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1.
Neuropeptides ; 36(1): 13-21, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12147210

RESUMEN

The neurokinin-1 (NK-1) receptor interacts with peptides that belong to the tachykinin family. NK-1 is inducible in bone marrow (BM) stroma. In neural cells, its expression is high to constitutive. Screening of three cDNA libraries indicated that this different in NK-1 expression in neural and BM cells could not be explained by differences in the cDNA sequence. Analyses the 5' flanking sequence in BM stroma and three neural cell lines indicated that sequence +1/+358 relative to the transcription start (TS) site could account for the differences in NK-1 expression. Particular cytokines could reverse the repressive effects of region +1/+358 in BM stroma. The effects of NF-kappa B and cAMP activators were studied in stromal cells using a dominant negative inhibitor of NF-kappa B (I kappa B) or a repressor of CRE activators (ICERII gamma). The results showed that their effects of these transcription factors depended on the stimulating cytokine. This study provides insight into the tissue-specific differences in the expression of the NK-1 gene.


Asunto(s)
Células de la Médula Ósea/metabolismo , Regulación de la Expresión Génica/fisiología , Mesodermo/metabolismo , Neuronas/metabolismo , Receptores de Neuroquinina-1/biosíntesis , Regiones no Traducidas 5'/genética , Línea Celular/metabolismo , AMP Cíclico/metabolismo , Citocinas/farmacología , ADN Complementario/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Mesodermo/citología , FN-kappa B/metabolismo , Neuroblastoma/patología , Especificidad de Órganos , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes de Fusión/biosíntesis , Sistemas de Mensajero Secundario/efectos de los fármacos , Células del Estroma/metabolismo , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo
2.
J Immunol ; 167(8): 4600-8, 2001 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-11591789

RESUMEN

The bone marrow (BM), which is the major site of immune cell development in the adult, responds to different stimuli such as inflammation and hemorrhagic shock. Substance P (SP) is the major peptide encoded by the immune/hemopoietic modulator gene, preprotachykinin-1 (PPT-I). Differential gene expression using a microarray showed that SP reduced hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA levels in BM stroma. Because long-term hypoxia induced the expression of PPT-I in BM mononuclear cells, we used timeline studies to determine whether PPT-I is central to the biologic responses of BM stroma subjected to 30-min hypoxia (pO(2) = 35 mm Hg) followed by reoxygenation. HIF-1alpha mRNA and protein levels were increased up to 12 h. At this time, beta-PPT-I mRNA was detected with the release of SP at 16 h. SP release correlated with down-regulation of HIF-1alpha to baseline. A direct role for SP in HIF-1alpha expression was demonstrated as follows: 1) transient knockout of beta-PPT-I showed an increase in HIF-1alpha expression up to 48 h of reoxygenation; and 2) HIF-1alpha expression remained baseline during reoxygenation when stroma was subjected to hypoxia in the presence of SP. Reoxygenation activated the PPT-I promoter with concomitant nuclear translocation of HIF-1alpha that can bind to the respective consensus sequences within the PPT-I promoter. SP reversed active caspase-3, an indicator of apoptosis and erythropoiesis, to homeostasis level after reoxygenation of hypoxic stroma. The results show that during reoxgenation the PPT-I gene acts as a negative regulator on the expression of HIF-1alpha and active caspase-3 in BM stroma subjected to reoxygenation.


Asunto(s)
Médula Ósea/metabolismo , Caspasas/metabolismo , Oxígeno/farmacología , Sustancia P/biosíntesis , Factores de Transcripción/biosíntesis , Regiones no Traducidas 5' , Adulto , Caspasa 3 , Hipoxia de la Célula , Activación Enzimática , Regulación de la Expresión Génica , Hematopoyesis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Regiones Promotoras Genéticas , Precursores de Proteínas/genética , Células del Estroma/metabolismo , Taquicininas/genética
3.
Blood ; 98(9): 2697-706, 2001 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-11675340

RESUMEN

Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)-beta and tumor necrosis factor-alpha in BM stroma. TGF-beta production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.


Asunto(s)
Retroalimentación Fisiológica , Hematopoyesis/efectos de los fármacos , Neprilisina/metabolismo , Factor de Células Madre/farmacología , Sustancia P/metabolismo , Sitios de Unión , Células de la Médula Ósea , Receptor con Dominio Discoidina 1 , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Proteínas de la Membrana , Modelos Moleculares , Neprilisina/farmacología , Biblioteca de Péptidos , ARN Mensajero/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Neuroquinina-1/efectos de los fármacos , Receptores de Neuroquinina-1/metabolismo , Receptores de Neuroquinina-2/efectos de los fármacos , Receptores de Neuroquinina-2/metabolismo , Factor de Células Madre/fisiología , Sustancia P/farmacología , Sustancia P/fisiología , Factor de Crecimiento Transformador beta/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo
4.
J Biol Chem ; 276(15): 12345-55, 2001 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-11152677

RESUMEN

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación hacia Abajo , Complejos Multienzimáticos/metabolismo , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Línea Celular , Humanos , Cinética , Fosforilación , Complejo de la Endopetidasa Proteasomal , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Ubiquitinas/metabolismo
5.
Brain Res Mol Brain Res ; 80(2): 166-76, 2000 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-11038249

RESUMEN

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.


Asunto(s)
Receptores Opioides delta , Secuencia de Aminoácidos , Analgésicos/metabolismo , Analgésicos/farmacología , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacología , Benzomorfanos/metabolismo , Benzomorfanos/farmacología , Unión Competitiva , Células Cultivadas , Clonación Molecular , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Leucina Encefalina-2-Alanina/farmacología , Proteínas de Unión al GTP/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Indoles/química , Indoles/farmacología , Isoquinolinas/química , Isoquinolinas/farmacología , Riñón/citología , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Morfina/metabolismo , Morfina/farmacología , Mutagénesis Sitio-Dirigida , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Quinolinas/química , Quinolinas/metabolismo , Quinolinas/farmacología , Ensayo de Unión Radioligante , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/genética , Tritio
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