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).