RESUMO
One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aß) peptide. In addition to Aß as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aß was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study, MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1 (BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.
Assuntos
Precursor de Proteína beta-Amiloide , Ligação Proteica , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Membrana Celular/metabolismo , Células CHO , Cricetulus , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Transporte Proteico , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , CricetinaeRESUMO
BACKGROUND: Cerebral stroke (CS) is the leading cause of death in China, and a complex disease caused by both alterable risk factors and genetic factors. This study intended to investigate the association of MMP3, MMP14, and MMP25 single nucleotide polymorphisms (SNPs) with CS risk in a Chinese Han population. METHODS: A total of 1,348 Han Chinese were recruited in this case-control study. Four candidate loci including rs520540 A/G and rs679620 T/C of MMP3, rs2236302 G/C of MMP14, and rs10431961 T/C of MMP25 were successfully screened. The correlation between the four SNPs and CS risk was assessed by logistic regression analysis. The results were analyzed by false-positive report probability (FPRP) for chance or significance. The interactions between four SNPs associated with CS risk were assessed by multifactor dimensionality reduction (MDR). RESULTS: rs520540 A/G and rs679620 C/T SNP in MMP3 were associated with risk of CS in allele, codominant, dominant and log-additive models. Ischemic stroke risk were significantly lower in carriers with rs520540-A allele and rs679620-T allele than those with G/G or C/C genotypes. However, rs520540-A allele and rs679620-T allele were associated with higher risk of hemorrhagic stroke. Stratified analysis showed that these two SNPs were associated with reduced risk of CS in aged < 55 years, non-smoking and non-drinking participants, and rs679620 SNP also reduced CS risk in male participants. The levels of uric acid, high-density lipoprotein cholesterol, and eosinophil were different among patients with different genotypes of rs520540 and rs679620. No statistically significant association was found between MMP14 rs2236302 G/C or MMP25 rs10431961 T/C with CS even after stratification by stroke subtypes, age, gender as well as smoking and drinking conditions in all the genetic models. CONCLUSION: MMP3 rs520540 A/G and rs679620 C/T polymorphisms were associated with CS risk in the Chinese Han population, which provides useful information for the prevention and diagnosis of CS.
Assuntos
Metaloproteinase 14 da Matriz , Metaloproteinase 3 da Matriz , Metaloproteinases da Matriz Associadas à Membrana , Acidente Vascular Cerebral , Estudos de Casos e Controles , Acidente Vascular Cerebral/genética , Humanos , Masculino , Feminino , Polimorfismo de Nucleotídeo Único , Predisposição Genética para Doença , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 14 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , AVC Isquêmico/genética , Acidente Vascular Cerebral Hemorrágico/genéticaRESUMO
BACKGROUND: Breast cancer is the most common malignancy in women, and is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers express normal levels of REST (repressor element 1 silencing transcription factor) mRNA but lack functional REST protein. Loss of REST function is seen in ~ 20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. METHODS: TCGA data was analyzed for the expression of REST target genes in breast cancer patient samples. We then utilized gene knockdown in MCF-7 cells in the presence or absence of steroid hormones estrogen and/ progesterone followed by RNA sequencing, as well as chromatin immunoprecipitation and PCR in an attempt to understand the tumor suppressor role of REST in breast cancer. RESULTS: We show that REST directly regulates CEMIP (cell migration-inducing and hyaluronan-binding protein, KIAA1199) and MMP24 (matrix metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cells leads to significant upregulation of CEMIP and MMP24. In addition, we found REST binds to RE-1 sites (repressor element-1) within the genes and influences their transcription. Furthermore, we found that the estrogen receptor (ESR1) signaling pathway is activated in the absence of REST, regardless of hormone treatment. CONCLUSIONS: We demonstrate a critical role for the loss of REST in aggressive breast cancer pathogenesis and provide evidence for REST as an important diagnostic marker for personalized treatment plans.
Assuntos
Neoplasias da Mama/genética , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Hialuronoglucosaminidase/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Biomarcadores Tumorais/genética , Feminino , Humanos , Mutação com Perda de Função/genética , Células MCF-7 , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Processos Neoplásicos , Fenótipo , Prognóstico , RNA Mensageiro/genética , Proteínas Repressoras , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
For some time, it has been accepted that the ß-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the ß-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer's disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100ß expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.
Assuntos
Astrócitos/metabolismo , Transportador 1 de Aminoácido Excitatório/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/genética , Potenciais de Ação/fisiologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/citologia , Sistemas CRISPR-Cas , Diferenciação Celular , Linhagem Celular , Transportador 1 de Aminoácido Excitatório/metabolismo , Edição de Genes , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Metaloproteinases da Matriz Associadas à Membrana/deficiência , Células-Tronco Neurais/citologia , Neurônios/citologia , Técnicas de Patch-Clamp , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Transdução de SinaisRESUMO
Background: Matrix Metalloproteinases (MMPs) play an indispensable role in the initial alteration and development of PCa. We tried to generate an MMP-related prognostic signature (MMPS) in prostate cancer (PCa). Methods: TCGA-PRAD, MSKCC/GSE21032, GSE116918, GSE70769 cohorts were enrolled to assess the prognostic value of MMPs. The least absolute shrinkage and selection operator (LASSO) Cox regression was employed to generate the MMPS signature. The log-rank test and Kaplan-Meier (K-M) survival curve were applied to show the difference RFS, The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) was plotted to predict the accuracy of signature. CIBERSORT was conducted to analyze the different immune infiltration in MMPS-H and MMPS-L groups. Potential signaling pathways activated in the MMPS-H groups by Metascape. Results: MMP1, MMP7, MMP11, MMP24 and MMP26 were selected by LASSO regression and established the MMPS predict signature. The MMPS showed the high prognostic value in TCGA-PRAD training cohort (AUC=0.714) and validation cohorts (GSE116918: AUC=0.976, GSE70769: AUC=0.738, MSKCC: AUC=0.793). Pid integrin1 pathway, G2M checkpoint, and response to growth factor signaling pathways were activated in MMPS-H group, patients with the high MMPS risk score and low M2 macrophage showed the worst recurrence-free survival (RFS). Conclusion: MMPs involved and played an essential role in the tumorigenesis and biochemical recurrence in PCa patients. The MMPS signature could accurately predict the recurrence of PCa patients and validated in several cohorts.
Assuntos
Metaloproteinases da Matriz/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Intervalo Livre de Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Estimativa de Kaplan-Meier , Masculino , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 11 da Matriz/genética , Metaloproteinase 11 da Matriz/metabolismo , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 7 da Matriz/metabolismo , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz Secretadas/metabolismo , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Neoplasias da Próstata/metabolismo , Curva ROCRESUMO
Recent studies have indicated the existence of an endogenous sulfur dioxide (SO2)generating system in the cardiovascular system. The present study aimed to discuss the function and regulatory mechanism of gaseous signal molecule SO2 in inhibiting apoptosis and endoplasmic reticulum stress (ERS) via the HippoMST signaling pathway to improve myocardial fibrosis of diabetic rats. A total of 40 male SpragueDawley rats were randomly divided into four groups (10 rats per group): Normal control group (control group), diabetic rats group [streptozotocin (STZ) group], SO2 intervention group (STZ+SO2 group) and diabetes mellitus rats treated with LAspartic acid ßhydroxamate (HDX) group (HDX group). Diabetic rats models were established by intraperitoneal injection of STZ (40 mg/kg) Following model establishment, intraperitoneal injection of Na2SO3/NaHSO3 solution (0.54 mmol/kg) was administered in the STZ+SO2 group, and HDX solution (25 mg/kg/week) was administered in the HDX group. A total of 4 weeks later, echocardiography was performed to evaluate rats' cardiac function; Masson staining, terminal deoxynucleotidyl transferase dUTP nick end labeling staining and transmission electron microscopy examinations were performed to observe myocardial morphological changes. ELISA was employed to determine the SO2 content. Western blot analysis was performed to detect the expression of proteins associated with apoptosis, ERS and the HippoMST signalling pathway. Compared with the control group, the STZ group and HDX group had a disordered arrangement of myocardial cells with apparent myocardial fibrosis, and echocardiography indicated that the cardiac function was lowered, there was an obvious increase of apoptosis in myocardial tissue, the expression levels of apoptosisassociated protein Bcell lymphoma associated protein X, caspase3 and caspase9 were upregulated, and Bcl2 expression was downregulated. The expression of ERS and HippoMST pathwayassociated proteins, including CHOP, GRP94, MST1 and MST2, were significantly upregulated. By contrast, these abovementioned changes were reversed by SO2 treatment. Compared with STZ group, the HDX group had a further increase of myocardial fibrosis and apoptosis, while there were no statistically significant differences in the expression of Bax/Bcl2, caspase3, caspase9 and ERS and HippoMST pathwayassociated proteins. The results of the present study demonstrated that the gaseous signal molecule SO2 can effectively improve the myocardial fibrosis of diabetic rats, and its mechanism may be associated with reduced apoptosis and ERS by downregulated HippoMST pathway.
Assuntos
MAP Quinase Quinase Quinases/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Dióxido de Enxofre/metabolismo , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental , Estresse do Retículo Endoplasmático , Fibrose , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Miocárdio/ultraestrutura , Ratos , Dióxido de Enxofre/farmacologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
BACKGROUND/AIMS: Membrane-type matrix metalloproteinases (MT-MMPs) are expressed on the cell surface and hydrolyze extracellular matrix components and signaling molecules by which they influence cancer cell migration and metastasis. Two of the six known MT-MMPs are anchored to the plasma membrane via a GPI anchor, one of which is MT4-MMP. Only little is known about MT4-MMP expression, synthesis, regulation and degradation. METHODS: We analyzed several human cancer cell lines as well as tissue homogenates using Western blotting and quantitative PCR for the expression of MT4-MMP. Organelles of SK-Mel-28 cells were separated using continuous Iodixanol gradients. Glycosylation of the SK-Mel-28 protein was studied via glucosidases and site directed mutagenesis of the MT4-MMP cDNA prior to transfection. RESULTS: We found the MT4-MMP highly expressed in human melanoma cell lines as well as skin and melanoma tissue samples. Three forms of MT4-MMP with molecular masses of 45 kDa, 58 kDa and 69 kDa were detected. Further, we demonstrate that the 58 kDa form is the mature protein in the cell membrane, while the 69 kDa form is its precursor found in intracellular compartments. The 69 kDa forms are processed by furin cleavage in the Golgi apparatus. Moreover, we identified Asn318 as the single N-glycosylation site of MT4-MMP. CONCLUSION: We demonstrate the novel expression of MT4-MMP in melanocytic tissues and propose a precursor/product-relationship of the different forms of MT4-MMP in melanoma cells.
Assuntos
Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/patologia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Estresse do Retículo Endoplasmático , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Metaloproteinases da Matriz Associadas à Membrana/genética , Melanoma/metabolismo , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
We have used a murine Achilles tendinopathy model to investigate whether tissue changes (such as collagen disorganization, chondroid metaplasia, and loss of tensile properties) which are broadly characteristic of human tendinopathies, are accompanied by changes in the expression of chromatin-modifying enzymes and the methylation status of promoter regions of tendon cell DNA. Tendinopathy was induced by two intra-tendinous TGF-ß1 injections followed by cage activity or treadmill running for up to 28 days. Activation of DNA methyltransferases occurred at 3 days after the TGF-ß1 injections and also at 14 days, but only with treadmill activity. Genome-wide Methyl Mini-Seq™ analysis identified 19 genes with differentially methylated promoters, five of which perform functions with an apparent direct relevance to tendinopathy (Leprel2, Foxf1, Mmp25, Igfbp6, and Peg12). The functions of the genes identified included collagen fiber assembly and pericellular interactions, therefore their perturbation could play a role in the characteristic disorganization of fibers in affected tendons. We postulate that a study of the functional genomics of these genes in animal and human tendon could further delineate the pathogenesis of this multi-factorial complex disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:947-955, 2017.
Assuntos
Metilação de DNA , Tendinopatia/metabolismo , Tendão do Calcâneo/patologia , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Proteínas Ligadas por GPI/genética , Expressão Gênica , Estudo de Associação Genômica Ampla , Masculino , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Regiões Promotoras Genéticas , Tendinopatia/patologiaRESUMO
Metastasis is the leading cause of death in people with lung cancer, yet the molecular effectors underlying tumor dissemination remain poorly defined. Through the development of an in vivo spontaneous lung cancer metastasis model, we show that the developmentally regulated transcriptional repressor Capicua (CIC) suppresses invasion and metastasis. Inactivation of CIC relieves repression of its effector ETV4, driving ETV4-mediated upregulation of MMP24, which is necessary and sufficient for metastasis. Loss of CIC, or an increase in levels of its effectors ETV4 and MMP24, is a biomarker of tumor progression and worse outcomes in people with lung and/or gastric cancer. Our findings reveal CIC as a conserved metastasis suppressor, highlighting new anti-metastatic strategies that could potentially improve patient outcomes.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Carcinoma Pulmonar de Células não Pequenas/secundário , Neoplasias Pulmonares/patologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas E1A de Adenovirus/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos , Camundongos SCID , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ets , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Tumorais CultivadasRESUMO
Matrix metalloproteases (MMPs) regulate innate immunity acting over proinflammatory cytokines, chemokines, and other immune-related proteins. MMP-25 (membrane-type 6-MMP) is a membrane-bound enzyme predominantly expressed in leukocytes whose biological function has remained largely unknown. We have generated Mmp25-deficient mice to elucidate the in vivo function of this protease. These mutant mice are viable and fertile and do not show any spontaneous phenotype. However, Mmp25-null mice exhibit a defective innate immune response characterized by low sensitivity to bacterial LPS, hypergammaglobulinemia, and reduced secretion of proinflammatory molecules. Moreover, these immune defects can be tracked to a defective NF-κB activation observed in Mmp25-deficient leukocytes. Globally, our findings provide new mechanistic insights into innate immunity through the activity of MMP-25, suggesting that this proteinase could be a potential therapeutic target for immune-related diseases.
Assuntos
Hipergamaglobulinemia/imunologia , Leucócitos/imunologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Imunidade Inata/genética , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
Membrane-type 5-matrix metalloproteinase (MT5-MMP) is a proteinase mainly expressed in the nervous system with emerging roles in brain pathophysiology. The implication of MT5-MMP in Alzheimer's disease (AD), notably its interplay with the amyloidogenic process, remains elusive. Accordingly, we crossed the genetically engineered 5xFAD mouse model of AD with MT5-MMP-deficient mice and examined the impact of MT5-MMP deficiency in bigenic 5xFAD/MT5-MMP(-/-) mice. At early stages (4 months) of the pathology, the levels of amyloid beta peptide (Aß) and its amyloid precursor protein (APP) C-terminal fragment C99 were largely reduced in the cortex and hippocampus of 5xFAD/MT5-MMP(-/-), compared to 5xFAD mice. Reduced amyloidosis in bigenic mice was concomitant with decreased glial reactivity and interleukin-1ß (IL-1ß) levels, and the preservation of long-term potentiation (LTP) and spatial learning, without changes in the activity of α-, ß- and γ-secretases. The positive impact of MT5-MMP deficiency was still noticeable at 16 months of age, as illustrated by reduced amyloid burden and gliosis, and a better preservation of the cortical neuronal network and synaptophysin levels in bigenic mice. MT5-MMP expressed in HEKswe cells colocalized and co-immunoprecipitated with APP and significantly increased the levels of Aß and C99. MT5-MMP also promoted the release of a soluble APP fragment of 95 kDa (sAPP95) in HEKswe cells. sAPP95 levels were significantly reduced in brain homogenates of 5xFAD/MT5-MMP(-/-) mice, supporting altogether the idea that MT5-MMP influences APP processing. MT5-MMP emerges as a new pro-amyloidogenic regulator of APP metabolism, whose deficiency alleviates amyloid pathology, neuroinflammation and cognitive decline.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/fisiopatologia , Hipocampo/enzimologia , Hipocampo/fisiopatologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cognição , Feminino , Deleção de Genes , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Potenciação de Longa Duração , Masculino , Metaloproteinases da Matriz Associadas à Membrana/análise , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Aprendizagem EspacialRESUMO
Membrane type 4 matrix metalloproteinase (MT4-MMP) [matrix metalloproteinase (MMP) 17] is a GPI-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple-negative breast cancer cells, MT4-MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG-tagged or Myc-tagged MT4-MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4-MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the clathrin-independent carriers/GPI-enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane-type MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Feminino , Humanos , Cinética , Metaloproteinases da Matriz Associadas à Membrana/genéticaRESUMO
BACKGROUND/AIM: The single nucleotide polymorphisms (SNPs) rs2282978 (CDK6), rs2425019 (MMP24), rs8081612 (MAP3K3), rs2871865 (IGF1R) and rs3782415 (SOCS2) were among the SNPs most strongly associated with height in a meta-analysis of 47 genome-wide association studies (GWAS) involving 114,223 adults from six ethnic groups. The present study aimed to examine associations between these SNPs and height in Brazilian children. METHODS: Cross-sectional heights of 1,008 healthy unrelated 4.4- to 9.7-year-old children were evaluated. All genotypes were determined by allele-specific polymerase chain reactions. Height standard deviation scores (SDS) were generated for this population and regressed on allele counts. Linear regressions were performed to estimate the effect of individual SNPs or a polygenic allelic score on height. RESULTS: The T allele of rs8081612 (MAP3K3), the C allele of rs2871865 (IGF1R) and the G allele of rs2425019 (MMP24) were significantly associated with a 0.091-SDS greater height (95% CI 0.089-0.093, p = 0.001) by polygenic analysis. The mean height SDS difference between children with 2 'tall' alleles and children with 4 'tall' alleles was 0.24 SDS (95% CI 0.05-0.43, p = 0.01). The observed allelic effect is consistent with that found in previous GWAS. CONCLUSIONS: Polymorphisms in MAP3K3, MMP24 and IGF1R act additively on height in children of an admixed population. These results demonstrate the importance of these loci for children's height.
Assuntos
Estatura/genética , Loci Gênicos , MAP Quinase Quinase Quinase 3/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Polimorfismo de Nucleotídeo Único , Receptores de Somatomedina/genética , Alelos , Brasil , Criança , Pré-Escolar , Estudos Transversais , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Receptor IGF Tipo 1RESUMO
RATIONALE: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.
Assuntos
Aneurisma da Aorta Torácica/genética , Dissecção Aórtica/genética , Metaloproteinases da Matriz Associadas à Membrana/fisiologia , Mutação de Sentido Incorreto , Adulto , Substituição de Aminoácidos , Angiotensina II , Animais , Aorta/embriologia , Aorta/patologia , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/terapia , Ruptura Aórtica/etiologia , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Predisposição Genética para Doença , Terapia Genética , Vetores Genéticos/uso terapêutico , Células HEK293 , Humanos , Lentivirus/genética , Masculino , Metaloproteinases da Matriz Associadas à Membrana/química , Metaloproteinases da Matriz Associadas à Membrana/deficiência , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Osteopontina/metabolismo , Conformação ProteicaRESUMO
OBJECTIVE: We examined the role of thrombus recanalization and ongoing blood flow in the process of thrombus resolution by comparing two murine in vivo models of deep venous thrombosis. METHODS: In CD1 mice, we performed surgical inferior vena cava ligation (stasis thrombosis), stenosis (thrombosis with recanalization), or sham procedure. We analyzed thrombus weight over time as a measure of thrombus resolution and quantified the messenger RNA and protein levels of membrane-type matrix metalloproteinases (MT-MMPs) as well as effectors of the plasmin complex at days 4, 8, and 12 after surgery. RESULTS: Despite similar initial thrombus size, the presence of ongoing blood flow (stenosis model) was associated with a 45.91% subsequent improvement in thrombus resolution at day 8 and 12.57% at day 12 compared with stasis thrombosis (ligation model). Immunoblot and real-time polymerase chain reaction analysis demonstrated a difference in MMP-2 and MMP-9 activity at day 8 between the two models (P = .03 and P = .006, respectively) as well as a difference in MT2-MMP gene expression at day 8 (P = .044) and day 12 (P = .03) and MT1-MMP protein expression at day 4 (P = .021). Histologic analyses revealed distinct areas of recanalization in the thrombi of the stenosis model compared with the ligation model as well as the recruitment of inflammatory cells, especially macrophages, and a focal pattern of localized expression of MT1-MMP and MT3-MMP proteins surrounding the areas of recanalization in the stenosis model. CONCLUSIONS: Recanalization and ongoing blood flow accelerate deep venous thrombus resolution in vivo and are associated with distinct patterns of MT1-MMP and MT3-MMP expression and macrophage localization in areas of intrathrombus recanalization.
Assuntos
Metaloproteinases da Matriz Associadas à Membrana/sangue , Veia Cava Inferior/cirurgia , Trombose Venosa/enzimologia , Trombose Venosa/cirurgia , Animais , Modelos Animais de Doenças , Metaloproteinase 2 da Matriz , Metaloproteinases da Matriz , Metaloproteinases da Matriz Associadas à Membrana/genética , Metalotioneína 3 , Camundongos , RNA Mensageiro/análise , Trombose Venosa/fisiopatologiaRESUMO
Matrix metalloproteinases (MMPs) are a large and complex family of zinc-dependent endoproteinases widely recognized for their roles in remodeling the extracellular matrix (ECM) during embryonic development, wound healing, and tissue homeostasis. Their misregulation is central to many pathologies, and they have therefore been the focus of biomedical research for decades. These proteases have also recently emerged as mediators of neural development and synaptic plasticity in vertebrates, however, understanding of the mechanistic basis of these roles and the molecular identities of the MMPs involved remains far from complete. We have identified a zebrafish orthologue of mmp25 (a.k.a. leukolysin; MT6-MMP), a membrane-type, furin-activated MMP associated with leukocytes and invasive carcinomas, but which we find is expressed by a subset of the sensory neurons during normal embryonic development. We detect high levels of Mmp25ß expression in the trigeminal, craniofacial, and posterior lateral line ganglia in the hindbrain, and in Rohon-Beard cells in the dorsal neural tube during the first 48 h of embryonic development. Knockdown of Mmp25ß expression with morpholino oligonucleotides results in larvae that are uncoordinated and insensitive to touch, and which exhibit defects in the development of sensory neural structures. Using in vivo zymography, we observe that Mmp25ß morphant embryos show reduced Type IV collagen degradation in regions of the head traversed by elongating axons emanating from the trigeminal ganglion, suggesting that Mmp25ß may play a pivotal role in mediating ECM remodeling in the vicinity of these elongating axons.
Assuntos
Gânglios Sensitivos/enzimologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Células Receptoras Sensoriais/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Colágeno Tipo IV/metabolismo , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Matriz Extracelular/enzimologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Sensitivos/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Masculino , Metaloproteinases da Matriz Associadas à Membrana/genética , Especificidade de Órgãos , Homologia de Sequência de Aminoácidos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
An intrafollicular increase in proteolytic activity drives ovulatory events. Surprisingly, the periovulatory expression profile of the membrane-type matrix metalloproteinases (MT-MMPs), unique proteases anchored to the cell surface, has not been extensively examined. Expression profiles of the MT-MMPs were investigated in ovarian tissue from well-characterized rat and macaque periovulatory models and naturally cycling women across the periovulatory period. Among the six known MT-MMPs, mRNA expression of Mmp14, Mmp16, and Mmp25 was increased after human chorionic gonadotropin (hCG) administration in rats. In human granulosa cells, mRNA expression of MMP14 and MMP16 increased following hCG treatment. In contrast, mRNA levels of MMP16 and MMP25 in human theca cells were unchanged before ovulation but declined by the postovulatory stage. In macaque granulosa cells, hCG increased mRNA for MMP16 but not MMP14. Immunoblotting showed that protein levels of MMP14 and MMP16 in rats increased, similar to their mRNA expression. In macaque granulosa cells, only the active form of the MMP14 protein increased after hCG, unlike its mRNA or the proprotein. By immunohistochemistry, both MMP14 and MMP16 localized to the different ovarian cell types in rats and humans. Treatment with hCG resulted in intense immunoreactivity of MMP14 and MMP16 proteins in the granulosa and theca cells. The present study shows that MMP14 and MMP16 are increased by hCG administration in the ovulating follicle, demonstrating that these MMPs are conserved among rats, macaques, and humans. These findings suggest that MT-MMPs could have an important role in promoting ovulation and remodeling of the ovulated follicle into the corpus luteum.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Macaca fascicularis/fisiologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 16 da Matriz/metabolismo , Ovário/enzimologia , Ovulação/fisiologia , Animais , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 16 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , TranscriptomaRESUMO
CD16 (FcγRIIIa), the low-affinity receptor for IgG, expressed by the majority of human NK cells, is a potent activating receptor that facilitates Ab-dependent cell-mediated cytotoxicity (ADCC). ADCC dysfunction has been linked to cancer progression and poor prognosis for chronic infections, such as HIV; thus, understanding how CD16 expression is regulated by NK cells has clinical relevance. Importantly, CD16 cell-surface expression is downmodulated following NK cell activation and, in particular, exposure to stimulatory cytokines (IL-2 or IL-15), likely owing to the action of matrix metalloproteinases (MMPs). In this article, we identify membrane-type 6 (MT6) MMP (also known as MMP25) as a proteinase responsible for CD16 downmodulation. IL-2-induced upregulation of MT6/MMP25 cell-surface expression correlates with CD16 downmodulation. MT6/MMP25, sequestered in intracellular compartments in unstimulated NK cells, translocates to the cell surface after stimulation; moreover, it polarizes to the effector-target cell interface of the CD16-mediated immunological synapse. siRNA-mediated disruption of MT6/MMP25 expression enhances the ADCC capacity of NK cells, emphasizing the important functional role of MT6/MMP25 in the regulation of ADCC activity. Thus, this study uncovers a previously unknown role of MT6/MMP25 in human NK cells, and suggests that inhibition of MT6/MMP25 activity could improve ADCC efficacy of therapeutically administered NK cells that require IL-2 for culture and expansion.
Assuntos
Sinapses Imunológicas , Interleucina-2/farmacologia , Células Matadoras Naturais/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/fisiologia , Receptores de IgG/biossíntese , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Comunicação Celular , Compartimento Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Polaridade Celular , Células Cultivadas , Dipeptídeos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-15/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/ultraestrutura , Ativação Linfocitária/efeitos dos fármacos , Metaloproteinases da Matriz Associadas à Membrana/biossíntese , Metaloproteinases da Matriz Associadas à Membrana/genética , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
INTRODUCTION: Pre-eclampsia is a serious complication of pregnancy, characterized by severe endothelial dysfunction resulting in hypertension, proteinuria and maternal end-organ damage. Soluble endoglin is an anti-angiogenic factor released from placenta that has been linked to severe pre-eclampsia. We recently reported MMP-14 is capable of cleaving endoglin to release soluble endoglin from placenta, however inhibition studies only partially repressed production. To this end we have sought to identify other proteases that mediate endoglin shedding from placenta. MMP-14 is one of six-membrane-type (MT-) MMPs, a sub-family of the MMP superfamily, so named because they are membrane bound. MMP-15 is phylogenetically the closest MMP relative to MMP-14, however its inhibition has no effect on soluble endoglin production from placenta. METHODS: Here we aimed to characterize the remaining four MT-MMPs (MMP-16, -17, -24 and -25) in severe early-onset pre-eclamptic placenta and assess their relative contribution to soluble endoglin production. RESULTS: Immunolocalisation studies revealed MMP-16, -24 and -25 were localized to the syncytiotrophoblast, the same site as endoglin, whilst MMP-17 was predominantly localized to fetal vessels and underlying stroma. MMP-17 protein was significantly (p < 0.05) up-regulated in pre-eclamptic placentas compared to gestationally matched pre-term controls, whilst MMP-25 mRNA was significantly (p < 0.05) down regulated. siRNA knockdown of MMP-16, -17, -24 and -25 in syncytialised BeWo cells did not alter soluble endoglin production in vitro. CONCLUSION: This is the first study to characterize MT-MMP protein localization in human placenta and indicates that MMP-14 is the only MT-MMP that contributes to soluble endoglin production in pre-eclampsia.
Assuntos
Antígenos CD/biossíntese , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Placenta/metabolismo , Pré-Eclâmpsia/metabolismo , Receptores de Superfície Celular/biossíntese , Adulto , Estudos de Casos e Controles , Endoglina , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Recém-Nascido , Masculino , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 16 da Matriz/genética , Metaloproteinase 16 da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana/genética , Pré-Eclâmpsia/genética , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Solubilidade , Trofoblastos/metabolismoRESUMO
INTRODUÇÃO E OBJETIVO: O conhecimento do comportamento biológico de lesões de natureza odontogênica é essencial para tornar a abordagem terapêutica adequada e estabelecer um prognóstico. A produção de metaloproteinases da matriz extracelular (MMPs), a angiogênese e a proliferação celular fornecem subsídios para o crescimento tumoral. O presente artigo tem como objetivo fazer uma revisão de literatura de pesquisas em tumores odontogênicos (TOs) selecionados a partir da nova classificação da Organização Mundial da Saúde (OMS) de 2005 sobre a expressão de MMPs, marcadores angiogênicos e proliferação celular e verificar, nestes estudos, a relação desses marcadores quanto ao comportamento biológico dessas lesões. RESULTADOS: Nota-se que as MMPs -1, -2, -7, -9 e -26 encontram-se mais expressas no componente epitelial e estroma e, particularmente, a -13 em estroma. Uma maior angiogênese é observada em TOs mais agressivos. CD 105 foi mais expresso no TO ceratocístico (TOC) e CD34 em ameloblastomas sólidos (ASs). Relata-se elevada expressão do Ki-67 e p53 no TOC e no AS e baixo índice de proliferação celular no TO adenomatoide (TOA). CONCLUSÃO: Esses resultados mostram que as MMPs participam no processo de invasão e recorrência de algumas lesões odontogênicas, estando associadas ao comportamento biológico desses tumores. A angiogênese é fundamental para fornecer suporte à proliferação celular e esses dois eventos em conjunto estão correlacionados com diferentes níveis de comportamento biológico nos TOs, quando comparados com cistos de natureza odontogênica, o que pode sugerir o uso de inibidores angiogênicos como provável abordagem terapêutica nessas lesões.
INTRODUCTION AND OBJECTIVE: The study of biological behavior of odontogenic lesions is essential to the establishment of appropriate therapeutic approach and prognosis. The production of extracellular matrix metalloproteinases (MMPs), angiogenesis and cell proliferation contribute to tumor growth. This paper aims to review the literature on odontogenic tumors (OT) selected according to the new World Health Organization classification (WHO- 2005) by evaluating the expression of MMPs, angiogenic and cell proliferation. Furthermore, it aims to verify the relation between these markers and the biological behavior of these lesions. RESULTS: it was found that MMPs -1, -2, -7, -9 and -26 had a higher expression in both epithelial component and stroma, and 13 particularly in the stroma. Increased angiogenesis was observed in more aggressive OT. CD105 expression was higher in keratocystic odontogenic tumour (KOT) and CD34 in solid ameloblastomas (SA). It was observed a higher expression of Ki-67 and p53 in SA and KOT and a low cell proliferation rate in the adenomatoid odontogenic tumour (AOT). CONCLUSION: These results show that MMPs are involved in invasion and recurrence of some odontogenic lesions and are associated with the biological behavior of these tumors. Angiogenesis is critical to provide support to cell proliferation and these concomitant events are correlated with different levels of biological behavior in OT when compared to odontogenic cysts, hence the use of angiogenic inhibitors may be a potential therapeutic approach in these lesions.