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1.
J Neurosci ; 30(6): 2138-49, 2010 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-20147541

RESUMEN

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2(+/-) mice with an approximately 50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre-Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Na(v)1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2(+/-) mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2(+/-) mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor alpha (TNF-alpha) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-alpha activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2(+/-) mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Dolor/enzimología , Dolor/fisiopatología , Animales , Astrocitos/metabolismo , Células Cultivadas , Quimiocina CCL3/farmacología , Quimiocina CCL3/fisiología , Femenino , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Hiperalgesia/enzimología , Hiperalgesia/fisiopatología , Inflamación/enzimología , Inflamación/fisiopatología , Macrófagos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/enzimología , Dolor/inmunología , Enfermedades del Sistema Nervioso Periférico/enzimología , Enfermedades del Sistema Nervioso Periférico/inmunología , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/enzimología , Médula Espinal/enzimología , Canales Catiónicos TRPV/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología
2.
J Neurochem ; 104(4): 979-92, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17971124

RESUMEN

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1beta-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1beta for 24 h induced a 2-3-fold increase in GRK2 and decreased C-C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/- animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.


Asunto(s)
Calcio/metabolismo , Quimiocina CCL2/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Astrocitos/enzimología , Astrocitos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación
3.
Brain Behav Immun ; 22(2): 200-8, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17869478

RESUMEN

Many neurotransmitters involved in pain perception transmit signals via G protein-coupled receptors (GPCRs). GPCR kinase 2 (GRK2) regulates agonist-induced desensitization and signaling of multiple GPCRs and interacts with downstream molecules with consequences for signaling. In general, low GRK2 levels are associated with increased responses to agonist stimulation of GPCRs. Recently, we reported that in mice with reduced GRK2 levels, inflammation-induced mechanical allodynia was increased. In addition, mice with impaired interleukin (IL)-1 beta signaling did not develop mechanical allodynia after L5 spinal nerve transection (SNT). We hypothesized that in the L5 SNT model mechanical allodynia would be associated with reduced neuronal GRK2 levels in the spinal cord dorsal horn and that IL-1 beta signaling would be required to induce both the decrease in GRK2 and mechanical allodynia. We show here that in wild type (WT) mice L5 SNT induces a bilateral decrease in neuronal GRK2 expression in the lumbar spinal cord dorsal horn, 1 and 2 weeks after L5 SNT. No changes in GRK2 were observed in the thoracic segments. Moreover, spinal cord GRK2 expression was not decreased in IL-1R(-/-) mice after L5 SNT. These data show that IL-1 beta signaling is not only required for the development of mechanical allodynia, but also to reduce neuronal GRK2 expression. These results suggest a functional relation between the L5 SNT-induced IL-1 beta-mediated decrease in GRK2 and development of mechanical allodynia.


Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Hiperalgesia/inmunología , Interleucina-1beta/metabolismo , Células del Asta Posterior/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Nervios Espinales/lesiones , Animales , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Hiperalgesia/metabolismo , Vértebras Lumbares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Umbral del Dolor/fisiología , Estimulación Física , Receptores Tipo I de Interleucina-1/genética , Transducción de Señal/fisiología
4.
Virology ; 488: 96-107, 2016 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-26615334

RESUMEN

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatible with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication.


Asunto(s)
Antibacterianos/metabolismo , Doxiciclina/metabolismo , VIH-1/fisiología , Activación Transcripcional , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/biosíntesis , Animales , Células Cultivadas , Virus de la Fiebre Aftosa , VIH-1/genética , Humanos , Ratones Transgénicos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética
5.
Hum Gene Ther ; 20(5): 524-30, 2009 May.
Artículo en Inglés | MEDLINE | ID: mdl-19203210

RESUMEN

Doxycycline (DOX) is widely used as a pharmacological agent and as an effector molecule in inducible gene expression systems. For most applications, it is important to determine whether the DOX concentration reaches the level required for optimal efficacy. We developed a sensitive bioassay for measuring the DOX concentration in biological samples. We used a modified HeLa cell line with the luciferase reporter gene under the control of the DOX-inducible Tet-On system for regulation of gene expression. These HeLaDOX cells constitutively express a novel variant of the rtTA transcriptional activator protein that is highly DOX-sensitive. Incubation of the cells with a DOX-containing biological sample triggers luciferase expression, which can be quantitated by standard methods. This bioassay is sensitive, with a DOX detection limit of 22 ng/ml in plasma. The assay was used to determine the DOX concentration in plasma derived from DOX-treated rhesus macaques and mice. Furthermore, we found that the DOX concentration in murine cerebrospinal fluid is 31-fold lower than the concurrent plasma DOX level. This bioassay for the quantification of DOX concentration in biological samples has several advantages over high-performance liquid chromatography-based and microbiological assays: (1) multiple samples can be assayed in a single experiment; (2) only small sample volumes are required; (3) the assay has a low detection limit; and (4) the assay can be performed in any cell culture laboratory.


Asunto(s)
Doxiciclina/análisis , Expresión Génica/efectos de los fármacos , Animales , Bioensayo , Proteínas de Unión al ADN/genética , Doxiciclina/sangre , Doxiciclina/líquido cefalorraquídeo , Doxiciclina/farmacología , Genes Reporteros , Células HeLa , Humanos , Límite de Detección , Luciferasas/genética , Macaca mulatta , Ratones , Ratones Mutantes
6.
Eur J Neurosci ; 25(6): 1696-704, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17408432

RESUMEN

Inflammation and nerve injury can both induce mechanical allodynia via mechanisms involving the production of pro-inflammatory cytokines and increased neuronal activity. Many neurotransmitters involved in pain signal via G protein-coupled receptors (GPCRs). GPCR kinase (GRK)2 is a member of the GRK family that regulates agonist-induced desensitization and signalling of GPCRs. Low intracellular GRK2 levels are associated with increased receptor signalling. The aim of this study was to investigate whether mechanical allodynia is associated with decreased spinal cord GRK2 expression and whether reduced GRK2 increases inflammation-induced mechanical allodynia. Mechanical allodynia was induced in rats by chronic constriction injury of the sciatic nerve. After 2 weeks, neuronal GRK2 expression was decreased bilaterally in the superficial layers of the lumbar spinal cord dorsal horn. Moreover, interleukin-1beta significantly reduced GRK2 expression ex vivo in spinal cord slices. To investigate whether reduced GRK2 potentiates inflammation-induced mechanical allodynia, we used GRK2(+/-) animals expressing decreased GRK2. At baseline, the threshold for mechanical stimulation did not differ between GRK2(+/-) and wild-type mice. However, GRK2(+/-) animals were more sensitive to mechanical stimulation than wild-type animals after intraplantar lambda-carrageenan injection. We propose cytokine-induced down-regulation of spinal cord neuronal GRK2 expression as a novel mechanism that contributes to increased neuronal signalling in mechanical allodynia.


Asunto(s)
Hiperestesia/metabolismo , Umbral del Dolor/fisiología , Quinasas de Receptores Adrenérgicos beta/metabolismo , Animales , Conducta Animal , Constricción , Lateralidad Funcional , Quinasa 2 del Receptor Acoplado a Proteína-G , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteína Ácida Fibrilar de la Glía/metabolismo , Hiperestesia/etiología , Hiperestesia/patología , Interleucina-1beta/farmacología , Masculino , Proteínas Asociadas a Microtúbulos/metabolismo , Técnicas de Cultivo de Órganos , Umbral del Dolor/efectos de los fármacos , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Sprague-Dawley , Ciática/complicaciones , Ciática/etiología , Médula Espinal/efectos de los fármacos , Factores de Tiempo
7.
J Biol Chem ; 280(3): 2020-7, 2005 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-15542591

RESUMEN

Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is excreted by the majority of S. aureus strains and is a potent inhibitor of C5a- and formylated peptide-mediated chemotaxis of neutrophils and monocytes. Recently, we reported that CHIPS binds to the C5a receptor (C5aR) and the formylated peptide receptor, thereby blocking activation by C5a and formylated peptides, respectively. The anaphylatoxin C5a plays an important role in host immunity and pathological inflammatory processes. For C5a a two-site binding model is proposed in which C5a initially binds the C5aR N terminus, followed by interaction of the C5a C-terminal tail with an effector domain on the receptor. We have shown here that CHIPS does not affect activation of the C5aR by a peptide mimic of the C5a C terminus. Moreover, CHIPS was found to bind human embryonic kidney 293 cells expressing only the C5aR N terminus. Deletion and mutation experiments within this C5aR N-terminal expression system revealed that the binding site of CHIPS is contained in a short stretch of 9 amino acids (amino acids 10-18), of which the aspartic acid residues at positions 10, 15, and 18 plus the glycine at position 12 are crucial. Binding studies with C5aR/C3aR and C5aR/IL8RA chimeras confirmed that CHIPS binds only to the C5aR N terminus without involvement of its extracellular loops. CHIPS may provide new strategies to block the C5aR, which may lead to the development of new C5aR antagonists.


Asunto(s)
Proteínas Bacterianas/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Complemento C5a/metabolismo , Cartilla de ADN , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/genética
8.
J Immunol ; 173(9): 5704-11, 2004 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-15494522

RESUMEN

Staphylococcus aureus excretes a factor that specifically and simultaneously acts on the C5aR and the formylated peptide receptor (FPR). This chemotaxis inhibitory protein of S. aureus (CHIPS) blocks C5a- and fMLP-induced phagocyte activation and chemotaxis. Monoclonal anti-CHIPS Abs inhibit CHIPS activity against one receptor completely without affecting the other receptor, indicating that two distinct sites are responsible for both actions. A CHIPS-derived N-terminal 6 aa peptide is capable of mimicking the anti-FPR properties of CHIPS but has no effect on the C5aR. Synthetic peptides in which the first 6 aa are substituted individually for all other naturally occurring amino acids show that the first and third residue play an important role in blocking the FPR. Using an Escherichia coli expression system, we created mutant CHIPS proteins in which these amino acids are substituted. These mutant proteins have impaired or absent FPR- but still an intact C5aR-blocking activity, indicating that the loss of the FPR-blocking activity is not caused by any structural impairment. This identifies the first and third amino acid, both a phenylalanine, to be essential for CHIPS blocking the fMLP-induced activation of phagocytes. The unique properties of CHIPS to specifically inhibit the FPR with high affinity (kd=35.4 +/- 7.7 nM) could be an important new tool to further stimulate the fundamental research on the mechanisms underlying the FPR and its role in disease processes.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Inhibición de Migración Celular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Receptor de Anafilatoxina C5a , Receptores de Formil Péptido/antagonistas & inhibidores , Staphylococcus aureus/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Animales , Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión de Anticuerpos , Unión Competitiva/genética , Unión Competitiva/inmunología , Femenino , Humanos , Radioisótopos de Yodo/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Formil Péptido/metabolismo , Células U937
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