Data presented in this report provide first evidence that δ-opioid receptor activation by SNC-121 treatment significantly increases the protein acetylation of histone H2B, H3, and H4. We found the most robust acetylation of histone H3 in response to SNC-121 treatment. We further confirmed that SNC-121 increases histone H3 acetylation by HATs activation because pretreatment of cells with Garcinol (i.e., a selective HATs inhibitor) fully blocked SNC-121–induced histone H3 acetylation. Garcinol is a highly selective inhibitor of histone acetyl transferases, principally p300 and p300/CBP-associated factor, which is known to inhibit HATs activity in other systems.
52–54 Furthermore, δ-opioid receptor activation by SNC-121 treatment significantly decreases the activities of class I HDACs (e.g., HDAC 1, 2, and 3) and class IIb HDAC (HDAC 6). In contrast, we did not see any inhibitory effect of SNC-121 on class IIa and IV HDACs (HDAC 4, 5, 7, and 9–11) activities in ONH astrocytes, suggesting that δ-opioid receptor agonist selectively regulates the class I and class IIb HDACs activities in ONH astrocytes. This SNC-121–induced decrease in HDACs activities is due to δ-opioid receptor activation because a selective δ-opioid receptor antagonist (e.g., naltrindole) fully blocked SNC-121–induced reduction in HDAC activities. Moreover, we found that SNC-121 treatment attenuated the mRNA expression of HDAC 3 and 6 and decreases the protein expression levels of HDAC 1, 2, 3, and 6. Interestingly, mRNA expression of HDAC 1 and 2 was not inhibited by SNC-121 treatment. These data further suggest that δ-opioid receptor agonist is targeting upstream transcription factors that are regulating the transcription of specific HDACs (i.e., HDAC 3 and 6), whereas it can regulate other HDACs (i.e., HDAC-1 and 2) post-transcriptionally. To confirm this observation, we have used another set of HDAC 1 and 2 primers (
Table 2) and we did not see any changes in mRNA expression of HDAC 1 and 2 in response to SNC-121 treatment (data not shown), suggesting that δ-opioids differentially regulate HDACs mRNA expression at transcriptional level. Additionally, we did not see any decrease in protein expression of class IIa (4, 5, 7, and 9) and IV (11) HDACs in response to SNC-121 treatment. In other systems, studies have shown that TNF-α treatment suppressed HDAC 1 protein expression owing to proteasomal degradation without affecting its mRNA levels in ZR-75-1 cells.
55 Based on our observation and previously published work in other systems, we speculate that SNC-121–induced reduction in HDAC 1 and 2 protein expression are due to ubiquitination and/or proteosomal degradation process and not due to decrease in transcriptional activity. We also believe that SNC-121–induced increase in histone acetylation is due to both activation of HATs and decrease in HDACs expression in ONH astrocytes. However, this claim requires further experimental support, which will be tested in our future studies. In this study, we did not measure the effects of SNC-121 on HDAC class III (sirtuins 1–7); however, this work is under progress and it will be published in future studies. We also determined if δ-opioid receptor activation regulates the production of proinflammatory cytokines (e.g., TNF-α) and astrocyte activation. We provided evidence that LPS-induced TNF-α is fully blocked by SNC-121 treatment. LPS activates TLR-4 receptors and it has been frequently used to mimic stress/inflammatory conditions in numerous cell types.
19,56 In addition, in vivo studies herein showed that SNC-121 treatment decrease the astrocytes activation as measured by GFAP immunostaining, suggesting that δ-opioid receptor activation lowers inflammation and astrocytes activation under glaucomatous condition.