RESUMEN
Rheumatoid arthritis (RA) is a common chronic autoimmune disease featured by synovial inflammation. miR-496 is closely involved in various pathologic conditions. However, its role in RA has not yet been elucidated. Expression of miR-496 and MMP10 was determined based on the clinical samples with RA retrieved from the Gene Expression Omnibus (GEO) datasets. In vitro model of RA was constructed in MH7A cells stimulated by IL-1ß (10 ng/mL). Cell counting kit 8 (CCK-8) and flow cytometry experiments were implemented to investigate the cell viability and apoptosis rate of MH7A cells. TargetScan was applied to identify the targets of miR-496, and the regulation of miR-496 on MMP10 expression was validated by a dual-luciferase reporter gene assay. qRT-PCR and western blot analyses were conducted to examine the expression. miR-496 expression was decreased in RA tissues and MH7A cells after IL-1ß treatment. Overexpression of miR-496 significantly inhibited IL-1ß-treated MH7A cell viability. MMP10 was identified as a target of miR-496 and its expression was negatively regulated by miR-496. The effects of miR-496 on MH7A cell proliferation and apoptosis were reversed by MMP10. The activity of NF-κB pathway was associated with the miR-496/MMP10 axis in IL-1ß-stimulated MH7A cells. To summarize, this study demonstrated that miR-496 can impair the proliferative ability and facilitate the apoptosis of IL-1ß-treated MH7A through regulating MMP10 expression and NF-κB signaling pathway.
Asunto(s)
Fibroblastos/metabolismo , Interleucina-1beta/toxicidad , Metaloproteinasa 10 de la Matriz/biosíntesis , MicroARNs/biosíntesis , FN-kappa B/metabolismo , Sinoviocitos/metabolismo , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Fibroblastos/efectos de los fármacos , Humanos , Transducción de Señal/fisiología , Sinoviocitos/efectos de los fármacosRESUMEN
Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active ß-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation-associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical ß-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear ß-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/ß-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/ß-catenin signaling, which may offer prognostic and therapeutic opportunities.
Asunto(s)
MicroARNs/fisiología , Neoplasias de la Vejiga Urinaria/patología , Proteínas Wnt/fisiología , Vía de Señalización Wnt , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Espectrometría de Masas , Metaloproteinasa 10 de la Matriz/biosíntesis , Invasividad Neoplásica , Metástasis de la Neoplasia , Oncogenes , Pronóstico , Neoplasias de la Vejiga Urinaria/metabolismo , Proteínas Wnt/genética , beta Catenina/metabolismoRESUMEN
INTRODUCTION: Tissue inhibitors of metalloproteinases (TIMP) and the matrix metalloproteinases (MMP) are involved in the spread of cancer. METHODS: We have evaluated the matrix metalloproteinases' (MMP-10, MMP-7) and their inhibitors' (tissue inhibitors of metalloproteinases - TIMP-1, TIMP-2) mRNA expression in 61 esophageal cancer samples from patients who had undergone surgery, by using real-time quantitative RT-PCR, and correlated the results with the patient clinicopathologic features. RESULTS: MMP-10, MMP-7, TIMP-1, TIMP-2 were overexpressed in 73%, 85%, 55% and 42% of esophageal cancer samples, respectively. The expression of MMP-10, TIMP-1, and TIMP-2 correlated with the tumor size. The MMP-7 overexpression was associated with the tumour stage (I, II vs III, p=0.05) and lymph node metastasis (N0 vs N1, p=0.037). CONCLUSIONS: We conclude that in the resected esophageal cancer an increased mRNA expression of MMP-7, MMP-10 and TIMP-1 correlated with clinicopathologic features. We suggest that these genes may play a role during progression of the disease.
Asunto(s)
Neoplasias Esofágicas/genética , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 7 de la Matriz/biosíntesis , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Anciano , Neoplasias Esofágicas/patología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Masculino , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/genética , Persona de Mediana Edad , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-2/genéticaRESUMEN
BACKGROUND: Some matrix metalloproteinases (MMPs) are involved in invasion and metastasis of head and neck squamous cell carcinoma (HNSCC). However, there are few studies on association between stromelysin-2 (ST-2) and invasive behavior of HNSCC. The purpose of this study was to investigate Stromelysin-2 expression by immunohistochemistry. MATERIALS AND METHODS: This study was conducted on 81 specimens, including 61 HNSCC and 20 non neoplastic epithelium. Sections with 5 micron thickness were prepared and stained with immunohistochemistry technique. Then expression of ST-2 was evaluated according to percentage of stained cells and intensity of staining. Data were analyzed by SPSS (V.21) using Kruskal-Wallis and Tukey tests (P<0.05). RESULTS: The 61 HNSCC specimens were grades I 36.1%, II 34.4% and III 29.5%. The level of ST-2 expressions were moderate (++) and intensive (+++) in 21.3% and 78.7% of tumors, respectively. The ST-2 expression level was only significant between the tumors with grade I and grade III (P=0.016). Tumors presented ST-2 expression with staining intensity of mild 6.6%, moderate 26.2% and strong 67.2%. Staining intensity of ST-2 in grade I tumors was significantly lower than grade II and grade III (P<0.05), and there was no significant difference between grades II and III (P=0.99). CONCLUSIONS: According to this study, the expression of ST-2 is associated with histopathological grade and tumor differentiation in HNSCCs.
Asunto(s)
Carcinoma de Células Escamosas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/patología , Metaloproteinasa 10 de la Matriz/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: Cancer invasion and metastasis develops through a series of steps that involve the loss of cell to cell and cell to matrix adhesion, degradation of extracellular matrix and induction of angiogenesis. Different protease systems (e.g., matrix metalloproteinases, MMPs) are involved in these steps. MMP-10, one of the lesser studied MMPs, is limited to epithelial cells and can facilitate tumor cell invasion by targeting collagen, elastin and laminin. Enhanced MMP-10 expression has been linked to poor clinical prognosis in some cancers, however, mechanisms underlying a role for MMP-10 in tumorigenesis and progression remain largely unknown. Here, we report that MMP-10 expression is positively correlated with the invasiveness of human cervical and bladder cancers. METHODS: Using commercial tissue microarray (TMA) of cervical and bladder tissues, MMP-10 immunohistochemical staining was performed. Furthermore using a panel of human cells (HeLa and UROtsa), in vitro and in vivo experiments were performed in which MMP-10 was overexpressed or silenced and we noted phenotypic and genotypic changes. RESULTS: Experimentally, we showed that MMP-10 can regulate tumor cell migration and invasion, and endothelial cell tube formation, and that MMP-10 effects are associated with a resistance to apoptosis. Further investigation revealed that increasing MMP-10 expression stimulates the expression of HIF-1α and MMP-2 (pro-angiogenic factors) and PAI-1 and CXCR2 (pro-metastatic factors), and accordingly, targeting MMP-10 with siRNA in vivo resulted in diminution of xenograft tumor growth with a concomitant reduction of angiogenesis and a stimulation of apoptosis. CONCLUSION: Taken together, our findings show that MMP-10 can play a significant role in tumor growth and progression, and that MMP-10 perturbation may represent a rational strategy for cancer treatment.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Metaloproteinasa 10 de la Matriz/biosíntesis , Invasividad Neoplásica/genética , Neoplasias de la Vejiga Urinaria/genética , Neoplasias del Cuello Uterino/genética , Apoptosis , Movimiento Celular , Matriz Extracelular/genética , Matriz Extracelular/patología , Femenino , Células HeLa , Humanos , Metaloproteinasa 10 de la Matriz/genética , Terapia Molecular Dirigida , ARN Interferente Pequeño , Neoplasias de la Vejiga Urinaria/patología , Neoplasias del Cuello Uterino/patologíaRESUMEN
PURPOSE: Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression. METHODS: Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections. RESULTS: Proteinase silencing reduced epithelial wound healing time versus rAV-enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α3ß1 integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain. CONCLUSIONS: Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
Asunto(s)
Catepsina F/genética , Enfermedades de la Córnea/genética , Diabetes Mellitus/genética , Silenciador del Gen , Metaloproteinasa 10 de la Matriz/genética , Células Madre/metabolismo , Cicatrización de Heridas/genética , Anciano , Anciano de 80 o más Años , Catepsina F/biosíntesis , Células Cultivadas , Enfermedades de la Córnea/metabolismo , Enfermedades de la Córnea/patología , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Epitelio Corneal/metabolismo , Femenino , Terapia Genética/métodos , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasa 10 de la Matriz/biosíntesis , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Células Madre/citologíaRESUMEN
Nucleocytoplasmic shuttling of class IIa of histone deacetylases (HDACs) is a key mechanism that controls cell fate and animal development. We have identified the filamin B (FLNB) as a novel HDAC7-interacting protein that is required for temporal and spatial regulation of vascular endothelial growth factor (VEGF)-mediated HDAC7 cytoplasmic sequestration. This interaction occurs in the cytoplasm and requires monoubiquitination of an evolutionarily conserved lysine 1147 (K1147) in the immunoglobulin (Ig)-like repeat 10 (R10) of FLNB and the nuclear localization sequence of HDAC7. Inhibition of protein kinase C (PKC) blocks VEGF-induced ubiquitination of FLNB and its interaction with HDAC7. Small interfering RNA (siRNA) knockdown of FLNB or ubiquitin (Ub) in human primary endothelial cells blocks VEGF-mediated cytoplasmic accumulation of HDAC7, reduces VEGF-induced expression of the HDAC7 target genes Mmp-10 and Nur77, and inhibits VEGF-induced vascular permeability. Using dominant negative mutants and rescue experiments, we demonstrate the functional significance of FLNB K1147 to interfere with the ability of phorbol myristate acetate (PMA) to promote FLNB-mediated cytoplasmic accumulation of HDAC7. Taken together, our data show that VEGF and PKC promote degradation-independent protein ubiquitination of FLNB to control intracellular trafficking of HDAC7.
Asunto(s)
Proteínas Contráctiles/metabolismo , Histona Desacetilasas/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteína Quinasa C/metabolismo , Factores de Crecimiento Endotelial Vascular/metabolismo , Secuencia de Aminoácidos , Carbazoles/farmacología , Línea Celular Tumoral , Movimiento Celular , Proteínas Contráctiles/genética , Células Endoteliales/metabolismo , Filaminas , Células HeLa , Humanos , Indoles/farmacología , Maleimidas/farmacología , Metaloproteinasa 10 de la Matriz/biosíntesis , Proteínas de Microfilamentos/genética , Neovascularización Fisiológica , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Proteína Quinasa C/antagonistas & inhibidores , Estructura Terciaria de Proteína , Transporte de Proteínas , Interferencia de ARN , ARN Interferente Pequeño , Acetato de Tetradecanoilforbol/farmacología , Ubiquitina/genética , Ubiquitinación , Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVES: The aim of this study was to immunohistochemically evaluate the expression of matrix metalloproteinase (MMP)-1, MMP- 2, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, and podoplanin in oral squamous cell carcinoma (OSCC). Immunohistochemical staining of podoplanin-positive lymphatic vessel density (LVD) was also assessed. STUDY DESIGN: Forty cases of OSCC were analyzed by immunohistochemistry. RESULTS: MMP-2, MMP-10, TIMP-1, TIMP-2, and podoplanin were detected in each of the 40 OSCC cases. The expression of MMP-2 was significantly correlated with histologic grade. The expression of podoplanin was positively correlated with gender and negatively correlated with tumor size. A significant positive correlation was also detected between LVD and the presence of lymph node metastases, gender, age, and diameter of the lymph node (if involved), as well as histologic grade. CONCLUSIONS: The results are suggestive of important roles that MMP-2, MMP-10, TIMP-2, and podoplanin play in pathologic processes of OSCC, including invasion. Our findings also suggest that LVD may play a role in lymphatic metastasis and tumor progression.
Asunto(s)
Carcinoma de Células Escamosas/química , Metaloproteinasas de la Matriz/análisis , Glicoproteínas de Membrana/análisis , Neoplasias de la Boca/química , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Femenino , Humanos , Irán , Metástasis Linfática , Vasos Linfáticos/patología , Masculino , Metaloproteinasa 10 de la Matriz/análisis , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/análisis , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasas de la Matriz/biosíntesis , Glicoproteínas de Membrana/biosíntesis , Persona de Mediana Edad , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/patología , Invasividad Neoplásica , Análisis de Regresión , Estudios Retrospectivos , Factores Sexuales , Estadísticas no Paramétricas , Inhibidor Tisular de Metaloproteinasa-1/análisis , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/análisis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesisRESUMEN
Cytokines present in the tumor microenvironment can promote the invasiveness and metastatic potential of cancer cells. We therefore investigated the effects of interleukin-1 beta (IL-1B) and transforming growth factor beta-3 (TGFB3) on the non-small cell lung carcinoma (NSCLC) cell line A549. We found that these cytokines synergistically activated matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10 gene expression in these cells through mitogen-activated protein kinase (MAPK)-dependent pathways. Consistent with this, both cytokines stimulated epithelial to mesenchymal transition and MAPK-dependent invasion through Matrigel™. These studies identify IL-1B and TGFB3 as pro-invasive factors in NSCLC and potential therapeutic targets for tumor progression.
Asunto(s)
Adenocarcinoma/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Interleucina-1beta/farmacología , Neoplasias Pulmonares/patología , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 1 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/biosíntesis , Proteínas de Neoplasias/biosíntesis , Factor de Crecimiento Transformador beta3/farmacología , Carcinoma de Células Renales/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Línea Celular Tumoral/patología , Colágeno , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Inducción Enzimática/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Renales/patología , Laminina , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , ProteoglicanosRESUMEN
Discoidin domain receptors (DDRs) DDR1 and DDR2 are receptor tyrosine kinases with the unique ability among receptor tyrosine kinases to respond to collagen. Several signaling molecules have been implicated in DDR signaling, including Shp-2, Src, and MAPK pathways, but a detailed understanding of these pathways and their transcriptional targets is still lacking. Similarly, the regulation of the expression of DDRs is poorly characterized with only a few inflammatory mediators, such as lipopolysaccharide and interleukin-1ß identified as playing a role in DDR1 expression. DDRs have been reported to induce the expression of various genes including matrix metalloproteinases and bone morphogenetic proteins, but the regulatory mechanisms underlying DDR-induced gene expression remain to be determined. The aim of the present work was to elucidate the molecular mechanisms implicated in the expression of DDRs and to identify DDR-induced signaling pathways and target genes. Our data show that collagen I induces the expression of DDR1 in a dose- and time-dependent manner in primary human lung fibroblasts. Furthermore, activation of DDR2, JAK2, and ERK1/2 MAPK signaling pathways was essential for collagen I-induced DDR1 and matrix metalloproteinase 10 expression. Finally, inhibition of the ERK1/2 pathway abrogated DDR1 expression by blocking the recruitment of the transcription factor polyoma enhancer A-binding protein 3 to the DDR1 promoter. Our data provide new insights into the molecular mechanisms of collagen I-induced DDR1 expression and demonstrate an important role for ERK1/2 activation and the recruitment of polyoma enhancer-A binding protein 3 to the DDR1 promoter.
Asunto(s)
Colágeno Tipo I/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Janus Quinasa 2/metabolismo , Pulmón/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptores Mitogénicos/biosíntesis , Colágeno Tipo I/genética , Receptores con Dominio Discoidina , Fibroblastos/citología , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Janus Quinasa 2/genética , Lipopolisacáridos/farmacología , Pulmón/citología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores Mitogénicos/genéticaRESUMEN
Rhinovirus (RV) infections trigger asthma exacerbations. Genome-wide expression analysis of RV1A-infected primary bronchial epithelial cells from normal and asthmatic donors was performed to determine whether asthma is associated with a unique pattern of RV-induced gene expression. Virus replication rates were similar in cells from normal and asthmatic donors. Overall, RV downregulated 975 and upregulated 69 genes. Comparisons of transcriptional profiles generated from microarrays and confirmed by quantitative reverse transcription PCR and cluster analysis showed some up- and downregulated genes in asthma cells involved in immune responses (IL1B, IL1F9, IL24, and IFI44) and airway remodeling (LOXL2, MMP10, FN1). Notably, most of the asthma-related differences in RV-infected cells were also present in the cells before infection. These findings suggest that differences in RV-induced gene expression profiles of cells from normal and mild asthmatic subjects could affect the acute inflammatory response to RV, and subsequent airway repair and remodeling.
Asunto(s)
Asma/inmunología , Infecciones por Picornaviridae/inmunología , Mucosa Respiratoria/metabolismo , Rhinovirus/fisiología , Adulto , Remodelación de las Vías Aéreas (Respiratorias)/genética , Aminoácido Oxidorreductasas/biosíntesis , Aminoácido Oxidorreductasas/genética , Asma/complicaciones , Asma/genética , Asma/patología , Células Cultivadas , Citocinas/biosíntesis , Citocinas/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Análisis por Micromatrices , Infecciones por Picornaviridae/complicaciones , Infecciones por Picornaviridae/genética , Infecciones por Picornaviridae/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Replicación ViralRESUMEN
We previously reported that matrix metalloproteinase (MMP)-10 mRNA levels were significantly lower in tumor tissues than in adjacent normal tissues in human non-small cell lung cancer (NSCLC), whereas protein levels of MMP-10 were higher in the tumor tissues than the adjacent tissues. The mechanism of this divergence is still unknown. In the present study the role of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) on interleukin (IL)-6 mediated regulation of MMP-10 expression was investigated in a human lung adenocarcinoma cell line (A549 cells) and the molecular regulatory mechanism of MMP-10 expression was explored. A549 cells were stimulated by different concentrations of IL-6 with or without AG490, a specific JAK2 inhibitor. It was demonstrated that IL-6 moderately reduced the MMP-10 mRNA levels, whereas it significantly enhanced the MMP-10 protein mass in the A549 cells. This phenomenon mimicked the divergence of mRNA level and protein mass of MMP-10 in human NSCLC. Moreover, the present study indicated that IL-6 regulation of MMP-10 expression was via the JAK2/STAT3 pathway. STAT3 mRNA levels were significantly increased when the cells were treated with IL-6, whereas when AG490 (50 muM) was added to the cell cultures, IL-6-induced increase of STAT3 mRNA levels was abolished. Meanwhile, AG490 blocked the IL-6-induced inhibition of MMP-10 mRNA as well as blocking the IL-6-induced increase of MMP-10 protein mass in the A549 cells. Neither IL-6 nor AG490 influenced JAK2 mRNA levels in the A549 cell cultures. It is concluded that the JAK2/STAT3 pathway is involved in the IL-6-mediated regulation of MMP-10, and IL-6 can moderately reduce MMP-10 mRNA levels and strongly increase MMP-10 protein mass in human lung adenocarcinoma A549 cells. Contrasting effects of IL-6 on MMP-10 mRNA level and protein concentration in A549 cells may partially explain the divergence of MMP-10 mRNA level and protein mass in human NSCLC.
Asunto(s)
Adenocarcinoma/metabolismo , Interleucina-6/farmacología , Janus Quinasa 2/metabolismo , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 10 de la Matriz/biosíntesis , Factor de Transcripción STAT3/metabolismo , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Línea Celular Tumoral , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Janus Quinasa 2/antagonistas & inhibidores , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Metaloproteinasa 10 de la Matriz/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT3/genética , Transducción de Señal , Tirfostinos/farmacologíaRESUMEN
Before metastasis, certain organs have already been influenced by primary tumors. However, the exact alterations and regulatory mechanisms of the premetastatic organs remain poorly understood. Here, we report that, in the premetastatic stage, angiopoietin 2 (Angpt2), matrix metalloproteinase (MMP) 3, and MMP10 are up-regulated in the lung by primary B16/F10 tumor, which leads to the increased permeability of pulmonary vasculatures and extravasation of circulating tumor cells. Subsequent studies show that Angpt2, MMP3, and MMP10 have a synergistic effect on disrupting vascular integrity in both in vitro and in vivo models. Lentivirus-based in vivo RNA interference of Angpt2, MMP3, and MMP10 attenuates the pulmonary vascular permeability and suppresses the infiltration of myeloid cells in the premetastatic lung. Moreover, knocking down these factors significantly inhibits the spontaneous lung metastasis in the model by orthotopic implantation of MDA-MB-231-Luc-D3H1 cells in nude mice. Further investigations reveal that the malignancy of tumor cells is positively correlated with their capabilities to induce the expression of Angpt2, MMP3, and MMP10. Luciferase reporter assay and chromatin immunoprecipitation assay also suggest that transforming growth factor-beta1 and tumor necrosis factor-alpha signaling are involved in the regulation of these premetastatic factors. Our study shows that pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of tumor cells and facilitates lung metastasis, which may provide potential targets for clinical prevention of metastasis.
Asunto(s)
Neoplasias Pulmonares/secundario , Pulmón/irrigación sanguínea , Angiopoyetina 2/antagonistas & inhibidores , Angiopoyetina 2/biosíntesis , Angiopoyetina 2/genética , Animales , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Permeabilidad Capilar , Femenino , Expresión Génica , Humanos , Pulmón/enzimología , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/enzimología , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Melanoma Experimental/enzimología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Interferencia de ARN , Regulación hacia ArribaRESUMEN
OBJECTIVE: Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. METHODS AND RESULTS: Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. CONCLUSIONS: Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/metabolismo , Trombina/biosíntesis , Trombina/farmacología , Animales , Estudios de Casos y Controles , Bovinos , Células Cultivadas , Coagulación Intravascular Diseminada/enzimología , Humanos , Sistema de Señalización de MAP Quinasas , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/sangre , Ratones , Ratones Noqueados , Infarto del Miocardio/enzimología , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Transcripción AP-1/antagonistas & inhibidores , Factor de Transcripción AP-1/metabolismo , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Matrix metalloproteinase-1 (MMP-1) is an inflammation-inducible neutral protease that mediates extracellular matrix remodeling and promotes tumor invasion. In this study, we examined the activation of MMP-1 gene expression in A549 lung carcinoma cells stimulated with the inflammatory cytokine interleukin-1beta (IL-1beta). We found that MMP-1 mRNA levels were maximal following 16 hours of IL-1beta stimulation and that this correlated with the expression of the transcription factor CCAAT enhancer-binding protein-beta (CEBPB). Knockdown of CEBPB expression with short hairpin RNA abrogated the expression of MMP-1, MMP-3, and MMP-10 in IL-1beta-stimulated A549 cells. An established CEBP element in the MMP-1 promoter was found to be required for basal and IL-1beta-induced transcription. Electrophoresis mobility shift assays showed that CEBPB binds to this promoter element maximally 16 hours after IL-1beta stimulation. DNA affinity chromatography studies showed that the LAP1, LAP2, and LIP isoforms of CEBPB bind to the IL-1beta-responsive CEBPB site in the MMP-1 promoter. Exogenous expression of the LAP1 and LAP2 isoforms stimulated the MMP-1 promoter, whereas LIP had no effect. Phosphorylation of CEBPB at Thr(235) peaked at 16 hours in IL-1beta-stimulated cells. The MEK inhibitor U0126 inhibited this phosphorylation and reduced MMP-1 gene induction. These studies establish CEBPB as an important mediator of metalloproteinase gene activation during inflammatory responses in lung cancer cells and highlight the different regulatory roles of CEBPB isoforms.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Interleucina-1beta/farmacología , Neoplasias Pulmonares/metabolismo , Metaloproteinasa 1 de la Matriz/biosíntesis , Secuencia de Bases , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Oligonucleótidos/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Interferencia de ARNRESUMEN
INTRODUCTION: Matrix metalloproteinases (MMPs) are known to be involved in the degradation of the nucleus pulposus (NP) during intervertebral disc (IVD) degeneration. This study investigated MMP-10 (stromelysin-2) expression in the NP during IVD degeneration and correlated its expression with pro-inflammatory cytokines and molecules involved in innervation and nociception during degeneration which results in low back pain (LBP). METHODS: Human NP tissue was obtained at postmortem (PM) from patients without a history of back pain and graded as histologically normal or degenerate. Symptomatic degenerate NP samples were also obtained at surgery for LBP. Expression of MMP-10 mRNA and protein was analysed using real-time polymerase chain reaction and immunohistochemistry. Gene expression for pro-inflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-alpha), nerve growth factor (NGF) and the pain-associated neuropeptide substance P were also analysed. Correlations between MMP-10 and IL-1, TNF-alpha and NGF were assessed along with NGF with substance P. RESULTS: MMP-10 mRNA was significantly increased in surgical degenerate NP when compared to PM normal and PM degenerate samples. MMP-10 protein was also significantly higher in degenerate surgical NP samples compared to PM normal. IL-1 and MMP-10 mRNA demonstrated a significant correlation in surgical degenerate samples, while TNF-alpha was not correlated with MMP-10 mRNA. NGF was significantly correlated with both MMP-10 and substance P mRNA in surgical degenerate NP samples. CONCLUSIONS: MMP-10 expression is increased in the symptomatic degenerate IVD, where it may contribute to matrix degradation and initiation of nociception. Importantly, this study suggests differences in the pathways involved in matrix degradation between painful and pain-free IVD degeneration.
Asunto(s)
Disco Intervertebral/patología , Dolor de la Región Lumbar/metabolismo , Metaloproteinasa 10 de la Matriz/biosíntesis , Factor de Crecimiento Nervioso/biosíntesis , Sustancia P/biosíntesis , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-1/biosíntesis , Disco Intervertebral/metabolismo , Dolor de la Región Lumbar/etiología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
PURPOSE: To investigate the changes in gene expression induced by cyclic mechanical stress (CMS) in trabecular meshwork (TM) cells. METHODS: Human TM cultures from three donors were plated on type I collagen-coated flexible silicone bottom plates and subjected to 15% stretching, one cycle per second for 6 h. Non-stressed parallel control cultures were incubated under the same conditions in the absence of CMS. Total RNA from each culture was amplified (1 round of amplification) and hybridized to Operon Human Oligo Arrays version 3.0 (35 K probes). Differences in gene expression induced by CMS were analyzed using Genespring 7.2. quantitative polymerase chain reaction (Q-PCR) was used to confirm changes in the expressions of 12 selected genes. The effects of chemical inhibitors for p38, ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal kinase), PKA (protein kinase A), PI3K (phosphoinositide 3-kinase), and P2 (purinergic 2) receptors on the induction of MMP3 (matrix metalloproteinase 3), HSP70 (heat shock protein 70), ECSM1 (endothelial cell specific molecule 1), BMP2 (bone morphogenetic protein 2), VEGFC (vascular endothelial growth factor C), and IL-8 (interleukin 8) were evaluated in porcine TM cells subjected to the same regime of CMS as that used in human cells. RESULTS: CMS induced extensive gene expression changes (664 genes, p < or = 0.05) twofold or higher in cultured TM cells. Many of these changes were related to extracellular matrix (ECM) synthesis and remodeling including the upregulation of two metalloproteinases (MMP3 and MMP10). Cytoskeleton and cell adhesion genes were also affected by CMS as well as genes known to be involved in cellular protection against stress including several members of the HSP70 family. Inhibition of PI3K/AKT and P2 receptors pathways significantly reduced the induction of MMP3 and IL-8 whereas the inhibition of the PKA/cAMP pathway decreased ECSM1 and BMP2. CONCLUSIONS: CMS activated many genes that could influence the aqueous humor outflow facility, specifically genes involved in ECM synthesis and remodeling (e.g. MMPs), cytoskeletal organization, and cell adhesion. Induction of MMP3 has the potential to increase the aqueous humor outflow facility and could be part of a homeostatic mechanism involved in the maintenance of normal intraocular pressure (IOP) levels. Other observed changes are more likely to be related to general cellular responses to stress (e.g., HSP70, ECSM1, and BMP2). Although these latter changes may initially help to repair mechanical damage, some of them such as the induction of BMP2 could eventually increase tissue rigidity and compromise the ability of the TM to maintain normal levels of outflow resistance.
Asunto(s)
Expresión Génica , Estrés Mecánico , Malla Trabecular , Adolescente , Adulto , Animales , Humor Acuoso/metabolismo , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Técnicas de Cultivo de Célula , Supervivencia Celular , Citoprotección/genética , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Inhibidores Enzimáticos/farmacología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Perfilación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas HSP70 de Choque Térmico/genética , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/biosíntesis , Metaloproteinasa 3 de la Matriz/genética , Porcinos , Malla Trabecular/citología , Malla Trabecular/fisiologíaRESUMEN
Proteinase-activated receptors (PARs) are G-protein-coupled receptors that have been linked to an array of cellular processes, including inflammation, migration, and proliferation. Although signal transduction downstream of PARs has been actively investigated, little is known about the mechanisms that lead to changes in transcriptional programs. Here we show that the CUX1 homeodomain protein is a downstream effector of PAR2. Treatment of epithelial and fibroblastic cells with trypsin or the PAR2-activating peptide (PAR2-AP) caused a rapid increase in CUX1 DNA binding activity. The stimulation of CUX1 was specific to PAR2 because no effect was observed with thrombin or the PAR1-AP. Using a panel of recombinant CUX1 proteins, the regulation was found to involve the cut repeat 3 (CR3) and the cut homeodomain, two DNA binding domains that are present in all CUX1 isoforms. Expression analysis in cux1(-/-) mouse embryo fibroblasts led to the identification of three genes that are regulated downstream of both PAR2 and CUX1 as follows: interleukin-1alpha, matrix metalloproteinase-10, and cyclo-oxygenase-2. p110 CUX1 was able to activate each of these genes, both in reporter assays and following the infection of cells. Moreover, the treatment of Hs578T breast tumor cells with trypsin led to a rapid recruitment of p110 CUX1 to the promoter of these genes and to a concomitant increase in their mRNA steady-state levels. Altogether, these results suggest a model whereby activation of PAR2 triggers a signaling cascade that culminates with the stimulation of p110 CUX1 DNA binding and the transcriptional activation of target genes.
Asunto(s)
Fibroblastos/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Modelos Biológicos , Proteínas Nucleares/metabolismo , Receptor PAR-2/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/efectos de los fármacos , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Embrión de Mamíferos , Fibroblastos/citología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Inflamación/genética , Inflamación/metabolismo , Interleucina-1alfa/biosíntesis , Interleucina-1alfa/genética , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Ratones , Ratones Noqueados , Mutación , Células 3T3 NIH , Proteínas Nucleares/genética , Péptidos/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Proteínas Represoras/genética , Transducción de Señal/efectos de los fármacos , Tripsina/farmacologíaRESUMEN
The effects of endothelins (ETs) on the production of stromelysins, a sub-family of matrix metalloproteinases, were examined in cultured astrocytes. The treatment of cultured rat astrocytes with ET-1 increased stromelysin-1 mRNA levels, while stromelysin-2 and -3 mRNAs were not affected. Immunocytochemical observations showed that cultured astrocytes produced stromelysin-1 protein. ET-1 and Ala(1,3,11,15)-ET-1, an ET(B) receptor selective agonist, stimulated the release of stromelysin-1 from cultured astrocytes. Accompanying the increase in protein release, the peptidase activity of stromelysin-1 in the medium was also increased by ET-1. The effects of ET-1 on astrocytic stromelysin-1 expression were inhibited by PD98059, staurosporine, and Ca(2+) chelation, but not by SB203580 or pyrrolidine dithiocarbamate. These results show that activation of astrocytic ET receptors stimulates the production of stromelysin-1, suggesting a role for ETs in stromelysin production in brain pathologies.
Asunto(s)
Astrocitos/enzimología , Antagonistas de los Receptores de la Endotelina B , Endotelinas/fisiología , Metaloproteinasa 3 de la Matriz/biosíntesis , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Endotelina-1/farmacología , Endotelina-1/fisiología , Endotelinas/farmacología , Flavonoides/farmacología , Metaloproteinasa 10 de la Matriz/biosíntesis , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 11 de la Matriz/biosíntesis , Metaloproteinasa 11 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/genética , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Ratas , Estaurosporina/farmacologíaRESUMEN
We showed previously that the attachment of synovial fibroblasts to laminin (LM)-111 in the presence of transforming growth factor-beta induces significant expression of the matrix metalloproteinase (MMP)-3. Here we go on to investigate the regulation of additional MMPs and their specific tissue inhibitors of matrix proteases (TIMPs). Changes in steady-state mRNA levels encoding TIMPs and MMPs were investigated by quantitative reverse transcription-polymerase chain reaction. Production of MMPs was monitored by a multiplexed immunoarray. Signal transduction pathways were studied by immunoblotting. Attachment of synovial fibroblasts to LM-111 in the presence of transforming growth factor-beta induced significant increases in MMP-3 mRNA (12.35-fold, p < 0.001) and protein (mean 62 ng/ml, sixfold, p < 0.008) and in expression of MMP-10 mRNA (11.68-fold, p < 0.05) and protein (54 ng/ml, 20-fold, p > or = 0.02). All other TIMPs and MMPs investigated failed to show this LM-111-facilitated transforming growth factor-beta response. No phosphorylation of nuclear factor-kappaB was observed. We conclude that co-stimulation of synovial fibroblasts by LM-111 together with transforming growth factor-beta suffices to induce significant expression of MMP-3 and MMP-10 by synovial fibroblasts and that this induction is independent of nuclear factor-kappaB phosphorylation.