Adenovirus-mediated expression of a dominant negative mutant of p65/RelA inhibits proinflammatory gene expression in endothelial cells without sensitizing to apoptosis.
We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor kappaB (NF-kappaB), would prolong survival of vascularized xenografts. Our previous studies have shown that inhibition of NF-kappaB by adenovirus-mediated overexpression of I kappaB alpha suppresses the induction of proinflammatory genes in EC. However, I kappaB alpha sensitizes EC to TNF-alpha-mediated apoptosis, presumably by suppressing the induction of the NF-kappaB-dependent anti-apoptotic genes A20, A1, manganese superoxide dismutase (MnSOD), and cellular inhibitor of apoptosis 2. We report here that adenovirus mediated expression of a dominant negative C-terminal truncation mutant of p65/RelA (p65RHD) inhibits the induction of proinflammatory genes, such as E-selectin, ICAM-1, VCAM-1, IL-8, and inducible nitric oxide synthase, in EC as efficiently as does I kappaB alpha. However, contrary to I kappaB alpha, p65RHD does not sensitize EC to TNF-alpha-mediated apoptosis although both inhibitors suppressed the induction of the anti-apoptotic genes A20, A1, and MnSOD equally well. We present evidence that this difference in sensitization of EC to apoptosis is due to the ability of p65RHD, but not I kappaB alpha, to inhibit the constitutive expression of c-myc, a gene involved in the regulation of TNF-alpha-mediated apoptosis. These data demonstrate that it is possible to block the expression of proinflammatory genes during EC activation by targeting NF-kappaB, without sensitizing EC to apoptosis and establishes the role of c-myc in controlling induction of apoptosis during EC activation. Finally, these data provide the basis for a potential approach to suppress EC activation in vivo in transgenic pigs to be used as donors for xenotransplantation.
Soares
M P
MP
Immunobiology Research Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02115, USA.
Muniappan
A
A
Kaczmarek
E
E
Koziak
K
K
Wrighton
C J
CJ
Steinhäuslin
F
F
Ferran
C
C
Winkler
H
H
Bach
F H
FH
Anrather
J
J
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D005822:Genetic Vectors / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D015778:Minor Histocompatibility Antigens; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000502:physiology; D009687:Nuclear Proteins; D009928:Organ Specificity; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013482:Superoxide Dismutase / Q000096:biosynthesis / Q000235:genetics; D013552:Swine; D051996:Transcription Factor RelA; D014158:Transcription, Genetic; D014162:Transfection; D019076:Transgenes; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D020672:I-kappa B Proteins; D047908:Intracellular Signaling Peptides and Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D009687:Nuclear Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D051996:Transcription Factor RelA; D000072000:NF-KappaB Inhibitor alpha; D013482:Superoxide Dismutase; C076529:TNFAIP3 protein, human; D000072598:Tumor Necrosis Factor alpha-Induced Protein 3; D003546:Cysteine Endopeptidases; C513261:Tnfaip3 protein, mouse; D051818:Replication Protein C