Sumoylation of IkB attenuates NF-kB-induced nitrosative stress at rostral ventrolateral medulla and cardiovascular depression in experimental brain death

Abstract

Background: Small ubiquitin-related modifier (SUMO) is a group of proteins that participates in post-translational modifications. One known SUMO target is the transcription factor nuclear factor-kB (NF-kB) that plays a pivotal role in many disease processes; sumoylation inactivates NF-kB by conjugation with inhibitors of NF-kB (IkB). Our laboratory demonstrated previously that transcriptional upregulation of nitric oxide synthase II (NOS II) by NF-kB, leading to nitrosative stress by the formation of peroxynitrite in the rostral ventrolateral medulla (RVLM), underpins the defunct brain stem cardiovascular regulation that precedes brain death. Based on an experimental endotoxemia model, this study evaluated the hypothesis that sumoylation plays a pro-life role in brain death by interacting with the NF-kB/NOS II/peroxynitrite signaling pathway in the RVLM.

Results: In Sprague-Dawley rats, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 10 mg kg^-1) elicited an augmentation of SUMO-1 and ubiquitin-conjugase 9 (Ubc9) mRNA or protein levels, alongside SUMO-1-conjugated proteins in the RVLM. Immunoneutralization of SUMO-1 or Ubc9 in the RVLM significantly potentiated the already diminished sumoylation of IkBα and intensified NF-kB activation and NOS II/peroxynitrite expression in this brain stem substrate, together with exacerbated fatality, cardiovascular depression and reduction of an experimental index of a life-and-death signal detected from arterial pressure that disappears in comatose patients signifying failure of brain stem cardiovascular regulation before brain death.

Conclusion: We conclude that sumoylation of IkB in the RVLM ameliorates the defunct brain stem cardiovascular regulation that underpins brain death in our experimental endotoxemia modal by reducing nitrosative stress via inhibition of IkB degradation that diminishes the induction of the NF-kB/NOS II/peroxynitrite signaling cascade.

Keywords: Defunct brain stem cardiovascular regulation; Nitrosative stress; Rostral ventrolateral medulla; Small ubiquitin-related modifier; Ubiquitin-conjugase 9.

Fig. 1

Temporal changes in mean arterial pressure (MAP), heart rate (HR) or power density of the low-frequency (BLF) component of AP spectrum in rats that received pretreatment by microinjection bilaterally into the RVLM of normal rat serum (NRS; 1:20), anti-SUMO-1 antiserum (SUMO-1 Ab; 1:20) or anti-Ubc9 antiserum (Ubc9 Ab; 1:20), followed by intravenous administration of saline or LPS (10 mg kg⁻¹). The three distinct phases based on reduced (Phase I), augmented (Phase II) and a secondary decreased (Phase III) power density of the BLF spectral component induced by LPS are denoted on top of the figure. Values are mean ± SEM of 7–8 animals per experimental group. *P < 0.05 versus NRS + saline group, and ⁺ P < 0.05 versus NRS + LPS group at corresponding time-points in the post hoc Scheffé multiple-range test. B = preinjection baseline. Note that for clarity of illustration, data on pretreatment with normal mouse serum in this and Figs. 4 to 6 were omitted because they were comparable to those with NRS pretreatment

Fig. 2

Phasic fold-changes relative to sham-controls of sumo1 or ubc9 mRNA in the RVLM determined by real-time PCR after intravenous administration of saline or LPS (10 mg kg⁻¹). Tissue samples were collected during the peak of Phases I, II or III in the LPS group; or 20 (I), 150 (II) or 300 (III) min in the saline group. Values are mean ± SEM of triplicate analyses on individual samples obtained from 4–5 animals per experimental group. *P < 0.05 versus sham-control group or corresponding saline group in the post hoc Scheffé multiple-range test

Fig. 3

Representative western blots (insets) or percentage of SUMO-1 monomer and SUMO-1 conjugate (a) or Ubc9 (b) relative to β-actin detected in the RVLM during Phases I, II or III after intravenous administration of saline or LPS (10 mg kg⁻¹). Values are mean ± SEM of duplicate analyses on individual samples obtained from 5–6 animals per experimental group. *P < 0.05 versus corresponding saline group in the post hoc Scheffé multiple-range test

Fig. 4

Survival rate over 300 min of animals that received pretreatment by microinjection bilaterally into the RVLM of an antiserum directed against SUMO-1 (SUMO-1 Ab; 1:20) or Ubc9 (Ubc9 Ab; 1:20), or normal rabbit serum (NRS; 1:20), followed by intravenous administration of saline or LPS (10 mg kg⁻¹). Each group contained 11–14 animals at the beginning of the experiment

Fig. 5

Results from immunoblot (IB) using an antiserum against SUMO-1 on proteins immunoprecipitated (IP) by an anti-IkBα antiserum from the RVLM (a); western blot analysis of expression of IkBα relative to β-actin in the RVLM (b); transcriptional activity of NF-kB p65 in the RVLM (c) after intravenous administration of saline or LPS (10 mg kg⁻¹); or animals that received pretreatment by microinjection bilaterally into RVLM of NRS (1:20), anti-SUMO-1 antiserum (SUMO-1 Ab; 1:20) or anti-Ubc9 antiserum (Ubc9 Ab; 1:20) during Phase III in the experimental endotoxemia model of brain death. Values are mean ± SEM of duplicate analyses on individual samples obtained from 5–6 animals per experimental group. *P < 0.05 versus corresponding saline group and ⁺ P < 0.05 versus NRS + LPS group in the post hoc Scheffé multiple-range test

Fig. 6

Representative western blots (insets), or percentage of NOS II or nitrotyrosine (NT) relative to β-actin in the RVLM detected after intravenous administration of LPS (10 mg kg⁻¹) in animals (a); or animals that received pretreatment by microinjection bilaterally into the RVLM of NRS (1:20), anti-SUMO-1 antiserum (SUMO-1 Ab; 1:20) or anti-Ubc9 antiserum (Ubc9 Ab; 1:20) during phase III in the experimental endotoxemia model of brain death (b). Values are mean ± SEM of duplicate analyses on individual samples obtained from 5–6 animals per experimental group. *P < 0.05 versus corresponding saline group in the post hoc Scheffé multiple-range test. Note that in (a), dividing lines are placed on the representative gel images to denote re-grouping of images from different parts of the same gel