BACKGROUNDS & AIMS Chronic stress alters the hypothalamic–pituitary–adrenal axis, increases gut motility, and increases 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 hr 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 was also associated with upregulation in DNMT1-associated methylation of the Cnr1 promoter and downregulation 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.

Background Chronic psychological stress (CPS) is associated with increased intestinal epithelial permeability and visceral hyperalgesia. It is unknown whether corticosterone (CORT) plays a role in mediating alterations of epithelial permeability in response to CPS. Methods Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous CORT injection daily for 10 consecutive days in the presence or absence of corticoid-receptor antagonist RU-486. The visceromotor response (VMR) to colorectal distension (CRD) was measured. The in situ single-pass intestinal perfusion was used to measure intestinal permeability in jejunum and colon simultaneously. Key Results We observed significant decreases in the levels of glucocorticoid receptor (GR) and tight junction proteins in the colon but not the jejunum in stressed rats. These changes were largely reproduced by serial CORT injections in control rats and were significantly reversed by RU-486. Stressed and CORT-injected rats demonstrated a 3-fold increase in permeability for PEG-400 (MW) in colon but not jejunum and significant increase in VMR to CRD, which was significantly reversed by RU-486. In addition, no differences in permeability to PEG-4,000 and PEG-35,000 were detected between control and WA groups. Conclusions & Inferences Our findings indicate that CPS was associated with region-specific decrease in epithelial tight junction protein levels in the colon, increased colon epithelial permeability to low-molecular weight macromolecules which were largely reproduced by CORT treatment in control rats and prevented by RU-486. These observations implicate a novel, region-specific role for CORT as a mediator of CPS-induced increased permeability to macromolecules across the colon epithelium.

Background & Aims Chronic stress is associated with visceral hyperalgesia in functional gastrointestinal disorders. We investigated whether corticosterone plays a role in chronic psychological stress-induced visceral hyperalgesia. Methods Male rats were subjected to 1-hour water avoidance (WA) stress or subcutaneous corticosterone injection daily for 10 consecutive days in the presence or absence of corticoid receptor antagonist RU-486 and cannabinoid receptor agonist WIN55,212-2. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Receptor protein levels were measured and whole-cell patch-clamp recordings employed to assess TRPV1 currents in L6-S2 DRG neurons. Mass spectrometry was used to measure endocannabinoid anandamide content. Results Chronic WA stress was associated with visceral hyperalgesia in response to CRD, increased stool output and reciprocal changes in CB1 (decreased) and TRPV1 (increased) receptor expression and function. Treatment of WA stressed rats with RU-486 prevented these changes. Control rats treated with serial injections of corticosterone in situ demonstrated a significant increase in serum corticosterone associated with visceral hyperalgesia, enhanced anandamide content, increased TRPV1 and decreased CB1 receptor protein levels which were prevented by co-treatment with RU-486. Exposure of isolated control L6-S2 DRGs in vitro to corticosterone reproduced the changes in CB1 and TRPV1 receptors observed in situ which was prevented by co-treatment with RU-486 or WIN55,212-2. Conclusions These results support a novel role for corticosterone to modulate CB1 and TRPV1 receptor pathways in L6-S2 DRGs in the chronic WA stressed rat which contributes to visceral hyperalgesia observed in this model.

Chronic stress and elevated glucocorticoid hormone are associated with decreases in the intestinal epithelial tight junction protein claudin-1 (CLDN1). Human/rat CLDN1 promoters contain glucocorticoid response elements (GREs) and adjacent transcription repressor HES1 binding N-boxes. Notch signaling target HES1 expression was high and glucocorticoid receptor (NR3C1) low at the crypt base and the pattern reversed at the crypt apex. Chronic stress reduced overall rat colon HES1 and NR3C1 that was associated with CLDN1 downregulation. Chromatin-immunoprecipitation experiments showed that HES1 and NR3C1 bind to the CLDN1 promoter in rat colon crypts. The binding of NR3C1 but not HES1 to CLDN1 promoter significantly decreased in chronically stressed animals, which was prevented by the NR3C1 antagonist RU486. We employed the 21-day Caco-2/BBe cell model to replicate cell differentiation along the crypt axis. HES1 siRNA treatment early in differentiation increased CLDN1. In contrast, stress levels of cortisol decreased CLDN1 in late differentiation stage but not in the early stage. HES1 was high, whereas NR3C1 and CLDN1 were low in the early stage which reversed in the late stage, e.g. HES1/NR3C1 binding to CLDN1 promoter demonstrates a dynamic and reciprocal pattern. These results suggest that chronic stress impairs colon epithelium homeostasis and barrier function via different mechanisms along the crypt axis.

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.