Sumoylation of HIF-1a in Experimental Brain Death Figure 3. Preferential nucleus-bound translocation of HIF-1a in RVLM during the pro-life phase. (A and B) Illustrative gels or summary of fold changes against sham (S) controls in ratio of total protein of HIF-1a, HIF-1b or HIF-2a relative to b-actin protein (A), or nuclear or cytosolic content of HIF-1a or HIF-1b protein (B) detected in ventrolateral medulla during Phases EMI, MI and MII Mev intoxication or in aCSF (V) controls. Values are mean 6 SEM of triplicate analyses on samples pooled from 4–6 animals in each group. *P,0.05 versus aCSF group in the post hoc Scheff e´ multiple-range analysis. Note numbers on top of the gels correspond to columns in the data summary. (C) Representative laser scanning confocal microscopic images showing cells in RVLM that were immunoreactive to NeuN (green fluorescence) and additionally stained positively for HIF-1a or HIF-1b subunit (yellow fluorescence or colocalization of red and green fluorescence) in sham controls or during Phases EMI, MI and MII Mev intoxication. These results are typical of 4 animals from each experimental group. Scale bar, 8 mm. doi:10.1371/journal.pone.0017375.g003 A novel contribution of the present study is the identification of a vital role for sumoylation of HIF-1a in RVLM in ameliorating brain stem cardiovascular regulatory dysfunction during brain death. The covalent conjugation of SUMOs to proteins has received much attention since its discovery [45,46] because the affected proteins are involved in gene expression, chromatin structure, signal transduction or maintenance of the genome [31]. Transient global cerebral ischemia induces massive increases in sumoylation of proteins, including transcription factors [29]. In particular, hypoxia increases SUMO-1 mRNA and protein levels in brain, and the induced SUMO-1 co-expresses with HIF-1a in neurons [32]. However, controversy exists on the cellular sequel of modulation of HIF-1a through sumoylation. It is generally contended that sumoylation of transcription factors blocks their activation [37,47]. Thus, conjugation of SUMO to HIF-1a decreases its activity [34], and hypoxia-induced sumoylation of HIF-1a leads to its ubiquitination and degradation [36]. On the other hand, Bae et al. [33] reported that upregulation of HIF-1a through SUMO-1 modification at Lys(391)/Lys(477) residues increases its stability and enhances its transcriptional activity, and sumoylation of HIF-1a blocks its degradation [29]. Whereas the present study was not designed to address this controversy, complementary results from our biochemical and physiological experiments strongly indicated that elevation in SUMO-1 and Ubc9 levels that paralleled temporally with hypoxia, followed by an increase in conjugation between SUMO-1 and HIF-1a, took place in RVLM preferentially during the pro-life phase. More importantly, we demonstrated in our model that this enhanced sumoylation of HIF-1a in RVLM ameliorates the dysfunction of brain stem cardiovascular regulation associated with brain death. The return of SUMO-1, Ubc9 or HIF-1a expression in RVLM to control levels during phase II despite a sustained oxygen level at 462 mmHg below baseline suggests that stronger hypoxic drive is required to trigger sumoylation of HIF-1a and its transcriptional activities. Although unrelated directly to brain death, the engagement of sumoylation in cardiovascular regulation is reported in a recent study [48]. Sumoylation of phosducin inhibits postganglionic sympathetic neuron activity and prevents stress- induced hypertension. Gill [49] reviewed that SUMO shares structural homology with ubiquitin, and the machinery that attaches SUMO to substrate proteins and the enzymes that participate in ubiquitination are strikingly similar. As such, SUMO may compete with ubiquitin for the same lysine residues and function essentially as an antiubiqui- tin [47]. We reason that this modus operandi is at work in RVLM during brain death, noting that degradation of HIF-1a by the ubiquitin-proteasome system determines its prevalence [41]. In support of this notion, we demonstrated a preferential increase of association between SUMO-1 and HIF-1a in RVLM during the pro-life phase, and that immunoneutralization of SUMO-1 or Ubc9 significantly antagonized the augmented expression of HIF- 1a in RVLM and exacerbated the dysfunction of brain stem cardiovascular regulation. We noted that the level of augmented HIF-1a detected from proteins immunoprecipitated by SUMO-1 during the pro-life phase (Figure 6A) was not overtly more than the increase in HIF-1a determined from total protein (Figure 3A). It is likely that this seeming discrepancy is a demonstration of the phenomenon of SUMO enigma [31], which stipulates that only a small proportion of the available substrate protein needs to be sumoylated to achieve maximal effects. A well-known hypoxia responsive gene product is NOS II [19,26]. Melillo et al. [50] showed that a sequence homologous to a hypoxia-responsive enhancer (NOS II-HRE) is responsible for activation of nos II gene in murine macrophages. A putative HIF-1 site (CTACGTGCT) in the murine NOS II gene was subsequently shown to be crucial for hypoxia-induced transcription in pulmonary artery endothelial cells [51] and cardiomyocytes [52]. Results from our previous studies [11–14] indicated that NOS II/ peroxynitrite signaling in RVLM underlies the significant dysfunction of cardiovascular regulation seen during the pro- death phase of brain death. Since immunoneutralization of HIF- 1a or HIF-1b did not significantly affect the progressive augmentation of NOS II or nitrotyrosine levels in ventrolateral medulla in our animal model, the participation of HIF-1 as a cellular signal upstream to NOS II/peroxynitrite pathways is deemed minimal. Our results did lend credence to the notion that HIF-1 acts as the upstream cellular signal to NOS I/PKG pathways. Hypoxia increases NOS I expression that parallels activation of HIF-1a in piglet ventricular tissues [17]. An increase in NOS I and HIF-1a expression also occurs in cerebral cortex of anemic rats [18]. The present study further showed that immunoneutralization of HIF-1a or HIF-1b blunted the surge of NOS I or PKG expression during Phase I Mev intoxication. In conclusion, the present study provided novel demonstration that enhanced sumoylation and stabilization of HIF-1a in response to hypoxia in RVLM ameliorates the dysfunction of brain stem cardiovascular regulation in our Mev intoxication model of brain death via augmented expression selectively of the pro-life NOS I/ PKG signaling cascade. It is generally contended that hypoxia serves as the culprit for many abnormal brain functions. Nonetheless, working in conjunction with a neural substrate whose neuronal activity is reflected in the waxing and waning of the life-and-death signal, it is intriguing that our results demonstrated that enhanced protein sumoylation in response to hypoxia in RVLM in fact ameliorates brain stem cardiovascular regulatory failure associated with experimental brain death. This information, along with the signaling cascades identified in the present study, should provide further mechanistic insights into the etiology of brain death and offer new directions for the development of therapeutic strategy against this fatal eventuality aiming at preventing brain stem cardiovascular regulatory failure. Materials and Methods Ethics Statement All experimental procedures carried out in this study have been approved by the Laboratory Animal Committee of the Kaohsiung Chang Gung Memorial Hospital (CGMH97006), and were in compliance with the guidelines for animal care set forth by this Committee. PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17375