Epigenetic regulation of genes that modulate chronic stress-induced visceral pain in the peripheral nervous system

Abstract

Background & aims: Chronic stress alters the hypothalamic-pituitary-adrenal axis, increases gut motility, and increases the perception of visceral pain. We investigated whether epigenetic mechanisms regulate chronic stress-induced visceral pain in the peripheral nervous systems of rats.

Methods: Male rats were subjected to 1 hour of water avoidance stress each day, or given daily subcutaneous injections of corticosterone, for 10 consecutive days. L4-L5 and L6-S2 dorsal root ganglia (DRG) were collected and compared between stressed and control rats (placed for 1 hour each day in a tank without water). Levels of cannabinoid receptor 1 (CNR1), DNA (cytosine-5-)-methyltransferase 1 (DNMT1), transient receptor potential vanilloid type 1 (TRPV1), and EP300 were knocked down in DRG neurons in situ with small interfering RNAs. We measured DNA methylation and histone acetylation at genes encoding the glucocorticoid receptor (NR3C1), CNR1, and TRPV1. Visceral pain was measured in response to colorectal distention.

Results: Chronic stress was associated with increased methylation of the Nr3c1 promoter and reduced expression of this gene in L6-S2, but not L4-L5, DRGs. Stress also was associated with up-regulation in DNMT1-associated methylation of the Cnr1 promoter and down-regulation of glucocorticoid-receptor-mediated expression of CNR1 in L6-S2, but not L4-L5, DRGs. Concurrently, chronic stress increased expression of the histone acetyltransferase EP300 and increased histone acetylation at the Trpv1 promoter and expression of the TRPV1 receptor in L6-S2 DRG neurons. Knockdown of DNMT1 and EP300 in L6-S2 DRG neurons of rats reduced DNA methylation and histone acetylation, respectively, and prevented chronic stress-induced increases in visceral pain.

Conclusions: Chronic stress increases DNA methylation and histone acetylation of genes that regulate visceral pain sensation in the peripheral nervous system of rats. Blocking epigenetic regulatory pathways in specific regions of the spinal cord might be developed to treat patients with chronic abdominal pain.

Keywords: Epigenetic Modification; HPA; Pain Sensitivity; Visceral Hyperalgesia.

Figure 1

Chronic water avoidance (WA) stress induced region-specific DNA methylation at the Nr3c1 promoter region and down-regulation in NR3C1 expression. (A) Pyrosequencing analysis of DNA methylation in the Nr3c1 exon 1₇ promoter region of L6-S2 DRG neurons from stressed and control rats. PCR neg: no input DNA; pos: MeDIP positive control using input DNA prepared from lumbar DRGs of control rats; neg: MeDIP negative control using normal mouse IgG; input: 4% input DNA from relevant DRGs for normalization; IP: MeDIP using anti-mouse 5-methylcytosine antibody. (B) Region-specific changes in DNA methylation of the Nr3c1 exon 1₇ promoter region measured by MeDIP and PCR. (C) Percentage change in Nr3c1 promoter methylation in control and stressed rats. (D) Relative mRNA levels of Nr3c1 in DRG neurons from control and stressed rats determined by qPCR. (E) Western blots for NR3C1 in different spinal (DRG) regions. (F) Quantification of the percentage of neurons that stained positive for NR3C1 in small vs. large L6-S2 DRG neurons. Data are expressed as mean ± stand error, n = 6 in each group. *P < 0.05, **P < 0.01 between control and WA stress rats.

Figure 2

Cnr1 transcription was regulated by NR3C1 in F11 cells. (A) DNA pull-down assay in F11 cells using synthesized glucocorticoid response elements (GREs). GRE1 (−95 to −124) and GRE2 (−247 to −276) represent two GREs near the Cnr1 TSS. (B) Cnr1 promoter transcription activity in F11 cells that were transfected with four different Cnr1 constructs: −1545 to +87, −1022 to +87, −376 to +87, and −203 to +87 from TSS. Transfected F11 cells were treated with Nr3c1 siRNA or vehicle. (C) Western immunoblots for NR3C1 in transfected F11 cells treated with/without Nr3c1-siRNA. * P<0.05; n = 4 for each treatment.

Figure 3

NR3C1 regulated Cnr1 expression and function in DRG neurons in chronic stress in a region-specific manner. (A) ChIP using anti-NR3C1 antibody followed by PCR for Cnr1 promoter (−145 to −42) in L6-S2 and L4–L5 DRG neurons from control and stressed rats. PCR neg: no input DNA; ChIP pos: ChIP using input DNA prepared from lumbar DRGs of control rats; ChIP neg: ChIP with normal rabbit IgG. Input: 4% input DNA from relevant DRGs for normalization. (B) ChIP analysis of NR3C1 binding to Cnr1 promoter in L6-S2 DRG neurons in RU486-treated stressed rats. (C) Percentage of NR3C1 binding to Cnr1 promoter in L6-S2 DRG neurons. (D) Q-PCR analysis of relative Cnr1 mRNA levels in L6-S2 DRG neurons. (E) Western blots for NR3C1 and CNR1 in L6-S2 DRG neurons of rats treated with Nr3c1 siRNA or random sequence siRNA (control) delivered in situ. (F) Visceral motor response to colorectal distension in stressed and control rats treated with Cnr1 siRNA or vehicle (veh) delivered in situ. *P < 0.05 for stressed rats compared to controls or RU486-treated stressed rats; ^#P < 0.05 for stressed rats or Cnr1 siRNA-treated controls compared to vehicle-treated controls; two-way ANOVA followed by Bonferroni test; n = 6–8 per group.

Figure 4

Increased DNMT1 expression and function in L6-S2 DRGs in chronic WA stress. (A) Western blots for DNMTs in L6-S2 DRG neurons from control and stressed rats. (B) Western immunoblots for DNMT1 in isolated nuclei fragmentation from L6-S2 DRGs of the stressed and control rats. (C) DNMT1 activity in nuclei of L6-S2 DRG neurons. (D) Immunofluorescence staining of DNMT1 (shown red) in L6-S2 DRG neurons innervating the colon. CTB-FITC (shown green) was used for retrograde labeling. Scale bar: 40 μm. (E) CORT treatment of isolated DRGs from control rats with/without RU486. n = 5 in each group.

Figure 5

DNMT1 mediates Cnr1 promoter methylation and visceral pain perception in chronic stress. (A) MeDIP of Cnr1 promoter region 1 (−376 to −146) and 2 (−145 to −42) in L6-S2 DRG neurons of rats with/without RU486. (B) Cnr1 promoter transcription activity in constructs treated with CORT or vehicle with/without pre-methylation. *P < 0.05 for CORT-treated compared to vehicle; ^#P < 0.01 for pre-methylated compared to control; ^¤P < 0.01 for CORT-treated compared to pre-methylated CORT-treated. (C) MeDIP of Cnr1 promoter region 2 in L6-S2 DRG neurons from rats treated with/without Dnmt1 siRNA during WA stress. (D) Western blots for DNMT1 and CNR1 in L6-S2 DRG neurons from rats treated with/without Dnmt1 siRNA. (E) Visceral pain in stressed rats treated with Dnmt1 siRNA compared to vehicle. *P < 0.05 for stressed rats compared to controls; ^#P < 0.05 for stressed rats compared to Dnmt1 siRNA–treated stressed rats; two-way ANOVA followed by Bonferroni test. n = 6–8 in each group.

Figure 6

Histone acetyltransferase EP300 regulates Trpv1 histone H3 acetylation, protein expression and visceral pain perception in chronic stress. (A) ChIP using anti–acetyl-histone H3 antibody followed by PCR for Trpv1 promoter in L6-S2 DRG neurons. (B) Percentage of H3 acetylation (normalized to control) at Trpv1 promoter in groups treated with/without RU486. *P < 0.05 for the stressed group compared to the control or RU486-treated group. (C) Western blots for EP300 in L6-S2 DRG neurons from control and stressed rats (n = 4 rats per group). (D) ChIP analysis using anti-EP300 antibody followed by PCR for Trpv1 promoter in L6-S2 DRG neurons from stressed rats treated with Ep300 siRNA compared to untreated. (E) ChIP analysis using anti–acetyl-histone H3 antibody followed by PCR for Trpv1 promoter in L6-S2 DRG neurons from stressed rats treated with Ep300 siRNA compared to untreated rats. (F) Visceral pain assessment in stressed rats treated with Ep300 siRNA or Trpv1 siRNA compared to untreated rats. *P < 0.05 for stressed rats compared to the control or siRNA-treated group; two-way ANOVA followed by Bonferroni test. n = 6–8 in each group.

Figure 7

Proposed epigenetic regulation of CNR1 and TRPV1 pathways underlying chronic stress–induced visceral hyperalgesia in primary nociceptive DRG neurons. Chronic stress increases serum corticosterone, which activates glucocorticoid receptors (NR3C1) and mineralocorticoid receptors (NR3C2). NR3C1 and NR3C2 activation enhances transcription of DNA methyltransferases (DNMTs), i.e. DNMT1, and histone acetyltransferases (HATs), i.e. EP300. This leads to increased methylation of Cnr1 promoter region, culminating in decreased transcription factor (NR3C1) binding and thus reduced Cnr1 transcription and expression, and increased histone acetylation of Trpv1 promoter, resulting in increased Trpv1 transcription and function. These events occur preferentially in nociceptive DRG neurons innervating the GI tract.