RESUMEN
The human hCLCA1 gene is a member of the CLCA gene family that has a well-documented role in inflammatory airway diseases. Previously, we demonstrated that secreted hCLCA1 plays a role in regulating the innate immune response by activating airway macrophages. However, the mechanism of this regulation remains unclear. In this present study, recombinant proteins containing different hCLCA1 domains are expressed to determine the specific hCLCA1 domain(s) responsible for macrophage activation. Specifically, hCLCA1 constructs containing the hydrolase domain (HYD), the von Willebrand Factor Type A (VWA) domain, and the fibronectin type III (FN3) domain were heterologously expressed and affinity purified through fast protein liquid chromatography. Circular dichroism spectroscopy revealed that the purified hCLCA1 constructs exhibited secondary structure consistent with folded proteins. The VWA domain clearly demonstrated an ability to activate macrophages, inducing an increase in both IL-1ß mRNA and protein expression. This activation was associated with the activation of MAPKs and NF-κB pathways, identifying potential mechanistic pathways by which hCLCA1's VWA domain exerts its signaling effect. Altogether, this work identifies a domain with signaling function within hCLCA1, providing a specific target to one of the most highly induced gene products of airway inflammatory disease.
RESUMEN
In plants, strict regulation of stomatal pores is critical for modulation of CO2 fixation and transpiration. Under certain abiotic and biotic stressors, pore closure is initiated through anionic flux, with calcium (Ca2+) playing a central role. The aluminum-activated malate transporter 12 (ALMT12) is a malate-activated, voltage-dependent member of the aluminum-activated malate transporter family that has been implicated in anionic flux from guard cells controlling the stomatal aperture. Herein, we report the characterization of the regulatory mechanisms mediating channel activities of an ALMT from the grass Brachypodium distachyon (BdALMT12) that has the highest sequence identity to Arabidopsis thaliana ALMT12. Electrophysiological studies in a heterologous cell system confirmed that this channel is malate- and voltage-dependent. However, this was shown to be true only in the presence of Ca2+ Although a general kinase inhibitor increased the current density of BdALMT12, a calmodulin (CaM) inhibitor reduced the Ca2+-dependent channel activation. We investigated the physiological relevance of the CaM-based regulation in planta, where stomatal closure, induced by exogenous Ca2+ ionophore and malate, was shown to be inhibited by exogenous application of a CaM inhibitor. Subsequent analyses revealed that the double substitutions R335A/R338A and R335A/K342A, within a predicted BdALMT12 CaM-binding domain (CBD), also decreased the channels' ability to activate. Using isothermal titration calorimetry and CBD-mimetic peptides, as well as CaM-agarose affinity pulldown of full-length recombinant BdALMT12, we confirmed the physical interaction between the CBD and CaM. Together, these findings support a co-regulatory mechanism of BdALMT12 activation by malate, and Ca2+/CaM, emphasizing that a complex regulatory network modulates BdALMT12 activity.
Asunto(s)
Brachypodium , Calcio , Calmodulina , Transportadores de Anión Orgánico , Proteínas de Plantas , Estomas de Plantas , Sustitución de Aminoácidos , Brachypodium/química , Brachypodium/genética , Brachypodium/metabolismo , Calcio/química , Calcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Activación del Canal Iónico/fisiología , Malatos/química , Malatos/metabolismo , Mutación Missense , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estomas de Plantas/química , Estomas de Plantas/genética , Estomas de Plantas/metabolismoRESUMEN
BACKGROUND: Congenital stationary night-blindness (CSNB) is a recessive autosomal defect in low-light vision in Appaloosa and other horse breeds. This condition has been mapped by linkage analysis to a gene coding for the Transient Receptor Potential cation channel Member 1 (TRPM1). TRPM1 is normally expressed in the ON-bipolar cells of the inner nuclear layer of the retina. Down-regulation of TRPM1 expression in CSNB results from a transposon-like insertion in intron 1 of the TRPM1 gene. Stop transcription signals in this transposon significantly reduce TRPM1 primary transcript levels in CSNB horses. This study describes additional contributions by a second mutation of the TRPM1 gene, the ECA1 108,249,293 C > T SNP, to down-regulation of transcription of the TRPM1 gene in night-blind horses. This TRPM1 SNP introduces a consensus binding site for neuro-oncological ventral antigen 1 (Nova-1) protein in the primary transcript. Nova-1 binding disrupts normal splicing signals, producing unstable, non-functional mRNA transcripts. RESULTS: Retinal bipolar cells express both TRPM1 and Nova-1 proteins. In vitro addition of Nova-1 protein retards electrophoretic migration of TRPM1 RNA containing the ECA1 108,249,293 C > T SNP. Up-regulating Nova-1 expression in primary cultures of choroidal melanocytes carrying the intron 11 SNP caused an average log 2-fold reduction of ~6 (64-fold) of TRPM1 mRNA expression. CONCLUSIONS: These finding suggest that the equine TRPM1 SNP can act independently to reduce survival of TRPM1 mRNA escaping the intron 1 transcriptional stop signals in CSNB horses. Coexistence and co-inheritance of two independent TRPM1 mutations across 1000 equine generations suggests a selective advantage for the apparently deleterious CSNB trait.
Asunto(s)
Enfermedades Hereditarias del Ojo/veterinaria , Enfermedades Genéticas Ligadas al Cromosoma X/veterinaria , Enfermedades de los Caballos/genética , Miopía/veterinaria , Ceguera Nocturna/veterinaria , Polimorfismo de Nucleótido Simple , Canales Catiónicos TRPM/genética , Animales , Sitios de Unión , Células Cultivadas , Exones , Enfermedades Hereditarias del Ojo/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Caballos , Miopía/genética , Proteínas del Tejido Nervioso/genética , Antígeno Ventral Neuro-Oncológico , Ceguera Nocturna/genética , ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
BACKGROUND: Lipopolysaccharide (LPS)-mediated sickness behaviour is known to be a result of increased inflammatory cytokines in the brain. Inflammatory cytokines have been shown to mediate increases in brain excitation by loss of GABAA-mediated inhibition through receptor internalization or inactivation. Inflammatory pathways, reactive oxygen species and stress are also known to increase monoamine oxidase-A (MAO-A) and acetylcholinesterase (ACh-E) activity. Given that neuromodulator actions on neural circuits largely depend on inhibitory pathways and are sensitive to alteration in corresponding catalytic enzyme activities, we assessed the impact of systemic LPS on neuromodulator-mediated shaping of a simple cortical network. METHODS: Extracellular field recordings of evoked postsynaptic potentials in adult mouse somatosensory cortical slices were used to evaluate effects of a single systemic LPS challenge on neuromodulator function 1 week later. Neuromodulators were administered transiently as a bolus (100 µl) to the bath perfusate immediately upstream of the recording site to mimic phasic release of neuromodulators and enable assessment of response temporal dynamics. RESULTS: Systemic LPS administration resulted in loss of both spontaneous and evoked inhibition as well as alterations in the temporal dynamics of neuromodulator effects on a paired-pulse paradigm. The effects on neuromodulator temporal dynamics were sensitive to the Monoamine oxidase-A (MAO-A) antagonist clorgyline (for norepinephrine and serotonin) and the ACh-E inhibitor donepezil (for acetylcholine). This is consistent with significant increases in total MAO and ACh-E activity found in hemi-brain samples from the LPS-treated group, supporting the notion that systemic LPS administration may lead to longer-lasting changes in inhibitory network function and enzyme (MAO/ACh-E) activity responsible for reduced neuromodulator actions. CONCLUSIONS: Given the significant role of neuromodulators in behavioural state and cognitive processes, it is possible that an inflammatory-mediated change in neuromodulator action plays a role in LPS-induced cognitive effects and could help define the link between infection and neuropsychiatric/degenerative conditions.
Asunto(s)
Acetilcolinesterasa/metabolismo , Corteza Cerebral , Lipopolisacáridos/farmacología , Monoaminooxidasa/metabolismo , Neuronas/efectos de los fármacos , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Bicuculina/farmacología , Monoaminas Biogénicas/farmacología , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Antagonistas de Receptores de GABA-A/farmacología , Suspensión Trasera/métodos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/metabolismo , Tiempo de Reacción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
The objectives of this study were to determine the effect of an increase in diet fermentability on 1) the rate and extent to which short-chain fatty acid (SCFA) absorption pathways adapt relative to changes in Na(+) transport, 2) the epithelial surface area (SA), and 3) the barrier function of the bovine ruminal epithelium. Twenty-five Holstein steer calves were assigned to either the control diet (CON; 91.5% hay and 8.5% supplement) or a moderately fermentable diet (50% hay; 41.5% barley grain (G), and 8.5% supplement) fed for 3 (G3), 7 (G7), 14 (G14), or 21 days (G21). All calves were fed at 2.25% body weight at 0800. Calves were killed (at 1000), and ruminal tissue was collected to determine the rate and pathway of SCFA transport, Na(+) transport and barrier function in Ussing chambers. Tissue was also collected for SA measurement and gene expression. Mean reticular pH decreased from 6.90 for CON to 6.59 for G7 and then increased (quadratic P < 0.001). While effective SA of the ruminal epithelium was not affected (P > 0.10) by dietary treatment, the net Na(+) flux increased by 125% within 7 days (quadratic P = 0.016). Total acetate and butyrate flux increased from CON to G21, where passive diffusion was the primary SCFA absorption pathway affected. Increased mannitol flux, tissue conductance, and tendencies for increased expression of IL-1ß and TLR2 indicated reduced rumen epithelium barrier function. This study indicates that an increase in diet fermentability acutely increases Na(+) and SCFA absorption in the absence of increased SA, but reduces barrier function.
Asunto(s)
Epitelio/metabolismo , Ácidos Grasos Volátiles/metabolismo , Sodio/metabolismo , Adaptación Fisiológica , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Animales , Transporte Biológico/fisiología , Bovinos , Dieta/veterinaria , Suplementos Dietéticos , Rumen/fisiología , Factores de TiempoRESUMEN
The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage-monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation.
Asunto(s)
Canales de Cloruro/metabolismo , Activación de Macrófagos , Macrófagos Alveolares/metabolismo , Transducción de Señal , Animales , Bovinos , Canales de Cloruro/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/patología , Estructura Terciaria de Proteína , Porcinos , Células U937RESUMEN
Influenza infects the epithelial cells lining the airways. Normally epithelial cells move solutes through ion channels to create the osmotic drive to hydrate the airways. Viral alteration of this process could explain, in part, the fluid imbalance in the lungs and the resulting pulmonary edema that occurs during severe influenza infections. Using western blot and RT-qPCR, we measured ion channel and cytokine expression in the Calu3 airway cell line after infection with influenza virus (H1N1) for 48 h. We simultaneously measured chloride and potassium channel function by means of a short-circuit current (I(sc)) produced in an Ussing chamber. At a multiplicity of infection (MOI) of 10, viral M1 protein and pro-inflammatory cytokine expression was observed 24h post-infection, despite a lack of measurable change in Isc. However, we observed a decreased secretory response in cAMP- and calcium-induced Isc 48 h post-infection. This correlated with a decrease in CFTR and KCNN4 protein levels. Interestingly, a viral dose of an MOI 0.6 revealed an increased secretory response that correlated with pro-inflammatory cytokine expression. This increased secretory response seemed to be primarily driven through KCNN4. We detected an increase in KCNN4 mRNA and protein, while CFTR function and expression remained unchanged. Furthermore, inhibition of the KCNN4-stimulated I(sc) with TRAM-34, a specific inhibitor, ameliorated the response, implicating KCNN4 as the main driving force behind the secretory phenotype.