RESUMEN
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.
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Adenosina Trifosfatasas/química , Proteínas de Unión al ADN/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Histonas/química , Isoxazoles/química , Fragmentos de Péptidos/química , Procesamiento Proteico-Postraduccional , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Biotinilación , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Isoxazoles/síntesis química , Isoxazoles/farmacología , Cinética , Ligandos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Docilidad , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , metaminobenzoatos/síntesis química , metaminobenzoatos/química , metaminobenzoatos/farmacologíaRESUMEN
The microRNA (miR)-200s and their negative regulator ZEB1 have been extensively studied in the context of the epithelial-mesenchymal transition. Loss of miR-200s has been shown to enhance cancer aggressiveness and metastasis, whereas replacement of miR-200 miRNAs has been shown to inhibit cell growth in several types of tumors, including lung cancer. Here, we reveal a novel function of miR-200c, a member of the miR-200 family, in regulating intracellular reactive oxygen species signaling and explore a potential application for its use in combination with therapies known to increase oxidative stress such as radiation. We found that miR-200c overexpression increased cellular radiosensitivity by direct regulation of the oxidative stress response genes PRDX2, GAPB/Nrf2, and SESN1 in ways that inhibits DNA double-strand breaks repair, increase levels of reactive oxygen species, and upregulate p21. We used a lung cancer xenograft model to further demonstrate the therapeutic potential of systemic delivery of miR-200c to enhance radiosensitivity in lung cancer. Our findings suggest that the antitumor effects of miR-200c result partially from its regulation of the oxidative stress response; they further suggest that miR-200c, in combination with radiation, could represent a therapeutic strategy in the future.
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Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/terapia , MicroARNs/metabolismo , Fármacos Sensibilizantes a Radiaciones/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Desnudos , MicroARNs/genética , Trasplante de Neoplasias , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiaciónRESUMEN
Neurotensin (NT) is a gastrointestinal neuropeptide that modulates intestinal inflammation and healing by binding to its high-affinity receptor NTR1. The dual role of NT in inflammation and healing is demonstrated in models of colitis induced by Clostridium difficile toxin A and dextran sulfate sodium, respectively, and involves NF-κB-dependent IL-8 expression and EGF receptor-mediated MAPK activation in human colonocytes. However, the detailed signaling pathways involved in these responses remain to be elucidated. We report here that NT/NTR1 coupling in human colonic epithelial NCM460 cells activates tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) in a time- and dose-dependent manner. NT also rapidly induces Src tyrosine phosphorylation, whereas pretreatment of cells with the Src inhibitor PP2 before NT exposure decreases NT-induced IGF-1R phosphorylation. In addition, inhibition of IGF-1R activation by either its specific antagonist AG1024 or siRNA against IGF-1 significantly reduces NT-induced IL-8 expression and NF-κB-dependent reporter gene expression. Pretreatment with AG1024 also inhibits Akt activation and apoptosis induced by NT. Silencing of Akt expression by siRNA also substantially attenuates NT-induced IL-8 promoter activity and NF-κB-dependent reporter gene expression. This is the first report to indicate that NT transactivates IGF-1R and that this response is linked to Akt phosphorylation and NF-κB activation, contributing to both pro-inflammatory and tissue repair signaling pathways in response to NT in colonic epithelial cells. We propose that IGF-1R activation represents a previously unrecognized key pathway involved in the mechanisms by which NT and NTR1 modulate colonic inflammation and inflammatory bowel disease.
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Colon/metabolismo , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Neurotensina/farmacología , Receptor IGF Tipo 1/metabolismo , Apoptosis/efectos de los fármacos , Toxinas Bacterianas/toxicidad , Línea Celular , Colitis/inducido químicamente , Colitis/metabolismo , Colitis/patología , Colon/patología , Sulfato de Dextran/efectos adversos , Sulfato de Dextran/farmacología , Relación Dosis-Respuesta a Droga , Enterotoxinas/toxicidad , Activación Enzimática/efectos de los fármacos , Células Epiteliales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucina-8/biosíntesis , Mucosa Intestinal/patología , FN-kappa B/metabolismo , Neurotensina/metabolismo , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptor IGF Tipo 1/agonistas , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Factores de Tiempo , Tirfostinos/farmacologíaRESUMEN
PURPOSE: For many tumor cells, de novo lipogenesis is a requirement for growth and survival. A considerable body of work suggests that inhibition of this pathway may be a powerful approach to antineoplastic therapy. It has recently been shown that inhibition of various steps in the lipogenic pathway individually can induce apoptosis or loss of viability in tumor cells. However, it is not clear whether quantitative differences exist in the ability of lipogenic enzymes to control tumor cell survival. We present a systematic approach that allows for a direct comparison of the control of lipogenic pathway enzymes over tumor cell growth and apoptosis using different cancer cells. EXPERIMENTAL DESIGN: RNA interference-mediated, graded down-regulation of fatty acid synthase (FAS) pathway enzymes was employed in combination with measurements of lipogenesis, apoptosis, and cell growth. RESULTS: In applying RNA interference titrations to two lipogenic enzymes, acetyl-CoA carboxylase 1 (ACC1) and FAS, we show that ACC1 and FAS both significantly control cell growth and apoptosis in HCT-116 cells. These results also extend to PC-3 and A2780 cancer cells. CONCLUSIONS: Control of tumor cell survival by different steps in de novo lipogenesis can be quantified. Because ACC1 and FAS both significantly control tumor cell growth and apoptosis, we propose that pharmacologic inhibitors of either enzyme might be useful agents in targeting cancer cells that critically rely on fatty acid synthesis. The experimental approach described here may be extended to other targets or disease-relevant pathways to identify steps suitable for therapeutic intervention.
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Acetiltransferasas/metabolismo , Neoplasias del Colon/enzimología , Ácido Graso Sintasas/metabolismo , Línea Celular , Proliferación Celular , Supervivencia Celular , Inhibidores Enzimáticos/farmacología , Células HCT116 , Humanos , Lipogénesis , Transducción de Señal , TransfecciónRESUMEN
The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bencimidazoles/química , Bencimidazoles/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Diseño de Fármacos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Bencimidazoles/farmacocinética , Proteínas Portadoras/química , Proteínas de Unión al ADN , Femenino , Humanos , Metilación , Ratones , Simulación del Acoplamiento Molecular , Proteínas Nucleares/química , Unión ProteicaRESUMEN
The SWI/SNF multisubunit complex modulates chromatin structure through the activity of two mutually exclusive catalytic subunits, SMARCA2 and SMARCA4, which both contain a bromodomain and an ATPase domain. Using RNAi, cancer-specific vulnerabilities have been identified in SWI/SNF-mutant tumors, including SMARCA4-deficient lung cancer; however, the contribution of conserved, druggable protein domains to this anticancer phenotype is unknown. Here, we functionally deconstruct the SMARCA2/4 paralog dependence of cancer cells using bioinformatics, genetic, and pharmacologic tools. We evaluate a selective SMARCA2/4 bromodomain inhibitor (PFI-3) and characterize its activity in chromatin-binding and cell-functional assays focusing on cells with altered SWI/SNF complex (e.g., lung, synovial sarcoma, leukemia, and rhabdoid tumors). We demonstrate that PFI-3 is a potent, cell-permeable probe capable of displacing ectopically expressed, GFP-tagged SMARCA2-bromodomain from chromatin, yet contrary to target knockdown, the inhibitor fails to display an antiproliferative phenotype. Mechanistically, the lack of pharmacologic efficacy is reconciled by the failure of bromodomain inhibition to displace endogenous, full-length SMARCA2 from chromatin as determined by in situ cell extraction, chromatin immunoprecipitation, and target gene expression studies. Furthermore, using inducible RNAi and cDNA complementation (bromodomain- and ATPase-dead constructs), we unequivocally identify the ATPase domain, and not the bromodomain of SMARCA2, as the relevant therapeutic target with the catalytic activity suppressing defined transcriptional programs. Taken together, our complementary genetic and pharmacologic studies exemplify a general strategy for multidomain protein drug-target validation and in case of SMARCA2/4 highlight the potential for drugging the more challenging helicase/ATPase domain to deliver on the promise of synthetic-lethality therapy.
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Compuestos de Azabiciclo/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Proteínas Cromosómicas no Histona/deficiencia , ADN Helicasas/antagonistas & inhibidores , Terapia Molecular Dirigida , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Proteínas Nucleares/antagonistas & inhibidores , Piridinas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/deficiencia , Unión Competitiva , Catálisis , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/química , ADN Helicasas/deficiencia , ADN Complementario/genética , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Humanos , Neoplasias Pulmonares/patología , Análisis por Micromatrices , Neoplasias/genética , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Tumor Rabdoide/genética , Tumor Rabdoide/patología , Sarcoma Sinovial/genética , Sarcoma Sinovial/patología , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Proteins that 'read' the histone code are central elements in epigenetic control and bromodomains, which bind acetyl-lysine motifs, are increasingly recognized as potential mediators of disease states. Notably, the first BET bromodomain-based therapies have entered clinical trials and there is a broad interest in dissecting the therapeutic relevance of other bromodomain-containing proteins in human disease. Typically, drug development is facilitated and expedited by high-throughput screening, where assays need to be sensitive, robust, cost-effective and scalable. However, for bromodomains, which lack catalytic activity that otherwise can be monitored (using classical enzymology), the development of cell-based, drug-target engagement assays has been challenging. Consequently, cell biochemical assays have lagged behind compared to other protein families (e.g., histone deacetylases and methyltransferases). RESULTS: Here, we present a suite of novel chromatin and histone-binding assays using AlphaLISA, in situ cell extraction and fluorescence-based, high-content imaging. First, using TRIM24 as an example, the homogenous, bead-based AlphaScreen technology was modified from a biochemical peptide-competition assay to measure binding of the TRIM24 bromodomain to endogenous histone H3 in cells (AlphaLISA). Second, a target agnostic, high-throughput imaging platform was developed to quantify the ability of chemical probes to dissociate endogenous proteins from chromatin/nuclear structures. While overall nuclear morphology is maintained, the procedure extracts soluble, non-chromatin-bound proteins from cells with drug-target displacement visualized by immunofluorescence (IF) or microscopy of fluorescent proteins. Pharmacological evaluation of these assays cross-validated their utility, sensitivity and robustness. Finally, using genetic and pharmacological approaches, we dissect domain contribution of TRIM24, BRD4, ATAD2 and SMARCA2 to chromatin binding illustrating the versatility/utility of the in situ cell extraction platform. CONCLUSIONS: In summary, we have developed two novel complementary and cell-based drug-target engagement assays, expanding the repertoire of pharmacodynamic assays for bromodomain tool compound development. These assays have been validated through a successful TRIM24 bromodomain inhibitor program, where a micromolar lead molecule (IACS-6558) was optimized using cell-based assays to yield the first single-digit nanomolar TRIM24 inhibitor (IACS-9571). Altogether, the assay platforms described herein are poised to accelerate the discovery and development of novel chemical probes to deliver on the promise of epigenetic-based therapies.
RESUMEN
INTRODUCTION: The radiation doses used to treat unresectable lung cancer are often limited by the proximity of normal tissues. Overexpression of c-Met, a receptor tyrosine kinase, occurs in about half of non-small-cell lung cancers (NSCLCs) and has been associated with resistance to radiation therapy and poor patient survival. We hypothesized that inhibiting c-Met would increase the sensitivity of NSCLC cells to radiation, enhancing the therapeutic ratio, which may potentially translate into improved local control. METHODS: We tested the radiosensitivity of two high-c-Met-expressing NSCLC lines, EBC-1 and H1993, and two low-c-Met-expressing lines, A549 and H460, with and without the small-molecule c-Met inhibitor MK-8033. Proliferation and protein expression were measured with clonogenic survival assays and Western blotting, respectively. γ-H2AX levels were evaluated by immunofluorescence staining. RESULTS: MK-8033 radiosensitized the high-c-Met-expressing EBC-1 and H1993 cells but not the low-c-Met-expressing cell lines A549 and H460. However, irradiation of A549 and H460 cells increased the expression of c-Met protein at 30 minutes after the irradiation. Subsequent targeting of this up-regulated c-Met by using MK-8033 followed by a second radiation dose reduced the clonogenic survival of both A549 and H460 cells. MK-8033 reduced the levels of radiation-induced phosphorylated (activated) c-Met in A549 cells. CONCLUSIONS: These results suggest that inhibition of c-Met could be an effective strategy to radiosensitize NSCLC tumors with high basal c-Met expression or tumors that acquired resistance to radiation because of up-regulation of c-Met.
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Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Neoplasias Pulmonares/radioterapia , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Apoptosis/efectos de la radiación , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Radioisótopos de Cesio , Técnica del Anticuerpo Fluorescente , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Proteínas Proto-Oncogénicas c-met/metabolismo , Ensayo de Tumor de Célula MadreRESUMEN
We examined the hypothesis that substance P (SP) and the neurokinin-1 receptor (NK-1R), both in vitro and in vivo, promote mucosal healing during recovery from colitis by stimulating antiapoptotic pathways in human colonic epithelial cells. For the in vitro experiments, human nontransformed NCM460 colonocytes stably transfected with NK-1R (NCM460-NK-1R cells) were exposed to SP, and cell viability assays, TUNEL assays, and Western blot analyses were used to detect apoptotic and antiapoptotic pathways. SP exposure of NCM460-NK-1R colonocytes stimulated phosphorylation of the antiapoptotic molecule Akt and inhibited tamoxifen-induced cell death and apoptosis evaluated by the cell viability assay and poly(ADP-ribose) polymerase cleavage, respectively. SP-induced phosphorylation of Akt and cleavage of poly(ADP-ribose) polymerase were inhibited by blockade of integrin alphaVbeta3, Jak2, and activation of phosphatidylinositol 3-kinase. For the in vivo experiments, C57BL/6 mice, administered 5% dextran sulfate (DSS) dissolved in tap water for 5 days followed by a 5-day recovery period, were treated with the NK-1R antagonist CJ-12,255 or vehicle. Vehicle-treated mice showed increased colonic Akt phosphorylation and apoptosis compared with mice that received no DSS. In contrast, daily i.p. administration of CJ-12,255 for 5 days post-DSS suppressed Akt activation, exacerbated colitis, and enhanced apoptosis, and pharmacologic inhibition of Akt, either alone or together with CJ-12,255, produced a similar effect. Thus, SP, through NK-1R, possesses antiapoptotic effects in the colonic mucosa by activating Akt, which prevents apoptosis and mediates tissue recovery during colitis.
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Apoptosis/fisiología , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sustancia P/fisiología , Animales , Línea Celular , Colitis/metabolismo , Colon/citología , Colon/metabolismo , Humanos , Mucosa Intestinal/citología , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Neurotensin (NT) is a highly expressed gastrointestinal (GI) neuropeptide, which modulates GI motility, secretion and cell growth as well as intestinal inflammation. Since EGF receptor is highly expressed in human colon cancer cells, we sought to examine whether NT stimulation contributes to the EGFR overexpression using nontransformed colonocyte NCM460 cells. The results show that NT treatment caused a significant increase in EGFR protein expression and gene transcription. Pretreatment with MAP kinase pathway inhibitor PD98059 blocked NT-induced EGFR expression. As the EGFR promoter has a functional Egr-1 site, previously shown to mediate its transcription in response to hypoxia, we examined the role of Egr-1 in the NT response. We first show that NT stimulated Egr-1 expression, which can be inhibited by PD98059. We also determined whether NT increases Egr-1 binding to its site within the EGFR promoter. The data indicate that NT enhanced the amount of Egr-1 binding to the EGFR Egr-1 site and that this binding was significantly decreased by PD98059. To verify that Egr-1 mediated NT-induced EGFR transcription, Egr-1 siRNA was used to knock down its expression. The data show that transfection of Egr-1 siRNA significantly inhibited NT-stimulated EGFR transcription. Together, our results suggest that NT can stimulate MAP kinase-mediated Egr-1 and EGFR gene expression in human colonocytes. Our results may be relevant to the mechanisms by which NT participates in the development of colon cancer.
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Colon/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Receptores ErbB/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neurotensina/farmacología , Línea Celular , Colon/enzimología , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Células Epiteliales/enzimología , Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Humanos , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Transducción de Señal/efectos de los fármacos , Activación TranscripcionalRESUMEN
Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Ctheta (PKCtheta), but not PKCepsilon and PKCdelta, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.
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Colon/efectos de los fármacos , Colon/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/biosíntesis , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción STAT/metabolismo , Sustancia P/farmacología , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Ciclooxigenasa 2/genética , ADN/genética , ADN/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Janus Quinasa 2 , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Antagonistas del Receptor de Neuroquinina-1 , Regiones Promotoras Genéticas , Receptores de Neuroquinina-1/metabolismo , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Ghrelin, a newly identified gastric peptide, is known for its potent activity in growth hormone (GH) release and appetite. Although ghrelin is involved in several other responses such as stress and intestinal motility, its potential role in intestinal inflammation is not clear. Here, we show that expression of ghrelin and its receptor mRNA is significantly increased during acute experimental colitis in mice injected intracolonically with trinitrobenzene sulfate (TNBS). We found by PCR that ghrelin receptor mRNA is expressed in non-transformed human colonic epithelial NCM460 cells. Exposure of NCM460 cells stably transfected with ghrelin receptor mRNA to ghrelin, increased IkappaBalpha phosphorylation and its subsequent degradation. In addition, ghrelin stimulated NF-kappaB-binding activity and NF-kappaB p65 subunit phosphorylation, and induced IL-8 promoter activity and IL-8 protein secretion. Furthermore, our data show that ghrelin-induced IkappaBalpha and p65 phosphorylation was markedly reduced by pharmacological inhibitors of intracellular calcium mobilization (BAPTA/AM) and protein kinase C (GF 109203X). Pretreatment with BAPTA/AM or GF109203X also significantly attenuated ghrelin-induced IL-8 production. Together, our results strongly suggest that ghrelin may be a proinflammatory peptide in the colon. Ghrelin may participate in the pathophysiology of colonic inflammation by inducing PKC-dependent NF-kappaB activation and IL-8 production at the colonocyte level.
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Colon/metabolismo , Células Epiteliales/metabolismo , Interleucina-8/genética , FN-kappa B/metabolismo , Hormonas Peptídicas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Colitis/metabolismo , Citocinas/metabolismo , Ghrelina , Humanos , Inflamación , Interleucina-8/metabolismo , Ratones , Ratones Endogámicos , Hormonas Peptídicas/farmacología , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Ghrelina , Transducción de Señal , Trinitrobencenos/metabolismo , Trinitrobencenos/farmacología , Regulación hacia ArribaRESUMEN
Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor kappa B (NF-kappaB)-driven proinflammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-kappaB activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10(-7) M) induced an early (1 min) phosphorylation of the PKC isoforms PKCdelta, PKC, and PKCepsilon, followed by I-kappaB kinase, IkappaBalpha, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 microM) blocked SP-induced IkappaBalpha and p65 phosphorylation and IL-8 production. The PKCdelta inhibitor rottlerin at a low concentration (1 microM), but not pseudosubstrate PKC and PKCepsilon inhibitors (10 microM), significantly reduced IL-8 secretion. PKCdelta silencing by RNA interference reduced PKCdelta protein expression and SP-induced PKCdelta phosphorylation that was associated with diminished IL-8 promoter and NF-kappaB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCdelta increased SP-induced IL-8 promoter- and NF-kappaB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCdelta plays an important role in SP-induced proinflammatory signaling in human colonocytes.
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Colon/metabolismo , Interleucina-8/genética , Proteína Quinasa C/fisiología , Sustancia P/farmacología , Células Cultivadas , Activación Enzimática , Expresión Génica/efectos de los fármacos , Humanos , Proteínas I-kappa B/metabolismo , Mucosa Intestinal/metabolismo , Inhibidor NF-kappaB alfa , FN-kappa B/fisiología , Fosforilación , Regiones Promotoras Genéticas , Proteína Quinasa C-delta , Proteína Quinasa C-epsilonRESUMEN
Neurotensin (NT) is released in the gastrointestinal tract and participates in the pathophysiology of colonic inflammation. We have shown that NT mediates acute intestinal inflammation in vivo and stimulates nuclear factor-kappaB-dependent interleukin (IL)-8 expression in nontransformed human colonocytes in vitro. However, the exact mechanisms by which NT induces IL-8 expression have not been elucidated. In this study, we first show that NT stimulates IkappaBalpha phosphorylation and degradation and p65 phosphorylation and transcriptional activity. Inhibition of protein kinase C (PKC) activation significantly attenuates NT-induced IL-8 expression. This effect seems to be mediated through inhibition of IkappaBalpha phosphorylation and degradation and by p65 phosphorylation and transcriptional activity. We also show that intracellular calcium mobilization is necessary for NT-induced phosphorylation of IkappaBalpha and p65, suggesting that a conventional PKC is involved. Furthermore, transfection of a dominant-negative form of PKCalpha significantly reduces NT-induced IL-8 promoter activity. These results indicate that the conventional PKCalpha is an important mediator in the proinflammatory signaling pathway elicited by NT at the colonocyte level.
Asunto(s)
Proteínas I-kappa B/metabolismo , Interleucina-8/biosíntesis , FN-kappa B/metabolismo , Neurotensina/fisiología , Proteína Quinasa C/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas I-kappa B/genética , Insectos , Interleucina-8/genética , Inhibidor NF-kappaB alfa , FN-kappa B/genética , Fosforilación/efectos de los fármacos , Proteína Quinasa C/genética , Proteína Quinasa C-alfa , Factor de Transcripción ReIARESUMEN
Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.