Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 72
Filtrar
1.
EMBO Rep ; 15(11): 1154-62, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25216944

RESUMO

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Membrana Celular/metabolismo , Galinhas , Proteínas Associadas aos Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Ligação Proteica
2.
Matrix Biol ; 36: 5-14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24681327

RESUMO

α4-Laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by vascular and lymphatic endothelial cells, leukocytes and other normal cell types. These laminins are recognized by α6ß1 and α6ß4 integrins and MCAM (CD146), and promote adhesion and migration of the cells. α4-Laminins are also expressed and secreted by some tumor cells and strongly promote tumor cell migration. Moreover, the abluminal side of blood and/or lymphatic vessels and the nerve perineurium, common tracks of tumor cell dissemination, express α4-laminins, and these laminin isoforms, when expressed in the stroma, may contribute to tumor invasion. In the present study, we examined ten mAbs to human laminin α4 chain for their reactivity with the isolated laminin α4 globular domain, their ability to inhibit tumor cell adhesion and migration on laminins 411 and 421, and their effect on the binding of α6ß1 integrin and MCAM to both α4-laminins. Most of the mAbs reacted with the laminin α4 globular domain, but only two, mAbs FC10 and 084, significantly inhibited tumor cell adhesion and migration on laminin-411. When used in combination, these antibodies practically abolished the cell adhesion and migration on laminin-411 and significantly reduced the cellular responses on laminin-421. Accordingly, mAbs FC10 and 084 significantly inhibited the binding of purified α6ß1 integrin and MCAM to laminins 411 and 421. These results indicate that mAbs to the laminin α4 globular domain are able to inhibit tumor cell adhesion and migration on laminins 411 and 421, and that α6ß1 integrin and MCAM bind α4-laminins at very close sites on the globular domain. These reagents contribute to a better understanding of the biology of α4-laminins and may have a therapeutic potential in malignant and inflammatory diseases.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Integrina alfa6beta1/metabolismo , Laminina/metabolismo , Neoplasias/genética , Antígeno CD146/metabolismo , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Humanos , Laminina/imunologia , Neoplasias/metabolismo , Neoplasias/patologia , Estrutura Terciária de Proteína
3.
J Histochem Cytochem ; 61(10): 719-30, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23900596

RESUMO

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-ß1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α6ß1. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α6ß1 were seen using IF imaging at day 14. Laminin-411 and Int α6ß1 were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.


Assuntos
Adipócitos/citologia , Adipogenia , Membrana Basal/metabolismo , Laminina/biossíntese , Laminina/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adipócitos/metabolismo , Adulto , Compostos Azo/metabolismo , Regulação da Expressão Gênica , Humanos , Laminina/genética , Células-Tronco Mesenquimais/citologia , Transporte Proteico
4.
PLoS One ; 8(1): e53648, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23308268

RESUMO

Laminins, a large family of αßγ heterotrimeric proteins mainly found in basement membranes, are strong promoters of adhesion and migration of multiple cell types, such as tumor and immune cells, via several integrin receptors. Among laminin α (LMα) chains, α5 displays the widest tissue distribution in adult life and is synthesized by most cell types. Here, we have generated and characterized five novel monoclonal antibodies (mAbs) to the human LMα5 chain to further study the biological relevance of α5 laminins, such as laminins 511 (α5ß1γ1) and 521 (α5ß2γ1). As detected by ELISA, immunohistochemistry, immunoprecipitation and Western blotting, each antibody displayed unique properties when compared to mAb 4C7, the prototype LMα5 antibody. Of greatest interest, mAb 8G9, but not any other antibody, strongly inhibited α3ß1/α6ß1 integrin-mediated adhesion and migration of glioma, melanoma, and carcinoma cells on laminin-511 and, together with mAb 4C7, on laminin-521. Accordingly, mAb 8G9 abolished the interaction of soluble α3ß1 integrin with immobilized laminins 511 and 521. Binding of mAb 8G9 to laminin-511 was unaffected by the other mAbs to the LMα5 chain but largely hindered by mAb 4E10 to a LMß1 chain epitope near the globular domain of laminin-511. Thus, mAb 8G9 defines a novel epitope localized at or near the integrin-binding globular domain of the LMα5 chain, which is essential for cell adhesion and migration, and identifies a potential therapeutic target in malignant and inflammatory diseases.


Assuntos
Anticorpos Monoclonais/farmacologia , Integrinas/antagonistas & inibidores , Laminina/antagonistas & inibidores , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Epitopos/imunologia , Feminino , Humanos , Hibridomas/imunologia , Integrinas/imunologia , Integrinas/metabolismo , Laminina/imunologia , Laminina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica
5.
Neurobiol Dis ; 50: 107-19, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23069676

RESUMO

Cathepsin D deficiency is a fatal neurodegenerative disease characterized by extreme loss of neurons and myelin. Our previous studies have demonstrated that structural and functional alterations in synapses are central to the disease pathogenesis. Therefore, we took a systematic approach to examine the synaptic proteome in cathepsin D knock-out mice, where the synaptic pathology resembles that of human patients. We applied quantitative mass spectrometry analysis on synaptosomal fractions isolated from cathepsin D knock-out and control mice at the age of 24 days. From the approximately 600 identified proteins, 43 were present in different amounts (P<0.05, measured in triple biological replicates) in cathepsin D knock-out mice compared to controls. We connected and bridged these 43 proteins using protein interaction data, and overlaid the network with brain specific gene expression information. Subsequently, we superimposed the network with Gene Ontology, pathway, phenotype and disease involvement, allowing construction of a dynamic, disease-protein centered network and prediction of functional modules. The measured changes in the protein levels, as well as some of the bioinformatically predicted ones, were confirmed by quantitative Western blotting or qualitative immunohistochemistry. This combined approach indicated alterations in distinct cellular entities, previously not associated with the disease, and including microtubule associated cytoskeleton and cell projection organization. Cell spreading and wound healing assays confirmed strongly compromised spatial orientation, associated with changes in distribution of focal adhesions and integrin assembly, in cathepsin D deficient cells. These changes might contribute to commencement of synaptic alterations and neuronal degeneration observed in cathepsin D deficiency.


Assuntos
Encéfalo/metabolismo , Catepsina D/deficiência , Movimento Celular , Citoesqueleto/metabolismo , Animais , Western Blotting , Encéfalo/patologia , Catepsina D/metabolismo , Análise por Conglomerados , Biologia Computacional , Citoesqueleto/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Lipofuscinoses Ceroides Neuronais/metabolismo , Lipofuscinoses Ceroides Neuronais/patologia , Proteoma , Proteômica , Sinapses
6.
J Cereb Blood Flow Metab ; 32(12): 2171-80, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22948298

RESUMO

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia caused by mutations in NOTCH3 gene. Pathology is manifested in small- and middle-sized arteries throughout the body, though primarily in cerebral white matter. Hemodynamics is altered in CADASIL and NOTCH3 is suggested to regulate actin filament polymerization and thereby vascular tone. We analyzed NOTCH3 expression and morphology of actin cytoskeleton in genetically genuine cultured human CADASIL vascular smooth muscle cells (VSMCs) (including a cell line homozygous for p.Arg133Cys mutation) derived from different organs, and in control VSMCs with short hairpin RNA (shRNA)-silenced NOTCH3. NOTCH3 protein level was higher in VSMCs derived from adult than newborn arteries in both CADASIL and control VSMCs. CADASIL VSMCs showed altered actin cytoskeleton including increased branching and node formation, and more numerous and smaller adhesion sites than control VSMCs. Alterations in actin cytoskeleton in shRNA-silenced VSMCs were similar as in CADASIL VSMCs. Severity of the alterations in actin filaments corresponded to NOTCH3 expression level being most severe in VSMCs derived from adult cerebral arteries. These observations suggest that hypomorphic NOTCH3 activity causes alterations in actin organization in CADASIL. Furthermore, arteries from different organs have specific characteristics, which modify the effects of the NOTCH3 mutation and which is one explanation for the exceptional susceptibility of cerebral white matter arteries.


Assuntos
Citoesqueleto de Actina/metabolismo , CADASIL/metabolismo , Inativação Gênica , Músculo Liso Vascular/metabolismo , Mutação de Sentido Incorreto , Miócitos de Músculo Liso/metabolismo , RNA Interferente Pequeno/biossíntese , Receptores Notch/biossíntese , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/patologia , Actinas/genética , Actinas/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Substituição de Aminoácidos , Animais , CADASIL/patologia , Linhagem Celular , Feminino , Humanos , Recém-Nascido , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Especificidade de Órgãos/genética , RNA Interferente Pequeno/genética , Receptor Notch3 , Receptores Notch/genética , Transdução Genética
7.
Mod Pathol ; 25(6): 859-68, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22343787

RESUMO

Laminin a non-collagenous glycoprotein is a major component of the renal glomerular basement membrane and mesangium. Thus far eleven distinct chains have been described, permutations of which make up 15 laminin isoforms. Laminin molecules interact with cells and other matrix molecules during organ development and differentiation. We studied the distribution of laminin isoforms in patients with type 1 diabetic nephropathy, membranous nephropathy, membranoproliferative glomerulonephritis and IgA nephropathy/ Henoch-Schönlein purpura. Immunofluorescence microscopic studies with laminin-chain-specific antibodies to the α1, α2, α5, ß1, ß2 and γ1 chains detected α2, ß1 and γ1 chain expression in the normal mesangium and α5, ß2 and γ1 in normal glomerular basement membrane. Significantly, constituents of the glomerular basement membrane, α5, ß2 and γ1 chains were overexpressed in kidneys with diabetic nephropathy. Initially the constituents of the mesangium increased commensurate with the degree of mesangial expansion and degree of diabetic nephropathy. Reduction in α2 chain intensity was observed with severe mesangial expansion and in the areas of nodular glomerulosclerosis. In addition, with late disease aberrant expression of α2 and ß2 chains was observed in the mesangium. Glomerular basement membrane in renal disease overexpressed molecules normally present in that location. In summary, the alterations in basement membrane composition in various renal diseases seem to not only reflect the balance between synthesis and degradation of normal basement membrane constituents, but also their aberrant expression.


Assuntos
Nefropatias Diabéticas/metabolismo , Nefropatias/metabolismo , Rim/química , Laminina/análise , Adolescente , Adulto , Biomarcadores/análise , Criança , Pré-Escolar , Nefropatias Diabéticas/patologia , Membrana Basal Glomerular/química , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite Membranoproliferativa/metabolismo , Glomerulonefrite Membranosa/metabolismo , Humanos , Vasculite por IgA/metabolismo , Rim/patologia , Nefropatias/patologia , Microscopia de Fluorescência , Pessoa de Meia-Idade , Isoformas de Proteínas , Índice de Gravidade de Doença , Adulto Jovem
8.
Mol Cell Endocrinol ; 355(1): 78-86, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22314207

RESUMO

We have previously characterized the molecular composition of human islet basement membranes and shown that human beta cells bind to laminin 511 (LM511) through integrin α3ß1 and Lutheran glycoprotein. We have now investigated the impact of physical contact between cultured human beta cells and the laminin isoforms occurring in their natural niche. Human islet preparations derived from 15 donors were used, beta cells and duct cells were purified by magnetic sorting. Overall beta-cell proliferation was low or undetectable. However, in many experiments the only proliferating beta cells were detected in contact with the laminin isoforms that are found in the human islets in vivo (511 and 411). Purified ductal and beta cells underwent epithelial-mesenchymal transition (EMT). LM511 partially blocked this dedifferentiation of purified beta cells, and did not affect purified duct cells. Interactions with the surrounding basement membrane are important for the growth and function of human beta cells. However, only a very limited level of beta-cell proliferation can be induced by exogenous factors. LM511 may be a useful substrate for human beta-cell maintenance in vitro.


Assuntos
Células Secretoras de Insulina/citologia , Laminina/metabolismo , Ductos Pancreáticos/citologia , Membrana Basal/metabolismo , Adesão Celular , Movimento Celular , Proliferação de Células , Separação Celular , Células Cultivadas , Transição Epitelial-Mesenquimal , Humanos , Células Secretoras de Insulina/fisiologia , Isoformas de Proteínas/metabolismo
9.
Stem Cell Res ; 8(1): 97-108, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22099024

RESUMO

Fibroblast feeder cells play an important role in supporting the derivation and long term culture of undifferentiated, pluripotent human embryonic stem cells (hESCs). The feeder cells secrete various growth factors and extracellular matrix (ECM) proteins into extracellular milieu. However, the roles of the feeder cell-secreted factors are largely unclear. Animal feeder cells and use of animal serum also make current feeder cell culture conditions unsuitable for derivation of clinical grade hESCs. We established xeno-free feeder cell lines using human serum (HS) and studied their function in hESC culture. While human foreskin fibroblast (hFF) feeder cells were clearly hESC supportive, none of the established xeno-free human dermal fibroblast (hDF) feeder cells were able to maintain undifferentiated hESC growth. The two fibroblast types were compared for their ECM protein synthesis, integrin receptor expression profiles and key growth factor secretion. We show that hESC supportive feeder cells produce laminin-511 and express laminin-binding integrins α3ß1, α6ß1 and α7ß1. These results indicate specific laminin isoforms and integrins in maintenance of hESC pluripotency in feeder-dependent cultures. In addition, several genes with a known or possible role for hESC pluripotency were differentially expressed in distinct feeder cells.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Embrionárias/metabolismo , Células Alimentadoras/metabolismo , Laminina/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Derme/citologia , Regulação para Baixo , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células Alimentadoras/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Prepúcio do Pênis/citologia , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Subunidades Proteicas/metabolismo , Fator de Crescimento Transformador beta/metabolismo
10.
Histol Histopathol ; 26(4): 443-9, 2011 04.
Artigo em Inglês | MEDLINE | ID: mdl-21360437

RESUMO

Snail1, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in tumour progression. Previous studies of snail1 have mainly focused on the epithelial tumour cells. The objective of this study was to evaluate the expression of snail1 protein in endothelial cells, stromal myofibroblasts and malignant epithelial cells of pharyngeal squamous cell carcinomas (PSCC), as well as its relation to clinicopathological features and survival. One hundred and ten tissue microarray samples were analyzed for snail1 expression using immunohistochemistry. In endothelial cells snail1 expression was observed in 51 (48%) of 107 cases and it predicted reduced disease specific survival (DSS) (p=0.009). In 49 (46%) tumour samples snail1 immunostaining was detected in stromal myofibroblasts and there was a tendency to poorer DSS in that group (p=0.067). Snail1 expression in endothelial cells and stromal myofibroblasts is also associated with hypopharyngeal tumours (p=0.01 and p=0.038 respectively), increasing T category (T3-4) (p=0.005, p=0.037 respectively) and poorer general condition of the patient (Karnofsky performance status score <70; p=0.029, p=0.039 respectively). Moreover endothelial expression correlated with advanced stage (III-IV) (p=0.005) and poorer differentiation (grade 2-3; p=0.012). In malignant epithelial cells snail1 immunostaining was detected in 75 of 110 cases (68%). Expression of the protein was more common in hypopharyngeal tumours (p=0.044). Snail1 positive tumours associated with a lower Karnofsky performance status score (p=0.039) and regional failure (p=0.042). Our findings indicate that snail1 protein expression in endothelial cells and to some extent also in tumour stromal myofibroblasts seems to be a predictor of poor survival in PSCC. The presence of snail1 protein in tumour microenvironment rather than in malignant epithelial tumour cells may induce tissue remodelling and tumour progression.


Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Faríngeas/patologia , Fatores de Transcrição/metabolismo , Dedos de Zinco/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Progressão da Doença , Endotélio Vascular/metabolismo , Feminino , Finlândia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Miofibroblastos/metabolismo , Neoplasias Faríngeas/metabolismo , Neoplasias Faríngeas/mortalidade , Fatores de Transcrição da Família Snail , Células Estromais/metabolismo , Taxa de Sobrevida , Análise Serial de Tecidos
11.
BMC Cancer ; 11: 73, 2011 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-21324165

RESUMO

BACKGROUND: Epitheliomesenchymal transition (EMT) is the process where cancer cells attain fibroblastic features and are thus able to invade neighboring tissues. Transcriptional factors zeb1, snai1 and twist regulate EMT. METHODS: We used immunohistochemistry to investigate the expression of zeb1, twist and snai1 in tumor and stromal compartments by in a large set of breast carcinomas. The results were compared with estrogen and progesterone receptor status, HER2 amplification, grade, histology, TNM status and survival of the patients. RESULTS: Nuclear expression for twist was seen in the epithelial tumor cell compartment in 3.6% and for snai1 in 3.1% of the cases while zeb1 was not detected at all in these areas. In contrast, the tumor stromal compartment showed nuclear zeb1 and twist expression in 75% and 52.4% of the cases, respectively. Although rare, nuclear expression of twist in the epithelial tumor cell compartment was associated with a poor outcome of the patients (p = 0.054 log rank, p = 0.013, Breslow, p = 0.025 Tarone-Ware). Expression of snai1, or expression of zeb1 or twist in the stromal compartment did not have any prognostic significance. Furthermore, none of these factors associated with the size of the tumors, nor with the presence of axillary or distant metastases. Expression of zeb1 and twist in the stromal compartment was positively associated with a positive estrogen or progesterone receptor status of the tumors. Stromal zeb1 expression was significantly lower in ductal in situ carcinomas than in invasive carcinomas (p = 0.020). Medullary carcinomas (p = 0.017) and mucinous carcinomas (p = 0.009) had a lower stromal expression of zeb1 than ductal carcinomas. Stromal twist expression was also lower in mucinous (p = 0.017) than in ductal carcinomas. CONCLUSIONS: Expression of transcriptional factors zeb1 and twist mainly occur in the stromal compartment of breast carcinomas, possibly representing two populations of cells; EMT transformed neoplastic cells and stromal fibroblastic cells undergoing activation of zeb1 and twist due to growth factors produced by the tumor. However, epithelial expression of twist was associated with a poor prognosis, hinting at its importance in the spread of breast carcinoma.


Assuntos
Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Carcinoma/diagnóstico , Carcinoma/mortalidade , Carcinoma/patologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Proteínas de Homeodomínio/fisiologia , Humanos , Imuno-Histoquímica , Estadiamento de Neoplasias , Proteínas Nucleares/fisiologia , Prognóstico , Fatores de Transcrição da Família Snail , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Fatores de Transcrição/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco
12.
Respir Res ; 12: 2, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21205293

RESUMO

BACKGROUND: Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity. METHODS: Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro. RESULTS: OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings. CONCLUSIONS: Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.


Assuntos
Remodelação das Vias Aéreas , Asma/metabolismo , Pulmão/metabolismo , Fator de Transcrição STAT4/deficiência , Tenascina/metabolismo , Animais , Asma/genética , Asma/imunologia , Asma/fisiopatologia , Hiper-Reatividade Brônquica , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/imunologia , Fibroblastos/metabolismo , Humanos , Interferon gama/metabolismo , Pulmão/imunologia , Pulmão/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina , RNA Mensageiro/metabolismo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Tenascina/genética , Células Th1/imunologia , Células Th2/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
13.
Hum Pathol ; 42(1): 57-67, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20970160

RESUMO

Several matrix metalloproteinases (MMPs) are implicated in the degradation of the epithelial basement membrane (BM), invasiveness, and malignancy of endometrial and ovarian carcinomas. We have recently proposed a cooperative role for RUNX1/AML1 and ETV5/ERM in myometrial infiltration during endometrioid endometrial invasiveness. In the present work, we have characterized the occurrence, levels of expression, and codistribution of gelatinases MMP-2 and -9, and the transcription factors RUNX1/AML1 and ETV5/ERM, together with collagen type IV and laminin chains of the epithelial BM in endometrioid endometrial (EEC) and ovarian endometrioid carcinoma (OEC). MMP-2 and -9 expression levels were up-regulated at the invasive front of both carcinomas, and they showed a relatively high degree of volume codistribution with RUNX1/AML1 and ETV5/ERM. EEC tissue microarrays showed similar significant expression and correlation for MMPs and the transcription factors. When the array samples were grouped according to the carcinoma stages, there was significant correlation in the expression levels for both MMP-2 and -9 with ETV5/ERM. Colocalization of MMP-2 and -9 with epithelial basement membrane component collagen type IV showed close spatial association for both MMPs and discontinuation of collagen type IV expression at the invasive front in both EEC and OEC. BM components laminin α1, α2, α3, α5, and γ2 chains, laminin α5 receptor basal cell adhesion molecule (BCAM), and laminin 332 were all detected both in EEC and OEC. Highest expression levels in EEC were for laminin α3 and in OEC for laminin α5 chain. Laminin γ2 chain and laminin 332 showed discontinuous immunoreactivity in the epithelial basement membrane suggestive of proteolytic degradation. These results indicate concurrent mechanisms in expression of MMP-2 and -9, RUNX1/AML1 and ETV5/ERM, and several of the basement membrane components, which are likely to associate with the invasive stage of EEC and OEC.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fatores de Transcrição/metabolismo , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/metabolismo , Carcinoma Epitelial do Ovário , Colágeno Tipo IV/metabolismo , Feminino , Humanos , Laminina/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Regulação para Cima
14.
Acta Ophthalmol ; 89(6): 569-74, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19900203

RESUMO

PURPOSE: Climatic droplet keratopathy (CDK) is an acquired corneal disease characterized by progressive scarring of the cornea. In several corneal diseases, matrix metalloproteinases (MMPs) are upregulated during the degradation of epithelial and stromal tissues. We investigated the levels, degree of activation and molecular forms of MMP-2, MMP-9, MMP-8 and MMP-13 and their tissue inhibitors TIMP-1 and TIMP-2 in tear fluid of patients with CDK. METHODS: Seventeen CDK patients and 10 controls living in Argentine Patagonia received a complete eye examination, and MMPs and TIMP-1/2 were determined by immunofluorometric assay (IFMA), gelatin zymography and quantitative Western immunoblot analysis in tear samples. RESULTS: The MMPs were detected mostly in their latent forms. The levels of MMP-9 and MMP-2 were found to be significantly elevated in CDK patients, whereas latent and active MMP-8 levels were significantly enhanced in controls. There was no significant difference in the level of MMP-13. TIMPs were found as part of complexes, and the TIMP-1 levels were significantly lower in patients than controls. CONCLUSION: Elevated MMP-2 and MMP-9 levels have been implicated in the failure of corneal re-epithelialization, and enhanced MMP-2 and MMP-9 levels in CDK patients suggest that these MMPs may play a role in corneal scarring in CDK. Elevated levels of MMP-8 suggest a defensive role for this MMP in inflammatory reactions associated with recurring corneal traumas. Decreased expression of TIMP-1 in CDK patients suggest deficient antiproteolytic shield likely to render the corneas of CDK patients vulnerable to enhanced MMPs. Overall, these data suggest a mechanistic link between MMPs and TIMP-1 level in cornea and tears with corneal scarring in CDK.


Assuntos
Doenças da Córnea/enzimologia , Metaloproteinases da Matriz/metabolismo , Lágrimas/enzimologia , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Feminino , Fluorimunoensaio , Humanos , Masculino , Pessoa de Meia-Idade
15.
J Rheumatol ; 37(6): 1181-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20436081

RESUMO

OBJECTIVE: Laminin alpha1-chain normally induces intercalated duct progenitors to differentiate to acinar cells through integrin (INT) alpha1ss1 and alpha2ss1 receptors. Maintenance of acinar cells is impaired in Sjögren's syndrome (SS), which is also characterized by low levels of serum and salivary androgens. We hypothesized that androgens normally support salivary gland remodeling by upregulating either laminin alpha1 chain or its cellular alpha1 or alpha2 INT subunit-containing receptors. METHODS: Intercalated duct and acinar human salivary gland (HSG) cells and labial salivary gland (LSG) biopsies from healthy controls and patients with SS were cultured without or with sex steroids. Laminin alpha1 chain and INT alpha1 and alpha2 subunits were studied using quantitative reverse-transcription real-time polymerase chain reaction and INT alpha1 and alpha2 subunits using immunofluorescence staining. RESULTS: INT alpha1-subunit and alpha2-subunit messenger RNA (mRNA) levels were increased in intercalated duct and acinar cells by DHEA and testosterone. In contrast, laminin alpha1-chain mRNA levels were not affected. The upregulating effect of DHEA on INT subunits was also seen at the protein level. DHEA also increased mRNA levels of both INT subunits in healthy but not SS LSG. CONCLUSION: Androgens increased INT alpha1 and alpha2 subunits in tubuloepithelial cells and in healthy LSG, but in SS salivary glands this androgen regulation was defective, which is likely to contribute to defective outside-in signaling, acinar atrophy, and ductal cell hyperplasia.


Assuntos
Desidroepiandrosterona/metabolismo , Integrina alfa1/metabolismo , Integrina alfa2/metabolismo , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/metabolismo , Testosterona/metabolismo , Adolescente , Adulto , Idoso , Biópsia , Linhagem Celular , Desidroepiandrosterona/genética , Desidroepiandrosterona/farmacologia , Feminino , Expressão Gênica , Humanos , Integrina alfa1/genética , Integrina alfa2/genética , Laminina/genética , Laminina/metabolismo , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Ductos Salivares/efeitos dos fármacos , Ductos Salivares/metabolismo , Glândulas Salivares Menores/efeitos dos fármacos , Síndrome de Sjogren/genética , Testosterona/genética , Testosterona/farmacologia , Adulto Jovem
16.
J Oral Pathol Med ; 39(3): 275-8, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20359311

RESUMO

BACKGROUND: E-cadherin (E-Cad) is a 120-kDa adhesive protein found in adherens junctions of the digestive tract epithelium. We tested the ability of two Candida strains to degrade human E-Cad in the Candida virulence factor perspective. MATERIALS AND METHODS: We set out to study oral mucosal E-Cad degradation by clinical and reference strains of Candida albicans and Candida glabrata. We also included hyphal and secreted aspartic proteinase (Sap) mutants of C. albicans to test the effect of yeast/hyphal transition on the ability to degrade E-Cad. The tests were performed at pH 4 and pH 6 to determine the effect of local tissue acidity on the activation of Saps. The C. albicans strains used were: CCUG 32723; clinical strain SC5314 which is known to be strongly invasive; hyphal mutants of SC5314: HLC52 (efg1/efg1), HLC54 (cph1/cph1 efg1/efg1) and JKC19 (cph1/cph1); clinical strain B1134; Sap 1-3 and Sap 4-6 mutants of SC5314. The C. glabrata strains used were ATCC 90030, and the clinical strains 5WT and G212. RESULTS: The sonicated yeast cells of C. albicans JKC19 and SC5314, both in hyphal form, degraded E-Cad at pH 4. The 10x concentrated growth media of the strains HLC-52, HLC-54, 32723 and B1134; all in yeast form, caused degradation at pH 4, HLC-52 and HLC-54 also at pH 6. The C. glabrata strains did not degrade E-Cad. CONCLUSIONS: pH is a strain dependent triggering factor in activating yeast or hyphal form related Candida Saps in degrading epithelial cell associated E-Cads.


Assuntos
Caderinas/metabolismo , Candida albicans/metabolismo , Candida glabrata/metabolismo , Queratinócitos/metabolismo , Mucosa Bucal/citologia , Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/classificação , Candida albicans/patogenicidade , Candida glabrata/classificação , Candida glabrata/patogenicidade , Adesão Celular , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hifas/metabolismo , Queratinócitos/microbiologia , Mucosa Bucal/microbiologia , Virulência/fisiologia
17.
J Cell Mol Med ; 14(6B): 1569-93, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19656240

RESUMO

Podosomes and invadopodia are actin-based structures at the ventral cell membrane, which have a role in cell adhesion, migration and invasion. Little is known about the differences and dynamics underlying these structures. We studied podosome-like structures of oral squamous carcinoma cells and invadopodia of their invasive variant that has undergone a spontaneous epithelial-mesenchymal transition (EMT). In 3D imaging, podosomes were relatively large structures that enlarged in time, whereas invadopodia of invasive cells remained small, but were more numerous, degraded more extracellular matrix (ECM) and were morphologically strikingly different from podosomes. In live-cell imaging, highly dynamic, invadopodia-embedded actin tails were frequently released and rocketed through the cytoplasm. Resembling invadopodia, we found new club-ended cell extensions in EMT-experienced cells, which contained actin, cortactin, vinculin and MT1-matrix metalloproteinase. These dynamic cell extensions degraded ECM and, in field emission scanning electron microscopy, protruded from the dorsal cell membrane. Plectin, alphaII-spectrin, talin and focal adhesion kinase immunoreactivities were detected in podosome rings, whereas they were absent from invadopodia. Tensin potentially replaced talin in invadopodia. Integrin alpha(3)beta(1) surrounded both podosomes and invadopodia, whereas integrin alpha(v)beta(5) localized only to invadopodia heads. Pacsin 2, in conjunction with filamin A, was detected early in podosomes, whereas pacsin 2 was not found in invadopodia and filamin A showed delayed accumulation. Fluorescence recovery after photobleaching indicated faster reorganization of actin, cortactin and filamin A in podosomes compared to invadopodia. In conclusion, EMT affects the invasion machinery of oral squamous carcinoma cells. Non-invasive squamous carcinoma cells constitutively organize podosomes, whereas invasive cells form invadopodia. The club-ended cell extensions, or externalized invadopodia, are involved in ECM degradation and maintenance of contact to adhesion substrate and surrounding cells during invasion.


Assuntos
Actinas/metabolismo , Epitélio/patologia , Mesoderma/patologia , Neoplasias/patologia , Pseudópodes/patologia , Idoso , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Matriz Extracelular/metabolismo , Feminino , Recuperação de Fluorescência Após Fotodegradação , Humanos , Integrinas/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Moduladores de Tubulina/farmacologia , Cicatrização/efeitos dos fármacos
18.
Acta Ophthalmol ; 88(4): 431-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19758401

RESUMO

PURPOSE: The aim of the present study was to determine whether the eye tissues of arterial hypertensive rats evince expression of angiotensin receptors (AT(1) and AT(2)) as well as the novel Mas receptor, whose endogenous ligand is vasorelaxing Angiotensin (1-7) [Ang (1-7)]. METHODS: Enucleated eyes from spontaneously hypertensive rats (SHR) and double transgenic rats harbouring human renin and angiotensinogen genes (dTGR) and their normotensive controls were used. Half of the rats were pretreated orally with an Angiotensin II (Ang II) type 1 receptor blocker (ARB). The eyes were snap-frozen in isopentane at -40 degrees and stored at -70 degrees for subsequent reverse transcriptase polymerase chain reaction (RT-PCR) analysis or in vitro autoradiography. RESULTS: The mRNA expression of AT(1a) and AT(2) as well as the novel Mas receptor was detected in all rat groups, being markedly higher in the retina than in the ciliary body. dTGR had significantly more receptors than SHR, but no direct relation to blood pressure level was seen. According to the autoradiography, treatment with ARB blocked a part of AT(1) receptors but had no clear effect on AT(2) receptors. CONCLUSION: The novel Mas receptor was found by RT-PCR in eye tissue for the first time. Its specific ligand, Ang (1-7), may be involved in the regulation of intraocular pressure--as recently demonstrated by us--and in the pathogenesis of retinal diseases as a counter-regulatory component for the vascular and proliferative actions of Ang II. The results suggest that the density of AT(1) receptors in the eye is independent of the blood pressure level of the animal.


Assuntos
Corpo Ciliar/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipertensão/genética , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/genética , Retina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Angiotensinogênio/genética , Animais , Animais Geneticamente Modificados , Autorradiografia , Pressão Sanguínea , Pressão Intraocular , Masculino , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Renina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
J Oral Pathol Med ; 39(4): 290-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19889153

RESUMO

BACKGROUND: The contribution of stromal laminin chain expression to malignant potential, tumour stroma reorganization and vessel formation in oral squamous cell carcinoma (OSCC) is not fully understood. Therefore, the expression of the laminin chains alpha2, alpha3, alpha4, alpha5 and gamma2 in the stromal compartment/vascular structures in OSCC was analysed. METHODS: Frozen tissue of OSCC (9x G1, 24x G2, 8x G3) and normal (2x)/hyperplastic (11x) oral mucosa was subjected to laminin chain and alpha-smooth muscle actin (ASMA) immunohistochemistry. Results were correlated to tumour grade. The relation of laminin chain positive vessels to total vessel number was assessed by immunofluorescence double labelling with CD31. RESULTS: Stromal laminin alpha2 chain significantly decreases and alpha3, alpha4, alpha5 and gamma2 chains and also ASMA significantly increase with rising grade. The amount of stromal alpha3, alpha4 and gamma2 chains significantly increased with rising ASMA positivity. There is a significant decrease in alpha3 chain positive vessels with neoplastic transformation. CONCLUSIONS: Mediated by myofibroblasts, OSCC development is associated with a stromal up-regulation of laminin isoforms possibly contributing to a migration promoting microenvironment. A vascular basement membrane reorganization concerning alpha3 and gamma2 chain laminins during tumour angioneogenesis is suggested.


Assuntos
Fibroblastos/patologia , Laminina/análise , Mucosa Bucal/patologia , Neoplasias Bucais/patologia , Neovascularização Patológica/patologia , Actinas/análise , Membrana Basal/patologia , Transformação Celular Neoplásica/patologia , Tecido Conjuntivo/irrigação sanguínea , Tecido Conjuntivo/patologia , Células Endoteliais/patologia , Endotélio Vascular/patologia , Imunofluorescência , Humanos , Hiperplasia , Mucosa Bucal/irrigação sanguínea , Neoplasias Bucais/irrigação sanguínea , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Regulação para Cima
20.
PLoS One ; 4(9): e6879, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19721715

RESUMO

BACKGROUND: Tumor-stroma reaction is associated with activation of fibroblasts. Nemosis is a novel type of fibroblast activation. It leads to an increased production of growth factors and proinflammatory and proteolytic proteins, while at the same time cytoskeletal proteins are degraded. Here we used paired normal skin fibroblasts and cancer-associated fibroblasts (CAF) and primary and recurrent oral squamous cell carcinoma (SCC) cells to study the nemosis response. PRINCIPAL FINDINGS: Fibroblast nemosis was analyzed by protein and gene expression and the paracrine regulation with colony formation assay. One of the normal fibroblast strains, FB-43, upregulated COX-2 in nemosis, but FB-74 cells did not. In contrast, CAF-74 spheroids expressed COX-2 but CAF-43 cells did not. Alpha-SMA protein was expressed in both CAF strains and in FB-74 cells, but not in FB-43 fibroblasts. Its mRNA levels were downregulated in nemosis, but the CAFs started to regain the expression. FSP1 mRNA was downregulated in normal fibroblasts and CAF-74 cells, but not in CAF-43 fibroblasts. Serine protease FAP was upregulated in all fibroblasts, more so in nemotic CAFs. VEGF, HGF/SF and FGF7 mRNA levels were upregulated to variable degree in nemosis. CAFs increased the colony formation of primary tumor cell lines UT-SCC-43A and UT-SCC-74A, but normal fibroblasts inhibited the anchorage-independent growth of recurrent UT-SCC-43B and UT-SCC-74B cells. CONCLUSIONS: Nemosis response, as observed by COX-2 and growth factor induction, and expression of CAF markers alpha-SMA, FSP1 and FAP, varies between fibroblast populations. The expression of CAF markers differs between normal fibroblasts and CAFs in nemosis. These results emphasize the heterogeneity of fibroblasts and the evolving tumor-promoting properties of CAFs.


Assuntos
Carcinoma de Células Escamosas/fisiopatologia , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Bucais/fisiopatologia , Pele/patologia , Adulto , Idoso , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Humanos , Inflamação , Masculino , Neoplasias Bucais/metabolismo , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...