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B RelA Phosphorylation at Serine 2761
* Instituto Gulbenkian de Ciência, Apartado 14, Oeiras, Portugal; and
Department of Neurology and Neuroscience, Division of Neurobiology, Weill Medical College of Cornell University, New York, NY 10021
Heme oxygenase-1 (HO-1; encoded by the Hmox1 gene) catalyzes the degradation of free heme into biliverdin, via a reaction that releases iron (Fe) and carbon monoxide. We report that HO-1 down-regulates the proinflammatory phenotype associated with endothelial cell (EC) activation by reducing intracellular nonprotein-bound Fe (labile Fe). EC isolated from Hmox1–/– mice have higher levels of intracellular labile Fe and reactive oxygen species (ROS) as compared with EC isolated from Hmox1+/+ mice. Basal and TNF-induced expression of VCAM-1, ICAM-1, and E-selectin were increased in Hmox1–/– vs Hmox1+/+ EC, an effect reversed by Fe chelation using deferoxamine mesylate (DFO). Fe chelation inhibits TNF-driven transcription of Vcam-1, Icam-1, and E-selectin, as assessed using luciferase reporter assays. This effect is associated with inhibition of the transcription factor NF-
B via a mechanism that is not associated with the inhibition of IB
phosphorylation/degradation or NF-B (i.e., RelA) nuclear translocation, although it affects very modestly NF-B binding to DNA B consensus sequences in the Vcam-1 and E-selectin promoters. HO-1 inhibits NF-B (i.e., RelA) phosphorylation at Ser276, a phosphoacceptor that is critical to sustain TNF-driven NF-B activity in EC. This effect was mimicked by Fe chelation as well as by antioxidants (N-acetylcysteine). In conclusion, we demonstrate a novel mechanism via which HO-1 down-modulates the proinflammatory phenotype of activated EC, i.e., the inhibition of RelA phosphorylation at Ser276.
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.
1 This work was supported by the grants POCTI/BIA-BCM/56829/2004 and POCTI/SAU-MNO/56066/2004 (to M.S.), Ph.D. fellowships SFRH/BD/2990/2000 (to M.P.S.) and SFRH/BD/28016/2006 (to N.P.), postdoctoral fellowships SFRH/BPD/6303/2001 and SFRH/BPD/21072/2004 (to G.S.), SFRH/BPD/9380/2002 (to I.P.G.) and SFRH/BPD/25436/2005 (to R.L.), all from "Fundação para Ciência e a Tecnologia" Portugal, the European Commission’s Sixth Framework Programme contract no. LSHB-CT-2006-037377, Xenome (to M.P.S.). Research described in this article was supported in part by Philip Morris USA.
2 M.P.S. and G.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Miguel Soares, Instituto Gulbenkian de Ciencia, Rua da Quinta Grande 6, Oeiras, 2780-156, Portugal. E-mail address: mpsoares{at}igc.gulbenkian.pt
4 Abbreviations used in this paper: EC, endothelial cell; BAEC, bovine aortic EC; CM-H2DCFDA, 5-(and-6)-chloromethyl-2',7'-dichlorofluorescein diacetate, acetyl ester; DAPI, 4' 6-diamidino-2-phenylindole; DFO, deferoxamine mesylate; Fe, iron; HBSer, N-(2-hydroxybenzyl)-L-serine; HEK-293, human epithelial kidney 293 (cell); H-ferritin, heavy chain ferritin; HO-1, heme oxygenase 1; HPRT, hypoxanthine-guanine-phosphoribosyltransferase; luc, luciferase; MEC, mouse endothelial cell; MEF, mouse embryonic fibroblast; NAC, N-acetyl-L-cysteine; PKA, protein kinase A; RHD, Rel homology domain; ROS, reactive oxygen species; SIH, salicylaldehyde isonicotinoylhydrazone; TAD, transactivation domain; tet°, tetracycline operon; TET, tet° DNA binding domain; VP16, virion protein 16.
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