Sumoylation of Hypoxia-Inducible Factor-1α Ameliorates Failure of Brain Stem Cardiovascular Regulation in Experimental Brain Death

One aspect of brain death is cardiovascular deregulation because asystole invariably occurs shortly after its diagnosis. A suitable neural substrate for mechanistic delineation of this aspect of brain death resides in the rostral ventrolateral medulla (RVLM). RVLM is the origin of a life-and-death signal that our laboratory detected from blood pressure of comatose patients that disappears before brain death ensues. At the same time, transcriptional upregulation of heme oxygenase-1 in RVLM by hypoxia-inducible factor-1α (HIF-1α) plays a pro-life role in experimental brain death, and HIF-1α is subject to sumoylation activated by transient cerebral ischemia. It follows that sumoylation of HIF-1α in RVLM in response to hypoxia may play a modulatory role on brain stem cardiovascular regulation during experimental brain death.A clinically relevant animal model that employed mevinphos as the experimental insult in Sprague-Dawley rat was used. Biochemical changes in RVLM during distinct phenotypes in systemic arterial pressure spectrum that reflect maintained or defunct brain stem cardiovascular regulation were studied. Western blot analysis, EMSA, ELISA, confocal microscopy and immunoprecipitation demonstrated that drastic tissue hypoxia, elevated levels of proteins conjugated by small ubiquitin-related modifier-1 (SUMO-1), Ubc9 (the only known conjugating enzyme for the sumoylation pathway) or HIF-1α, augmented sumoylation of HIF-1α, nucleus-bound translocation and enhanced transcriptional activity of HIF-1α in RVLM neurons took place preferentially during the pro-life phase of experimental brain death. Furthermore, loss-of-function manipulations by immunoneutralization of SUMO-1, Ubc9 or HIF-1α in RVLM blunted the upregulated nitric oxide synthase I/protein kinase G signaling cascade, which sustains the brain stem cardiovascular regulatory machinery during the pro-life phase.We conclude that sumoylation of HIF-1α in RVLM ameliorates brain stem cardiovascular regulatory failure during experimental brain death via upregulation of nitric oxide synthase I/protein kinase G signaling. This information should offer new therapeutic initiatives against this fatal eventuality

Preferential nucleus-bound translocation of HIF-1α in RVLM during the pro-life phase.

<p>(<i>A</i> and <i>B</i>) Illustrative gels or summary of fold changes against sham (S) controls in ratio of total protein of HIF-1α, HIF-1β or HIF-2α relative to β-actin protein (<i>A</i>), or nuclear or cytosolic content of HIF-1α or HIF-1β protein (<i>B</i>) detected in ventrolateral medulla during Phases EMI, MI and MII Mev intoxication or in aCSF (V) controls. Values are mean ± SEM of triplicate analyses on samples pooled from 4–6 animals in each group. *<i>P</i><0.05 versus aCSF group in the post hoc Scheffé multiple-range analysis. Note numbers on top of the gels correspond to columns in the data summary. (<i>C</i>) Representative laser scanning confocal microscopic images showing cells in RVLM that were immunoreactive to NeuN (green fluorescence) and additionally stained positively for HIF-1α or HIF-1β 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 µm.</p

Severe hypoxia and preferential augmentation of protein sumoylation in RVLM during the pro-life phase.

<p>(<i>A</i>) 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 µg kg⁻¹, at dashed line) during early Phase I (EMI) and Phases I and II (MI and MII) Mev intoxication. BPU: blood perfusion unit. (<i>B</i> and <i>C</i>) Illustrative gels or summary of fold changes against sham (S) controls of nuclear or cytosolic proteins that were conjugated to SUMO-1 (<i>B</i>) or Ubc9 protein expression (<i>C</i>) 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 ± SEM of triplicate analyses on samples pooled from 4–6 animals in each group. *<i>P</i><0.05 versus aCSF group in the post hoc Scheffé multiple-range analysis. Note numbers on bottom of the gels correspond to columns in the data summary. (<i>D</i>) 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 µm.</p

Sumoylation of proteins in RVLM ameliorates failure of central cardiovascular regulation in Mev intoxication model of brain death.

<p>Temporal changes in mean systemic arterial pressure (MSAP), heart rate (HR) or power density of the low-frequency (LF) component of SAP signals in rats that received pretreatment by microinjection bilaterally into RVLM of an anti-SUMO-1 or anti-Ubc9 antiserum (1∶20), normal rabbit serum (NRS,1∶20; vehicle for anti-SUMO antiserum) or normal mouse serum (NMS, 1∶20; vehicle for anti-Ubc9 antiserum), 30 min before local application (at arrow) of artificial cerebrospinal fluid (aCSF) or mevinphos (Mev; 10 nmol) to the bilateral RVLM. Values are mean ± SEM, n = 5–7 animals per experimental group. *<i>P</i><0.05 versus NRS+aCSF or NMS+aCSF group, and ⁺<i>P</i><0.05 versus NRS+Mev or NMS+Mev group at corresponding time-points in the post hoc Scheffé multiple-range test. B, baseline.</p

Preferential sumoylation of HIF-1α in RVLM during the pro-life phase.

<p>(<i>A</i> and <i>B</i>) Illustrative gels or summary of fold changes against sham (S) controls of HIF-1α from proteins immunoprecipitated by anti-SUMO-1 antiserum (<i>A</i>); or SUMO-1 from proteins immunoprecipitated by anti-HIF-1α antiserum (<i>B</i>) in the nuclear or cytosolic fraction of samples collected from ventrolateral medulla of sham (S) or aCSF (V) controls during Phases MI and MII Mev intoxication. (<i>C</i> and <i>D</i>) Illustrative gels or summary of fold changes against sham (S) controls in ratio of HIF-1α relative to β-actin protein in whole cell lysate (<i>C</i>) or transcriptional activity of HIF-1α in nuclear extract measured by an ELISA-based HIF-1 transcription factor assay kit (<i>D</i>) that detects the amount of HIF-1 binding to an oligonucleotide containing the hypoxia response element sequence from rats that received pretreatment by microinjection bilaterally into RVLM of an anti-SUMO-1 or anti-Ubc9 antiserum (1∶20), NRS (1∶20) or NMS (1∶20), 30 min before local application of aCSF or Mev (10 nmol) to the bilateral RVLM. Values are mean ± SEM of triplicate analyses on samples pooled from 4–6 animals in each group. *<i>P</i><0.05 versus aCSF, NRS or NMS group in the post hoc Scheffé multiple-range test. Note numbers below (<i>A, B</i>) or on top (<i>C</i>) of the gels correspond to columns in the data summary.</p

Activation of HIF-1α in RVLM ameliorates failure of central cardiovascular regulation associated with experimental brain death.

<p>Temporal changes in MSAP, HR or power density of LF component of SAP signals in rats that received pretreatment by microinjection bilaterally into RVLM of NRS (1∶20), anti-HIF-1α, HIF-1β or HIF-2α antiserum (1∶20), 30 min before local application (at arrow) of aCSF or Mev (10 nmol) to the bilateral RVLM. Values are mean ± SEM, n = 5–7 animals per experimental group. *<i>P</i><0.05 versus NRS+aCSF group, and ⁺<i>P</i><0.05 versus NRS+Mev group at corresponding time-points in the post hoc Scheffé multiple-range test. B, baseline.</p

HDAC4 represses p21(WAF1/Cip1) expression in human cancer cells through a Sp1-dependent, p53-independent mechanism.

Cancer cells have complex, unique characteristics that distinguish them from normal cells, such as increased growth rates and evasion of anti-proliferative signals. Global inhibition of class I and II histone deacetylases (HDACs) stops cancer cell proliferation in vitro and has proven effective against cancer in clinical trials, at least in part, through transcriptional reactivation of the p21(WAF1/Cip1)gene. The HDACs that regulate p21(WAF1/Cip1) are not fully identified. Using small interfering RNAs, we found that HDAC4 participates in the repression of p21(WAF1/Cip1) through Sp1/Sp3-, but not p53-binding sites. HDAC4 interacts with Sp1, binds and reduces histone H3 acetylation at the Sp1/Sp3 binding site-rich p21(WAF1/Cip1) proximal promoter, suggesting a key role for Sp1 in HDAC4-mediated repression of p21(WAF1/Cip1). Induction of p21(WAF1/Cip1) mediated by silencing of HDAC4 arrested cancer cell growth in vitro and inhibited tumor growth in an in vivo human glioblastoma model. Thus, HDAC4 could be a useful target for new anti-cancer therapies based on selective inhibition of specific HDACs