The Expression of Transcription Factors Mecp2 and CREB Is Modulated in Inflammatory Pelvic Pain

Abstract

Early activation of transcription factors is one of the epigenetic mechanisms contributing to the induction and maintenance of chronic pain states. Previous studies identified the changes in a number of nociception-related genes, such as calcitonin gene-related peptide (CGRP), substance P (SP), and brain-derived neurotropic factor (BDNF) in the pelvic organs after transient colonic inflammation. The gene and protein expression of these neuropeptides could be modulated by transcription factors Methyl-CpG-binding protein 2 (Mecp2) and cAMP response element-binding protein (CREB). In this study, we aimed to evaluate time-dependent changes in the expression levels of Mecp2 and CREB in the lumbosacral (LS) spinal cord and sensory ganglia after inflammation-induced pelvic pain in rat. Adult Sprague-Dawley rats were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce transient colonic inflammation. LS (L6-S2) spinal cord segments and respective dorsal root ganglias (DRGs) were isolated from control and experimental animals at 1, 2, 6, 24 h and 3 days post-TNBS treatment. Immunohistochemical (IHC) labeling and Western blotting experiments were performed to assess the expression of Mecp2, CREB and their phosphorylated forms. Total Mecp2 expression, but not phosphorylated p-Mecp2 (pS421Mecp2) expression was detected in the cells of the spinal dorsal horn under control conditions. Colonic inflammation triggered a significant decrease in the number of Mecp2-expressing neurons in parallel with elevated numbers of pS421Mecp2-expressing cells at 2 h and 6 h post-TNBS. The majority of Mecp2-positive cells (80 ± 6%) co-expressed CREB. TNBS treatment caused a transient up-regulation of CREB-expressing cells at 1 h post-TNBS only. The number of cells expressing phosphorylated CREB (pS133CREB) did not change at 1 h and 2 h post-TNBS, but was down-regulated by three folds at 6 h post-TNBS. Analysis of DRG sections revealed that the number of Mecp2-positive neurons was up-regulated by TNBS treatment, reaching three-fold increase at 2 h post-TNBS, and eight-fold increase at 6 h post-TNBS (p ≤ 0.05 to control). These data showed early changes in Mecp2 and CREB expression in the dorsal horn of the spinal cord and sensory ganglia after colonic inflammation, suggesting a possible contribution Mecp2 and CREB signaling in the development of visceral hyperalgesia and pelvic pain following peripheral inflammation.

Keywords: CREB; Mecp2; TNBS; dorsal root ganglia; inflammation; spinal cord; visceral pain.

Figure 1

Intracolonic 2,4,6-trinitrobenzenesulfonic acid (TNBS) treatment led to rapid changes in cellular expression of methyl-CpG-binding protein 2 (Mecp2) and pS421Mecp2 in the spinal afferent and efferent region. (A) Schematic representation of rat lumbosacral (LS) spinal cord. Maroon region in the superficial dorsal horn (laminae I-III; afferent) and the blue region in the intermediolateral (IML) region (efferent) of the spinal cord indicate from where the data images were taken. (B) Representative immunohistochemical (IHC) images from the dorsal horn of the spinal cord from saline-treated and TNBS-treated rats at 2-h post instillation. TNBS instillation led to a decrease in the number of Mecp2-positive cells (left panels) and an increase in the number of pS421Mecp2-positive cells (right panels) compared to the saline-treated controls. Scale bars: 50 μm. (C) TNBS instillation led to a progressive decrease in the number of Mecp2-positive cells in laminae I-III (n = 16–20, N = 4 per group, *p ≤ 0.05 compared to control). (D) TNBS instillation led to a significant increase in the number of pS421Mecp2-positive cells in laminae I-III (n = 16–20, N = 4 per group, *p ≤ 0.05 compared to control). (E) TNBS instillation led to a transient increase and subsequently decrease in the number of Mecp2-positive cells in the IML region of the spinal cord at 1 h after instillation (n = 16–20, N = 4 per group, *p ≤ 0.05 compared to control). (F) TNBS instillation led to a progressive increase in the number of pS421Mecp2-positive cells in the IML region of the spinal cord (n = 16–20, N = 4 per group, *p ≤ 0.05 compared to control).

Figure 2

Intracolonic TNBS treatment led to rapid changes in nuclear expression of cAMP response element-binding protein (CREB) and pS133CREB in the dorsal horn of the spinal cord. (A) Representative IHC images from the dorsal horn of the spinal cord from control and TNBS-treated rats at 2-h post instillation. CREB-positive cells largely overlap with Mecp2 positive cells. TNBS instillation led to a decrease in the number of CREB-positive cells in the dorsal horn. Scale bars: 50 μm. (B) TNBS instillation led to a transient decrease in the number of CREB-positive cells in the dorsal horn of the spinal cord (n = 12, N = 4 per group, *p ≤ 0.05 compared to control). (C) TNBS instillation led to a significant decrease in the number of pS133CREB-positive cells in the dorsal horn of the spinal cord (n = 12, N = 4 per group, *p ≤ 0.05 to control).

Figure 3

Molecular analysis of nuclear expression and phosphorylation of Mecp2 and CREB in the LS spinal cord following intracolonic TNBS treatment. (A) Representative images of CREB and pS133CREB expression (left panels) co-labeled with nuclear marker DRAQ (right panels) in the dorsal horn at 1 h after TNBS instillation. Both CREB and pS133CREB expression are restricted in the nuclei. Scale bars: 50 μm. (B) Representative Western blot experiment showing nuclear protein isolated from LS spinal cord of saline-treated control group and TNBS-treated group. There was no significant change of nuclear Mecp2 up to 6 h after TNBS instillation. (C) Nuclear pS421Mecp2 and pS133CREB were significantly upregulated in most of the time points after TNBS instillation (normalized to α-actin; N = 4 per group, *p ≤ 0.05 compared to control).

Figure 4

Intracolonic TNBS treatment led to phasic changes in neuronal expression of Mecp2, CREB and pS133CREB in LS sensory ganglia. (A) Representative image of IHC co-labeling of cellular expression of Mecp2 and CREB in LS dorsal root ganglias (DRGs; 2 h post-TNBS). Mecp2-positive labeling were detected in both the nuclei and cytosol in neurons as well as in satellite glial cells (SGCs), whereas CREB-positive labeling was mostly restricted to nuclei of the cells. Scale bar: 50 μm. (B) TNBS instillation led to significant increases in the number of Mecp2-positive neurons in LS DRGs up to 3 days post-TNBS treatment (n = 16, N = 4, *p ≤ 0.05 to compared to control). (C) Representative images of immunohistochemistry labeling of CREB in LS DRGs from control group and 2 h post-TNBS-instillation (TNBS-2 h). Scale bar: 50 μm. (D) TNBS instillation led to short-term decrease and long-term increase in the number of CREB-positive neurons in LS DRGs (n = 12, N = 4 per group, *p ≤ 0.05 compared to control). (E) Quantitative analysis of nuclear pS133CREB expression in LS DRGs of control group and TNBS-treated group (N = 4, *p ≤ 0.05 to compared to control). TNBS instillation led to short-term upregulation in nuclear pS133CREB in LS DRGs. (F) Representative images of nuclear CREB expression in bladder-projecting sensory neurons (yellow arrows) in LS DRGs. Bladder-projecting sensory neurons were retrogradely labeled with Fast Blue. CREB expression was detected in ~60% of bladder-projecting sensory neurons in LS DRGs (bottom panel). Scale bar: 50 μm.

Figure 5

Intracolonic TNBS treatment triggers transient increases in nuclear phosphorylated-RAC-alpha serine/threonine-protein kinase (p-Akt) and p-extracellular signal-regulated kinases 1/2 (p-Erk1/2) in the LS spinal cord and nuclear p-Akt in LS-DRGs. (A) Representative Western blotting gels showing the nuclear content of p-Erk1/2 and p-Akt isolated from LS spinal cord from the controls and TNBS-treated groups at 1, 2, 6 and 24 h after TNBS instillation (N = 4 per group, *p ≤ 0.05 compared to control). (B) TNBS instillation induced changes in nuclear content of p-Erk1/2 and p-Akt (normalized to α-actin). Phosphorylation of ERK1/2 (p-ERK1/2) was significantly upregulated at 1 h post-TNBS treatment and down regulated at 6 h post-TNBS treatment in the LS spinal cords (N = 4 per group, *p ≤ 0.05 compared to control). (C) Representative Western Blotting experiment showing nuclear content of p-AKT and p-ERK1/2 isolated from LS DRG at 1 and 2 h post-TNBS instillation (N = 4 per group, *p ≤ 0.05 compared to control). (D) TNBS installation led to significant upregulation of nuclear p-Akt in LS DRGs at 1 h and 2 h post-TNBS treatment (N = 4 per group, *p ≤ 0.05 compared to control).