RESUMO
Integrin-mediated adhesion of cells to extracellular matrix proteins has been shown to activate various intracellular signaling events. In the present study, we demonstrate that the addition of a monoclonal antibody raised against the beta4 integrin subunit in the culture medium of a clone derived from the colon adenocarcinoma cell line LoVo specifically results in stimulation of cell migration and invasion through reconstituted basement membrane matrices. Moreover, an increase in MMP-2 activity is observed. Conversely, monoclonal anti-alpha6 and anti-beta1 have no effect on MMP-2 expression. The s. c. co-injection of adenocarcinoma cells with antibodies raised against the beta4 integrin subunit to immunosuppressed newborn rats gives rise to tumors displaying altered and disorganized peri-tumoral basement membranes compared with tumors obtained when cells are injected with adenocarcinoma cells alone. Higher metastatic capacity of cells results when they are co-injected with antibodies to the beta4 integrin subunit. Our results suggest that the beta4 subunit of alpha6beta4 integrin, a laminin receptor in colon adenocarcinoma, may be responsible for the specific signals which stimulate cell motility, expression of MMP-2 and tumor invasion.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos CD/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Antígenos CD/imunologia , Southern Blotting , Movimento Celular , Humanos , Imuno-Histoquímica , Integrina beta4 , Laminina/metabolismo , Microscopia Eletrônica , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Several integrin alpha subunits undergo post-translational endoproteolytic processing at pairs of basic amino acids that is mediated by the proprotein convertase furin. Here we ask whether other convertase family members can participate in these processing events. We therefore examined the endoproteolysis rate of the integrin subunits pro-alpha5, alpha6 and alphav by recombinant furin, proprotein convertase (PC)5A, paired basic amino acid converting enzyme (PACE)4, PC1, PC2 and PC7 in vitro and/or ex vivo after overexpression in LoVo cells that were deficient in furin activity. We found that 60-fold more PC1 than furin was needed to produce 50% cleavage of pro-alpha subunit substrates in vitro; the defective pro-alpha chain endoproteolysis in LoVo cells was not rescued by overexpression of PC1 or PC2. No endoproteolysis occurred with PC7 either in vitro or ex vivo, although similar primary sequences of the cleavage site are found in integrins and in proteins efficiently processed by PC7, which suggests that a particular conformation of the cleavage site is required for optimal convertase-substrate interactions. In vitro, 50% cleavage of pro-alpha subunits was obtained with one-third of the amount of PC5A and PACE4 than of furin. In LoVo cells, PC5A remained more active than furin, PACE4 activity was quite low, and PC5B, which differs from PC5A by a C-terminal extension containing a transmembrane domain, was very inefficient in processing integrin alpha-subunit precursors. In conclusion, these results indicate that integrin alpha-subunit endoproteolytic processing involves the redundant function of furin and PC5A and to a smaller extent PACE4, but not of PC1, PC2, PC5B or PC7.
Assuntos
Integrinas/metabolismo , Proteínas de Membrana , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Serina Endopeptidases/metabolismo , Subtilisinas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Cálcio/farmacologia , Furina , Expressão Gênica , Humanos , Hidrólise/efeitos dos fármacos , Integrinas/química , Cinética , Peso Molecular , Testes de Precipitina , Pró-Proteína Convertase 5 , Pró-Proteína Convertases , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Especificidade por Substrato , Subtilisinas/deficiência , Subtilisinas/genética , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND & AIMS: Homeobox genes are involved in establishing and maintaining differentiated patterns in adult tissues. Cdx1 might carry out that function in the intestinal epithelium because its expression is specific to that tissue and increases during development. METHODS: Cdx1 expression was induced in IEC-6 intestinal epithelial cells by stable transfection, and subsequent changes in cell growth, resistance to apoptosis, migration, and differentiation were monitored. RESULTS: Compared with control, IEC-6/Cdx1 cells proliferated more rapidly, were more resistant to apoptosis, and migrated 3-4 times faster, as shown by an in vitro wound assay. IEC-6/Cdx1 cells in culture formed multilayers. Morphology of the top layer was similar to that of columnar epithelium, with cells showing typical features of differentiated enterocytes, including complex junctions and well-developed microvilli with glycocalix. Expression of 2 markers of enterocyte differentiation, aminopeptidase N and villin, was induced in IEC-6/Cdx1 cells. Aminopeptidase N was targeted to the basolateral membrane, and villin was localized to the cytoplasm. Actin filaments, which were mostly present in transcytoplasmic stress fibers in control cells, were redistributed to the cortex in Cdx1-transfected cells. CONCLUSIONS: Cdx1 expression in IEC-6 cells induces phenotypic changes characteristic of differentiating enterocytes, suggesting an important role for Cdx1 in the transition from stem cells to proliferating/transit cells.
Assuntos
Proteínas Aviárias , Proteínas de Homeodomínio/fisiologia , Mucosa Intestinal/citologia , Actinas/metabolismo , Animais , Apoptose/fisiologia , Antígenos CD13/metabolismo , Proteínas de Transporte/metabolismo , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Ratos , Células-Tronco/citologia , Transfecção , Fator de Crescimento Transformador beta/farmacologiaRESUMO
The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.
Assuntos
Moléculas de Adesão Celular/metabolismo , Laminina/metabolismo , Osteócitos/citologia , Fragmentos de Peptídeos/metabolismo , Células-Tronco/citologia , Animais , Anticorpos Monoclonais , Ligação Competitiva , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/farmacologia , Diferenciação Celular , Células Cultivadas , Temperatura Alta , Integrina beta1/metabolismo , Laminina/química , Laminina/farmacologia , Camundongos , Osteócitos/efeitos dos fármacos , Osteócitos/metabolismo , Elastase Pancreática/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Desnaturação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacologia , Ratos , Crânio/citologia , Crânio/efeitos dos fármacos , Crânio/embriologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Células Tumorais Cultivadas , CalininaRESUMO
The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for (alpha) and beta chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds-1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-beta1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the (alpha)2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-beta1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of (alpha)2 chain function by inhibiting antibodies was corrected by anti-beta1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of beta1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-(alpha)2 chain antibodies. Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-beta1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.
Assuntos
Colágeno/metabolismo , Integrinas/química , Integrinas/metabolismo , Queratinócitos/metabolismo , Anticorpos Bloqueadores , Adesão Celular/fisiologia , Células Cultivadas , Humanos , Integrinas/imunologia , Cinética , Ligantes , Conformação Proteica , Receptores de ColágenoRESUMO
The heterodimer alpha6beta4 is a major integrin and the main laminin receptor in epithelia. The alpha6 integrin subunit is proteolytically cleaved, probably by furin, and glycosylated during its biosynthesis. In the present work, we have investigated the kinetics of the assembly process of alpha6beta4 heterodimers in the colonic adenocarcinoma cell line HT29-D4. We demonstrate that the association of alpha6 and beta4 precursors occurs within the ER, while the endoproteolytic cleavage of pro-alpha6 occurs later, probably in the trans-Golgi network. When pro-alpha6 was blocked within the ER by treatment with brefeldin A, its maturation processing was completely prevented without any consequence on its association with beta4 subunit. Low temperature (20 degrees C) also blocked pro-alpha6 maturation, like brefeldin A, but in addition impaired the integrin assembly. Calnexin, an ER resident protein chaperone, was found to be associated with both the alpha6 and beta4 subunit precursors. Our data suggest that calnexin might be responsible for the prolonged retention of pro-alpha6 within the ER compartment and for the defect of integrin subunit association observed at low temperature.
Assuntos
Adenocarcinoma/metabolismo , Antígenos de Superfície/biossíntese , Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias do Colo/metabolismo , Integrinas/biossíntese , Adenocarcinoma/patologia , Transporte Biológico , Calnexina , Neoplasias do Colo/patologia , Dimerização , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HT29 , Humanos , Hidrólise , Integrina alfa6beta4 , TemperaturaRESUMO
Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha5beta1 associates primarily with the zymogens proPC1, proPC1-DeltaC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha5beta1.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Oligopeptídeos/metabolismo , Receptores de Fibronectina/metabolismo , Sequência de Aminoácidos , Animais , Ácido Aspártico Endopeptidases/química , Sequência de Bases , Transporte Biológico , Células CHO , Cricetinae , Primers do DNA , Ativação Enzimática , Imuno-Histoquímica , Pró-Proteína Convertases , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/química , TransfecçãoRESUMO
We have previously described an inverse relationship between Cdx1 and Cdx2 mRNA levels and the extent of dysplasia and severity of clinical outcome in colorectal carcinoma, suggesting that altered expression of these genes was associated with colorectal carcinogenesis or tumor progression. To investigate further their involvement in the physiopathology of colorectal cancer, HT29 colon carcinoma cells that show very low Cdx expression were transfected with Cdx1 and/or Cdx2 cDNA to elicit their overexpression. Growth rate, tumorigenicity, resistance to apoptosis, and migration potential of the corresponding cells were analyzed. Growth rate of cells overexpressing Cdx2 decreased by half, whereas overexpression of Cdx1 had no effect. However, cells overexpressing both Cdxs had a growth rate reduced to 20% of control. In cells overexpressing Cdx1 or Cdx2, tumorigenicity and resistance to apoptosis induced by serum starvation, ceramide, or staurosporine were not changed compared with control cells; yet phorbol ester-stimulated cell migration was decreased by 50%. In cells overexpressing both Cdx1 and Cdx2, tumorigenicity was decreased by 50%, resistance to apoptosis was significantly lowered, and stimulated cell migration was further decreased to 15% of control compared with cells expressing Cdx1 or Cdx2. Finally, cells overexpressing both Cdxs showed strongly decreased Bcl-2 expression, which could account for their increased sensitivity to apoptosis. These findings show that, in HT29 cells, both Cdx1 and Cdx2 genes must be expressed to reduce tumorigenic potential, to increase sensitivity to apoptosis, and to reduce cell migration, suggesting that the two genes control the normal phenotype by independent pathways. This may explain why loss of Cdx1 or Cdx2 expression is associated with tumor development and invasiveness in colorectal tumors.
Assuntos
Proteínas Aviárias , Neoplasias do Colo/genética , Genes Homeobox , Proteínas de Homeodomínio/genética , Apoptose/efeitos dos fármacos , Sequência de Bases , Fator de Transcrição CDX2 , Divisão Celular , Movimento Celular/efeitos dos fármacos , Ceramidas/farmacologia , Neoplasias do Colo/patologia , Meios de Cultura Livres de Soro , Primers do DNA , Regulação para Baixo , Genes bcl-2 , Células HT29 , Humanos , Estaurosporina/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Transativadores , TransfecçãoRESUMO
The modulation by PKC activators and inhibitors of adhesion, spreading, migration, actin cytoskeleton organization, and focal complex formation in keratinocytes attaching to type I collagen was studied. Two actin microfilament networks, stress fibers and cortical actin, could be distinguished on the basis of cellular distribution and opposite regulation by growth factors, tyrosine kinase inhibitors, and PKC activators. Stress fiber formation was stimulated by growth factors and by PMA (100 ng/ml) and these stimulations were blocked by tyrosine kinase inhibitors (0.3 mM genistein and 1 microM herbimycin A). By contrast, the cortical network occurred in quiescent cells, was unaffected by tyrosine kinase inhibitors, and was broken down after PKC activation by PMA. Spreading, migration, and actin polymerization were completely blocked while adhesion efficacy was significantly decreased by three specific PKC inhibitors. Half-inhibition of migration was obtained with 0.025, 1, and 3 microM concentrations of calphostin C, chelerytrine chloride, and D-erythrosphingosine, respectively, which are concentrations close to those known to inhibit the PKC kinase function in vitro. Paxillin clustering, which was observed even in the presence of tyrosine kinase inhibitors, disappeared only when actin polymerization was completely impaired, i.e., in cells treated with PKC inhibitors or with both tyrosine kinase inhibitors and PMA, which indicated that focal complex formation was highly dependent on microfilament reorganization. The analysis of these data underscores a major regulation function of PKC in the molecular events involved in growth factor and adhesion-dependent regulation of microfilament dynamics.
Assuntos
Citoesqueleto de Actina/enzimologia , Movimento Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Queratinócitos/citologia , Proteína Quinase C/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Actinas/metabolismo , Carcinógenos/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/fisiologia , Tamanho Celular/fisiologia , Células Cultivadas , Colágeno/metabolismo , Colágeno/farmacologia , Ativação Enzimática , Substâncias de Crescimento/farmacologia , Humanos , Queratinócitos/química , Queratinócitos/enzimologia , Naftalenos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pele/citologia , Estresse Fisiológico/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In an attempt to define the molecular basis of the adherence of Aspergillus fumigatus conidia to the host tissues, a step which might be mediated by the recognition of basement membrane laminin or fibrinogen, we analyzed the binding of these glycoproteins by flow cytometry and a microtiter plate adherence assay. Flow cytometry revealed that the binding of fluorescein isothiocyanate-labeled laminin to conidia was saturable and specific. Moreover, the ability of conidia to bind laminin increased with their maturation. Competition experiments showed a cross-reactivity between laminin and fibrinogen binding and a lack of interactions with glycosaminoglycans. In addition, the binding of laminin was not inhibited by the different adhesive synthetic peptides tested. Furthermore, the microtiter plate assay of adherence to chymotrypsin degradation products of laminin or fibrinogen purified by gel filtration suggested a unique binding site common to sequential degradation fragments or the presence of multiple binding sites on the two ligands. Therefore, the role of carbohydrates in the recognition process was investigated. Among the carbohydrates tested, constitutive of the conidial wall or of the oligosaccharide side chains of laminin and fibrinogen, only N-acetylneuraminic acid and sialyllactose inhibited the binding of these glycoproteins to conidia. In conclusion, these results strengthen the idea that the laminin and fibrinogen receptors in A. fumigatus are identical and suggest an interaction mediated by a sialic acid-specific lectin of the conidial wall.
Assuntos
Aspergillus fumigatus/imunologia , Fibrinogênio/imunologia , Laminina/imunologia , Ácido N-Acetilneuramínico/fisiologia , Quimotripsina/farmacologia , Reações Cruzadas , Fibronectinas/imunologia , Citometria de Fluxo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/análise , Receptores de Laminina/análiseRESUMO
To understand the modulation and the behavior of glycocalyx elements during adhesion, we explored one of its components, the CD43 molecule, on human monocytic THP-1 cells exposed to cytokine stimulation and its redistribution during heterotypic adhesion to opsonized erythrocytes. First we demonstrated by immunofluorescence and immunoprecipitation that CD43 is dys-sialylated in monocytic THP-1 cells stimulated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and stimulation increased correlated to heterotypic adhesion. CD43 anti-adhesive effect seemed to be related to sialic acid moeties because an increase in adhesion was also induced by sialidase treatment and by monoclonal antibodies recognizing sialic acid-dependent epitopes on CD43. Second, a redistribution of CD43 molecules was observed after adhesion, resulting in the exclusion of CD43 molecules from contact areas as demonstrated by immunofluorescence and by ultrastructural immunogold localization. We therefore demonstrated in monocytic THP-1 cells that some glycocalyx molecules can be modulated by cytokines and redistributed during adhesion. These results support the concept that CD43 can regulate cell interactions.
Assuntos
Antígenos CD34/fisiologia , Eritrócitos/citologia , Monócitos/citologia , Anticorpos Monoclonais/farmacologia , Antígenos CD34/imunologia , Antígenos CD34/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Células Cultivadas , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Interferon gama/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Neuraminidase/farmacologia , Estimulação Química , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
In the present report the biosynthesis of the integrin alpha-chains endowed with constitutive endoproteolytic cleavage was evaluated in LoVo cells where furin, a subtilisin-like convertase involved in post-translational endoproteolytic processing, is not functional. It was found that cell-surface alpha 3, alpha 6 and alpha v subunits were not processed endoproteolytically into heavy and light chains as they were in HT29-D4 cells, a furin-competent cell line. Complete removal of N-linked oligosaccharides and pulse-chase experiments confirmed that the cleavage of the alpha 6 integrin subunit occurring 45 min after translation in HT29 cells did not take place in LoVo cells. Apart from cleavage deficiency, alpha 6 subunit glycosylation, association with beta 4 subunits and targeting to the plasma membrane seemed comparable in LoVo and HT29 cells. The pro-alpha 6 and the pro-alpha 3 subunits immunopurified from LoVo cells were highly sensitive to endoproteolysis by recombinant furin. Furin cleavage was calcium dependent and resulted in the conversion of the 140 kDa pro-alpha 6 into a 120 kDa heavy chain. These results suggest strongly that furin is involved in the endoproteolytic processing of cleavable integrin alpha subunits.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Integrinas/metabolismo , Subtilisinas/metabolismo , Fatores de Virulência , Adenocarcinoma , Amidoidrolases/metabolismo , Neoplasias do Colo , Exotoxinas/toxicidade , Furina , Humanos , Hidrólise , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Subtilisinas/farmacologia , Células Tumorais Cultivadas , Exotoxina A de Pseudomonas aeruginosaRESUMO
Fungi of the order Mucorales determine various infections involving principally the respiratory tract. In spite of their medical importance, little is known about their mechanisms of adherence to the host tissues. Thus we have attempted to define the morphological stages involved in the adherence process of Rhizopus oryzae which is the main causative agent of mucormycoses. The study of the kinetics of germination and adherence to plastic revealed that attachment occurred prior to germination and decreased dramatically with germ tube formation. This correlates with important modifications of the cell wall of the fungus with respect to both carbohydrate composition and distribution of anionic sites. Moreover, the attachment of spores to extracellular matrix components immobilized onto wells of polystyrene microtiter plates has been investigated. Spores adhered readily to immobilized laminin or type IV collagen, but not to fibronectin or the glycosaminoglycans. Attachment to laminin and collagen was dose-dependent and specific. Adhesion was not inhibited by the different carbohydrates tested, suggesting that a lectin was not involved in these interactions. Finally, immunofluorescence revealed that laminin and type IV collagen interacted exclusively with spores and mother cells of germ tubes. Thus, the recognition of laminin or collagen by spores may participate in their adherence to epithelial basement membranes exposed after epithelial tissue damage which frequently accompanies the predisposing factors for mucormycoses.
Assuntos
Rhizopus/citologia , Esporos Fúngicos/citologia , Anticorpos/análise , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Matriz Extracelular/fisiologia , Integrina beta1/imunologia , Integrinas/imunologia , Microscopia de Contraste de Fase , Receptores de Colágeno , Receptores de Laminina/imunologiaRESUMO
Mediterranean spotted fever due to infection by Rickettsia conorii, is characterized by a general vasculitis. This vasculitis is thought to be due to a direct injury to endothelial cells induced by R. conorii. However, production and activity of cytokines on endothelial cells is an important pathway in inflammation, and part of the underlying mechanism of vasculitis. In the present studies, human umbilical vein endothelial cells (HUVEC) infected with R. conorii actively secrete high levels of IL-8 and IL-6 (P < 0.002, and P < 0.03, respectively, compared with uninfected cells). IL-1alpha, IL-1beta, or TNFalpha were not detected in the culture supernates. Nevertheless, IL-6 and IL-8 production was due, in a large part, to a cell-associated form of IL-1 alpha expressed on R. conorii-infected HUVEC, since production of these cytokines was suppressed by 80% (P = 0.0001) and 85% (P < 0.04) by the addition of IL-1 receptor antagonist, or anti-IL-1alpha antibodies (60% inhibition, P < 0.01 and 65% inhibition, P < 0.05, respectively) and IL-1alpha was measured after lysis of R. conorii-infected HUVEC but not in uninfected cells (P < 0.01). Rickettsial lipopolysaccharide does not seem to be involved, since polymyxin B did not reduce cytokine secretion. On the contrary, infection by intracellular R. conorii appears to be necessary to induce IL-1alpha and subsequently IL-8, since formalin-fixed R. conorii did not induce cytokine production. These observations demonstrate that R. conorii-infected HUVEC secrete IL-6 and IL-8 via the induction of cell-associated IL-1alpha, providing a possible mechanism for the vasculitis observed in Mediterranean spotted fever.
Assuntos
Endotélio Vascular/imunologia , Interleucina-1/fisiologia , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Rickettsia/imunologia , Anticorpos/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Interleucina-1/imunologia , Interleucina-1/farmacologia , Cinética , Fatores de Tempo , Veias UmbilicaisRESUMO
BACKGROUND: Junctional epidermolysis bullosa (JEB) encompasses several genodermatoses characterized by skin blistering, and possibly disturbed wound healing. Although the molecular defects underlying JEB are not known, we have demonstrated previously that nicein, an adhesive laminin-related basement membrane component, is immunologically altered in the very severe JEB of Herlitz type (H-JEB), and was expressed to a lesser extent in skin from patients with inversa JEB (I-JEB). In this study, we assessed adhesion and migration of H-JEB and I-JEB keratinocytes on exogenous nicein and laminin to get insights on the biologic function defective in JEB skin. EXPERIMENTAL DESIGN: Adhesion of cultured epidermal keratinocytes from H-JEB and I-JEB patients was assayed by quantitation of cell attachment 1 hour after seeding into microtiter wells coated with nicein or laminin. Cell migration and modulation by function-blocking antibodies to integrins was quantified by computer-assisted image analysis of the tracks left by the cells in a phagokinetic assay using gold particles coated with nicein or laminin. RESULTS: In spite of the fact that H-JEB keratinocytes do not produce normal immunoreactive nicein, they were able to adhere on exogenous nicein similarly to normal and I-JEB keratinocytes which produce nicein. Adhesion of both JEB and normal keratinocytes to laminin was weak compared with nicein. At low and high concentrations of nicein, a reduced migration response occurred with H-JEB keratinocytes whereas I-JEB cells behaved like their normal counterparts. Integrin alpha 3 beta 1 was dominantly involved in adhesion and migration of all these cells. Laminin did not support the migration of either JEB or normal keratinocytes. CONCLUSIONS: H-JEB and I-JEB keratinocytes which produce no or less nicein than normal keratinocytes are able to adhere and migrate on exogenous nicein. Integrin alpha 3 beta 1 which is specifically involved in migration and adhesion of keratinocytes on nicein does not appear altered in JEB. These data indicate that defective nicein rather than modifications of the nicein-recognizing receptor play a central role in the pathogenesis of H-JEB.
Assuntos
Moléculas de Adesão Celular , Epidermólise Bolhosa Juncional/patologia , Integrinas , Queratinócitos/patologia , Adulto , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Epidermólise Bolhosa Juncional/metabolismo , Epidermólise Bolhosa Juncional/fisiopatologia , Feminino , Imunofluorescência , Humanos , Processamento de Imagem Assistida por Computador , Integrina alfa3beta1 , Integrinas/análise , Integrinas/fisiologia , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Laminina/farmacologia , CalininaRESUMO
Using whole viable human colon carcinoma HT29 cells as immunogen, we produced a monoclonal antibody (mAb) termed 69-6-5. The antibody was functionally selected on its anti-cell-spreading activity. By immunoprecipitation of surface radiolabeled cell lysates from HT29-D4 cells (an HT29 cell clone), mAb 69-6-5 recognized a molecular complex resembling integrin heterodimers. Sequential immunodepletions with mAb to the integrin alpha v subunit demonstrated that this complex was composed of alpha v-containing integrins. Accordingly, mAb 69-6-5 reacted with integrin alpha v beta 3 immunopurified from melanoma cells and integrins alpha v beta 5 and alpha v beta 6 immunopurified from pancreatic carcinoma cells. In cell adhesion assays, the 69-6-5 mAb was able to inhibit strongly in a dose-dependent manner arginine-glycine-aspartic acid-mediated adhesion of HT29-D4 cells to vitronectin, fibronectin, or ProNectin F but not to laminin or collagen. Immunoprecipitations with beta chain-specific antisera indicated that these cells express integrins alpha v beta 5 (receptor for vitronectin) and alpha v beta 6 (receptor for fibronectin) but neither alpha v beta 1 nor alpha v beta 3. In summary, these results indicated that mAb 69-6-5 reacts with several alpha v integrins and that it can effectively interfere with the adhesive functions of at least alpha v beta 5 and alpha v beta 6, which represent the major receptors on HT29-D4 cells responsible for their adhesion on vitronectin and fibronectin.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Adesão Celular/fisiologia , Fibronectinas , Glicoproteínas , Integrinas/fisiologia , Sequência de Aminoácidos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular , Neoplasias do Colo , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Epitopos/química , Epitopos/imunologia , Humanos , Integrinas/análise , Integrinas/imunologia , Dados de Sequência Molecular , Peso Molecular , Receptores de Citoadesina/imunologia , Receptores de Citoadesina/fisiologia , Receptores de Vitronectina , Células Tumorais Cultivadas , VitronectinaRESUMO
Two clones derived from the human adenocarcinoma cell line LoVo, E2 and C5 xenografted subcutaneously to immunosuppressed newborn rats, respectively produced well-differentiated and undifferentiated tumors. The comparative morphogenesis of these tumors was performed on xenografts explanted as early as 18 h and up to 21 days after grafting by studying the progressive setting of the enterocyte differentiation marker dipeptidylpeptidase IV, the basal lamina component laminin and the alpha 6 integrin subunit. E2 xenografts which were entirely undifferentiated 18 h after grafting, presented well-polarized acini-like tumoral islets 6 h later, i.e. only 1 day after injection. Basement membranes, which were not organized at this moment, may not be necessary for morphological polarization. The chronology of function antigens polarization was characterized by formation of a basement membrane 5 days after the graft with associated basal sorting of alpha 6 integrin. The polarization of alpha 6 integrin took, however, longer to be achieved while apical addressing of dipeptidylpeptidase IV was the last to be completed. In contrast, C5 tumors never differentiated. Even 21 days after grafting alpha 6 integrin remained pericellular, dipeptidylpeptidase IV was underexpressed and laminin was found as perilobular patches. Quantitative differences in laminin or alpha 6 integrin expression could not account for the differences in the polarization process observed in the two variants.
Assuntos
Adenocarcinoma/patologia , Animais Recém-Nascidos/imunologia , Neoplasias do Colo/patologia , Hospedeiro Imunocomprometido , Transplante Heterólogo , Adenocarcinoma/química , Adenocarcinoma/ultraestrutura , Animais , Antígenos/análise , Antígenos/imunologia , Membrana Basal/química , Membrana Basal/ultraestrutura , Transformação Celular Neoplásica/patologia , Células Clonais , Neoplasias do Colo/química , Neoplasias do Colo/ultraestrutura , Dipeptidil Peptidase 4 , Dipeptidil Peptidases e Tripeptidil Peptidases/análise , Humanos , Imuno-Histoquímica , Integrinas/análise , Integrinas/imunologia , Laminina/análise , Microscopia Eletrônica , Morfogênese , Ratos , Células Tumorais CultivadasRESUMO
Fibronectin is a major component of the extracellular matrix of adherent layers of human long-term marrow cultures where it may stabilize the extracellular matrix network and provide adhesion sites for primitive hemopoietic cells. This study was devised to analyze the role of adherent cell populations in fibronectin synthesis, matrix assembly, and degradation. In cultures performed under the conditions described by Gartner and Kaplan, immunoprecipitation after metabolic labeling showed that adherent cells synthesized a fibronectin variant comprising the EDa domain and lacking the EDb one. Vascular smooth muscle-like stromal cells were the cell subset responsible for this synthesis. Once synthesized by stromal cells, EDa+fibronectin was secreted into the supernatant and incorporated into the extracellular matrix. The cumulation in the extracellular matrix was predominant by weeks 5 and 6 of culture, when a decrease in the stromal cell intracytoplasmic content of fibronectin was observed. Stromal cells from a transformed cell line, L2Ori-, were also able to synthesize the EDa+fibronectin variant, although for these cells the assembly into the extracellular matrix was partly impaired. Besides stromal cells, other cell types participated in fibronectin synthesis: early-adhering granulomonocytic cells and macrophages appearing later in culture were able to synthesize an EDa-, EDb- fibronectin variant, clearly distinct from the EDa+ variant produced by stromal cells. Studies on cultures in which macrophage growth was stimulated at the expense of stromal cells by adding granulocyte-macrophage colony-stimulating factor (50 ng/mL) to the culture medium showed a striking decrease in amounts of fibronectin measured in the adherent layer. This decrease was caused by a lack of incorporation of fibronectin in the extracellular matrix, disclosing a major difference between stromal cells and macrophages in terms of matrix assembly. This study confirms the similarity between stromal cells and vascular smooth muscle cells, because in vivo subendothelial intimal aortic smooth muscle cells and cultured smooth muscle cells from the aortic media express the EDa+, EDb- fibronectin variant. Furthermore, our results suggest that the level of fibronectin in adherent layers is regulated by stromal cells and macrophages. The balance between these two cell populations may therefore be crucial for the local control of hemopoiesis by regulating the extracellular fibronectin available for the adhesion of hematopoietic cells. Our data indicate that it may be essential to study the adhesion of stem cells to EDa+, EDb- fibronectin instead of EDa-, EDb- soluble fibronectin, as found in human plasma.
Assuntos
Células da Medula Óssea , Medula Óssea/fisiologia , Fibronectinas/biossíntese , Macrófagos/fisiologia , Células Cultivadas/efeitos dos fármacos , Meios de Cultura/química , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/química , Fibronectinas/genética , Imunofluorescência , Variação Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Testes de PrecipitinaRESUMO
Aspergillus fumigatus, the causative agent of human aspergillosis, binds to and degrades basement membrane laminin. Using immunoelectron microscopy, laminin binding appeared to be associated with the cell wall expansions of resting conidia, and progressively extended to the outer electron dense layer of the conidial wall during the germination process. Labeling of thin sections revealed numerous binding sites in the cytoplasm, whereas the inner cell wall and the plasma membrane were not labeled. Attachment of A fumigatus conidia on microtiter plates coated with laminin and its fragments P1 and E8 was also investigated. Conidia cells showed good adhesion to wells coated with laminin. As indicated by inhibition experiments, the interaction was specific and fragment P1 represented the major binding site on the laminin molecule. In addition, since A fumigatus produced an extracellular serine protease, we determined the susceptibility of laminin to this enzyme. We demonstrated that protease extract was capable to degrade laminin in solution as well as in tissue sections. The laminin cleavage products were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All the three chains were extensively degraded within 1 h. Treatment of the crude protease extract with the enzyme inhibitors, phenylmethylsulfonyl-fluoride and chymostatin, blocked the degradation of laminin, indicating a chymotrypsin-like serine protease activity. Immunofluorescence microscopy of cryostat sections of mouse and rat kidneys treated with the protease extract showed widespread loss of laminin epitopes from basement membranes. Enzyme treatment also removed immunoreactivity from lungs as observed after immunoperoxidase performed on paraffin sections. Binding and proteolytic degradation of laminin may together facilitate initial interaction of A fumigatus with the host tissues.
Assuntos
Aspergillus fumigatus/metabolismo , Laminina/metabolismo , Aspergillus fumigatus/enzimologia , Membrana Basal/metabolismo , Laminina/isolamento & purificação , Ligação Proteica , Serina Endopeptidases/metabolismo , SolubilidadeRESUMO
The production of laminin by 14-day fetal rat intestinal endoderm and mesenchyme was investigated. The amount of neosynthesized laminin was measured after purification using affinity chromatography. Chain composition of laminin was analyzed by immunoblotting and immunofluorescence staining. The data show that both embryonic intestinal tissue components synthesize laminin and that A and B1/B2 chains were detected in both endodermal and mesenchymal cells. The cellular source of laminin found at the epithelial basement membrane has been studied by immunocytochemistry in rat/chick or mouse/chick interspecies hybrid intestines taken at various stages of development. Immunodetection of the whole laminin molecule and of the individual A and B1/B2 chains by rodent-specific polyclonal and monoclonal antibodies at the basement membrane level in these hybrid intestines revealed (a) laminin molecules, which originate from both mesenchymal and endodermal cells; (b) deposition of A and B1/B2 chains by endodermal cells, regardless of the stage of growth of the hybrid intestines; and (c) asynchronous deposition of the various chains of laminin into the basement membrane by the mesenchyme. B1/B2 chains are deposited concomitant with contact with the epithelium, whereas A chains appear only later (13 days after grafting). These data reinforce the suggestion from previous studies that cooperation between epithelium and mesenchyme is necessary for the formation of a complete basement membrane in the developing intestine.