Growth Arrest and DNA-damage-inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats
Growth Arrest and DNA-damage-inducible Protein 45β-mediated DNA Demethylation of Voltage-dependent T-type Calcium Channel 3.2 Subunit Enhances Neuropathic Allodynia after Nerve Injury in Rats
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DOI：
10.1097/aln.0000000000001610
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发表时间：
2017-06-01
期刊：
影响因子：
8.8
通讯作者：
Peng, Hsien-Yu
中科院分区：
文献类型：
--
作者：
Lai, Cheng-Yuan;Hsieh, Ming-Chun;Peng, Hsien-Yu
背景：生长停滞和 DNA 损伤诱导蛋白 45 beta 通过 DNA 去甲基化重新激活甲基化沉默的神经可塑性相关基因。然而，生长停滞和 DNA 损伤诱导蛋白 45 β 依赖性去甲基化是否会导致神经性异常性疼痛相关的脊柱可塑性仍不清楚。方法：成年雄性 Sprague-Dawley 大鼠（659 只中的 654 只）接受脊神经结扎或假手术，鞘内应用或不应用以下其中一种药物：生长停滞和 DNA 损伤诱导蛋白 45 β信使RNA靶向小干扰RNA、表达生长停滞和DNA损伤诱导蛋白45β的慢病毒载体、Ro 25-6981（一种带有NR2B的N-甲基-D-天冬氨酸受体拮抗剂）或KN-93（一种钙调蛋白依赖性蛋白激酶II拮抗剂）用于行为测量、蛋白质印迹、 免疫荧光、斑点印迹、检测胞嘧啶-磷酸-鸟嘌呤位点未修饰的胞嘧啶富集、染色质免疫沉淀定量聚合酶链式反应分析和切片记录。结果：同侧背角神经元中神经结扎增强的生长停滞和 DNA 损伤诱导蛋白 45 β 表达 (n = 6) 伴有行为异常性疼痛（n = 7）。局部抑制生长停滞和 DNA 损伤诱导蛋白 45 β 表达，通过减少生长停滞和 DNA 损伤诱导蛋白 45 β 与电压依赖性 T 型钙通道 3.2 亚基启动子 (n = 6) 的结合，减轻结扎诱导的异常性疼痛 (n = 7)，从而降低电压依赖性 T 型钙通道 3.2 亚基启动子的表达和电流介导的电流 钙通道 3.2 亚基（均为 n = 6）。此外，带有 NR2B 的 N-甲基-D-天冬氨酸受体和钙调蛋白依赖性蛋白激酶 II 在上游级联中发挥作用，增加生长停滞和 DNA 损伤诱导蛋白 45β 的表达，从而增强电压依赖性 T 型钙通道 3.2 亚基启动子的去甲基化，并上调电压依赖性 T 型钙通道 3.2 亚基表达。鞘内给予 Ro 25-6981、KN-93 或生长停滞和 DNA 损伤诱导蛋白 45 β 靶向小干扰 RNA (n = 6)，通过增加相关的 5-甲酰胞嘧啶和 5-羧基胞嘧啶水平。结论：通过将 5-甲酰基胞嘧啶或 5-羧基胞嘧啶转化为未修饰的胞嘧啶，带有 NR2B 的 N-甲基-D-天冬氨酸受体、钙调蛋白依赖性蛋白激酶 II 或生长停滞和 DNA 损伤诱导蛋白 45 β 途径可促进电压依赖性 T 型钙的形成 通道3.2亚基基因去甲基化介导神经性异常性疼痛。
Background: Growth arrest and DNA-damage-inducible protein 45 beta reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage-inducible protein 45 beta-dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear.Methods: Adult male Sprague-Dawley rats (654 out of 659) received a spinal nerve ligation or a sham operation with or without intrathecal application of one of the following: growth arrest and DNA-damage-inducible protein 45 beta messenger RNA-targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage-inducible protein 45 beta, Ro 25-6981 (an NR2B-bearing N-methyl-D-aspartate receptor antagonist), or KN-93 (a calmodulin-dependent protein kinase II antagonist) were used for behavioral measurements, Western blotting, immunofluorescence, dot blots, detection of unmodified cytosine enrichment at cytosine-phosphate-guanine site, chromatin immunoprecipitation quantitative polymerase chain reaction analysis, and slice recordings.Results: Nerve ligation-enhanced growth arrest and DNA-damage-inducible protein 45 beta expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage-inducible protein 45 beta expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage-inducible protein 45 beta to the voltage-dependent T-type calcium channel 3.2 subunit promoter (n = 6) that decreased expression of and current mediated by the voltage-dependent T-type calcium channel 3.2 subunit (both n = 6). In addition, NR2B-bearing N-methyl-D-aspartate receptors and calmodulin-dependent protein kinase II act in an upstream cascade to increase growth arrest and DNA-damage-inducible protein 45 beta expression, hence enhancing demethylation at the voltage-dependent T-type calcium channel 3.2 subunit promoter and up-regulating voltage-dependent T-type calcium channel 3.2 subunit expression. Intrathecal administration of Ro 25-6981, KN-93, or a growth arrest and DNA-damage-inducible protein 45 beta-targeting small interfering RNA (n = 6) reversed the ligation-induced enrichment of unmodified cytosine at the voltage-dependent T-type calcium channel 3.2 subunit promoter by increasing the associated 5-formylcytosine and 5-carboxylcytosine levels.Conclusions: By converting 5-formylcytosine or 5-carboxylcytosine to unmodified cytosine, the NR2B-bearing N-methyl-D- aspartate receptor, calmodulin-dependent protein kinase II, or growth arrest and DNA-damage-inducible protein 45 beta pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.