RESUMEN
To identify if genetic determinants of CXCL16 modulate the susceptibility and phenotype of inflammatory bowel diseases (IBD), we analyzed genomic DNA from 574 individuals (365 IBD patients, 209 healthy controls) for the CXCL16 p.Ala181Val polymorphism. In this study, we demonstrate that in Crohn's disease (CD), the CXCL16 p.Ala181Val polymorphism is not a disease susceptibility gene but associated with younger age at disease onset (p=0.016) and higher frequency of ileal involvement (p=0.024; OR 2.17; 95% CI 1.12-4.21) in ValVal carriers compared to a higher frequency of colonic involvement in AlaAla carriers (p=0.009; OR 2.60; CI 1.29-5.25). Carriers of at least one Val allele and one CARD15/NOD2 variant had a higher incidence of a stricturing and penetrating phenotype (p=0.030, OR 4.04, CI 1.27-12.84) and of stenoses (p=0.014; OR 3.97; CI 1.38-11.40) than patients carrying NOD2 variants only, suggesting that this polymorphism contributes to a severe disease phenotype in CD.
Asunto(s)
Quimiocinas CXC/genética , Predisposición Genética a la Enfermedad , Genotipo , Enfermedades Inflamatorias del Intestino/genética , Fenotipo , Polimorfismo Genético , Receptores Depuradores/genética , Adulto , Edad de Inicio , Quimiocina CXCL16 , Femenino , Frecuencia de los Genes , Humanos , Enfermedades Inflamatorias del Intestino/patología , Masculino , Persona de Mediana Edad , Mutación , Proteína Adaptadora de Señalización NOD2/genéticaRESUMEN
In this study, we analyzed if IL-22 displays, similar to other IL-10 like cytokines such as IL-28A, antiviral properties in hepatic cells. Using RT-PCR and immunoblotting, we demonstrated that hepatic cell lines and primary hepatocytes express the functional IL-22 receptor complex consisting of IL-22R1 and IL-10R2. Hepatic IL-22 mRNA expression as measured by quantitative PCR was up-regulated in autoimmune and viral hepatitis compared to cholestatic liver diseases, while IL-22 serum levels did not differ significantly between patients with viral hepatitis and normal controls. IL-22 did not significantly change the expression levels of IFN-alpha/-beta and of the antiviral proteins MxA and 2',5'-OAS. Consequently, it had in comparison to IFN-alpha no relevant antiviral activity in in vitro models of HCV replication and infection. Taken together, hepatic IL-22 expression is up-regulated in viral hepatitis but IL-22 does not directly regulate antiviral proteins and has, in contrast to IFN-alpha, no effect on HCV replication.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Hepatitis C/inmunología , Hepatocitos/inmunología , Interleucinas/metabolismo , Línea Celular , Humanos , Immunoblotting , Subunidad beta del Receptor de Interleucina-10/genética , Subunidad beta del Receptor de Interleucina-10/metabolismo , Interleucinas/sangre , Interleucinas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Interleucina-22RESUMEN
OBJECTIVES: We analyzed ATG16L1, a recently identified Crohn's disease (CD) susceptibility gene, in a large cohort with inflammatory bowel disease (IBD) including potential interactions with other IBD genes as well as factors regulating its gene expression. METHODS: Genomic DNA from 2,890 Caucasians including 768 patients with CD, 507 patients with ulcerative colitis (UC), and 1,615 healthy controls was analyzed for 9 different ATG16L1 single nucleotide polymorphisms (SNPs). Genotyping included CARD15/NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C-->T) and SLC22A5/OCTN2 (-207 G-->C) as well as 10 CD-associated IL23R variants. The transcriptional regulation of ATG16L1 was studied in intestinal epithelial cells following stimulation with Toll-like receptor (TLR) ligands and proinflammatory cytokines and in a murine ileitis model and CD biopsies. RESULTS: All nine ATG16L1 gene variants analyzed displayed highly significant associations with CD demonstrating a CD-protective effect for the minor allele. The strongest associations were found for rs2241879 and the coding SNP rs2241880 (T300A); P= 3.6 x 10(-6) and 3.7 x 10(-6), respectively (OR 0.74, 95% CI 0.65-0.84 for both variants). The genotype-phenotype analysis revealed no significant associations. In UC, only rs6431660 was weakly disease-associated. There was no evidence for epistasis between the ATG16L1 gene and other susceptibility genes (IL23R, CARD15, SLC22A4/5). ATG16L1 mRNA expression was not upregulated in CD and murine ileitis, and was less than threefold increased in cells stimulated with proinflammatory cytokines and TLR ligands. CONCLUSION: ATG16L1 is a CD susceptibility gene without epistatic interaction with other CD susceptibility genes and is not upregulated in intestinal inflammation.
Asunto(s)
Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad , Proteína Adaptadora de Señalización NOD2/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Niño , Colitis Ulcerosa/genética , Enfermedad de Crohn/patología , Epistasis Genética , Femenino , Expresión Génica , Genotipo , Alemania , Humanos , Ileítis/genética , Masculino , Ratones , Persona de Mediana Edad , FenotipoRESUMEN
BACKGROUND: The IL23R gene has been identified as a susceptibility gene for inflammatory bowel disease (IBD) in the North American population. The aim of our study was to test this association in a large German IBD cohort and to elucidate potential interactions with other IBD genes as well as phenotypic consequences of IL23R variants. METHODS: Genomic DNA from 2670 Caucasian individuals including 833 patients with Crohn's disease (CD), 456 patients with ulcerative colitis (UC), and 1381 healthy unrelated controls was analyzed for 10 IL23R SNPs. Genotyping included the NOD2 variants p.Arg702Trp, p.Gly908Arg, and p.Leu1007fsX1008 and polymorphisms in SLC22A4/OCTN1 (1672 C-->T) and SLC22A5/OCTN2 (-207 G-->C). RESULTS: All IL23R gene variants analyzed displayed highly significant associations with CD. The strongest association was found for the SNP rs1004819 [P = 1.92x10(-11); OR 1.56; 95 % CI (1.37-1.78)]. 93.2% of the rs1004819 TT homozygous carriers as compared to 78% of CC wildtype carriers had ileal involvement [P = 0.004; OR 4.24; CI (1.46-12.34)]. The coding SNP rs11209026 (p.Arg381Gln) was protective for CD [P = 8.04x10(-8); OR 0.43; CI (0.31-0.59)]. Similar, but weaker associations were found in UC. There was no evidence for epistasis between the IL23R gene and the CD susceptibility genes CARD15 and SLC22A4/5. CONCLUSION: IL23R is an IBD susceptibility gene, but has no epistatic interaction with CARD15 and SLC22A4/5. rs1004819 is the major IL23R variant associated with CD in the German population, while the p.Arg381Gln IL23R variant is a protective marker for CD and UC.
Asunto(s)
Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad , Proteína Adaptadora de Señalización NOD2/genética , Proteínas de Transporte de Catión Orgánico/genética , Polimorfismo de Nucleótido Simple/genética , Receptores de Interleucina/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Demografía , Epistasis Genética , Femenino , Frecuencia de los Genes/genética , Alemania , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Miembro 5 de la Familia 22 de Transportadores de Solutos , Simportadores , Adulto JovenRESUMEN
BACKGROUND/AIM: Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Ralpha) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation. METHODS: Expression studies were performed by RT-PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays. RESULTS: Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF-alpha, IL1beta, IFN-gamma, and sodium butyrate stimulation increased IL31, IL31Ralpha, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK-1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNFDeltaARE mouse model of ileitis but in inflamed colonic lesions compared to non-inflamed tissue in patients with Crohn's disease (CD; average 2.4-fold increase) and in patients with ulcerative colitis (UC; average 2.6-fold increase) and correlated with the IL-8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88). CONCLUSION: IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation.
Asunto(s)
Células Epiteliales/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucinas/inmunología , Proteínas Quinasas Activadas por Mitógenos/inmunología , Factores de Transcripción STAT/inmunología , Animales , División Celular/inmunología , Línea Celular , Movimiento Celular/inmunología , Colon/inmunología , Infecciones por Citomegalovirus/inmunología , Humanos , Interferón gamma/análisis , Interleucina-1beta/análisis , Mucosa Intestinal/inmunología , Intestinos/citología , Intestinos/inmunología , Ratones , Ratones Endogámicos C57BL , Fosforilación , ARN Mensajero/análisis , Receptores de Interleucina/análisis , Receptores de Oncostatina M/inmunología , Factor de Necrosis Tumoral alfa/análisis , Regulación hacia Arriba/inmunologíaRESUMEN
The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.
Asunto(s)
Hepatocitos/metabolismo , Interleucinas/metabolismo , Regeneración Hepática , Hígado/metabolismo , Transducción de Señal , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Hepatectomía , Hepatitis/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Mediadores de Inflamación/metabolismo , Interleucinas/farmacología , Hígado/citología , Hígado/cirugía , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/genética , Linfocitos T/metabolismo , Factores de Tiempo , Transfección , Regulación hacia Arriba , Interleucina-22RESUMEN
BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with increased expression in inflammatory bowel disease. The aim of the study was to analyze the role of the MIF -173G/C single nucleotide polymorphism in Crohn's disease (CD). METHODS: Using restriction fragment length polymorphism analysis, genomic DNA of 198 patients with CD and 159 unrelated controls was analyzed for the -173G/C SNP in the MIF promoter region. Colonic MIF mRNA expression was measured by quantitative polymerase chain reaction (PCR), serum MIF levels by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-six of the 146 G/G wildtype carriers (24.7%) but only 3 of the 45 G/C heterozygotes (6.7%) and only 1 of the C/C homozygotes (14.3%) were diagnosed with upper gastrointestinal tract involvement (P = 0.009, odds ratio [OR] = 0.22, 95% confidence interval [CI], 0.06-0.75 for wildtype versus hetero- and homozygous carriers). This result was confirmed in a second prospective study, in which all patients diagnosed with upper gastrointestinal involvement (n = 13) were G/G wildtype carriers (P = 0.01 versus controls). All patients (n = 12; 100%) with a Crohn's disease activity index (CDAI) > 300 were G/G wildtype carriers compared to only 65.6% wildtype carriers in the group with a CDAI < 150 (P = 0.016). MIF is expressed in the colonic mucosa of CD patients and intestinal epithelial cells but its mRNA expression does not correlate with the degree of inflammation and is not upregulated by proinflammatory cytokines. In CD, MIF serum levels are not influenced by the MIF -173G/C polymorphism. CONCLUSIONS: The MIF -173G/C polymorphism appears to be a factor contributing to a particular CD phenotype characterized by protection against upper gastrointestinal tract involvement and severe disease activity.
Asunto(s)
Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Factores Inhibidores de la Migración de Macrófagos/genética , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas/genética , Tracto Gastrointestinal Superior/patología , Adolescente , Adulto , Femenino , Humanos , Masculino , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisisRESUMEN
OBJECTIVE: The identification of CARD15 as a susceptibility gene for Crohn's disease (CD) offers new possibilities for patient classification and risk assessment. The purpose of this study was to carry out a CARD15 sequence analysis in a large single-center IBD cohort and to investigate the impact of different genotypes on disease phenotypes. MATERIAL AND METHODS: A total of 445 unrelated patients with IBD (68.1% CD, 28.5% ulcerative colitis (UC), 3.4% indeterminate colitis (IC)) were included in the study. Clinical data were recorded by detailed questionnaire and analysis of the charts. CARD15 variants (R702W, G908R, 1007fs (frameshift)) were identified by DNA sequence analysis. RESULTS: CARD15 variants were found in 142 inflammatory bowel disease (IBD) patients (31.9%) including 120 CD patients (39.6%). In CD, the presence of two CARD15 variants was associated with ileal disease (p=0.008 versus wild-type (wt); OR 4.04; 95% CI 1.36-11.96) and a fibrostenotic phenotype (p=0.002 versus wt; OR 5.47; 95% CI 1.61-18.58). Subgroup analysis of 19 patients (4.3%) homozygous for the CARD15 variant 1007fs (3020ins C) revealed an association with onset of CD at an early age (p=0.014 versus wt), ileal involvement (p=0.001), and intestinal stenoses in all patients (p=0.001) frequently requiring surgery (73.7%; p=0.093). Of these patients 78.6% developed re-stenoses after surgical resection; 52.6% of the homozygotes were diagnosed as having entero-enteral fistulas. CONCLUSIONS: Patients homozygous for the 1007fs mutation had an early disease onset with long-segment ileal stenoses and entero-enteral fistulas. They frequently needed surgical intervention and had a high risk of re-stenosis. Genotyping therefore appears to be an important diagnostic tool in identifying severely affected patients requiring individualized treatment strategies at an early stage of the disease.
Asunto(s)
Enfermedad de Crohn/genética , Mutación del Sistema de Lectura , Enfermedades del Íleon/genética , Fístula Intestinal/genética , Proteína Adaptadora de Señalización NOD2/genética , Adolescente , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Niño , Colitis/genética , Colitis Ulcerosa/genética , Femenino , Genotipo , Homocigoto , Humanos , Enfermedades del Íleon/complicaciones , Enfermedades del Íleon/cirugía , Fístula Intestinal/complicaciones , Masculino , Persona de Mediana Edad , RecurrenciaRESUMEN
IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-alpha, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-alpha, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn's disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and IEC migration.
Asunto(s)
Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Regulación de la Expresión Génica/inmunología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Movimiento Celular , Células Cultivadas , Células Epiteliales/inmunología , Células Epiteliales/patología , Humanos , Inflamación/genética , Interleucina-22RESUMEN
BACKGROUND: The fractalkine receptor CX3CR1 has been shown to be involved in inflammation and immune response. Recently, two polymorphisms of CX3CR1 (V249I and T280M) were reported. AIMS: Our aim was to analyze fractalkine expression and the role of CX3CR1 polymorphisms in Crohn's disease (CD). METHODS: We determined fractalkine mRNA expression in the intestinal epithelial cell (IEC) line SW480 after stimulation with proinflammatory cytokines as well as in inflamed (n = 14) and noninflamed (n = 14) CD lesions by quantitative PCR. By restriction fragment length polymorphism analysis, genomic DNA from 206 patients with CD and 211 unrelated controls was analyzed for the two single nucleotide polymorphisms in the CX3CR1 gene, which result in the V249I and T280M substitutions. RESULTS: All proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly increased fractalkine mRNA expression in IEC. There was also a significant increase in fractalkine mRNA expression in inflamed lesions of CD patients when compared to noninflamed colonic mucosa (p = 0.02). Intestinal fractalkine mRNA levels correlated highly with IL-8 mRNA expression levels (r = 0.931). However, there was no difference in the V249I and T280M genotype frequencies between CD patients and the control group. In the CD group, 33.0% were heterozygous and 8.3% homozygous for the V249I polymorphism, while 23.3% were heterozygous and 4.4% homozygous for the T280M polymorphism. All T280M homozygotes were diagnosed of intestinal stenosis (p = 0.03 vs wildtype and heterozygous carriers) and had significantly more often ileocolonic involvement more often than patients with wildtype and heterozygous genotypes (p = 0.01). These associations were independent of the CARD15 genotype status. CONCLUSIONS: The expression of the chemokine fractalkine is upregulated by proinflammatory cytokines and enhanced in inflamed CD lesions. The CX3CR1 T280M polymorphism appears to influence CD phenotype and localization.
Asunto(s)
Quimiocinas CX3C/metabolismo , Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad , Proteínas de la Membrana/metabolismo , Polimorfismo Genético , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Receptores del VIH/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Receptor 1 de Quimiocinas CX3C , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CX3CL1 , Quimiocinas CX3C/genética , Intervalos de Confianza , Enfermedad de Crohn/patología , Femenino , Marcadores Genéticos , Humanos , Obstrucción Intestinal/genética , Obstrucción Intestinal/patología , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Datos de Secuencia Molecular , Oportunidad Relativa , Fenotipo , Probabilidad , Pronóstico , ARN Mensajero/análisis , Receptores de Quimiocina/genética , Receptores de Citocinas/genética , Receptores del VIH/genética , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
The expression of CCL20 (MIP-3alpha), which chemoattracts leukocytes to sites of inflammation, has been shown in intestinal epithelial cells (IEC). Aim of this study was to analyze the role of the CCL20 receptor CCR6 in IEC and colorectal cancer (CRC) cells. Expression of CCR6 and CCL20 was analyzed by RT-PCR and immunohistochemistry. Signaling was investigated by Western blotting, proliferation by MTS assays and chemotactic cell migration by wounding assays. The effect of CCL20 on Fas-induced apoptosis was determined by flow cytometry. CCR6 and its ligand CCL20 are expressed in IEC. Moreover, CRC and CRC metastases express CCR6, which is upregulated during IEC differentiation. Stimulation of IEC with CCL20 and proinflammatory stimuli (TNF-alpha, IL-1beta, LPS) significantly upregulates CCL20 mRNA expression. CCL20 expression was significantly increased in inflamed colonic lesions in Crohn's disease and correlated significantly with the IL-8 mRNA expression in these lesions (r = 0.71) but was downregulated in CRC metastases. CCL20 activated Akt, ERK-1/2, and SAPK/JNK MAP kinases and increased IL-8 protein expression. The CCL20 mediated activation of these pathways resulted in a 2.6-fold increase of cell migration (P = 0.001) and in a significant increase of cell proliferation (P < 0.05) but did not influence Fas-induced apoptosis. In conclusion, IEC and CRC express CCL20 and its receptor CCR6. CCL20 expression is increased in intestinal inflammation, while CCR6 is upregulated during cell differentiation. CCR6 mediated signals result in increased IEC migration and proliferation suggesting an important role in intestinal homeostasis and intestinal inflammation by mediating chemotaxis of IEC but also in mediating migration of CRC cells.
Asunto(s)
Neoplasias Colorrectales/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Quimiocina/metabolismo , Células CACO-2 , Diferenciación Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Quimiocina CCL20 , Quimiocinas CC/metabolismo , Enfermedad de Crohn/metabolismo , Citocinas/fisiología , Proteína Ligando Fas , Células HCT116 , Células HT29 , Humanos , Inflamación/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , MAP Quinasa Quinasa 1/fisiología , Proteínas Inflamatorias de Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosforilación , Receptores CCR6 , Transducción de Señal , Factores de Necrosis Tumoral/metabolismoRESUMEN
Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting in increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.
Asunto(s)
Movimiento Celular , Quimiocinas CXC/metabolismo , Neoplasias Colorrectales/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Receptores CXCR4/metabolismo , Apoptosis/efectos de los fármacos , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Quimiocina CXCL12 , Quimiocinas CXC/genética , Quimiocinas CXC/farmacología , Neoplasias Colorrectales/patología , Activación Enzimática , Proteína Ligando Fas , Humanos , Mucosa Intestinal/patología , Glicoproteínas de Membrana/farmacología , Invasividad Neoplásica , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt , ARN Mensajero/biosíntesis , Transducción de Señal , Factores de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Human cytomegalovirus virus (CMV) is a major cause of morbidity and mortality in immunocompromised individuals. Recently, a novel group of cytokines [interleukin (IL)-28A/B and IL-29, also termed interferon (IFN)-lambdas] has been described. Here, we demonstrate that intestinal epithelial cell (IEC) lines as well as murine and human colonic tissue express the IFN-lambda receptor subunits IL-28R and IL-10R2. IL-28A and IL-29 binding to their receptor complex activates ERK-1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase MAPKs and Akt, resulting in increased IL-8 protein expression. IFN-lambdas also induce phosphorylation of signal transducer and activator of transcription 1 and significantly increase mRNA expression of suppressor of cytokine signaling 3 and the antiviral proteins myxovirus resistance A and 2',5'-oligoadenylate synthetase. These signals result in an up to 83% reduction of cells positive for human CMV immediate-early protein after human CMV infection. In mice, IL-28A mRNA expression is upregulated after infection with murine CMV in vivo. Both IL-28A and IL-29 significantly decrease cell proliferation but have no effect on Fas-induced apoptosis. In conclusion, IECs express functional receptors for IFN-lambdas, which mediate antiviral and antiproliferative signals in IECs, suggesting a potential for therapeutic use in certain viral infections and as (antiproliferative) anticancer therapy.
Asunto(s)
Colon/metabolismo , Citocinas/metabolismo , Infecciones por Citomegalovirus/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Interleucinas/metabolismo , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Interferones , Interleucina-8/biosíntesis , Mucosa Intestinal , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas de Resistencia a Mixovirus , Receptores de Citocinas/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: We investigated the influence of 2 common Toll-like receptor 4 (TLR4) polymorphisms on susceptibility and disease characteristics of Crohn's disease (CD). METHODS: Genomic DNA from 204 patients with CD and 199 unrelated controls was analyzed for the presence of 2 single nucleotide polymorphisms in the TLR4 gene, resulting in the amino acid substitutions Asp299Gly and Thr399Ile. In addition, the carrier status for the 3 common CD-associated CARD15/NOD2 gene mutations, Arg702Trp, Gly908Arg, and 1007fs, was determined. The frequency of the different genotypes was compared, and a detailed genotype-phenotype correlation was performed. RESULTS: An almost 2-fold increase in the frequency of the TLR4 Asp299Gly phenotype was observed in patients with CD (14.2%) compared with healthy controls (7.5%, P = 0.038, odds ratio = 2.03). The prevalence of a stricturing phenotype was increased in patients heterozygous for 1 of the TLR4 polymorphisms studied (Asp299Gly, 34.5%; Thr399Ile, 36.7%) compared with patients with wild-type TLR4 (17.1% and 16.7%; P = 0.04 and 0.02, respectively). The presence of the Asp299Gly polymorphism in the absence of CARD15/NOD2 mutations was a particularly strong predictor of the stricturing disease phenotype that was present in 47.4% of the patients with Asp299Gly+/NOD2- compared with 10.1% of the patients with the Asp299Gly-/NOD2+ status (P = 0.0009; P = 0.0004 for Thr399Ile+/NOD2- versus Thr399Ile-/NOD2+). In contrast, there was a trend toward a higher prevalence of the penetrating phenotype in the TLR4-/NOD2+ group (71.6%) compared with the TLR4+/NOD2- group (47.4%, P = 0.059). CONCLUSIONS: The TLR4 Asp299Gly polymorphism is a risk factor for CD. TLR4 and CARD15/NOD2 mutations may contribute to distinct disease phenotypes.
Asunto(s)
Enfermedad de Crohn/epidemiología , Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Glicoproteínas de Membrana/genética , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/genética , Adulto , Estudios de Casos y Controles , Femenino , Alemania/epidemiología , Heterocigoto , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Desequilibrio de Ligamiento , Modelos Logísticos , Masculino , Persona de Mediana Edad , Mutación , Proteína Adaptadora de Señalización NOD2 , Fenotipo , Prevalencia , Estadísticas no Paramétricas , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Recently, we have shown that SOCS-1/3 overexpression in hepatic cells abrogates signaling of type I interferons (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-alpha because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT3 phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/3 phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-alpha due to inhibition by SOCS proteins.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Proteínas de Unión al GTP/metabolismo , Regulación de la Expresión Génica , Interferones/metabolismo , Interleucinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Represoras/metabolismo , 2',5'-Oligoadenilato Sintetasa/genética , Antivirales/metabolismo , Línea Celular Tumoral , Citocinas , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/genética , Expresión Génica , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/virología , Humanos , Interferones/química , Interferones/genética , Interferones/inmunología , Interleucinas/genética , Interleucinas/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de Resistencia a Mixovirus , Fosforilación , Fosfotirosina/metabolismo , Proteínas Represoras/genética , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Proteína 1 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Transactivadores/metabolismoRESUMEN
Nuclear matrix attachment regions (MARs) are regulatory DNA sequences that are important for higher-order chromatin organization, long-range enhancer function, and extension of chromatin modifications. Here we characterize a novel cell type-specific MAR-binding protein, SATB2, which binds to the MARs of the endogenous immunoglobulin micro locus in pre-B cells and enhances gene expression. We found that SATB2 differs from the closely related thymocyte-specific protein SATB1 by modifications of two lysines with the small ubiquitive related modifier (SUMO), which are augmented specifically by the SUMO E3 ligase PIAS1. Mutations of the SUMO conjugation sites of SATB2 enhance its activation potential and association with endogenous MARs in vivo, whereas N-terminal fusions with SUMO1 or SUMO3 decrease SATB2-mediated gene activation. Sumoylation is also involved in targeting SATB2 to the nuclear periphery, raising the possibility that this reversible modification of a MAR-binding protein may contribute to the modulation of subnuclear DNA localization.