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1.
Br J Dermatol ; 153(3): 607-12, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16120151

RESUMO

BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.


Assuntos
Doenças do Tecido Conjuntivo/metabolismo , Derme/química , Proteínas da Matriz Extracelular/análise , Proteínas Recombinantes/análise , Envelhecimento da Pele/fisiologia , Raios Ultravioleta/efeitos adversos , Adolescente , Adulto , Idoso , Biomarcadores/análise , Proteínas de Ligação ao Cálcio/análise , Criança , Pré-Escolar , Derme/efeitos da radiação , Elastina/análise , Feminino , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Humanos , Imuno-Histoquímica/métodos , Masculino , Proteínas dos Microfilamentos/análise , Pessoa de Meia-Idade
2.
J Mol Biol ; 314(4): 751-63, 2001 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-11733994

RESUMO

Laminin-5 is a typical component of several epithelial tissues and contains a unique gamma2 chain which can be proteolytically processed by BMP-1. This occurs in the N-terminal half of the gamma2 chain (606 residues), which consists of two rod-like tandem arrays of LE modules, LE1-3 and LE4-6, that flank a globular L4m module containing the cleavage site. Recombinant analysis of L4m, which includes an additional imperfect LE module essential for proper folding, demonstrated an unusual pattern of disulfide bonding. These connectivities prevented the release of gamma2LE1-3L4 m after BMP-1 cleavage which required in addition disulfide reshuffling by isomerases. The liberated segment bound through its L4 m module to heparin, nidogen-1, fibulin-1 and fibulin-2. A further heparin/sulfatide-binding site could be attributed to some arginine residues in module LE1. The gamma2LE4-6 segment remaining in processed laminin-5 showed only a strong binding to fibulin-2. Immunological studies showed a similar partial processing in cell culture and tissues and the persistence of the released fragment in tissues. This indicated that both N-terminal regions of the gamma2 chain may have a function in vivo.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Heparina/metabolismo , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 1 , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Dissulfetos/química , Dissulfetos/metabolismo , Esôfago/química , Humanos , Soros Imunes/imunologia , Isomerismo , Ligantes , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , Subunidades Proteicas , Coelhos , Pele/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Calinina
3.
Eur J Haematol ; 67(3): 176-84, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11737251

RESUMO

Glucocorticoids regulate hematopoietic cell interactions with the bone marrow microenvironment, but the molecules involved in the regulation are still largely unknown. We have studied the effect of glucocorticoids on mRNA expression and protein synthesis of the major extracellular matrix adhesion protein fibronectin and three other extracellular proteins, fibulin-1, fibulin-2 and nidogen-1, in mouse bone marrow cultures and in a hematopoiesis supporting the stromal MC3T3-G2/PA6 cell line. Glucocorticoids suppressed mRNA expression and protein synthesis of fibronectin, fibulin-1 and fibulin-2, but not nidogen-1, in adherent cells of bone marrow cultures, as shown by Northern blot analysis and immunoprecipitation. mRNA levels of all four proteins were down-regulated by dexamethasone in MC3T3-G2/PA6 cells, indicating a direct glucocorticoid effect on cells synthesizing extracellular matrix proteins. Dexamethasone down-regulated fibronectin mRNA rapidly, within 2 h of treatment, in the stromal cells. This effect did not require mRNA or protein synthesis, as shown by Northern blot analysis after treatment by actinomycin D and cycloheximide. Interferon-alpha, which also has been reported to modulate haematopoietic cell-matrix interactions, did not affect mRNA expression of the proteins in MC3T3-G2/PA6 cells. Our results indicate that glucocorticoids down-regulate expression of several mesenchymal-type extracellular matrix molecules in bone marrow, but with a variable effect on different proteins. Thus one mechanism by which glucocorticoids regulate haematopoiesis may be by altering the relative proportions of extracellular matrix proteins.


Assuntos
Medula Óssea/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Dexametasona/farmacologia , Proteínas da Matriz Extracelular/biossíntese , Fibronectinas/biossíntese , Glucocorticoides/farmacologia , Animais , Linhagem Celular , Regulação para Baixo/efeitos dos fármacos , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , Células Estromais/metabolismo
4.
FEBS Lett ; 509(2): 181-5, 2001 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-11741585

RESUMO

The globular domain IVa from the short arm region of mouse laminin alpha5 chain was obtained by recombinant production and shown to be a cell-adhesive substrate and to bind alphaVbeta3 integrin in solid-phase assays. These interactions were blocked by RGD peptides and a restricted panel of anti-integrin antibodies. The two RGD sequences present in alpha5IVa were shown by site-directed mutagenesis to make different contributions to cell adhesion but were equivalent in binding alphaVbeta3 integrin. A quantitative radioimmuno-inhibition assay was established based on domain alpha5IVa which demonstrated distinct amounts of alpha5 chain in various tissues, particularly in vessel walls. There it could play a role in angiogenesis steps requiring RGD-dependent integrins.


Assuntos
Moléculas de Adesão Celular/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Oligopeptídeos/metabolismo , Receptores de Vitronectina/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/isolamento & purificação , Humanos , Rim/citologia , Laminina/genética , Laminina/isolamento & purificação , Melanoma Experimental , Camundongos , Músculo Esquelético/citologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo
5.
Eur J Biochem ; 268(19): 5119-28, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11589703

RESUMO

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.


Assuntos
Anticorpos Monoclonais/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Sequência de Bases , Membrana Basal/metabolismo , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Microscopia Eletrônica , Ligação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
EMBO J ; 20(19): 5342-6, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11574465

RESUMO

Nidogen and perlecan are large multifunctional basement membrane (BM) proteins conserved in all metazoa. Their high-affinity interaction, which is likely to contribute to BM assembly and function, is mediated by the central G2 domain in nidogen and the third immunoglobulin (IG)-like domain in perlecan, IG3. We have solved the crystal structure at 2.0 A resolution of the mouse nidogen-1 G2-perlecan IG3 complex. Perlecan IG3 belongs to the I-set of the IG superfamily and binds to the wall of the nidogen-1 G2 beta-barrel using beta-strands C, D and F. Nidogen-1 residues participating in the extensive interface are highly conserved, whereas the corresponding binding site on perlecan is more variable. We hypothesize that a second, as yet unidentified, activity of nidogen overlaps with perlecan binding and accounts for the unusually high degree of surface conservation in the G2 domain.


Assuntos
Proteínas da Matriz Extracelular/química , Proteoglicanas de Heparan Sulfato/química , Glicoproteínas de Membrana/química , Sequência de Aminoácidos , Animais , Membrana Basal , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Imunoglobulinas/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Propriedades de Superfície
7.
FEBS Lett ; 505(1): 173-8, 2001 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-11557064

RESUMO

The N-terminal domain of laminin alpha 4 chains corresponds to a short rod-like structure which after recombinant production was found to be modified by chondroitin sulfate. Substitution occurred mainly to a single serine in its N-terminal ASGDG sequence. A similar yet partial modification was also demonstrated for the alpha 4 chain present in extracts of adult mouse tissues. Antibodies to the fragment were useful to demonstrate a relatively high content of alpha 4 in several tissues and for the immunolocalization in various blood vessels, some basement membranes and interstitial regions.


Assuntos
Sulfatos de Condroitina/genética , Laminina/genética , Laminina/metabolismo , Animais , Anticorpos , Condroitina ABC Liase/metabolismo , Sulfatos de Condroitina/imunologia , Imunofluorescência , Soros Imunes , Laminina/imunologia , Camundongos , Fragmentos de Peptídeos/genética , Coelhos , Radioimunoensaio , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Distribuição Tecidual
8.
Mol Cell Biol ; 21(20): 7025-34, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11564885

RESUMO

The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/fisiologia , Capilares/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Animais , Aorta/embriologia , Aorta/patologia , Northern Blotting , Southern Blotting , Embrião de Mamíferos/metabolismo , Matriz Extracelular/metabolismo , Vetores Genéticos , Homozigoto , Imuno-Histoquímica , Rim/embriologia , Rim/metabolismo , Rim/patologia , Pulmão/embriologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Genéticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
9.
Dev Dyn ; 222(1): 89-100, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11507771

RESUMO

Previous studies showed that extracellular matrix protein, fibulin-2, is expressed during epithelial-mesenchymal transformation in the endocardial cushion matrix during embryonic heart development. Our current study revealed that, in addition to the cardiac valvuloseptal formation, fibulin-2 is synthesized by the smooth muscle precursor cells of developing aortic arch vessels and the coronary endothelial cells that are originated from neural crest cells and epicardial cells, respectively. In the cardiac valves and the aortic arch vessels, fibulin-2 expression shows robust up-regulation when the transformed mesenchymal cells migrate into the existing extracellular matrix. In the epicardium, epicardial cells produce fibulin-2 upon their migration over the myocardial surface and its expression persists throughout coronary vasculogenesis and angiogenesis. Fibulin-2 is produced by the endothelial cells of coronary arteries and veins but not by the capillary endothelial cells in the myocardium. Thus, fibulin-2 not only uniquely marks the transformed mesenchymal cells during mouse embryonic cardiovascular development, but also indicates vascular endothelial cells of coronary arteries and veins in postnatal life.


Assuntos
Aorta Torácica/embriologia , Aorta Torácica/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Vasos Coronários/embriologia , Vasos Coronários/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Valvas Cardíacas/embriologia , Valvas Cardíacas/metabolismo , Mesoderma/metabolismo , Animais , Divisão Celular , Células Cultivadas , DNA Complementar/metabolismo , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Coração/embriologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Biológicos , Miocárdio/metabolismo , Pericárdio/metabolismo , Fatores de Tempo , Regulação para Cima
10.
J Mol Biol ; 311(3): 529-41, 2001 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-11493006

RESUMO

Perlecan, a major basement membrane proteoglycan, has a complex modular structure designed for the binding of many cellular and extracellular ligands. Its domain IV, which consists of a tandem of immunoglobulin-like modules (IG2-IG15), is rich in such binding sites, which have been mapped to different modules obtained by recombinant production. Heparin/sulfatide binding was restricted to IG5 and shown to depend on four arginine residues that are close in space in beta strands B and E of the C-type IG fold. The nidogen-1 and nidogen-2 isoforms bind to IG3 with high affinity (K(d) approximately 10 nM). This interaction depends on the globular nidogen domain G2 and is crucial for the formation of ternary complexes with laminins. Two loops of IG3 located between beta strands B/C and F/G, which are spatially close, make a major contribution to binding. Fibronectin binding was localized to IG4-5 and fibulin-2 binds to IG2 and IG13-15 with different affinities. This implicates a complex cluster of heterotypic interaction sites apparently important for the supramolecular organization of perlecan in tissues.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/metabolismo , Imunoglobulinas/química , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Arginina/genética , Arginina/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular , Proteoglicanas de Heparan Sulfato/genética , Humanos , Cinética , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Ressonância de Plasmônio de Superfície , Termodinâmica
11.
Nat Struct Biol ; 8(7): 634-40, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11427896

RESUMO

Nidogen, an invariant component of basement membranes, is a multifunctional protein that interacts with most other major basement membrane proteins. Here, we report the crystal structure of the mouse nidogen-1 G2 fragment, which contains binding sites for collagen IV and perlecan. The structure is composed of an EGF-like domain and an 11-stranded beta-barrel with a central helix. The beta-barrel domain has unexpected similarity to green fluorescent protein. A large surface patch on the beta-barrel is strikingly conserved in all metazoan nidogens. Site-directed mutagenesis demonstrates that the conserved residues are involved in perlecan binding.


Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Mutação/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Dissulfetos/metabolismo , Glicosilação , Proteínas de Fluorescência Verde , Ligantes , Proteínas Luminescentes/química , Glicoproteínas de Membrana/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência
12.
J Biol Chem ; 276(22): 18947-52, 2001 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-11259413

RESUMO

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.


Assuntos
Colágeno/química , Colágeno/metabolismo , Proteoglicanas/metabolismo , Animais , Biglicano , Ligação Competitiva , Células CHO , Bovinos , Condroitina ABC Liase/farmacologia , Cricetinae , Decorina , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular , Glicosaminoglicanos/metabolismo , Células HeLa , Humanos , Cinética , Microscopia Eletrônica , Placenta/metabolismo , Testes de Precipitina , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície
13.
Nat Biotechnol ; 19(1): 29-34, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11135548

RESUMO

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Neoplasias Encefálicas/terapia , Colágeno/uso terapêutico , Glioma/terapia , Fragmentos de Peptídeos/uso terapêutico , Alginatos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/toxicidade , Animais , Apoptose , Materiais Biocompatíveis , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Cápsulas , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Transplante de Células , Colágeno/genética , Colágeno/toxicidade , Endostatinas , Glioma/irrigação sanguínea , Glioma/patologia , Ácido Glucurônico , Ácidos Hexurônicos , Humanos , Imageamento por Ressonância Magnética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/toxicidade , Ratos , Transfecção , Transplante Heterólogo
14.
J Biol Chem ; 276(2): 1253-61, 2001 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-11038354

RESUMO

Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.


Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Proteoglicanas/química , Proteoglicanas/metabolismo , Agrecanas , Fosfatase Alcalina/metabolismo , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/ultraestrutura , Proteoglicanas de Sulfatos de Condroitina/ultraestrutura , Reagentes de Ligações Cruzadas , Dimerização , Proteínas da Matriz Extracelular/ultraestrutura , Cinética , Lectinas , Lectinas Tipo C , Mamíferos , Proteoglicanas/ultraestrutura , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície , Versicanas
15.
Matrix Biol ; 19(7): 637-47, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11102753

RESUMO

The purpose of this study was to determine the mRNA and protein expression of laminin alpha chains at various stages of in vitro skin morphogenesis. Fibroblasts in mono-cultures express low levels of the mRNA of laminin alpha1,alpha2, alpha3 and alpha4 chains. When co-cultured with keratinocytes for 28 days, they expressed the mRNA for all these chains. Keratinocytes in monolayer expressed the laminin alpha3 chain mRNA and very low levels of the mRNA of the alpha1 and alpha2 chains, although, when recombined with fibroblasts they also expressed laminin alpha1and alpha2 mRNA, but not the laminin alpha4 mRNA. Immunocytochemistry of cells in co-culture showed that laminin alpha1, alpha3 and alpha5 chains were expressed in the epidermis, while the laminin alpha2, beta1, and gamma1 chains were noted in the dermis and at the epidermo-dermal interface. The laminin alpha1chain was first expressed during the proliferative stage (14-21 days) and the laminin alpha2 and alpha5 chains appeared later, during the differentiation stage (28-42 days). The above results suggest that epithelial-mesenchymal interactions are involved in the expression of laminin alpha chain mRNA during in vitro skin morphogenesis. In addition, there is distinct temporal and spatial expression of these chains during proliferative and differentiation stages, possibly reflecting different functions.


Assuntos
Laminina/genética , Pele/metabolismo , Diferenciação Celular , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Laminina/metabolismo , Morfogênese , RNA Mensageiro , Pele/citologia
16.
Cell ; 103(3): 511-24, 2000 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-11081637

RESUMO

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.


Assuntos
Antígenos de Bactérias , Parede Celular/metabolismo , Glicolipídeos/metabolismo , Mycobacterium leprae/citologia , Mycobacterium leprae/fisiologia , Nervo Isquiático/microbiologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Axônios/microbiologia , Axônios/ultraestrutura , Membrana Basal/efeitos dos fármacos , Membrana Basal/metabolismo , Membrana Basal/microbiologia , Membrana Basal/ultraestrutura , Sítios de Ligação , Parede Celular/química , Parede Celular/ultraestrutura , Células Cultivadas , Técnicas de Cocultura , Proteínas da Matriz Extracelular/metabolismo , Glicolipídeos/química , Humanos , Laminina/química , Laminina/metabolismo , Laminina/farmacologia , Microscopia Eletrônica , Microesferas , Mycobacterium leprae/patogenicidade , Mycobacterium leprae/efeitos da radiação , Fibras Nervosas/efeitos dos fármacos , Fibras Nervosas/metabolismo , Fibras Nervosas/microbiologia , Fibras Nervosas/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/microbiologia , Nervo Isquiático/citologia , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Trissacarídeos/metabolismo , Trissacarídeos/farmacologia , Células Tumorais Cultivadas
17.
FASEB J ; 14(15): 2373-6, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11024009

RESUMO

Endostatin is a cleavage product of collagen XVIII that strongly inhibits tumor angiogenesis. To determine if endostatin affects other angiogenic processes, we generated full-thickness excisional wounds on the back of mice that were systemically treated with recombinant murine endostatin. No macroscopic abnormalities of the wound healing process were observed. Histological analysis revealed normal wound contraction and re-epithelialization, but a slight reduction in granulation tissue formation and reduced matrix deposition at the wound edge. The blood vessel density in the wounds of endostatin-treated mice was not affected. However, ultrastructural analysis demonstrated severe abnormalities in blood vessel maturation. The wound vessels in the endostatin-treated mice were narrowed or closed with an irregular luminal surface, resulting in a severe reduction in the number of functional vessels and extravasation of erythrocytes. Endostatin treatment did not affect the expression level and localization of collagen XVIII mRNA and protein. Furthermore, the angiogenesis regulators vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 were normally expressed in the wounds of endostatin-treated mice. However, expression of the major wound matrix proteins fibronectin and collagens I and III was significantly reduced. This reduction is likely to explain the reduced density of the wound matrix. Our results demonstrate that endostatin treatment reduces the number of functional blood vessels and the matrix density in the granulation tissue, but does not significantly affect the overall wound healing process.


Assuntos
Inibidores da Angiogênese/farmacologia , Colágeno/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Cicatrização/efeitos dos fármacos , Angiopoietina-1 , Angiopoietina-2 , Animais , Colágeno/biossíntese , Colágeno/isolamento & purificação , Colágeno Tipo XVIII , Endostatinas , Fatores de Crescimento Endotelial/biossíntese , Proteínas da Matriz Extracelular/biossíntese , Linfocinas/biossíntese , Glicoproteínas de Membrana/biossíntese , Camundongos , Biossíntese de Proteínas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular
18.
J Mol Biol ; 301(5): 1179-90, 2000 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-10966814

RESUMO

Endostatin is a fragment of the C-terminal domain NC1 of collagen XVIII that inhibits angiogenesis and tumor growth. We report the characterization of a collagen XV endostatin analogue and its parent NC1 domain, obtained by recombinant expression in mammalian cells. Both NC1 domains contain a trimerization domain, a hinge region that is more sensitive to proteolysis in collagen XVIII and the endostatin domain. Unlike endostatin-XVIII, endostatin-XV does not bind zinc or heparin, which is explained by the crystal structure of endostatin-XV. The collagen XV and XVIII fragments inhibited chorioallantoic membrane angiogenesis induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF), but there are striking differences depending on which cytokine is used and whether free endostatins or NC1 domains are applied. The collagen XV and XVIII fragments showed a similar binding repertoire for extracellular matrix proteins. Differences were found in the immunohistological localization in vessel walls and basement membrane zones. Together, these data indentify endostatin-XV as an angiogenesis inhibitor, which differs from endostatin-XVIII in several important functional details.


Assuntos
Colágeno/química , Colágeno/metabolismo , Neovascularização Fisiológica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/análise , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Embrião de Galinha , Córion/irrigação sanguínea , Córion/efeitos dos fármacos , Colágeno/análise , Colágeno/farmacologia , Colágeno Tipo XVIII , Cristalografia por Raios X , Endostatinas , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/farmacologia , Imunofluorescência , Heparina/metabolismo , Humanos , Ligantes , Linfocinas/antagonistas & inibidores , Linfocinas/farmacologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Especificidade por Substrato , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Zinco/metabolismo
19.
Matrix Biol ; 19(4): 309-17, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10963991

RESUMO

Laminin G domain-like (LG) modules of approximately 180-200 residues are found in a number of extracellular and receptor proteins and often are present in tandem arrays. LG modules are implicated in interactions with cellular receptors (integrins, alpha-dystroglycan), sulfated carbohydrates and other extracellular ligands. The recently determined crystal structures of LG modules of the laminin alpha2 chain reveal a compact beta sandwich fold and identify a novel calcium binding site. Binding epitopes for heparin, sulfatides and alpha-dystroglycan have been mapped by site-directed mutagenesis and show considerable overlap. The epitopes are located in surface loops around the calcium site, which in other proteins (agrin, neurexins) are modified by alternative splicing. Efficient ligand binding often requires LG modules to be present in tandem. The close proximity of the N- and C-termini in the LG module, as well as a unique link region between laminin LG3 and LG4, impose certain constraints on the arrangement of LG tandems. Further modifications may be introduced by proteolytic processing of laminin G domains, which is known to occur in the alpha2, alpha3 and alpha4 chains.


Assuntos
Laminina/química , Laminina/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Endopeptidases/metabolismo , Heparina/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Relação Estrutura-Atividade , Sulfoglicoesfingolipídeos/metabolismo
20.
EMBO J ; 19(15): 3990-4003, 2000 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-10921880

RESUMO

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.


Assuntos
Folículo Piloso/patologia , Integrina beta1/genética , Queratinócitos/patologia , Pele/patologia , Animais , Membrana Basal/patologia , Sobrevivência Celular , Derme/patologia , Epiderme/patologia , Proteínas da Matriz Extracelular/isolamento & purificação , Fibrose , Hipotricose/etiologia , Inflamação , Camundongos , Camundongos Knockout , Anormalidades da Pele/etiologia
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