Chronic Stress-Induced Visceral Hyperalgesia: Evidence for Epigenetic Regulation of DNA Methyltransferase I (Dnmt1) in Dorsal Root Ganglion (DRG) Neurons Innervating the Colon in the Rat

Abstract

Introduction: Increasing evidence suggests that chronic stress alters behavior and modifies epigenetic regulation of genes in the central nervous system. DNA methylation, catalyzed by DNA methyltransferases (DNMTs), is an important epigenetic mechanism of transcriptional control of gene expression. We reported previously that chronic psychological stress induced visceral hyperalgesia and differential alterations in the expression of several genes in DRG neurons innervating the colon in the rat. A potential role for epigenetic regulation in peripheral sensory pathways has not been investigated. Objectives: We examined the hypothesis that DNMTs play an important role in the regulation of chronic stress-induced visceral hyperalgesia. Methods: Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days as a chronic stress paradigm. SiRNA for DNMT1 was administrated in situ to L6-S2 DRGs every other day during the stress procedure. The visceromotor response (VMR) to colorectal distension was measured. Retrograde labeling with cholera toxin B (CTB)-FITCwas employed to identify colonDRG neurons. Immunofluorescence andWestern blot analysis were used to assess protein expression. In Vitro studies were performed in isolated control DRGs in the presence or absence of corticosterone (CORT; 10 μM) and RU-486 (corticoid receptor antagonist, 500 nM). Results: WA stress rats demonstrated significant increases in the level of DNMT1 and DNMT3b but not DNMT3a in L6-S2 DRGs compared with the controls. Enzyme activity assessment showed a 42% increase in DNMT1 activity in L6-S2 DRGs in stressed rats. Immunofluorescence studies revealed a significant increase in DNMT1 in small-sized, C-fiber neurons in WA stressed rats (52.3±2.2%) compared with the control (31.7±1.6%). Retrograde labeling demonstrated that 72.0±2.1% of the CTB-FITC labeled colonic DRG neurons were positive for DNMT1 in stressed rats compared to 40.0±6.5% in controls (P<0.05; n=4). The VMR in WA stressed rats was increased 68% and 92% above control responses at pressures of 40 and 60 mm Hg, respectively. Treatment of stressed rats with siRNA for DNMT1 prevented the VMR enhancement and changes in DNMT1 proteins levels in L6-S2 DRGs. In addition, treatment of control L6-S2 DRGs In Vitro with CORT (10 μM) increased DNMT1 expression level that was prevented by RU-486 (500 nM) (P<0.05). Conclusions: These data support the novel and provocative interpretation that: 1. Chronic stress induces epigenetic regulation of genes in primary nociceptive neurons; 2. DNA methyltransferase 1 (DNMT1) plays an important role in modulation of chronic stress-induced visceral hyperalgesia; and 3. DNA methyltransferases represent a potential target for treatment of functional GI disorders associated with visceral hyperalgesia.