SUMO-1 modification of IκBα controls NFκB
activation. A, cell models of SUMO-1 overexpression (105%,
increase in total SUMO-1 (13 kDa) and 70% increase in SUMO-1/IκBα
(55 kDa) (p < 0.001) and knockdown (small hairpin RNA) results in
appreciable (90% decrease in total SUMO-1 and SUMO-1/IκBα,
p < 0.01) modulation of SUMO-1/IκBα and SUMO-1 protein
levels. Nonspecific small hairpin RNA (NS) is shown as control.
B, cells transfected with pNRE-Luc were examined for NFκB
activity during hypoxia (24 h) in the presence of NECA (10 μm).
WT cells demonstrated attenuation of NFκB activity during Ado receptor
stimulation (*, p < 0.05). SUMO-1 knockdown cells
exposed to same conditions evidence significant increased NFκB activity
compared with WT cells (**, p < 0.01). C,
expression levels of SUMO-1 regulate NFκB activity at base-line
normoxia, during hypoxia and H/R. SUMO-1 knockdown cells demonstrated increase
NFκB activity in all conditions (*, p < 0.001);
however, the increase was more impressive during hypoxia (∼8-fold).
Overexpressing cells showed attenuated NFκB activity in all conditions
(**, p < 0.01). D and E, as
confirmatory evidence of NFκB attenuation by H/R and adenosine signaling
the profile of p65 translocation in normoxic conditions (D) was
compared with 24 h of hypoxia exposure (E). Here, a significant
nuclear localization was observed as shown by the arrows. In
contrast, cells pre-exposed to H/R followed by hypoxia did not evidenced
significant p65 translocation (F). G, cells coincubated with
NECA demonstrated a predominant cytoplasmic localization of p65, despite
hypoxia exposure. WT, wild type.