Adenovirus-Mediated Expression of a Dominant Negative Mutant of p65/RelA Inhibits Proinflammatory Gene Expression in Endothelial Cells Without Sensitizing to Apoptosis

Abstract

We hypothesized that blocking the induction of proinflammatory genes associated with endothelial cell (EC) activation, by inhibiting the transcription factor nuclear factor κB (NF-κB), would prolong survival of vascularized xenografts. Our previous studies have shown that inhibition of NF-κB by adenovirus-mediated overexpression of IκBα suppresses the induction of proinflammatory genes in EC. However, IκBα sensitizes EC to TNF-α-mediated apoptosis, presumably by suppressing the induction of the NF-κB-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 IκBα. However, contrary to IκBα, p65RHD does not sensitize EC to TNF-α-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 IκBα, to inhibit the constitutive expression of c-myc, a gene involved in the regulation of TNF-α-mediated apoptosis. These data demonstrate that it is possible to block the expression of proinflammatory genes during EC activation by targeting NF-κB, 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.

Comprehensive Targeted Sequencing Identifies Monogenic Disorders in Patients With Early‐onset Refractory Diarrhea

“…E, Structure of RelA protein. Localization of nonsense mutation p.Q389X in the patient (red) and p65RHD dominant negative mutant (20) (parenthesis) are shown. Cont = normal control; Fa = father; Mo = mother; Pt = patient.…”

“…A heterozygous nonsense mutation was confirmed before the TAD, and the truncated p65RHD mutant that lacked the TAD was not degraded and detected by western blotting. p65RHD acts as a dominant negative and inhibits the induction of proinflammatory genes and TNFα‐meditated antiapoptotic genes in NFκB signaling (20). Normally, IL‐1β and TNF‐α produced by the stimulation of bacterial LPS activate NFκB, with this activated NFκB producing IL‐6.…”

Heme Oxygenase-1 Protects Pancreatic β Cells from Apoptosis Caused by Various Stimuli

“…The β-galactosidase gene (Clontech Laboratories, Palo Alto, Calif) was cloned into the pcDNA3 vector (Invitrogen, Carlsbad, Calif), as described before. 34 The full-length rat HO-1 cDNA (a kind gift from Augustine Choi, Pittsburgh, Pa) was subcloned into the pcDNA3 vector under the control of the cytomegalovirus enhancer-promoter (pcDNA3-HO-1). 32 The murine Bcl-2 cDNA expression vector (a kind gift from R. Gerard, University of Texas Southwestern, Dallas) has been described elsewhere.…”

“…β-galactosidase-transfected cells were detected, and the percentage of viable cells was assessed by evaluating the number of β-galactosidase-expressing cells that retained their normal morphology. 34,36 The number of random fields counted was determined to have a minimum of 200 viable transfected cells per control well. The percentage of viable cells was normalized for each DNA preparation to the number of transfected cells counted in the absence of the apoptosis-inducing agent (100% viability).…”