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. 2016 Dec 7:12:1744806916682242.
doi: 10.1177/1744806916682242. Print 2016.

G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury

Lingli Liang  1 , Jian-Yuan Zhao  1   2 , Xiyao Gu  1 , Shaogen Wu  1 , Kai Mo  1 , Ming Xiong  1 , Brianna Marie Lutz  1 , Alex Bekker  1 , Yuan-Xiang Tao  3   4   5
Affiliations

G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury

Lingli Liang et al. Mol Pain. .

Abstract

Neuropathic pain, a distressing and debilitating disorder, is still poorly managed in clinic. Opioids, like morphine, remain the mainstay of prescribed medications in the treatment of this disorder, but their analgesic effects are highly unsatisfactory in part due to nerve injury-induced reduction of opioid receptors in the first-order sensory neurons of dorsal root ganglia. G9a is a repressor of gene expression. We found that nerve injury-induced increases in G9a and its catalyzed repressive marker H3K9m2 are responsible for epigenetic silencing of Oprm1, Oprk1, and Oprd1 genes in the injured dorsal root ganglia. Blocking these increases rescued dorsal root ganglia Oprm1, Oprk1, and Oprd1 gene expression and morphine or loperamide analgesia and prevented the development of morphine or loperamide-induced analgesic tolerance under neuropathic pain conditions. Conversely, mimicking these increases reduced the expression of three opioid receptors and promoted the mu opioid receptor-gated release of primary afferent neurotransmitters. Mechanistically, nerve injury-induced increases in the binding activity of G9a and H3K9me2 to the Oprm1 gene were associated with the reduced binding of cyclic AMP response element binding protein to the Oprm1 gene. These findings suggest that G9a participates in the nerve injury-induced reduction of the Oprm1 gene likely through G9a-triggered blockage in the access of cyclic AMP response element binding protein to this gene.

Keywords: G9a; dorsal root ganglion; nerve injury; opioid receptor.

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Figures

Figure 1.
Figure 1.
G9a is essential for nerve injury-induced downregulation of opioid receptors in the injured DRG. (a–c) The amounts of Ehmt2, Oprm1, Oprk1, and Oprd1 mRNAs in the ipsilateral (Ipsi) and contralateral (Con) L4 DRG (a) and the levels of G9a’s two protein isoforms, MOR, KOR, and H3K9me2 proteins in the ipsilateral L4 DRG (b and c) from G9afl/fl mice with microinjection of AAV5-GFP (GFP) or AAV5-Cre (Cre) into the ipsilateral L4 DRG on day 7 post-SNL or sham surgery. L, long isoform; S, short isoform. n = 12 mice/group for RT-PCR and 6 mice/group for Western blots. *P < 0.05 or **P < 0.01 vs the sham mice injected with AAV5-GFP, #P < 0.05 or ##P < 0.01 vs the SNL mice injected with AAV5-GFP, one-way ANOVA followed by post hoc Tukey test. (d and e) The levels of G9a’s two protein isoforms, H3K9me2, MOR and KOR in the ipsilateral L4 DRG from G9a KO mice and G9afl/fl mice on day 7 post-SNL or sham surgery. n = 12 mice/group. *P < 0.05 or **P < 0.01 vs the corresponding sham G9afl/fl mice, ##P < 0.01 vs the corresponding SNL G9afl/fl mice, one-way ANOVA followed by post hoc Tukey test. (f and g) The level of H3K9me2 in the ipsilateral L4 DRG (f) and the amounts of Oprm1, Oprk1, and Oprd1 mRNAs in the ipsilateral (Ipsi) and contralateral (Con) L5 DRG (g) from mice injected intrathecally with vehicle (Veh) or BIX-01294 (BIX; 2.5 µg/day for 6 days starting at the surgical day) on day 7 after SNL or sham surgery. n = 6 mice/group for Western blots and 12 mice/group for RT-PCR. *P < 0.05 or **P < 0.01 vs the group treated with sham plus vehicle, #P < 0.05 or ##P < 0.01 vs the group treated with SNL plus vehicle, one-way ANOVA followed by post hoc Tukey test.
Figure 2.
Figure 2.
G9a overexpression induced the downregulation of opioid receptors in the DRG. (a and b) The amounts of Ehmt2, Oprm1, Oprk1, Oprd1 mRNAs (a) and G9a’s two protein isoforms, MOR, KOR, and H3K9me2 proteins (b) in the ipsilateral (Ipsi) and contralateral (Con) L3/4 DRG of mice with microinjection of HSV-G9a or HSV-GFP on day 7 post-viral injection. n = 12 mice/group for RT-PCR and 6 mice/group for Western blot. *P < 0.05 or **P < 0.01 vs the corresponding AAV5-GFP-treated group or the corresponding contralateral side by two-tailed paired t-test. (c) The expression of MOR- and KOR-labeled neurons in the ipsilateral L4 DRG from mice with microinjection of HSV-G9a or HSV-GFP into this DRG on day 7 after viral injection. Representative immunohistochemical images (left) and a summary of analysis on the number of MOR- and KOR-labeled neurons (right) are shown. n = 3 mice (6 sections/mouse)/group. **P < 0.01 vs the corresponding HSV-GFP-injected group by two-tailed paired t-test. Scale bar: 25 µm. (d) Co-expression of Ehmt2 mRNA with Oprm1, Oprk1, and Oprd1 mRNAs in individual small DRG neurons. Gapdh mRNA was used as a positive control. Con, no-DNA. (e and f) The amounts of Ehmt2, Oprm1, Oprk1, and Oprd1 mRNAs (e) and G9a’s two protein isoforms, MOR, KOR, and H3K9me2 proteins (f) in the cultured DRG neurons transduced with HSV-G9a or HSV-GFP. n = 3 repeats. *P < 0.05 or **P < 0.01 vs the corresponding AAV5-GFP-treated group by two-tailed paired t-test.
Figure 3.
Figure 3.
Increase of MOR-gated primary afferent neurotransmitter release in spinal dorsal horn promoted by G9a overexpression in the DRG. Patch clamp recording of lamina II spinal dorsal horn neurons on the ipsilateral side from mice on day 7 after microinjection of HSV-GFP or HSV-G9a into unilateral L3/4 DRG. (a) Examples of traces of miniature (m) EPSCs before and after DAMGO (1 μM) application and after DAMGO washout. (b) Effect of DAMGO (1 μM) on mEPSC frequencies in the HSV-GFP-injected group (n = 21 neurons, 13 mice) and the HSV-G9a-injected group (n = 21 neurons, 14 mice). *P < 0.05 vs the HSV-GFP-injected group before DAMGO; ##P < 0.01 vs the HSV-GFP-injected group after DAMGO, one-way ANOVA followed by post hoc Tukey test. (c) mEPSC amplitude was not altered in both groups before and after DAMGO application. (d) Examples of traces of the C-fiber input recorded before and after DAMGO (1 μM) application and after DAMGO washout. (e) Pair pulse ratio (PPR) was significantly decreased in the HSV-G9a-injected group (n = 23 neurons, 19 mice) compared to that in the HSV-GFP-injected group (n = 23 neurons, 20 mice) before DAMGO. DAMGO significantly increased the PPR in the HSV-GFP-injected group, but not in the HSV-G9a-injected group. *P < 0.05 or **P < 0.01 vs the HSV-GFP-injected group before DAMGO, one-way ANOVA followed by post hoc Tukey test. (f) The first peak amplitude was significantly increased in the HSV-G9a-injected group compared to the HSV-GFP-injected group before DAMGO. DAMGO dramatically decreased the first peak amplitude in the HSV-GFP-injected group compared to the HSV-G9a-injected group. *P < 0.05 or **P < 0.01 vs the HSV-GFP-injected group before DAMGO, one-way ANOVA followed by post hoc Tukey test.
Figure 4.
Figure 4.
DRG G9a knockout out improves morphine analgesia and prevents morphine tolerance and/or hyperalgesia in naïve or SNL mice. (a) The analgesic effect of morphine (1 mg/kg, s.c.) in naïve G9afl/fl mice with microinjection of AAV5-GFP or AAV5-Cre into unilateral L3/4 DRGs. n = 8–9 mice/group. **P < 0.01 vs the corresponding AAV5-GFP-injected group by two-tailed paired t-test. (b and c) Paw withdrawal responses to mechanical (b) and thermal (c) stimuli on the ipsilateral side after morphine withdrawal in the AAV5-GFP- or AAV5-Cre-injected naïve mice that received repeated morphine injections (20 mg/kg, s.c. twice daily for 8 days). PWF, paw withdrawal frequency. PWL, paw withdrawal latency. n = 5–6 mice/group. **P < 0.01 vs baseline (before morphine injection), two-way ANOVA followed by post hoc Tukey test. (d) Time course of morphine-induced analgesia in naïve G9afl/fl mice and G9aKO mice that received repeated morphine injections (20 mg/kg, s.c. twice daily for 8 days). MPAE, maximal possible analgesic effect. n = 5–6 mice/group. **P < 0.01 vs the corresponding G9afl/fl mice, two-way ANOVA followed by post hoc Tukey test. (e) The cumulative dose-response curves of morphine in naïve G9afl/fl mice and G9aKO mice on day 9 after repeated morphine injections as described above. n = 5–6 mice/group. (f) Time course of morphine-induced analgesia in the SNL G9afl/fl mice and G9aKO mice that received repeated morphine injections (20 mg/kg, s.c. twice daily for 4 days starting on day 6 after SNL). n = 5–6 mice/group. **P < 0.01 vs the corresponding G9afl/fl mice, two-way ANOVA followed by post hoc Tukey test. (g) The cumulative dose-response curves of morphine in the SNL G9afl/fl mice and G9aKO mice that received repeated morphine injections as described above.
Figure 5.
Figure 5.
DRG G9a knockout out improves loperamide analgesia and prevents loperamide tolerance in naïve or SNL mice. (a and b) The analgesic effect of loperamide (LOP, 2.5 mg/kg, s.c.) in the ipsilateral side (a) and contralateral side (b) of naïve G9afl/fl mice with microinjection of AAV5-GFP or AAV5-Cre into unilateral L3/4 DRGs. n = 5 mice/group. This effect can be reversed by Naltrexone (NAL, 5 mg/kg, i.p.). *P < 0.05 vs the corresponding AAV5-GFP-injected group or LOP alone group by two-tailed paired t-test. (c and d) Time course of loperamide-induced analgesia in the ipsilateral side (c) and contralateral side (d) of naïve G9afl/fl mice with microinjection of AAV5-GFP or AAV5-Cre into unilateral L3/4 DRGs that received repeated loperamide injections (10 mg/kg, s.c. twice daily for 5 days). n = 5 mice/group. **P < 0.01 vs the corresponding AAV5-GFP-injected group, two-way ANOVA followed by post hoc Tukey test. (e) Time course of loperamide-induced analgesia on the ipsilateral side of SNL G9afl/fl mice with microinjection of AAV5-GFP or AAV5-Cre into unilateral L4 DRGs that received repeated loperamide injections (10 mg/kg, s.c. twice daily for 3 days starting on day 7 after SNL). n = 5 mice/group. **P < 0.01 vs the corresponding AAV5-GFP-injected group, two-way ANOVA followed by post hoc Tukey test.
Figure 6.
Figure 6.
Increased G9a is associated with reduced binding of CREB to its binding motif within the Oprm1 gene in the injured DRG after SNL. (a) Two fragments (F1, −450/−280 bp; F5, +142/+312 bp), but not other fragments (F2, −310/−143 bp; F3, −164/−6 bp; F4, −7/+166 bp), from the promoter and 5’-end untranslated regions of the Oprm1 gene were immunoprecipitated by the rabbit anti-G9a (not by rabbit normal IgG) in rat DRGs. Input, total purified fragments. M, ladder marker. n = 3 repeats. (b) G9a bindings to F1 and F5 fragments within the Oprm1 gene in the injured DRGs on day 7 post-SNL or sham surgery. n = 9 rats/group. **P < 0.01 vs the corresponding sham group by two-tailed paired t-test. (c) H3K9me2 bindings to F1 (−483/−320) and F5 (+163/+358) fragments within the Oprm1 gene in the injured DRGs of G9afl/fl mice and G9aKO mice on day 7 post-SNL or sham surgery. n = 15 mice/group. **P < 0.01 vs the corresponding sham G9afl/fl mice. ##P < 0.05 vs the corresponding SNL G9afl/fl mice, one-way ANOVA followed by post hoc Tukey test. (d and e) The fragment of Oprm1 gene including putative CREB binding motif immunoprecipitated by the rabbit anti-CREB, but not by rabbit normal IgG, in the ipsilateral L4 DRG on day 7 after SNL or sham surgery (d) or in the cultured mouse DRG neurons transduced with HSV-G9a or HSV-GFP (e). Input, total purified fragments. M, ladder marker. n = 3 repeats/model or transduction. (f) The amounts of CREB, G9a long (L) and short (S) isoforms, and MOR proteins in the cultured mouse DRG neurons transduced with HSV-GFP plus AAV5-GFP, HSV-GFP plus AAV5-CREB, or HSV-G9a plus AAV5-CREB. Representative Western blots (left) and a summary of densitometric analysis (right) are shown. n = 3 repeats. *P < 0.05 or **P < 0.01 vs the corresponding HSV-GFP plus AAV5-GFP group, ##P < 0.01 vs the corresponding HSV-GFP plus AAV5-CREB group, one-way ANOVA followed by post hoc Tukey test. (g) Representative examples showing the co-localization of CREB with G9a or MOR in the DRG neurons (arrows). Arrowheads, single CREB- or MOR-labeled DRG neurons. Scale bar: 25 µm. (h) The levels of phosphorylated (p-) CREB and CREB proteins in the ipsilateral L4 DRG after SNL. Representative Western blots (left) and a summary of densitometric analysis (right) are shown. n = 6 mice/time point. *P < 0.05 or **P < 0.01 vs the corresponding control group (0 day), one-way ANOVA followed by post hoc Tukey test.
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