Heme Oxygenase-1-derived Carbon Monoxide Requires the Activation of Transcription Factor NF- B to Protect Endothelial Cells from Tumor Necrosis Factor- -mediated Apoptosis*

We have shown that carbon monoxide (CO) generated by heme oxygenase-1 (HO-1) protects endothelial cells (EC) from tumor necrosis (TNF)-mediated apoptosis. This effect relies on the activation of p38 MAPK. We now demonstrate that HO-1/CO requires the activation of the transcription factor NFB to exert this anti-apoptotic effect. Our data suggest that EC have basal levels of NFB activity that sustain the expression of NFB-dependent anti-apoptotic genes required to support the anti-apoptotic effect of HO-1/CO. Over-expression of the inhibitor of NFB (I B ) suppresses the anti-apoptotic action of HO-1/CO. Reconstitution of NFB activity, by co-expression of I B with different members of the NFB family, i.e. p65/RelA or p65/RelA plus c-Rel, restores the anti-apoptotic effect of HO-1/CO. Expression of the NFB family members p65/RelA or p65/RelA with p50 or c-Rel up-regulates the expression of the anti-apoptotic genes A1, A20, c-IAP2, and manganese superoxide dismutase (MnSOD). Inhibition of NFB activity by overexpression of I B suppresses the expression of some of these anti-apoptotic genes, i.e. c-IAP2. Under inhibition of NFB, co-expression of some of these anti-apoptotic genes, i.e. c-IAP2 and A1, restores the anti-apoptotic action of HO-1/CO, whereas expression of A20 or MnSOD cannot. The ability of c-IAP2 and/or A1 to restore the anti-apoptotic action of HO-1/CO is abolished when p38 MAPK activation is blocked by over-expression of a p38 MAPK dominant negative mutant. In conclusion, we demonstrate that HO-1/CO cooperates with NFB-dependent anti-apoptotic genes, i.e. c-IAP2 and A1, to protect EC from TNF-mediated apoptosis. This effect is dependent on the ability of HO-1/CO to activate the p38 MAPK signal transduction pathway. Signaling via “death receptors,” such as the tumor necrosis factor(TNF) receptor 1 (TNFR-1/CD120a), can trigger endothelial cell (EC) to undergo apoptosis. Cross-linking of TNFR-1 leads to the recruitment of intracytoplasmic signal transduction molecules, e.g. TRADD (TNF receptor-associated death domain), FADD (Fas-associated death domain), and RIP (receptor-interacting protein) (1, 2). These molecules form the death-inducing signaling complex (DISC; reviewed in Refs. 1 and 2), which activates serine proteases, referred to as caspases (3). Caspase activation by the death-inducing signaling complex occurs via FADD-dependent recruitment and proximal catalytic cleavage/activation of pro-caspase-8 into the active form of caspase-8 (4, 5), which activates additional procaspases into active caspases, e.g. caspase-3, that execute the terminal phase of apoptosis (reviewed in Ref. 3). Under physiologic conditions, signaling via the TNFR-1 does not lead to EC apoptosis because TNFR-1 triggers the expression of early responsive anti-apoptotic genes such as the zinc finger A20 (6), the bcl-2 family member A1 (7), the antioxidant manganese superoxide dismutase (MnSOD) (8), several members of the inhibitor of apoptosis (IAP) family (9), IEXL-1 (10), and PAI-2 (plasminogen activator inhibitor type-2) (11). These anti-apoptotic genes prevail over the pro-apoptotic signals thus preventing TNF-mediated EC apoptosis. Expression of these anti-apoptotic genes is dependent on the activation of the transcription factor nuclear factor B (NFB) (12). Inhibition of NFB activity prevents the expression of these anti-apoptotic genes and thus sensitizes most cell types (13–15), including EC (12), to undergo TNF-mediated apoptosis. The NFB family of transcription factors consists of several homoor heterodimeric complexes of the Rel family, i.e. p50/ NFB1, p65/RelA, c-Rel (Rel), p52/NFB2, and RelB (reviewed in Refs. 16 and 17). In quiescent EC, NFB is thought to be retained in the cytoplasm by a series of inhibitory proteins referred to as inhibitor of B (I B) (reviewed in Ref. 16). Binding of NFB to I B molecules masks the nuclear localization signal in the NFB dimers, thereby preventing NFB nuclear translocation and transcription activity (18). Signaling via TNFR-1 triggers the release of NFB dimers from I B molecules via site-specific phosphorylation, ubiquitination, and sub* This work was supported in part by Grant 998521355 from the Roche Organ Transplantation Research Foundation and by National Institutes of Health Grants HL67040 (to M. P. S.) and HL58688 (to F. H. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work. ¶ Supported by a grant from Institut National de la Santé et de la Recherche Médicale (INSERM), France. Supported by a fellowship of the Swiss National Science Foundation. ‡‡ Supported by Grant BD2990/00 from the “Fundaçao para a Ciencia e Tecnologia” Portugal. §§ The Lewis Thomas Professor at the Harvard Medical School and a paid consultant for Novartis Pharma. ¶¶ To whom correspondence should be addressed: Immunobiology Research Center, Dept. of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-632-0885; Fax: 617–632-0880; E-mail: msoares@caregroup. harvard.edu. 1 The abbreviations used are: TNF, tumor necrosis factor; TNFR-1, tumor necrosis factorreceptor 1; Act.D, actinomycin-D; BAEC, bovine aortic endothelial cells; CMV, cytomegalovirus; EC, endothelial cell; EMSA, electrophoretic mobility shift assay; HO-1, heme oxygenase-1; HUVEC, human umbilical vein endothelial cell; I Ba, inhibitor of nuclear factorB ; MAPK, mitogen-activated protein kinase(s); MnSOD, manganese superoxide dismutase; NFB, nuclear factorB; PAEC, porcine aortic endothelial cells; IAP, inhibitor of apoptosis; HA, hemagglutinin; LPS, lipopolysaccharide; RT, reverse transcriptase. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 20, Issue of May 17, pp. 17950–17961, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.