Sumoylation of HIF-1a in Experimental Brain Death Figure 2. Severe hypoxia and preferential augmentation of protein sumoylation in RVLM during the pro-life phase. (A) Representative continuous tracings showing temporal changes of tissue oxygen concentration (upper panel) or microvascular perfusion (lower panel) in RVLM of rats that received intravenous administration of Mev (640 mg kg21, at dashed line) during early Phase I (EMI) and Phases I and II (MI and MII) Mev intoxication. BPU: blood perfusion unit. (B and C) Illustrative gels or summary of fold changes against sham (S) controls of nuclear or cytosolic proteins that were conjugated to SUMO-1 (B) or Ubc9 protein expression (C) detected in ventrolateral medulla during Phases EMI, MI and MII Mev intoxication or in aCSF (V) controls. Since comparable results were obtained from all corresponding time intervals after animals received microinjection of aCSF, only one set of data is presented in this and subsequent figures for clarity. 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 bottom of the gels correspond to columns in the data summary. (D) Representative laser scanning confocal microscopic images showing cells in RVLM that were immunoreactive to SUMO-1 (green fluorescence) and additionally stained positively for a neuronal marker, neuron-specific nuclear protein (NeuN; red 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, 20 mm. doi:10.1371/journal.pone.0017375.g002 Nucleus-bound translocation of HIF implies enhanced tran- scription activity via the formation of heterodimer between HIF- 1b and HIF-1a or HIF-2a in the nucleus [39–41]. Results from electrophoresis mobility shift assay (EMSA) (Figure 4A and 4B) demonstrated a significant increase in the association of HIF-1 with its consensus DNA oligonucleotide in nuclear extracts from ventrolateral medulla during the pro-life Phase I, which returned during the pro-death Phase II to levels exhibited by sham or aCSF controls. We confirmed that this association was not due to non- specific binding when a competitive assay using unlabeled oligonucleotide probe resulted in discernible disappearance of HIF-1 DNA binding (Figure 4A and 4B). Supershift experiments (Figure 4B) revealed that an antiserum against HIF-1a, but not HIF-1b or HIF-2a (data not shown), retarded the migration of proteins that interacted with the double-stranded HIF-1 oligonu- cleotide. Immunoblot analysis of proteins immunoprecipitated by an anti-HIF-1b antiserum from the nuclear fraction of ventrolat- eral medullary samples additionally confirmed a preferential augmentation in the complex formed between HIF-1b and HIF- 1a during the pro-life phase of Mev intoxication (Figure 4C). On the other hand, the association between HIF-1b and HIF-2a during both phases was unaffected (Figure 4C). As a control (Figure 4D), minimal association between HIF-1b and HIF-1a or HIF-2a was detected from the cytosolic fraction during both phases. The activation of HIF-1a in RVLM is causally involved in central cardiovascular regulation in our model of brain death (Figure 5). Immunoneutralization of HIF-1a or HIF-1b in RVLM significantly enhanced the progressive hypotension and antago- nized the increase in power density of LF component of SAP signals during Phase I Mev intoxication, without affecting HR. Furthermore, the hypotension and reduced LF power already exhibited during Phase II was again significantly augmented. On the other hand, pretreatment with the same dose of anti-HIF-2a antiserum (Figure 5) was ineffective against the phasic cardiovas- cular responses induced by Mev. Preferential sumoylation of HIF-1a in RVLM during the pro-life phase As a well-documented target for SUMO-1 [33–36], our fourth series of experiments evaluated whether augmentation of sumoyla- tion of HIF-1a is causally related to its enhanced stability or transcriptional activity in RVLM during the pro-life phase. Results from immunoblot analysis of HIF-1a in proteins immunoprecip- itated by SUMO-1 showed a preferential increase during Phase I Mev intoxication in the association of SUMO-1 and HIF-1a in both the nuclear and cytosolic fractions from ventrolateral medulla (Figure 6A). Comparable results were obtained from immunoblot analysis of SUMO-1 from proteins immunoprecipitated by HIF- 1a (Figure 6B). Furthermore, microinjection bilaterally of an anti- SUMO-1 or anti-Ubc9 antiserum (1:20) in RVLM significantly blunted the preferentially augmented HIF-1a expression (Figure 6C) and antagonized the heightened transcription activity of HIF-1a in whole cell lysate of ventrolateral medulla during this pro-life phase (Figure 6D). Activation of HIF-1 leads to phasic upregulation of NOS I/ PKG signaling in RVLM We demonstrated previously [11–14] that whereas NOS I/ PKG signaling in RVLM is responsible for the pro-life phase, NOS II/peroxynitrite signaling underlies the pro-death phase of Mev intoxication. Our final series of experiments assessed whether the upregulation of HIF-1 may subserve its pro-life role via modulation of these two signaling pathways. Immunoneutraliza- tion of HIF-1a or HIF-1b (Figure 7) blunted significantly and selectively the Mev-induced Phase I increase in NOS I or PKG protein expression in ventrolateral medulla. None of these pretreatments affected the progressive increase in NOS II or nitrotyrosine (marker for peroxynitrite) during both phases of Mev intoxication. Again, anti-HIF-2a antiserum was ineffective (Figure 7) against the phasic Mev-induced NOS I, PKG, NOS II or nitrotyrosine protein expression in ventrolateral medulla. Discussion HIF-1 activation plays an important role in tissue preservation as a response to regional hypoxia [42]. Of the very limited amount of information available on the functional role of HIF-1 in the brain, HIF-1a activation in cortical neurons produces rescue proteins in response to intermittent hypoxia [43]. HIF-1 is a heterodimer of two basic helix-loop-helix/PAS proteins, HIF-1a and HIF-1b [40]. HIF-1a is rapidly degraded under normoxic conditions by the ubiquitin-proteasome system, and HIF-1b is constitutively ex- pressed in the nucleus and its level is not significantly affected by oxygen levels [41]. Hypoxia stabilizes HIF-1a, and nucleus-bound translocation of the stabilized HIF-1a allows for formation of the HIF-1ab heterodimer that becomes transcriptionally active [39]. Jewell et al. [44] showed that human epithelial carcinoma cell line HeLaS3 already exhibited nuclear HIF-1a protein induction and HIF-1 DNA binding after the introduction of hypoxia for less than 2 min, reaching maximal levels one hour after anoxic/hypoxic exposure. Based on complementary results from Western blot analysis, EMSA, confocal microscopy and immunoprecipitation, we demonstrated that the repertoire of cellular events that included nucleus-bound translocation of HIF-1a, followed by formation of HIF-1ab heterodimer in RVLM neurons during the pro-life phase of Mev intoxication exhibited a time-course that paralleled the elicited drastic hypoxia in RVLM. More importantly, loss-of- function manipulations in conjunction with monitors of circulatory events showed that the activated HIF-1a ameliorated brain stem cardiovascular regulatory failure during the progression towards experimental brain death. We further found that HIF-2a plays no part in this process. PLoS ONE | www.plosone.org 5 March 2011 | Volume 6 | Issue 3 | e17375