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(266) TLR2 activation induces NGF gene and protein expression via NF-κB in human intervertebral disc cells
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Abstracts
The Journal of Pain
D11 Neurotransmitters and Neurotransmitter Receptors (264) AMPA glutamate receptors containing the GluA4 subunit are expressed in epidermal keratinocytes and may be involved in chronic itch and painful peripheral neuropathies ~ero, T Irie, Z Melyan, F Rice, and J Moron-Concepcion; Columbia D Caban University, New York, NY Epidermal keratinocytes express numerous neurosignaling molecules that were only thought to regulate cell differentiation prior to the discovery of extensive intraepidermal innervation by c- and Ad-fibers implicated in thermal, pain, and itch sensation. Immunocytochemical and pharmacological evidence indicates that excitatory and inhibitory mechanisms are expressed in differentially stratified epidermal patterns indicating that they may transduce or modulate tactile stimuli including those implicated in pain. In an experiment designed to explore AMPAR expression in epidermal nerve fibers from mouse, we found prominent immunolabeling (IL) for the GluA4 AMPAR subunit in mouse and human epidermal keratinocytes. In glabrous and hairy skin of the mouse, GluA4-IL was confirmed by immunological and molecular biology techniques. In addition, using whole-cell patch clamp recordings in cultured human keratinocytes we isolated AMPAR-mediated currents in the presence of the NMDAR blocker 2-amino-5-phosphonovalerate. Finally, we conducted immunological studies in human skin biopsies from patients with atopic dermatitis (AD) and patients with postherpetic neuralgia (PHN). In AD skin GluA4-IL was especially intense among pathological plaques of parakeratosis in the stratum corneum which normally lacks GluA4-IL. Interestingly, this increase in GluA4 was accompanied by an increase in the cytokine thymic stromal lymphopoietin (TSLP) which is thought to trigger both the initiation and maintenance of AD. In contrast, GluA4 expression was reduced in keratinocytes of PHN patients. Altogether, this study documents for the first time the expression of functional AMPAR in epidermal keratinocytes and highlights a possible role for these receptors in the mechanisms underlying chronic itch and painful peripheral neuropathies. Supported by NIH grant DA027460 to J.M.C.
(265) Resolving the molecular composition of synapses in central nociceptive pathways with array tomography Z Baber, C Tong, D Sch€adlich, F Yang, W Janssen, S Tappan, P Hof, and J Scholz; Columbia University Medical Center, New York City, New York, United States of America Inhibitory control of nociceptive connections in the dorsal horn of the spinal cord is essential for the adequate processing of pain-related information and its projection to the brain. Phasic activity of inhibitory interneurons in the spinal cord depends on excitatory input from primary somatosensory afferents, neighboring interneurons and descending pathways from the brainstem. We examined the molecular components that determine the synaptic input to gaminobutyric acid (GABA)ergic interneurons associated with nociceptive pathways in the mouse spinal cord. For this purpose, we utilized transgenic mice that express enhanced green fluorescent protein (eGFP) under the promoter of the GABA-synthesizing enzyme glutamate decarboxylase 1. Excitatory synapses on fluorescently labeled dendrites of GABAergic interneurons were investigated with array tomography, a technique that allows for an in-depth, high-resolution imaging of neuronal protein components in three dimensions. Ultrathin sections (70 nm) of resin-embedded spinal cord tissue were immunostained with fluorescently tagged antibodies to achieve a spatial resolution that is superior (along the z-axis) to confocal microscopy. The sections were restained repeatedly with different combinations of antibodies to permit analysis of multiple synapse components. More specifically, we obtained stereological estimates of the AMPA and NMDA receptor subunit expression at postsynaptic densities by implementing a modified physical dissector approach. The ability to visualize and quantify AMPA and NMDA receptor subtypes on the dendrites of GABAergic interneurons will be useful for elucidating the complex regulation of nociceptive pathways in the spinal cord, and for investigating mechanisms of synaptic plasticity in conditions of increased pain sensitivity, for example, after inflammation or peripheral nerve injury.
D12 Neurotrophins (266) TLR2 activation induces NGF gene and protein expression via NF-kB in human intervertebral disc cells E Krock, J Currie, D Rosenzweig, J Ouellet, M Weber, L Stone, and L Haglund; Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) is upregulated in disc degeneration. The presence of NGF may promote disc innervation, neuronal sensitization and low back pain, making NGF a possible therapeutic target. However, the mechanisms underlying the dysregulation of NGF during degeneration are poorly understood. During disc degeneration proteoglycans and other extra-cellular matrix (ECM) proteins are degraded and fragmented. ECM fragments can act as endogenous danger signal ligands for toll-like receptors, which were originally characterized in the innate immune system. TLR activation induces cytokine and chemokine expression, and could regulate a sterile inflammation in disc degeneration. NGF is often upregulated in inflammation, therefore we hypothesized that TLR activation leads to NGF overexpression in intervertebral disc cells. To test this hypothesis we treated human disc cells with IL-1b (control), peptidoglycan (PGN, TLR2 agonist) and lipopolysaccharide (LPS, TLR4 agonist) and evaluated NGF gene and protein expression. IL-1b and PGN treatment increased NGF gene expression after 6, 12 and 24 hours and protein secretion after 24 and 48 hours, while LPS had little effect. To confirm that PGN is acting through TLR2, we knocked down TLR2 with siRNA, which greatly decreased PGN-induced NGF expression. We evaluated which signaling pathways regulate NGF by western blot and found PGN treatment of disc cells increased p38 MAPK and NF-kB phosphorylation. Using small molecule signaling inhibitors, we found p38 inhibition had little effect on PGN induction of NGF, while NF-kB inhibition blocked NGF upregulation. Taken together, these results suggest that TLR2 activation regulates NGF through NF-kB signaling. Identification of this new mechanism of NGF regulation in intervertebral disc cells could lead to the development of new therapeutic strategies for LBP associated with disc degeneration.
D13 Peptides (267) AT2-ROS signaling induces peripheral mechanical, but not thermal hypersensitivity elicited by Angiotensin-II A Shepherd and D Mohapatra; The University of Iowa Carver College of Medicine, Iowa City, IA Angiotensin II (ANG-II), a proteolytic fragment of angiotensin I, was initially described for its vasoconstrictive properties and accordant increase in blood pressure. Pro-inflammatory effects of ANG-II have been reported in leukocytes, endothelial cells and vascular smooth muscle cells. Furthermore, agents that reduce ANG-II signaling (by targeting either angiotensin converting enzyme or AT1/AT2-type ANG-II receptors) have been proposed to have analgesic effects. However, the mechanisms responsible for any pro-algesic effect of ANG-II remain unclear. We show that intraplantar injection of ANG-II in mice caused significant mechanical hypersensitivity in a dose-dependent manner, without any significant alteration in thermal sensitivity. This mechanical hypersensitivity was absent in AT2 receptor-null mice, and could also be blocked by the co-injection of AT2 receptor-specific antagonist PD123319. Interestingly, ANG-II-induced mechanical hypersensitivity was unaffected by the co-injection of AT1 antagonist losartan, suggesting that ANG-II’s effect on mechanical sensitivity is mediated exclusively by AT2 receptor signaling. Consistent with this notion, production of reactive oxygen and nitrogen species (ROS/RNS) was elevated at the site of ANG-II injection, relative to saline-injected controls. The ROS/RNS scavenger N-acetylcysteine attenuated this elevated level of ROS/RNS, and abolished ANG-II-induced mechanical hypersensitivity, indicating that ROS/RNS are a necessary downstream component of these effects. Despite the enhanced mechanical sensitivity observed in vivo, functional Ca2+ imaging of cultured mouse DRG neurons showed no effect of ANG-II on capsaicin- or allyl isothiocyanate (AITC)-evoked Ca2+ flux, suggesting that ANG-II does not directly potentiate the activation of TRPV1 and TRPA1 ion channels on sensory neurons. This in turn suggests the involvement of other nociceptive ion channels and receptors, and/or a non-neuronal mechanism contributing to ANG-II’s effects on peripheral mechanical sensitivity. Since components of the renin angiotensin system, including ANG-II, are present at elevated levels at tissue injury and inflammation sites, ANG-II could presumably serve as a common mediator of inflammatory mechanical hyperalgesia.
Abstracts
The Journal of Pain
D11 Neurotransmitters and Neurotransmitter Receptors (264) AMPA glutamate receptors containing the GluA4 subunit are expressed in epidermal keratinocytes and may be involved in chronic itch and painful peripheral neuropathies ~ero, T Irie, Z Melyan, F Rice, and J Moron-Concepcion; Columbia D Caban University, New York, NY Epidermal keratinocytes express numerous neurosignaling molecules that were only thought to regulate cell differentiation prior to the discovery of extensive intraepidermal innervation by c- and Ad-fibers implicated in thermal, pain, and itch sensation. Immunocytochemical and pharmacological evidence indicates that excitatory and inhibitory mechanisms are expressed in differentially stratified epidermal patterns indicating that they may transduce or modulate tactile stimuli including those implicated in pain. In an experiment designed to explore AMPAR expression in epidermal nerve fibers from mouse, we found prominent immunolabeling (IL) for the GluA4 AMPAR subunit in mouse and human epidermal keratinocytes. In glabrous and hairy skin of the mouse, GluA4-IL was confirmed by immunological and molecular biology techniques. In addition, using whole-cell patch clamp recordings in cultured human keratinocytes we isolated AMPAR-mediated currents in the presence of the NMDAR blocker 2-amino-5-phosphonovalerate. Finally, we conducted immunological studies in human skin biopsies from patients with atopic dermatitis (AD) and patients with postherpetic neuralgia (PHN). In AD skin GluA4-IL was especially intense among pathological plaques of parakeratosis in the stratum corneum which normally lacks GluA4-IL. Interestingly, this increase in GluA4 was accompanied by an increase in the cytokine thymic stromal lymphopoietin (TSLP) which is thought to trigger both the initiation and maintenance of AD. In contrast, GluA4 expression was reduced in keratinocytes of PHN patients. Altogether, this study documents for the first time the expression of functional AMPAR in epidermal keratinocytes and highlights a possible role for these receptors in the mechanisms underlying chronic itch and painful peripheral neuropathies. Supported by NIH grant DA027460 to J.M.C.
(265) Resolving the molecular composition of synapses in central nociceptive pathways with array tomography Z Baber, C Tong, D Sch€adlich, F Yang, W Janssen, S Tappan, P Hof, and J Scholz; Columbia University Medical Center, New York City, New York, United States of America Inhibitory control of nociceptive connections in the dorsal horn of the spinal cord is essential for the adequate processing of pain-related information and its projection to the brain. Phasic activity of inhibitory interneurons in the spinal cord depends on excitatory input from primary somatosensory afferents, neighboring interneurons and descending pathways from the brainstem. We examined the molecular components that determine the synaptic input to gaminobutyric acid (GABA)ergic interneurons associated with nociceptive pathways in the mouse spinal cord. For this purpose, we utilized transgenic mice that express enhanced green fluorescent protein (eGFP) under the promoter of the GABA-synthesizing enzyme glutamate decarboxylase 1. Excitatory synapses on fluorescently labeled dendrites of GABAergic interneurons were investigated with array tomography, a technique that allows for an in-depth, high-resolution imaging of neuronal protein components in three dimensions. Ultrathin sections (70 nm) of resin-embedded spinal cord tissue were immunostained with fluorescently tagged antibodies to achieve a spatial resolution that is superior (along the z-axis) to confocal microscopy. The sections were restained repeatedly with different combinations of antibodies to permit analysis of multiple synapse components. More specifically, we obtained stereological estimates of the AMPA and NMDA receptor subunit expression at postsynaptic densities by implementing a modified physical dissector approach. The ability to visualize and quantify AMPA and NMDA receptor subtypes on the dendrites of GABAergic interneurons will be useful for elucidating the complex regulation of nociceptive pathways in the spinal cord, and for investigating mechanisms of synaptic plasticity in conditions of increased pain sensitivity, for example, after inflammation or peripheral nerve injury.
D12 Neurotrophins (266) TLR2 activation induces NGF gene and protein expression via NF-kB in human intervertebral disc cells E Krock, J Currie, D Rosenzweig, J Ouellet, M Weber, L Stone, and L Haglund; Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) is upregulated in disc degeneration. The presence of NGF may promote disc innervation, neuronal sensitization and low back pain, making NGF a possible therapeutic target. However, the mechanisms underlying the dysregulation of NGF during degeneration are poorly understood. During disc degeneration proteoglycans and other extra-cellular matrix (ECM) proteins are degraded and fragmented. ECM fragments can act as endogenous danger signal ligands for toll-like receptors, which were originally characterized in the innate immune system. TLR activation induces cytokine and chemokine expression, and could regulate a sterile inflammation in disc degeneration. NGF is often upregulated in inflammation, therefore we hypothesized that TLR activation leads to NGF overexpression in intervertebral disc cells. To test this hypothesis we treated human disc cells with IL-1b (control), peptidoglycan (PGN, TLR2 agonist) and lipopolysaccharide (LPS, TLR4 agonist) and evaluated NGF gene and protein expression. IL-1b and PGN treatment increased NGF gene expression after 6, 12 and 24 hours and protein secretion after 24 and 48 hours, while LPS had little effect. To confirm that PGN is acting through TLR2, we knocked down TLR2 with siRNA, which greatly decreased PGN-induced NGF expression. We evaluated which signaling pathways regulate NGF by western blot and found PGN treatment of disc cells increased p38 MAPK and NF-kB phosphorylation. Using small molecule signaling inhibitors, we found p38 inhibition had little effect on PGN induction of NGF, while NF-kB inhibition blocked NGF upregulation. Taken together, these results suggest that TLR2 activation regulates NGF through NF-kB signaling. Identification of this new mechanism of NGF regulation in intervertebral disc cells could lead to the development of new therapeutic strategies for LBP associated with disc degeneration.
D13 Peptides (267) AT2-ROS signaling induces peripheral mechanical, but not thermal hypersensitivity elicited by Angiotensin-II A Shepherd and D Mohapatra; The University of Iowa Carver College of Medicine, Iowa City, IA Angiotensin II (ANG-II), a proteolytic fragment of angiotensin I, was initially described for its vasoconstrictive properties and accordant increase in blood pressure. Pro-inflammatory effects of ANG-II have been reported in leukocytes, endothelial cells and vascular smooth muscle cells. Furthermore, agents that reduce ANG-II signaling (by targeting either angiotensin converting enzyme or AT1/AT2-type ANG-II receptors) have been proposed to have analgesic effects. However, the mechanisms responsible for any pro-algesic effect of ANG-II remain unclear. We show that intraplantar injection of ANG-II in mice caused significant mechanical hypersensitivity in a dose-dependent manner, without any significant alteration in thermal sensitivity. This mechanical hypersensitivity was absent in AT2 receptor-null mice, and could also be blocked by the co-injection of AT2 receptor-specific antagonist PD123319. Interestingly, ANG-II-induced mechanical hypersensitivity was unaffected by the co-injection of AT1 antagonist losartan, suggesting that ANG-II’s effect on mechanical sensitivity is mediated exclusively by AT2 receptor signaling. Consistent with this notion, production of reactive oxygen and nitrogen species (ROS/RNS) was elevated at the site of ANG-II injection, relative to saline-injected controls. The ROS/RNS scavenger N-acetylcysteine attenuated this elevated level of ROS/RNS, and abolished ANG-II-induced mechanical hypersensitivity, indicating that ROS/RNS are a necessary downstream component of these effects. Despite the enhanced mechanical sensitivity observed in vivo, functional Ca2+ imaging of cultured mouse DRG neurons showed no effect of ANG-II on capsaicin- or allyl isothiocyanate (AITC)-evoked Ca2+ flux, suggesting that ANG-II does not directly potentiate the activation of TRPV1 and TRPA1 ion channels on sensory neurons. This in turn suggests the involvement of other nociceptive ion channels and receptors, and/or a non-neuronal mechanism contributing to ANG-II’s effects on peripheral mechanical sensitivity. Since components of the renin angiotensin system, including ANG-II, are present at elevated levels at tissue injury and inflammation sites, ANG-II could presumably serve as a common mediator of inflammatory mechanical hyperalgesia.