Footnotes

↵* This work was supported by the Deutsche Forschungsgemeinschaft, in part by the European Community, by National Institutes of Health Grant CA60181, and a grant from the University of California Breast Cancer Research program. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

NO

nitric oxide

NOS

nitric oxide synthase

iNOS

inducible nitric oxide synthase

GSNO

S-nitrosoglutathione

IFN-γ

murine interferon-γ

LPS

lipopolysaccharide.
- Received January 11, 1996.
- Revision received May 21, 1996.