RESUMEN
Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica , Complejos Multiproteicos/metabolismo , Transducción de Señal , Proteínas de Anclaje a la Quinasa A/genética , Proteínas de Anclaje a la Quinasa A/metabolismo , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Transporte Activo de Núcleo Celular/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Angiopoyetina 1/biosíntesis , Angiopoyetina 1/genética , Línea Celular , Cromatina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Cadenas alfa de Integrinas/biosíntesis , Cadenas alfa de Integrinas/genética , Cadenas beta de Integrinas/biosíntesis , Cadenas beta de Integrinas/genética , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta2/biosíntesis , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta3/biosíntesis , Factor de Crecimiento Transformador beta3/genéticaRESUMEN
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.
Asunto(s)
Neoplasias Colorrectales/sangre , Cadenas beta de Integrinas/sangre , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Neoplasias Colorrectales/genética , Humanos , Cadenas beta de Integrinas/biosíntesis , Cadenas beta de Integrinas/genética , Pronóstico , Prueba de Estudio Conceptual , Modelos de Riesgos Proporcionales , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reproducibilidad de los ResultadosRESUMEN
Overexpression of integrin αvß6 is believed to play an important role in the invasion and metastasis of oral squamous cell carcinoma (OSCC). However, little is known about the molecular mechanisms leading to αvß6 upregulation in OSCC. As the integrin ß6 (ITGB6) is the only partner with αv, the expression of αvß6 is dependent on ITGB6, it is, therefore, pivotal to investigate the mechanisms underlying ITGB6 overexpression in OSCC. We previously reported the cloning and characterization of human ITGB6 gene. In the current study, we further investigated the molecular mechanisms of ITGB6 expression and the upregulation by carcinogenesis related cytokine-transforming growth factor-ß1 (TGF-ß1) in OSCC cells. We first demonstrated that TGF-ß1 can induce ITGB6 mRNA and protein express in a time and concentration dependent manner, and the induced-ITGB6 mRNA was not due to increase the mRNA stability, but regulated at transcriptional level. By using a luciferase reporter assay, site-mutation, RNA interference, and chromatin immunoprecipitation assay, we revealed for the first time that JunB, a member of the activator protein-1 (AP-1) family, is involved in the positive regulation to the ITGB6 transcription induced by TGF-ß1 in OSCC cells. Furthermore, our data also demonstrated that histone acetyltransferase (HAT) CBP mediated histone H3 and H4 hyperacetylation, and RNA Polymerase II recruitment to ITGB6 promoter, facilitated the binding of transcription factor JunB to ITGB6 promoter after TGF-ß1 stimulation. Collectively, these findings demonstrate that JunB and CBP-mediated histone hyperacetylation are responsible for TGF-ß1 induced ITGB6 transcription in OSCC cells, suggesting that epigenetic mechanisms are responsible for the active transcription expression of ITGB6 induced by TGF-ß1 in OSCC cells.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Cadenas beta de Integrinas/biosíntesis , Neoplasias de la Boca/metabolismo , Proteínas de Neoplasias/metabolismo , Transcripción Genética , Factor de Crecimiento Transformador beta1/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Humanos , Cadenas beta de Integrinas/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Proteínas de Neoplasias/genética , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
The objective of this study was to conduct an analysis of peripheral blood Th17 cells with the ability to home to gut mucosa (CD4+ Th17+ ß7+ ) during recent or chronic human immunodeficiency virus (HIV) infections. The relationship between HIV load and systemic inflammation markers was studied. Twenty-five patients with recent (n = 10) or chronic (n = 15) untreated HIV infections; 30 treated HIV-infected patients with undetectable HIV load at the time of inclusion and 30 healthy controls were included. Bacterial translocation markers (16S rDNA), soluble CD14 (sCD14) and interleukin (IL)-6 monocyte activation parameters, CD4/CD8 ratio and T helper type 17 (Th17) subpopulations [CD4+ Th17+ expressing the IL-23 receptor (IL-23R) or ß7] were analysed at baseline and after 6 and 12 months of anti-retroviral therapy (ART). 16S rDNA was detected in all patients. Significantly increased serum levels of sCD14 and IL-6 and a decreased CD4/CD8 ratio were observed in patients. Similar percentages of CD4+ IL-23R+ and CD4+ Th17+ ß7+ cells were observed in healthy controls and patients at baseline. After 12 months of therapy, patients with a recent HIV infection showed significant increases of CD4+ IL-23R+ and CD4+ Th17+ ß7+ cell percentages and a decrease in IL-6 levels, although 16S rDNA continued to be detectable in all patients. No significant differences were observed in Th17 subpopulations in patients with chronic HIV infection after therapy. Early initiation of ART helps to increase the number of Th17 cells with the ability to home to the intestinal mucosa and to partially restore gut mucosal homeostasis. These results provide a rationale for initiating ART during the acute phase of HIV infection.
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Terapia Antirretroviral Altamente Activa/métodos , Infecciones por VIH/inmunología , VIH-1/inmunología , Cadenas beta de Integrinas/biosíntesis , Mucosa Intestinal/inmunología , Células Th17/metabolismo , Adulto , Antirretrovirales/uso terapéutico , Relación CD4-CD8 , ADN Ribosómico/análisis , Femenino , Infecciones por VIH/virología , Humanos , Interleucina-6/análisis , Mucosa Intestinal/citología , Receptores de Lipopolisacáridos/análisis , Masculino , Persona de Mediana Edad , Receptores de Interleucina/biosíntesis , Células Th17/inmunología , Carga ViralRESUMEN
Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti- integrin ß7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in ß7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. © 2016 International Society for Advancement of Cytometry.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Citometría de Flujo/métodos , Cadenas beta de Integrinas/biosíntesis , Leucocitos Mononucleares/metabolismo , Anticuerpos Monoclonales/química , Regulación de la Expresión Génica , Humanos , Cadenas beta de Integrinas/inmunología , Leucocitos Mononucleares/ultraestructuraRESUMEN
BACKGROUND: It is now well established that IL-4 has a central role in the development of monocytes to multinucleated giant cells (MGCs) by inducing the expression of integrins on the surface of monocytes. The aim of this study was to investigate the potential role of IL-4 in induction of ß5 integrin expression in the peripheral blood samples of patients with giant cell granuloma. MATERIAL AND METHODS: Monocytes were isolated from peripheral blood samples of patients with central giant cell granuloma (CGCG) and healthy controls using human Monocyte Isolation Kit II. Isolated monocytes were then cultured in the absence or presence of IL-4 (10 and 20 ng/mL), and following RNA extraction and cDNA synthesis, Real-time PCR was performed to determine the level of ß5 integrin expression. The formation of CGCGs and morphological analyses were done under light microscopy. For confirmation of CGCGs, immunocytochemistry technique was also carried out by anti-RANK (receptor-activator of NF-κB ligand) antibody. RESULTS: In both patient and control groups, ß5 levels were significantly enhanced by increasing the IL-4 dose from 10 to 20 ng/mL. In addition, these differences were significant between patient and control groups without IL-4 treatment. On the other hand, the number of cells which expressed RANK and therefore the number of giant cells were significantly higher in the patient group in comparison to controls, as assessed by immunohistochemistry evaluations. CONCLUSIONS: In this study, we showed an elevation in the expression levels of ß5 integrin when stimulated by IL-4. It is strongly indicated that this integrin acts as an important mediator during macrophage to macrophage fusion and development of giant cells.
Asunto(s)
Células Gigantes/citología , Granuloma de Células Gigantes/sangre , Cadenas beta de Integrinas/biosíntesis , Interleucina-4/fisiología , Monocitos/metabolismo , Células Cultivadas , HumanosRESUMEN
OBJECTIVES: Polymerase delta interacting protein 2 (Poldip2) has previously been implicated in migration, proliferation and extracellular matrix (ECM) production in vascular smooth muscle cells. To better understand the role of Poldip2 in ECM regulation, we investigated the mechanism responsible for collagen I accumulation in Poldip2(+/-) mouse aortic smooth muscle cells (MASMs). APPROACH AND RESULTS: Protein degradation and protein synthesis pathways were investigated. Depletion of Poldip2 had no effect on proteasome activity, but caused a partial reduction in autophagic flux. However, the rate of collagen I degradation was increased in Poldip2(+/-) vs. Poldip2(+/+) MASMs. Conversely, activation of the PI3K/Akt/mTOR signaling pathway, involved in regulation of protein synthesis, was significantly elevated in Poldip2(+/-) MASMs as was ß1-integrin expression. Suppressing mTOR signaling using Akt inhibitor or rapamycin and reducing ß1-integrin expression using siRNA prevented the increase in collagen I production. While collagen I and fibronectin were increased in Poldip2(+/-) MASMs, overall protein synthesis was not different from that in Poldip2(+/)(+)MASMs, suggesting selectivity of Poldip2 for ECM proteins. CONCLUSIONS: Poldip2(+/-) MASMs exhibit higher ß1-integrin expression and activity of the PI3K/Akt/mTOR signaling pathway, leading to increased ECM protein synthesis. These findings have important implications for vascular diseases in which ECM accumulation plays a role.
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Colágeno Tipo I/metabolismo , Matriz Extracelular/genética , Cadenas beta de Integrinas/biosíntesis , Proteínas Mitocondriales/genética , Músculo Liso Vascular/metabolismo , Proteínas Nucleares/genética , Animales , Aorta/crecimiento & desarrollo , Aorta/metabolismo , Proliferación Celular/genética , Fibronectinas/metabolismo , Cadenas beta de Integrinas/metabolismo , Ratones , Proteínas Mitocondriales/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/biosíntesis , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas/genética , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly cancers and is expected to become the second leading cause of cancer death by 2030. Despite extensive efforts to improve surgical treatment, limited progress has been made. Increasing evidence indicates that integrin ß6 plays a crucial role in carcinoma invasion and metastasis. However, the expression and role of ß6 in PDAC remain largely unknown. In the present study, we investigated the expression of ß6 in PDAC and its potential value as a prognostic factor and therapeutic target. ß6 upregulation was identified as an independent unfavorable prognostic indicator. Integrin ß6 markedly promoted the proliferation and invasion of pancreatic carcinoma cells and induced ETS1 phosphorylation in an ERK-dependent manner, leading to the upregulation of matrix metalloprotease-9, which is essential for ß6-mediated invasiveness of pancreatic carcinoma cells. Accordingly, small interfering RNA-mediated silencing of integrin ß6 markedly suppressed xenograft tumor growth in vivo. Taken together, our results suggest that integrin ß6 plays important roles in the progression of pancreatic carcinoma and contributes to reduced survival times, and may serve as a novel therapeutic target for the treatment of PDAC.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Cadenas beta de Integrinas/biosíntesis , Metaloproteinasa 9 de la Matriz/biosíntesis , Proteína Proto-Oncogénica c-ets-1/genética , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Cadenas beta de Integrinas/genética , Sistema de Señalización de MAP Quinasas/genética , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Persona de Mediana Edad , Invasividad Neoplásica/genética , Pronóstico , Proteína Proto-Oncogénica c-ets-1/biosíntesis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.
Asunto(s)
Endometrio/efectos de los fármacos , Cadenas beta de Integrinas/biosíntesis , Factor Inhibidor de Leucemia/metabolismo , Perilla frutescens/química , Extractos Vegetales/farmacología , Antracenos/farmacología , Butadienos/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Imidazoles/farmacología , Cadenas beta de Integrinas/metabolismo , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/antagonistas & inhibidores , Subunidad alfa del Receptor del Factor Inhibidor de Leucemia/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nitrilos/farmacología , Hojas de la Planta/química , Raíces de Plantas/química , Piridinas/farmacología , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Activated T regulatory cells (Tregs) express latent TGF-ß1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-ß1 from the complex of latent TGF-ß1 and latent TGF-ß1 binding protein, their role in processing latent TGF-ß1 from the latent TGF-ß1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin ß8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-ß1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-ß1 on their cell surface consistent with defective processing. Thus, integrin αvß8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-ß1 from the latent TGF-ß1/GARP complex on the surface of tTregs.
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Cadenas beta de Integrinas/inmunología , Integrinas/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Biomarcadores , Diferenciación Celular/inmunología , Colitis/inmunología , Proteínas de Unión al ADN/biosíntesis , Factores de Transcripción Forkhead/biosíntesis , Proteínas Fluorescentes Verdes/genética , Cadenas beta de Integrinas/biosíntesis , Cadenas beta de Integrinas/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Th17/inmunología , Factores de Transcripción/biosíntesis , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
BACKGROUND: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. METHODS: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. RESULTS: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin ß8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. CONCLUSIONS: These results reveal that EphB4 regulates integrin ß8 expression and that integrin ß8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin ß8 may be a new treatment strategy for prostate cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Cadenas beta de Integrinas/biosíntesis , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Receptor EphB4/fisiología , Línea Celular Tumoral , Humanos , Masculino , Proteínas Tirosina Quinasas Receptoras/fisiologíaRESUMEN
We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and ß5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.
Asunto(s)
Carcinoma de Células Escamosas/patología , Movimiento Celular , Galectina 1/fisiología , Integrina alfa2/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Comunicación Autocrina , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Galectina 1/genética , Galectina 1/farmacología , Humanos , Invasividad Neoplásica , Regulación hacia ArribaRESUMEN
The integrin αvß8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-ß. Through its association with LAP, TGF-ß is maintained in a latent form that must be activated to function. Binding to the integrin αvß8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-ß activation in vivo. Altered expression of the integrin ß8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1ß (IL-1ß) increases ITGB8 expression on lung fibroblasts, which increases αvß8-mediated TGF-ß activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1ß. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvß8 surface expression, and αvß8-mediated TGFß activation.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Cadenas beta de Integrinas/biosíntesis , Interleucina-1beta/biosíntesis , Regiones Promotoras Genéticas , Factor de Crecimiento Transformador beta/metabolismo , Acetilación , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , ADN/genética , ADN/metabolismo , Células HeLa , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Integrina alfa5/biosíntesis , Integrina alfa5/genética , Cadenas beta de Integrinas/genética , Interleucina-1beta/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Telomerase, the enzyme that elongates our telomeres, is crucial for cancer development based on extensive analyses of human cells, human cancers, and mouse models. New data now suggest that a viral telomerase RNA gene encoded by Marek's disease virus (MDV), an oncogenic herpesvirus of chickens, promotes tumor formation. These findings highlight the importance of telomerase in cancer and raise new questions regarding the mechanisms by which the telomerase RNA component supports tumorigenesis.
Asunto(s)
Transformación Celular Viral , Linfoma de Células T/enzimología , Mardivirus/enzimología , Telomerasa/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Pollos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Regulación Leucémica de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Genoma , Humanos , Cadenas alfa de Integrinas/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Linfoma de Células T/genética , Linfoma de Células T/patología , Linfoma de Células T/virología , Mardivirus/genética , Enfermedad de Marek/enzimología , Enfermedad de Marek/genética , Enfermedad de Marek/patología , Enfermedad de Marek/virología , Ratones , Mutación , Homología de Secuencia de Aminoácido , Telomerasa/genética , Proteínas Virales/genéticaRESUMEN
Telomerase is a ribonucleoprotein complex consisting of two essential core components: a reverse transcriptase and an RNA subunit (telomerase RNA [TR]). Dysregulation of telomerase has been associated with cell immortalization and oncogenesis. Marek's disease herpesvirus (MDV) induces a malignant T cell lymphoma in chickens and harbors in its genome two identical copies of a viral TR (vTR) with 88% sequence identity to chicken TR. MDV mutants lacking both copies of vTR were significantly impaired in their ability to induce T cell lymphomas, although lytic replication in vivo was unaffected. Tumor incidences were reduced by >60% in chickens infected with vTR- viruses compared with animals inoculated with MDV harboring at least one intact copy of vTR. Lymphomas in animals infected with the vTR- viruses were also significantly smaller in size and less disseminated. Constitutive expression of vTR in the chicken fibroblast cell line DF-1 resulted in a phenotype consistent with transformation as indicated by morphological alteration, enhanced anchorage-independent cell growth, cell growth beyond saturation density, and increased expression levels of integrin alpha v. We concluded that vTR plays a critical role in MDV-induced T cell lymphomagenesis. Furthermore, our results provide the first description of tumor-promoting effects of TR in a natural virus-host infection model.
Asunto(s)
Transformación Celular Viral , Linfoma de Células T/enzimología , Mardivirus/enzimología , Telomerasa/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Pollos , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Regulación Leucémica de la Expresión Génica/fisiología , Regulación Viral de la Expresión Génica/fisiología , Genoma , Cadenas alfa de Integrinas/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Linfoma de Células T/genética , Linfoma de Células T/patología , Linfoma de Células T/virología , Mardivirus/genética , Enfermedad de Marek/enzimología , Enfermedad de Marek/genética , Enfermedad de Marek/patología , Enfermedad de Marek/virología , Mutación , Homología de Secuencia de Aminoácido , Telomerasa/genética , Proteínas Virales/genéticaRESUMEN
BACKGROUND: Cell properties, such as attachment, adhesion and invasion, are important for the normal function of the endometrium. However, it is believed that the same properties may also be involved in the development of gynaecological diseases, such as endometriosis. Endometrial cells, shed by retrograde menstruation, may have an aberrant expression of molecules involved in these functions, leading to endometriosis. Therefore, the aim of this study was to investigate the expression of proteins involved in adhesion, attachment and invasion in eutopic and ectopic endometrium. METHODS: Endometrial biopsy specimens were collected from healthy volunteers (controls: proliferative phase, n = 10; secretory phase, n = 15) and from endometriosis patients (proliferative phase: n = 9, secretory phase: n = 10). Biopsy specimens from endometriomas were also collected (proliferative phase: n = 9, secretory phase: n = 10). Expression of apolipoprotein E (ApoE), integrin ß-2 (ITGB2), integrin ß-7 (ITGB7), Laminin γ-1 (LAMC1), CD24 molecule (CD24) and junctional adhesion molecule-1 (JAM-1) was evaluated with real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. RESULTS: The endometrium from controls and women with endometriosis expressed ApoE, ITGB2, ITGB7, LAMC1, CD24 and JAM-1. Gene expression of ApoE and JAM-1 was decreased in both proliferative and secretory phase in the endometrium from women with endometriosis compared with control endometrium. Also, mRNA expression of LAMC1 was reduced in the endometrium from endometriosis patients compared with controls in the proliferative phase. An altered gene expression of CD24 was seen between the endometrium from endometriosis patients and endometriomas in the secretory phase. The ITGB2 protein expression was altered in epithelia cells between the endometrium from healthy volunteers and endometriosis patients in the secretory phase. CONCLUSIONS: We have shown differential expression of adhesion, attachment and invasion proteins in proliferative and secretory endometrium from controls and endometriosis patients and in endometriomas. This study suggests that molecules with these properties may have a role in the anchoring of endometrial cells at ectopic sites, thus initiating the development of endometriosis.
Asunto(s)
Endometriosis/patología , Endometrio/fisiopatología , Adulto , Apolipoproteínas E/biosíntesis , Biopsia , Antígenos CD18/biosíntesis , Antígeno CD24/biosíntesis , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Proliferación Celular , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Cadenas beta de Integrinas/biosíntesis , Laminina/biosíntesis , Ciclo Menstrual , Receptores de Superficie Celular/biosíntesisRESUMEN
High-grade cervical dysplasia caused by human papillomavirus (HPV) type 16 is a lesion that should be susceptible to an HPV-specific immune response; disease initiation and persistence is predicated on expression of two viral Ags, E6 and E7. In immune-competent subjects, at least 25% of HPV16(+) high-grade cervical dysplasia lesions undergo complete regression. However, in the peripheral blood, naturally occurring IFN-γ T cell responses to HPV E6 and E7 are weak, requiring ex vivo sensitization to detect, and are not sufficiently sensitive to predict regression. In this study, we present immunologic data directly assessing cervical lymphocytes from this cohort. We found that nearly all cervical tissue T cells express the mucosal homing receptor, α(4)ß(7) surface integrin. T cells isolated from dysplastic mucosa were skewed toward a central memory phenotype compared with normal mucosal resident T cells, and dysplastic lesions expressed transcripts for CCL19 and CCL21, raising the possibility that the tissue itself sustains a response that is not detectable in the blood. Moreover, lesion regression in the study window could retrospectively be predicted at study entry by the ability of CD8(+) T cells to gain access to lesional epithelium. Vascular endothelial expression of mucosal addressin cell adhesion molecule-1, the ligand that supports entry of α(4)ß(7)(+) T cells into tissues, colocalized tightly with the distribution of CD8 T cells and was not expressed in persistent dysplastic epithelium. These findings suggest that dysregulated expression of vascular adhesion molecules plays a role in immune evasion very early in the course of HPV disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Células Epiteliales/inmunología , Papillomavirus Humano 16/inmunología , Infecciones por Papillomavirus/inmunología , Displasia del Cuello del Útero/inmunología , Neoplasias de la Vulva/inmunología , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Movimiento Celular/inmunología , Estudios de Cohortes , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Endotelio Vascular/virología , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Humanos , Integrina alfa4/biosíntesis , Cadenas beta de Integrinas/biosíntesis , Proteínas Oncogénicas Virales/biosíntesis , Proteínas E7 de Papillomavirus/biosíntesis , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Estudios Prospectivos , Proteínas Represoras/biosíntesis , Estudios Retrospectivos , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virología , Neoplasias de la Vulva/patología , Neoplasias de la Vulva/virologíaRESUMEN
Bile duct epithelial cells (BDECs) contribute to liver fibrosis by expressing αVß6 integrin, a critical activator of latent transforming growth factor ß (TGF-ß). ß6 integrin (Itgß6) mRNA induction and αVß6 integrin expression in BDECs are partially TGF-ß-dependent. However, the signaling pathways required for TGF-ß-dependent Itgß6 mRNA induction in BDECs are not known. We tested the hypothesis that the p38 mitogen-activated protein kinase (MAPK) signaling pathway contributes to TGF-ß1 induction of Itgß6 mRNA by activating SMAD and activator protein 1 (AP-1) transcription factors. Pretreatment of transformed human BDECs (MMNK-1 cells) with two different p38 MAPK inhibitors, but not a control compound, inhibited TGF-ß1 induction of Itgß6 mRNA. Inhibition of p38 also reduced TGF-ß1 activation of a SMAD-dependent reporter construct. Expression of a dominant-negative SMAD3 (SMAD3ΔC) significantly reduced TGF-ß1-induced Itgß6 mRNA expression. Expression of JunB mRNA, but not other AP-1 proteins, increased in TGF-ß1-treated MMNK-1 cells, and induction of JunB expression was p38-dependent. Consistent with a requirement for de novo induction of JunB protein, cycloheximide pretreatment inhibited TGF-ß1 induction of Itgß6 mRNA. Expression of a dominant-negative AP-1 mutant (TAM67) also inhibited TGF-ß1 induction of Itgß6 mRNA. Overall, the results suggest that p38 contributes to TGF-ß1-induced Itgß6 mRNA expression in MMNK-1 cells by regulating activation of both SMAD and AP-1 transcription factors.
Asunto(s)
Conductos Biliares/metabolismo , Células Epiteliales/metabolismo , Cadenas beta de Integrinas/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Conductos Biliares/citología , Western Blotting , Línea Celular , Citosol/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Densitometría , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , ARN/biosíntesis , ARN/genética , ARN/aislamiento & purificación , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Proteína smad3/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Crecimiento Transformador beta1/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
PURPOSE: Multipurpose solutions (MPS) are used daily to clean and disinfect silicone hydrogel (SiHy) contact lenses. This in vitro study was undertaken to identify the potential for interaction between MPS, SiHy surface treatments, and lens materials, which may lead to changes in the response of human corneal epithelial cells (HCEC) to MPS-soaked lenses. METHODS: The MPS tested were renu fresh (formerly known as ReNu MultiPlus; ReNu), OptiFree Express (OFX), OptiFree RepleniSH, SoloCare Aqua, and Complete Moisture Plus. The SiHy materials evaluated were lotrafilcon A, lotrafilcon B, comfilcon A, galyfilcon A, and balafilcon A (BA). MPS-soaked lenses were placed on top of adherent HCEC. The effect of MPS dilutions (0.1 to 10% final concentration in medium) was also characterized. Cell viability, adhesion phenotype and caspase activation were studied after 24-h cell exposure. OFX released from lenses was determined using UV absorbance. RESULTS: A significant reduction in viability (between 30 to 50%) was observed with cells exposed to lenses soaked in ReNu and OFX. A significant downregulation of α(3) and ß(1) integrins, with integrin expression ranging from 60% to 75% of control (cells with no lens), was also observed with OFX and ReNu-soaked lenses. With the exception of BA, all other lenses soaked in OFX resulted in significant caspase activation, whereby over 18% of cells stained positive for caspases. Minimal caspase activation was observed in cells exposed to ReNu and Solo soaked lenses. For both OFX and ReNu, exposing cells to at least a 5% dilution had a significant effect on viability and integrin expression. While Complete and Solo did not lead to reduction in viability, cells exposed to a 10% dilution showed reduced integrin expression down to less than 70% of control value. Comparing cell response to diluted MPS solutions and various MPS-soaked lenses showed that it is not possible to reliably use cell response to MPS dilution alone to assess MPS biocompatibility. CONCLUSIONS: Our results demonstrate that the reaction of HCEC to MPS are affected by the type of lenses the MPS is released from and may potentially be influenced by the surface treatment (or lack of it) of SiHy materials.
Asunto(s)
Soluciones para Lentes de Contacto/toxicidad , Lentes de Contacto Hidrofílicos , Córnea/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Apoptosis/efectos de los fármacos , Caspasas/biosíntesis , Supervivencia Celular/efectos de los fármacos , Córnea/citología , Córnea/metabolismo , Regulación hacia Abajo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Hidrogeles , Integrina alfa3/biosíntesis , Cadenas beta de Integrinas/biosíntesis , SiliconasRESUMEN
BACKGROUND: The formation of metastases includes the separation of tumor cells from the primary tumor, cell migration into subendothelial tissue and cell proliferation in secondary organ. In this process, cell adhesion of tumor cells to the endothelium is an essential requirement for formation of metastases. Protein kinase C (PKC) regulates adhesion and proliferation. To identify a relation between PKC isoforms and tumor progression in renal cell carcinoma (RCC), the influence of PKC isoforms on cell adhesion and proliferation, and possible influences of integrins were analyzed in RCC cells. METHODS: The experiments were performed in the RCC cell lines CCF-RC1 and CCF-RC2 after pre-incubation (16 h) with the PKC inhibitors GF109203X (inhibits PKCalpha, betaI, betaII, gamma, delta and epsilon), GO6976 (inhibits PKCalpha, betaI and mu), RO31-8220 (inhibits PKCalpha, betaI, betaII, gamma and epsilon) and rottlerin (inhibits PKCdelta). Cell adhesion was assessed through adherence of RCC cells to an endothelial monolayer. Cell proliferation was analyzed by a BrdU incorporation assay. The expression of beta1 integrins was analyzed by flow cytometry. RESULTS: In CCF-RC1 cells, cell adhesion was significantly reduced by GO6976 to 55% and by RO31-8220 to 45% of control. In CCF-RC2 cells, only GO6976 induced a significant reduction of cell adhesion to 50% of control levels. Proliferation of both cell lines was reduced by rottlerin to 39% and 45% of control, respectively. The beta1 integrin expression on the cell surface of CCF-RC1 and CCR-RC2 cells was decreased by RO31-8220 to 8% and 7% of control, respectively. beta2 and beta3 integrins were undetectable in both cell lines. CONCLUSIONS: The combination of the PKC inhibitors leads to the assumption that PKCmu influences cell adhesion in CCF-RC1 and CCF-RC2 cells, whereas in CCF-RC1 cells PKCepsilon also seems to be involved in this process. The expression of beta1 integrins appears to be regulated in particular by PKCepsilon. Cell proliferation was inhibited by rottlerin, so that PKCdelta might be involved in cell proliferation in these cells.