GA inhibits the activation and expression of Jmjd3 after SCI
To determine whether GA inhibits the activity and expression of Jmjd3
and thereby exhibits the neuroprotective effect after SCI, we first
examined a structure-based docking study to explore whether GA directly
inhibits the H3K27me3 demethylation activity of Jmjd3. A co-crystal
structure of GA bound to Jmjd3 revealed the critical interactions within
the active site (Fig. 2A). GA binds to Jmjd3 by maintaining interactions
with Asn1393A, which is located at the entrance to the binding pocket
(Fig. 2A; right), and GA revealed a high total docking score, polar
score, and low crash score, indicating the presence of noncovalent
interactions, such as hydrogen bond interactions (Total Score: 5.46,
Polar Score: 5.45, and Crash Score: -0.87). To confirm the inhibitory
effect of GA on Jmjd3 enzymatic activity, we performed an in
vitro Jmjd3 inhibition assay by using GSK-J4, an inhibitor of Jmjd3 as
a positive control. As expected, the level of H3K27me3 was higher in
GSK-J4-treated group than in control group, indicating that GSK-J4
inhibits Jmjd3 activity (Fig. 2B; J4). GA treatment also increased the
level of H3K27me3 in a dose-dependent manner. (Fig. 2B; GA). Thus, these
results suggest that GA directly binds to the active site of Jmjd3 and
inhibits Jmjd3 activity (Fig. 2B).
Next, we examined the effect of GA on Jmjd3 expression by RT-PCR and
Western blot analysis. The level of Jmjd3 mRNA and protein was increased
after injury as compared with that of the sham control. Furthermore,
SCI-induced increase of Jmjd3 mRNA and protein expression was
significantly inhibited by GA treatment at 6 h and 1 d after injury as
compared with those of the vehicle control (Fig. 3A, B). Because Jmjd3
encodes a histone demethylase that specifically mediates the removal of
methyl groups from H3K27me3/me, the effect of GA on the level of histone
H3K27me3 was then examined. The result showed that the protein level of
H3K27me3 was markedly decreased at 8 h and 1 d after injury, indicating
that the activity of Jmjd3 is increased after SCI. In contrast, the
level of H3K27me3 was significantly higher in the GA-treated group than
in the vehicle group (Fig. 3C). Furthermore, double immunofluorescence
with the endothelial cell marker RECA1 clearly revealed that Jmjd3
expression was upregulated in the blood vessels of injured spinal cord
at 1 d after injury (Fig. 3D; Veh), whereas not observed in the blood
vessels of uninjured control spinal cord (Fig. 3D; Sham).