The integral interaction of signaling components in the regulation of visceral inflammation-induced central sensitization in the spinal cord has not been well studied. Here we report that phosphoinositide 3-kinase (PI3K)-dependent Akt activation and N-Methyl-D-aspartic acid receptor (NMDAR) in lumbosacral spinal cord independently regulates the activation of cAMP response element-binding protein (CREB) in vivo in a rat visceral pain model of cystitis induced by intraperitoneal injection of cyclophosphamide (CYP). We demonstrate that suppression of endogenous PI3K/Akt activity with a potent PI3K inhibitor LY294002 reverses CYP-induced phosphorylation of CREB, however, it has no effect on CYP-induced phosphorylation of NR1 at Ser897 and Ser896; conversely, inhibition of NMDAR in vivo with MK801 fails to block CYP-induced Akt activation but significantly attenuates CYP-induced CREB phosphorylation in lumbosacral spinal cord. This novel interrelationship of PI3K/Akt, NMDAR, and CREB activation in lumbosacral spinal cord is further confirmed in an ex vivo spinal slice culture system exposed to an excitatory neurotransmitter calcitonin gene-related peptide (CGRP). Consistently we found that CGRP-triggered CREB activation can be blocked by both PI3K inhibitor LY294002 and NMDAR antagonists MK801 and D-AP5. However, CGRP-triggered Akt activation cannot be blocked by MK801 or D-AP5; vice versa, LY294002 pretreatment that suppresses the Akt activity fails to reverse CGRP-elicited NR1 phosphorylation. These results suggest that PI3K/Akt and NMDAR independently regulates spinal plasticity in visceral pain model, and target of a single pathway is necessary but not sufficient in treatment of visceral hypersensitivity.

Brain-derived neurotrophic factor (BDNF) plays an essential role in sensory neuronal activation in response to visceral inflammation. Here we report that BDNF up-regulation in the primary afferent neurons in the dorsal root ganglia (DRG) in a rat model of colitis is mediated by the activation of endogenous extracellular signal-regulated protein kinases (ERK) 5 and by nerve growth factor (NGF) retrograde signaling. At 7 days of colitis, the expression level of BDNF is increased in conventional neuronal tracing dye Fast Blue labeled primary afferent neurons project to the distal colon. In these neurons, the phosphorylation (activation) level of ERK5 is also increased. In contrast, the level of phospho-ERK1/2 is not changed in the DRG during colitis. Prevention of the ERK5 activation in vivo with an intrathecal application of the MEK inhibitor PD98059 significantly attenuates the colitis-induced increases in BDNF expression in the DRG. Further studies show that BDNF up-regulation in the DRG is triggered by NGF retrograde signaling which also involves activation of the MEK/ERK pathways. Application of exogenous NGF exclusively to the compartment containing DRG nerve terminals in an ex vivo ganglia-nerve preparation has markedly increased the BDNF expression level in the DRG neuronal cell body that is placed in a different compartment; this BDNF elevation is attenuated by U0126, PD98059 and a specific ERK5 inhibitor BIX02188. These results demonstrate the mechanisms and pathways by which BDNF expression is elevated in primary sensory neurons following visceral inflammation that is mediated by increased activity of ERK5 and is likely to be triggered by the elevated NGF level in the inflamed viscera.

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPAR␥ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPAR␥ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPAR␥ and cytosol IB and decreased nuclear NF-B protein levels. PPAR␥ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPAR␥ competitor assay showed that the Ki for 25HC3S was ϳ1 M, similar to those of other known natural ligands. NF-B-dependent promoter reporter gene assays showed that 25HC3S suppressed TNF␣-induced luciferase activities only when cotransfected with pcDNAI-PPAR␥ plasmid. In addition, 25HC3S decreased LPSinduced expression and release of IL-1␤. In the PPAR␥-specific siRNA transfected macrophages or in the presence of PPAR␥-specific antagonist, 25HC3S failed to increase IB and to suppress TNF␣ and IL-1␤ expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPAR␥ and cytosol IB and increased nuclear NF-B protein levels. We conclude that 25HC3S acts in macrophages as a PPAR␥ ligand and suppresses inflammatory responses via the PPAR␥/IB/NF-B signaling pathway.peroxisome proliferator-activated receptor-␥; oxysterols; oxysterol sulfation; cholesterol metabolites; inflammatory response; macrophages; nuclear factor-B signaling pathway MACROPHAGES ARE THE KEY CELLULAR PLAYERS in the pathogenesis of atherosclerosis. In the early stage of atherosclerosis, macrophages in arterial walls accumulate lipids. These lipid-loaded macrophages, termed foam cells, are characteristic of a reversible early cellular phase of atherosclerotic lesions. Progressive lipid accumulation leads to further escalation of inflammatory responses and infiltration of inflammatory cells (26). Through this process, early cellular lesions are transformed to late, fibrous, atherosclerotic plaques. Physiological or pharmacological maneuvers that reduce macrophage lipids and inflammatory responses may be effective in preventing or reversing atherosclerosis.Nuclear receptors are ligand-activated transcription factors that regulate the expression of target genes to affect processes as diverse as reproduction, inflammation, development, and metabolism (17). Nuclear receptor peroxisome proliferatoractivated receptors (PPARs) play major roles in the regulation of lipid metabolism, glucose homeostasis, and inflammatory processes and may be ideal targets for therapeutic management strategies for cardiovascular diseases (4,6,11,16,23,42). PPAR␥ appears particularly important in regulating genes involved in lipid metabolism and inflammation (1,8,20,21). The PPAR␥...

BackgroundSpinal central sensitization is an important process in the generation and maintenance of visceral hypersensitivity. The release of brain-derived neurotrophic factor (BDNF) from the primary afferent neurons to the spinal cord contributes to spinal neuronal plasticity and increases neuronal activity and synaptic efficacy. The N-Methyl-D-aspartic acid (NMDA) receptor possesses ion channel properties, and its activity is modulated by phosphorylation of its subunits including the NMDA receptor 1 (NR1).MethodsColonic inflammation was induced by a single dose of intracolonic instillation of tri-nitrobenzene sulfonic acid (TNBS). NR1 phosphorylation by BDNF in vivo and in culture was examined by western blot and immunohistochemistry. Signal transduction was studied by direct examination and use of specific inhibitors.ResultsDuring colitis, the level of NR1 phospho-Ser896 was increased in the dorsal horn region of the L1 and S1 spinal cord; this increase was attenuated by injection of BDNF neutralizing antibody to colitic animals (36 μg/kg, intravenous (i.v.)) and was also reduced in BDNF+/− rat treated with TNBS. Signal transduction examination showed that the extracellular signal-regulated kinase (ERK) activation was not involved in BDNF-induced NR1 phosphorylation. In contrast, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediated BDNF-induced NR1 phosphorylation in vivo and in culture; this is an additional pathway to the phospholipase C-gamma (PLCγ) and the protein kinase C (PKC) that was widely considered to phosphorylate NR1 at Ser896. In spinal cord culture, the inhibitors to PLC (U73122), PKC (bisindolylmaleimide I), and PI3K (LY294002), but not MEK (PD98059) blocked BDNF-induced NR1 phosphorylation. In animals with colitis, treatment with LY294002 (50 μg/kg, i.v.) blocked the Akt activity as well as NR1 phosphorylation at Ser896 in the spinal cord.ConclusionBDNF participates in colitis-induced spinal central sensitization by up-regulating NR1 phosphorylation at Ser896. The PI3K/Akt pathway, in addition to PLCγ and PKC, mediates BDNF action in the spinal cord during colitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0371-z) contains supplementary material, which is available to authorized users.

The transient receptor potential cation channel subfamily V member 1 (TRPV1), also known as the capsaicin receptor or vanilloid receptor 1 (VR1), is expressed in nociceptive neurons in the dorsal root ganglia (DRG) and participates in the transmission of pain. The present study investigated the underlying molecular mechanisms by which TRPV1 was regulated by nerve growth factor (NGF) signaling pathways in colonic hypersensitivity in response to colitis. We found that during colitis TRPV1 protein levels were significantly increased in specifically labeled colonic afferent neurons in both L1 and S1 DRGs. TRPV1 protein up-regulation in DRG was also enhanced by NGF treatment. We then found that TRPV1 protein up-regulation in DRG was regulated by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway both in vivo and in vitro. Suppression of endogenous PI3K/Akt activity during colitis or NGF treatment with a specific PI3K inhibitor LY294002 reduced TRPV1 protein production in DRG neurons, and also reduced colitis-evoked TRPV1-mediated visceral hypersensitivity tested by hyper-responsiveness to colorectal distention (CRD) and von Frey filament stimulation of abdomen. Further studies showed that TRPV1 mRNA levels in the DRG were not regulated by either colitis or NGF. We then found that an up-regulation of the protein synthesis pathway was involved by which both colitis and NGF caused a PI3K-dependent increase in the phosphorylation level of eukaryotic translation initiation factor 4E-binding protein (4E-BP)1. These results suggest a novel mechanism in colonic hypersensitivity which involves PI3K/Akt-mediated TRPV1 protein, not mRNA, up-regulation in primary afferent neurons, likely through activation of the protein synthesis pathways.