RESUMO
Cutaneous TRPV1+ neurons directly sense noxious stimuli, inflammatory cytokines, and pathogen-associated molecules and are required for innate immunity against some skin pathogens. Important unanswered questions are whether TRPV1+ neuron activation in isolation is sufficient to initiate innate immune responses and what is the biological function for TRPV1+ neuron-initiated immune responses. We used TRPV1-Ai32 optogenetic mice and cutaneous light stimulation to activate cutaneous neurons in the absence of tissue damage or pathogen-associated products. We found that TRPV1+ neuron activation was sufficient to elicit a local type 17 immune response that augmented host defense to C. albicans and S. aureus. Moreover, local neuron activation elicited type 17 responses and augmented host defense at adjacent, unstimulated skin through a nerve reflex arc. These data show the sufficiency of TRPV1+ neuron activation for host defense and demonstrate the existence of functional anticipatory innate immunity at sites adjacent to infection that depends on antidromic neuron activation.
Assuntos
Imunidade Inata/imunologia , Interleucina-23/metabolismo , Interleucina-6/metabolismo , Células Receptoras Sensoriais/imunologia , Pele/imunologia , Canais de Cátion TRPV/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Candida albicans/imunologia , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Optogenética/métodos , Pele/microbiologia , Staphylococcus aureus/imunologia , Canais de Cátion TRPV/genéticaRESUMO
Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 â¼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.
Assuntos
Dopamina/análise , Drosophila/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Comportamento Animal , Dopamina/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/genética , Canais de Cátion TRPV/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.
Assuntos
Neurônios/metabolismo , Neutrófilos/metabolismo , Infecções Estreptocócicas/patologia , Streptococcus pyogenes/patogenicidade , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Botulínicas Tipo A/administração & dosagem , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Caspase 1/deficiência , Caspase 1/genética , Diterpenos/farmacologia , Fasciite Necrosante/etiologia , Fasciite Necrosante/patologia , Fasciite Necrosante/veterinária , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neutrófilos/imunologia , Dor/etiologia , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Infecções Estreptocócicas/complicações , Infecções Estreptocócicas/veterinária , Streptococcus pyogenes/metabolismo , Estreptolisinas/imunologia , Estreptolisinas/metabolismo , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
The sensation of pain is associated with increased mortality, but it is unknown whether pain perception can directly affect aging. We find that mice lacking TRPV1 pain receptors are long-lived, displaying a youthful metabolic profile at old age. Loss of TRPV1 inactivates a calcium-signaling cascade that ends in the nuclear exclusion of the CREB-regulated transcriptional coactivator CRTC1 within pain sensory neurons originating from the spinal cord. In long-lived TRPV1 knockout mice, CRTC1 nuclear exclusion decreases production of the neuropeptide CGRP from sensory endings innervating the pancreatic islets, subsequently promoting insulin secretion and metabolic health. In contrast, CGRP homeostasis is disrupted with age in wild-type mice, resulting in metabolic decline. We show that pharmacologic inactivation of CGRP receptors in old wild-type animals can restore metabolic health. These data suggest that ablation of select pain sensory receptors or the inhibition of CGRP are associated with increased metabolic health and control longevity.
Assuntos
Longevidade , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Animais , Proteína de Ligação a CREB/metabolismo , Caenorhabditis elegans , Células Cultivadas , Dieta , Feminino , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neurônios/metabolismo , Nociceptores/metabolismo , Canais de Cátion TRPV/genética , Fatores de Transcrição/metabolismoRESUMO
Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.
Assuntos
Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/química , Humanos , Células HEK293 , Animais , Ratos , Simulação de Dinâmica Molecular , Microscopia Crioeletrônica , Cálcio/metabolismo , Técnicas de Patch-Clamp , Ácidos/metabolismoRESUMO
Intestinal macrophages are critical for gastrointestinal (GI) homeostasis, but our understanding of their role in regulating intestinal motility is incomplete. Here, we report that CX3C chemokine receptor 1-expressing muscularis macrophages (MMs) were required to maintain normal GI motility. MMs expressed the transient receptor potential vanilloid 4 (TRPV4) channel, which senses thermal, mechanical, and chemical cues. Selective pharmacologic inhibition of TRPV4 or conditional deletion of TRPV4 from macrophages decreased intestinal motility and was sufficient to reverse the GI hypermotility that is associated with chemotherapy treatment. Mechanistically, stimulation of MMs via TRPV4 promoted the release of prostaglandin E2 and elicited colon contraction in a paracrine manner via prostaglandin E receptor signaling in intestinal smooth muscle cells without input from the enteric nervous system. Collectively, our data identify TRPV4-expressing MMs as an essential component required for maintaining normal GI motility and provide potential drug targets for GI motility disorders.
Assuntos
Colo/fisiologia , Motilidade Gastrointestinal , Macrófagos/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Animais , Receptor 1 de Quimiocina CX3C/metabolismo , Colo/fisiopatologia , Ciclo-Oxigenase 1/deficiência , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/análise , Dinoprostona/metabolismo , Feminino , Mucosa Gástrica/citologia , Expressão Gênica , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Contração Muscular , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genéticaRESUMO
PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.
Assuntos
Metabolismo Energético , Canais de Cátion TRPV/metabolismo , Termogênese , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Feminino , Técnicas de Silenciamento de Genes , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Transativadores/metabolismo , Fatores de Transcrição , Proteína Desacopladora 1RESUMO
TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Canais de Cátion TRPV/genética , Anilidas/farmacologia , Animais , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Canais de Cálcio/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Capsaicina/farmacologia , Células Cultivadas , Cinamatos/farmacologia , Colite/imunologia , Humanos , Interleucina-10/genética , Intestinos/imunologia , Intestinos/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/biossínteseRESUMO
BACKGROUND: Neutrophil extracellular traps (NETs) are composed of DNA, enzymes, and citrullinated histones that are expelled by neutrophils in the process of NETosis. NETs accumulate in the aorta and kidneys in hypertension. PAD4 (protein-arginine deiminase-4) is a calcium-dependent enzyme that is essential for NETosis. TRPV4 (transient receptor potential cation channel subfamily V member 4) is a mechanosensitive calcium channel expressed in neutrophils. Thus, we hypothesize that NETosis contributes to hypertension via NET-mediated endothelial cell (EC) dysfunction. METHODS: NETosis-deficient Padi4-/- mice were treated with Ang II (angiotensin II). Blood pressure was measured by radiotelemetry, and vascular reactivity was measured with wire myography. Neutrophils were cultured with or without ECs and exposed to normotensive or hypertensive uniaxial stretch. NETosis was measured by flow cytometry. ECs were treated with citrullinated histone H3, and gene expression was measured by quantitative reverse transcription PCR. Aortic rings were incubated with citrullinated histone H3, and wire myography was performed to evaluate EC function. Neutrophils were treated with the TRPV4 agonist GSK1016790A. Calcium influx was measured using Fluo-4 dye, and NETosis was measured by immunofluorescence. RESULTS: Padi4-/- mice exhibited attenuated hypertension, reduced aortic inflammation, and improved EC-dependent vascular relaxation in response to Ang II. Coculture of neutrophils with ECs and exposure to hypertensive uniaxial stretch increased NETosis and accumulation of neutrophil citrullinated histone H3. Histone H3 and citrullinated histone H3 exposure attenuates EC-dependent vascular relaxation. Treatment of neutrophils with the TRPV4 agonist GSK1016790A increases intracellular calcium and NETosis. CONCLUSIONS: These observations identify a role of NETosis in the pathogenesis of hypertension. Moreover, they define an important role of EC stretch and TRPV4 as initiators of NETosis. Finally, they define a role of citrullinated histones as drivers of EC dysfunction in hypertension.
Assuntos
Armadilhas Extracelulares , Hipertensão , Camundongos Knockout , Proteína-Arginina Desiminase do Tipo 4 , Canais de Cátion TRPV , Animais , Armadilhas Extracelulares/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Camundongos , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Neutrófilos/metabolismo , Camundongos Endogâmicos C57BL , Masculino , Angiotensina II/farmacologia , Humanos , Histonas/metabolismo , Pressão Sanguínea , Células Cultivadas , Células Endoteliais/metabolismoRESUMO
Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.
Assuntos
Antineoplásicos , Canais de Potencial de Receptor Transitório , Canais de Cátion TRPV/genética , Rutênio Vermelho/farmacologia , Microscopia Crioeletrônica , Cálcio/metabolismoRESUMO
Upon its mucosal transmission, HIV type 1 (HIV-1) rapidly targets genital antigen-presenting Langerhans cells (LCs), which subsequently transfer infectious virus to CD4+ T cells. We previously described an inhibitory neuroimmune cross talk, whereby calcitonin gene-related peptide (CGRP), a neuropeptide secreted by peripheral pain-sensing nociceptor neurons innervating all mucosal epithelia and associating with LCs, strongly inhibits HIV-1 transfer. As nociceptors secret CGRP following the activation of their Ca2+ ion channel transient receptor potential vanilloid 1 (TRPV1), and as we reported that LCs secret low levels of CGRP, we investigated whether LCs express functional TRPV1. We found that human LCs expressed mRNA and protein of TRPV1, which was functional and induced Ca2+ influx following activation with TRPV1 agonists, including capsaicin (CP). The treatment of LCs with TRPV1 agonists also increased CGRP secretion, reaching its anti-HIV-1 inhibitory concentrations. Accordingly, CP pretreatment significantly inhibited LCs-mediated HIV-1 transfer to CD4+ T cells, which was abrogated by both TRPV1 and CGRP receptor antagonists. Like CGRP, CP-induced inhibition of HIV-1 transfer was mediated via increased CCL3 secretion and HIV-1 degradation. CP also inhibited direct CD4+ T cells HIV-1 infection, but in CGRP-independent manners. Finally, pretreatment of inner foreskin tissue explants with CP markedly increased CGRP and CCL3 secretion, and upon subsequent polarized exposure to HIV-1, inhibited an increase in LC-T cell conjugate formation and consequently T cell infection. Our results reveal that TRPV1 activation in human LCs and CD4+ T cells inhibits mucosal HIV-1 infection, via CGRP-dependent/independent mechanisms. Formulations containing TRPV1 agonists, already approved for pain relief, could hence be useful against HIV-1.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Infecções por HIV , Humanos , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Linfócitos T/metabolismo , Células de Langerhans/metabolismo , Mucosa/metabolismo , Capsaicina/farmacologia , Dor/metabolismo , Infecções por HIV/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
High-threshold dorsal root ganglion (HT DRG) neurons fire at low frequencies during inflammatory injury, and low-frequency stimulation (LFS) of HT DRG neurons selectively potentiates excitatory synapses onto spinal neurons projecting to the periaqueductal gray (spino-PAG). Here, in male and female mice, we have identified an underlying peripheral sensory population driving this plasticity and its effects on the output of spino-PAG neurons. We provide the first evidence that Trpv1-lineage sensory neurons predominantly induce burst firing, a unique mode of neuronal activity, in lamina I spino-PAG projection neurons. We modeled inflammatory injury by optogenetically stimulating Trpv1+ primary afferents at 2â Hz for 2â min (LFS), as peripheral inflammation induces 1-2â Hz firing in high-threshold C fibers. LFS of Trpv1+ afferents enhanced the synaptically evoked and intrinsic excitability of spino-PAG projection neurons, eliciting a stable increase in the number of action potentials (APs) within a Trpv1+ fiber-induced burst, while decreasing the intrinsic AP threshold and increasing the membrane resistance. Further experiments revealed that this plasticity required Trpv1+ afferent input, postsynaptic G protein-coupled signaling, and NMDA receptor activation. Intriguingly, an inflammatory injury and heat exposure in vivo also increased APs per burst, in vitro These results suggest that inflammatory injury-mediated plasticity is driven though Trpv1+ DRG neurons and amplifies the spino-PAG pathway. Spinal inputs to the PAG could play an integral role in its modulation of heat sensation during peripheral inflammation, warranting further exploration of the organization and function of these neural pathways.
Assuntos
Interneurônios , Substância Cinzenta Periaquedutal , Ratos , Animais , Camundongos , Feminino , Masculino , Ratos Sprague-Dawley , Células Receptoras Sensoriais , Inflamação , Canais de Cátion TRPV/genéticaRESUMO
Magnetogenetics was developed to remotely control genetically targeted neurons. A variant of magnetogenetics uses magnetic fields to activate transient receptor potential vanilloid (TRPV) channels when coupled with ferritin. Stimulation with static or RF magnetic fields of neurons expressing these channels induces Ca2+ transients and modulates behavior. However, the validity of ferritin-based magnetogenetics has been questioned due to controversies surrounding the underlying mechanisms and deficits in reproducibility. Here, we validated the magnetogenetic approach Ferritin-iron Redistribution to Ion Channels (FeRIC) using electrophysiological (Ephys) and imaging techniques. Previously, interference from RF stimulation rendered patch-clamp recordings inaccessible for magnetogenetics. We solved this limitation for FeRIC, and we studied the bioelectrical properties of neurons expressing TRPV4 (nonselective cation channel) and transmembrane member 16A (TMEM16A; chloride-permeable channel) coupled to ferritin (FeRIC channels) under RF stimulation. We used cultured neurons obtained from the rat hippocampus of either sex. We show that RF decreases the membrane resistance (Rm) and depolarizes the membrane potential in neurons expressing TRPV4FeRIC RF does not directly trigger action potential firing but increases the neuronal basal spiking frequency. In neurons expressing TMEM16AFeRIC, RF decreases the Rm, hyperpolarizes the membrane potential, and decreases the spiking frequency. Additionally, we corroborated the previously described biochemical mechanism responsible for RF-induced activation of ferritin-coupled ion channels. We solved an enduring problem for ferritin-based magnetogenetics, obtaining direct Ephys evidence of RF-induced activation of ferritin-coupled ion channels. We found that RF does not yield instantaneous changes in neuronal membrane potentials. Instead, RF produces responses that are long-lasting and moderate, but effective in controlling the bioelectrical properties of neurons.
Assuntos
Ferritinas , Neurônios , Animais , Ferritinas/metabolismo , Ratos , Neurônios/fisiologia , Masculino , Feminino , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Células Cultivadas , Campos Magnéticos , Ratos Sprague-Dawley , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Hipocampo/fisiologia , Hipocampo/citologiaRESUMO
Chronic, persistent itch is a devastating symptom that causes much suffering. In recent years, there has been great progress made in understanding the molecules, cells, and circuits underlying itch sensation. Once thought to be carried by pain-sensing neurons, itch is now believed to be capable of being transmitted by dedicated sensory labeled lines. Members of the Mas-related G protein-coupled receptor (Mrgpr) family demarcate an itch-specific labeled line in the peripheral nervous system. In the spinal cord, the expression of other proteins identifies additional populations of itch-dedicated sensory neurons. However, as evidence for labeled-line coding has mounted, studies promoting alternative itch-coding strategies have emerged, complicating our understanding of the neural basis of itch. In this review, we cover the molecules, cells, and circuits related to understanding the neural basis of itch, with a focus on the role of Mrgprs in mediating itch sensation.
Assuntos
Sistema Nervoso Periférico/metabolismo , Prurido/genética , Receptores Acoplados a Proteínas G/genética , Células Receptoras Sensoriais/metabolismo , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Nociceptividade/fisiologia , Sistema Nervoso Periférico/fisiopatologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Prurido/metabolismo , Prurido/fisiopatologia , Receptores do Fator Natriurético Atrial/genética , Receptores do Fator Natriurético Atrial/metabolismo , Receptores da Bombesina/genética , Receptores da Bombesina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/patologia , Transdução de Sinais , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Mucosal immunity is critical to host protection from enteric pathogens and must be carefully controlled to prevent immunopathology. Regulation of immune responses can occur through a diverse range of mechanisms including bi-directional communication with neurons. Among which include specialized sensory neurons that detect noxious stimuli due to the expression of transient receptor potential vanilloid receptor 1 (TRPV1) ion channel and have a significant role in the coordination of host-protective responses to enteric bacterial pathogens. Here we have used the mouse-adapted attaching and effacing pathogen Citrobacter rodentium to assess the specific role of TRPV1 in coordinating the host response. TRPV1 knockout (TRPV1-/-) mice had a significantly higher C. rodentium burden in the distal colon and fecal pellets compared to wild-type (WT) mice. Increased bacterial burden was correlated with significantly increased colonic crypt hyperplasia and proliferating intestinal epithelial cells in TRPV1-/- mice compared to WT. Despite the increased C. rodentium burden and histopathology, the recruitment of colonic T cells producing IFNγ, IL-17, or IL-22 was similar between TRPV1-/- and WT mice. In evaluating the innate immune response, we identified that colonic neutrophil recruitment in C. rodentium infected TRPV1-/- mice was significantly reduced compared to WT mice; however, this was independent of neutrophil development and maturation within the bone marrow compartment. TRPV1-/- mice were found to have significantly decreased expression of the neutrophil-specific chemokine Cxcl6 and the adhesion molecules Icam1 in the distal colon compared to WT mice. Corroborating these findings, a significant reduction in ICAM-1 and VCAM-1, but not MAdCAM-1 protein on the surface of colonic blood endothelial cells from C. rodentium infected TRPV1-/- mice compared to WT was observed. These findings demonstrate the critical role of TRPV1 in regulating the host protective responses to enteric bacterial pathogens, and mucosal immune responses.
Assuntos
Infecções por Enterobacteriaceae , Mucosa Intestinal , Camundongos , Animais , Mucosa Intestinal/metabolismo , Colo/patologia , Citrobacter rodentium , Células Endoteliais/metabolismo , Imunidade Inata , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
The TRPV3 channel plays vital roles in skin physiology. Dysfunction of TRPV3 causes skin diseases, including Olmsted syndrome. However, the lack of potent and selective inhibitors impedes the validation of TRPV3 as a therapeutic target. In this study, we identified Trpvicin as a potent and subtype-selective inhibitor of TRPV3. Trpvicin exhibits pharmacological potential in the inhibition of itch and hair loss in mouse models. Cryogenic electron microscopy structures of TRPV3 and the pathogenic G573S mutant complexed with Trpvicin reveal detailed ligand-binding sites, suggesting that Trpvicin inhibits the TRPV3 channel by stabilizing it in a closed state. Our G573S mutant structures demonstrate that the mutation causes a dilated pore, generating constitutive opening activity. Trpvicin accesses additional binding sites inside the central cavity of the G573S mutant to remodel the channel symmetry and block the channel. Together, our results provide mechanistic insights into the inhibition of TRPV3 by Trpvicin and support TRPV3-related drug development.
Assuntos
Canais de Cátion TRPV , Camundongos , Animais , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/química , Mutação , Sítios de LigaçãoRESUMO
The mechanosensitive ion channels Transient Receptor Potential Vanilloid 4 (TRPV4) and PIEZO1 transduce physiologic and supraphysiologic magnitudes of mechanical signals in the chondrocyte, respectively. TRPV4 activation promotes chondrogenesis, while PIEZO1 activation by supraphysiologic deformations drives cell death. The mechanisms by which activation of these channels discretely drives changes in gene expression to alter cell behavior remain to be determined. To date, no studies have contrasted the transcriptomic response to activation of these channels nor has any published data attempted to correlate these transcriptomes to alterations in cellular function. This study used RNA sequencing to comprehensively investigate the transcriptomes associated with activation of TRPV4 or PIEZO1, revealing that TRPV4 and PIEZO drive distinct transcriptomes and also exhibit unique co-regulated clusters of genes. Notably, activation of PIEZO1 through supraphysiologic deformation induced a transient inflammatory profile that overlapped with the interleukin (IL)-1-responsive transcriptome and contained genes associated with cartilage degradation and osteoarthritis progression. However, both TRPV4 and PIEZO1 were also shown to elicit anabolic effects. PIEZO1 expression promoted a pro-chondrogenic transcriptome under unloaded conditions, and daily treatment with PIEZO1 agonist Yoda1 significantly increased sulfated glycosaminoglycan deposition in vitro. These findings emphasize the presence of a broad "mechanome" with distinct effects of TRPV4 and PIEZO1 activation in chondrocytes, suggesting complex roles for PIEZO1 in both the physiologic and pathologic responses of chondrocytes. The identification of transcriptomic profiles unique to or shared by PIEZO1 and TRPV4 (distinct from IL-1-induced inflammation) could inform future therapeutic designs targeting these channels for the management and treatment of osteoarthritis.
Assuntos
Condrócitos , Canais Iônicos , Canais de Cátion TRPV , Transcriptoma , Animais , Condrócitos/metabolismo , Condrogênese , Canais Iônicos/metabolismo , Canais Iônicos/genética , Mecanotransdução Celular , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , SuínosRESUMO
Itching is an aversive somatosensation that triggers the desire to scratch. Transient receptor potential (TRP) channel proteins are key players in acute and chronic itch. However, whether the modulatory effect of fibroblast growth factor 13 (FGF13) on acute and chronic itch is associated with TRP channel proteins is unclear. Here, we demonstrated that conditional knockout of Fgf13 in dorsal root ganglion neurons induced significant impairment in scratching behaviors in response to acute histamine-dependent and chronic dry skin itch models. Furthermore, FGF13 selectively regulated the function of the TRPV1, but not the TRPA1 channel on Ca2+ imaging and electrophysiological recordings, as demonstrated by a significant reduction in neuronal excitability and current density induced by TRPV1 channel activation, whereas TRPA1 channel activation had no effect. Changes in channel currents were also verified in HEK cell lines. Subsequently, we observed that selective modulation of TRPV1 by FGF13 required its microtubule-stabilizing effect. Furthermore, in FGF13 knockout mice, only the overexpression of FGF13 with a tubulin-binding domain could rescue TRP channel function and the impaired itch behavior. Our findings reveal a novel mechanism by which FGF13 is involved in TRPV1-dependent itch transduction and provide valuable clues for alleviating pathological itch syndrome.
Assuntos
Fatores de Crescimento de Fibroblastos , Camundongos Knockout , Microtúbulos , Prurido , Canais de Cátion TRPV , Animais , Humanos , Masculino , Camundongos , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Gânglios Espinais/metabolismo , Células HEK293 , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Prurido/metabolismo , Prurido/genética , Canal de Cátion TRPA1/metabolismo , Canal de Cátion TRPA1/genética , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.
Assuntos
Sinalização do Cálcio , Fibroblastos , Gengiva , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Animais , Humanos , Camundongos , Fibroblastos/metabolismo , Gengiva/metabolismo , Gengiva/citologia , Cálcio/metabolismo , Sistema de Sinalização das MAP Quinases , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-1/metabolismo , Interleucina-1/farmacologia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologiaRESUMO
Melanoma is a highly aggressive cancer endowed with a unique capacity of rapidly metastasizing, which is fundamentally driven by aberrant cell motility behaviors. Discovering "migrastatics" targets, specifically controlling invasion and dissemination of melanoma cells during metastasis, is therefore of primary importance. Here, we uncover the prominent expression of the plasma membrane TRPV2 calcium channel as a distinctive feature of melanoma tumors, directly related to melanoma metastatic dissemination. In vitro as well as in vivo, TRPV2 activity is sufficient to confer both migratory and invasive potentials, while conversely TRPV2 silencing in highly metastatic melanoma cells prevents aggressive behavior. In invasive melanoma cells, TRPV2 channel localizes at the leading edge, in dynamic nascent adhesions, and regulates calcium-mediated activation of calpain and the ensuing cleavage of the adhesive protein talin, along with F-actin organization. In human melanoma tissues, TRPV2 overexpression correlates with advanced malignancy and poor prognosis, evoking a biomarker potential. Hence, by regulating adhesion and motility, the mechanosensitive TRPV2 channel controls melanoma cell invasiveness, highlighting a new therapeutic option for migrastatics in the treatment of metastatic melanoma.