title: 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.
abstract: Growth arrest and DNA-damage-inducible protein 45β reactivates methylation-silenced neural plasticity-associated genes through DNA demethylation. However, growth arrest and DNA-damage-inducible protein 45β-dependent demethylation contributes to neuropathic allodynia-associated spinal plasticity remains unclear.
  
  
  
  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 45β messenger RNA-targeted small interfering RNA, lentiviral vector expressing growth arrest and DNA-damage-inducible protein 45β, 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.
  
  
  
  Nerve ligation-enhanced growth arrest and DNA-damage-inducible protein 45β expression (n = 6) in ipsilateral dorsal horn neurons accompanied with behavioral allodynia (n = 7). Focal knockdown of growth arrest and DNA-damage-inducible protein 45β expression attenuated ligation-induced allodynia (n = 7) by reducing the binding of growth arrest and DNA-damage-inducible protein 45β 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 45β 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 45β-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.
  
  
  
  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 45β pathway facilitates voltage-dependent T-type calcium channel 3.2 subunit gene demethylation to mediate neuropathic allodynia.
authors.affiliation: From the Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan (C.-Y.L.); Department of Medicine, Mackay Medical College, New Taipei, Taiwan (C.-Y.L., M.-C.H., Y.-C.H., A.-S.L., H.-H.W., J.-K.C., Y.-P.C., H.-Y.P.); Department of Physiology, College of Medicine, National Taiwan University, Taipei, Taiwan (M.-C.H.); and Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan (J.-K.C.).
authors.forename: Cheng-Yuan
authors.initials: CY
authors.lastname: Lai
authors.forename: Ming-Chun
authors.initials: MC
authors.lastname: Hsieh
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Ho
authors.forename: An-Sheng
authors.initials: AS
authors.lastname: Lee
authors.forename: Hsueh-Hsiao
authors.initials: HH
authors.lastname: Wang
authors.forename: Jen-Kun
authors.initials: JK
authors.lastname: Cheng
authors.forename: Yat-Pang
authors.initials: YP
authors.lastname: Chau
authors.forename: Hsien-Yu
authors.initials: HY
authors.lastname: Peng
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004195:Disease Models, Animal; D005455:Fluorescent Antibody Technique; D006930:Hyperalgesia / Q000235:genetics / Q000378:metabolism*; D008297:Male; D009437:Neuralgia / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D017207:Rats, Sprague-Dawley; D013127:Spinal Nerves / Q000293:injuries* / Q000378:metabolism; D000097642:GADD45 Proteins
chemical_list: D000943:Antigens, Differentiation; D020747:Calcium Channels, T-Type; C508094:Gadd45b protein, rat; D000097642:GADD45 Proteins