RESUMEN
Renal ischemia reperfusion injury (IRI) is associated with inflammation, including neutrophil infiltration that exacerbates the initial ischemic insult. The molecular pathways involved are poorly characterized and there is currently no treatment. We performed an in silico analysis demonstrating changes in NFκB-mediated gene expression in early renal IRI. We then evaluated NFκB-blockade with a BRD4 inhibitor on neutrophil adhesion to endothelial cells in vitro, and tested BRD4 inhibition in an in vivo IRI model. BRD4 inhibition attenuated neutrophil adhesion to activated endothelial cells. In vivo, IRI led to increased expression of cytokines and adhesion molecules at 6 h post-IRI with sustained up-regulated expression to 48 h post-IRI. These effects were attenuated, in part, with BRD4 inhibition. Absolute neutrophil counts increased significantly in the bone marrow, blood, and kidney 24 h post-IRI. Activated neutrophils increased in the blood and kidney at 6 h post-IRI and remained elevated in the kidney until 48 h post-IRI. BRD4 inhibition reduced both total and activated neutrophil counts in the kidney. IRI-induced tubular injury correlated with neutrophil accumulation and was reduced by BRD4 inhibition. In summary, BRD4 inhibition has important systemic and renal effects on neutrophils, and these effects are associated with reduced renal injury.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Activación Neutrófila/efectos de los fármacos , Neutrófilos/inmunología , Proteínas Nucleares/antagonistas & inhibidores , Daño por Reperfusión/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Riñón/citología , Riñón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Neutrófilos/efectos de los fármacos , Proteínas Nucleares/metabolismo , Daño por Reperfusión/inmunología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismoRESUMEN
Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1ß receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1ß-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1ß as a potential therapeutic target in ROP.
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Enfermedades de la Coroides/fisiopatología , Interleucina-1beta/metabolismo , Retinopatía de la Prematuridad/fisiopatología , Animales , Animales Recién Nacidos , Coroides/metabolismo , Coroides/fisiopatología , Enfermedades de la Coroides/etiología , Enfermedades de la Coroides/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Electrorretinografía , Endotelio/metabolismo , Humanos , Recién Nacido , Oxígeno/efectos adversos , Células Fotorreceptoras/metabolismo , Ratas , Ratas Sprague-Dawley , Retina/metabolismo , Retina/fisiopatología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/fisiopatología , Retinopatía de la Prematuridad/inducido químicamente , Retinopatía de la Prematuridad/etiología , Retinopatía de la Prematuridad/metabolismoRESUMEN
SLIT/ROBO signaling impacts many aspects of tissue development and homeostasis, in part, through the regulation of cell growth and proliferation. Recent studies have also linked SLIT/ROBO signaling to the regulation of diverse phagocyte functions. However, the mechanisms by which SLIT/ROBO signaling acts at the nexus of cellular growth control and innate immunity remain enigmatic. Here, we show that SLIT2-mediated activation of ROBO1 leads to inhibition of mTORC1 kinase activity in macrophages, leading to dephosphorylation of its downstream targets, including transcription factor EB and ULK1. Consequently, SLIT2 augments lysosome biogenesis, potently induces autophagy, and robustly promotes the killing of bacteria within phagosomes. Concordant with these results, we demonstrate decreased lysosomal content and accumulated peroxisomes in the spinal cords of embryos from Robo1 -/- , Robo2 -/- double knockout mice. We also show that impediment of auto/paracrine SLIT-ROBO signaling axis in cancer cells leads to hyperactivation of mTORC1 and inhibition of autophagy. Together, these findings elucidate a central role of chemorepellent SLIT2 in the regulation of mTORC1 activity with important implications for innate immunity and cancer cell survival.
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Proteínas del Tejido Nervioso , Receptores Inmunológicos , Animales , Ratones , Proteínas del Tejido Nervioso/genética , Receptores Inmunológicos/genética , Lisosomas , Bacterias , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.
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Infecciones Estafilocócicas , Staphylococcus aureus , Animales , Humanos , Ratones , Quimiotaxis de Leucocito , Inmunidad Innata , Neutrófilos , Infecciones Estafilocócicas/microbiologíaRESUMEN
The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.
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Receptores Acoplados a Proteínas G , Ácido Succínico , Animales , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Hipoxia , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Succinatos , Ácido Succínico/metabolismoRESUMEN
Macropinocytosis is essential for myeloid cells to survey their environment and for growth of RAS-transformed cancer cells. Several growth factors and inflammatory stimuli are known to induce macropinocytosis, but its endogenous inhibitors have remained elusive. Stimulation of Roundabout receptors by Slit ligands inhibits directional migration of many cell types, including immune cells and cancer cells. We report that SLIT2 inhibits macropinocytosis in vitro and in vivo by inducing cytoskeletal changes in macrophages. In mice, SLIT2 attenuates the uptake of muramyl dipeptide, thereby preventing NOD2-dependent activation of NF-κB and consequent secretion of pro-inflammatory chemokine, CXCL1. Conversely, blocking the action of endogenous SLIT2 enhances CXCL1 secretion. SLIT2 also inhibits macropinocytosis in RAS-transformed cancer cells, thereby decreasing their survival in nutrient-deficient conditions which resemble tumor microenvironment. Our results identify SLIT2 as a physiological inhibitor of macropinocytosis and challenge the conventional notion that signals that enhance macropinocytosis negatively regulate cell migration, and vice versa.
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Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Quimiocina CXCL1/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Péptidos y Proteínas de Señalización Intercelular/genética , Macrófagos/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/genética , Fagocitos/metabolismo , Pinocitosis/genética , Pinocitosis/fisiología , Receptores Inmunológicos/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Proteínas RoundaboutRESUMEN
The G-protein coupled receptor (GPCR) signaling was long believed to involve activation of receptor exclusively at the cell surface, followed by its binding to heterotrimeric G-proteins and arrestins to trigger various intracellular signaling cascades, and termination of signaling by internalization of the receptor. It is now accepted that many GPCRs continue to signal after internalization in the endosomes. Since the breakthrough discoveries of nuclear binding sites for their ligands in 1980s, several GPCRs have been detected at cell nuclei. But mechanisms of nuclear localization of GPCRs, many of whom contain putative nuclear localization signals, remain poorly understood to date. Nevertheless, it is known that subcellular trafficking of GPCRs is regulated by members of Ras superfamily of small GTPases, most notably by Rab and Arf GTPases. In this commentary, we highlight several recent studies which suggest novel roles of small GTPases, importins and sorting nexin proteins in the nuclear translocation of GPCRs via vesicular transport pathways. Taken together with increasing evidence for in vivo functionality of the nuclear GPCRs, better understanding of their trafficking will provide valuable clues in cell biology.
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Núcleo Celular/metabolismo , Carioferinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Humanos , Señales de Localización Nuclear , Transporte de Proteínas , Receptores Acoplados a Proteínas G/química , Transducción de SeñalRESUMEN
Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs.
RESUMEN
[This corrects the article DOI: 10.1038/celldisc.2016.17.].
RESUMEN
G-protein coupled receptors (GPCRs) evolved as specialized sensors of the extracellular environment. Comprising the largest family of cell surface receptors, GPCRs are common therapeutic targets. Over the last 25 years, several GPCRs have been observed at the cell nucleus, suggesting the presence of intracrine GPCR signaling beyond the plasma membrane. Yet specific physiological functions of nuclear GPCRs had not been reported, until lately. We recently uncovered distinct but complementary angiogenic roles of F2rl1 (formerly known as PAR2) depending on its subcellular localization at the plasma membrane or at the nucleus. Targeting subcellular compartments to improve drug selectivity may therefore inspire novel therapeutic strategies for transmembrane receptors.
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Diseño de Fármacos , Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Animales , Membrana Celular/metabolismo , Humanos , Terapia Molecular DirigidaRESUMEN
The traditional view of G protein-coupled receptors (GPCRs) being inactivated upon their internalization has been repeatedly challenged in recent years. GPCRs, in addition to forming the largest family of cell surface receptors, can also be found on intracellular membranes such as nuclear membranes. Since the first experimental evidence of GPCRs at the nucleus in the early 1990s, approximately 30 different GPCRs have been localized at the nucleus by independent research groups, including ours. In this chapter, we describe several techniques commonly used for immuno-detection of nuclear GPCRs focusing on subcellular fractionation of proteins based on their localization and transmission electron microscopy (TEM) using primary cultured cells as well as tissue sections. We also describe the use of confocal microscopy to study nuclear calcium currents, which can further affect downstream events such as gene transcription, nuclear envelope breakdown, or its reconstruction and nucleocytoplasmic protein transport.