RESUMO
An active involvement of blood-brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE). Endothelial basement membranes contained laminin 8 (alpha4beta1gamma1) and/or 10 (alpha5beta1gamma1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin alpha6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.
Assuntos
Barreira Hematoencefálica , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Endotélio/metabolismo , Laminina/metabolismo , Linfócitos T/imunologia , Animais , Especificidade de Anticorpos , Membrana Basal/metabolismo , Membrana Basal/patologia , Encéfalo/irrigação sanguínea , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Citocinas/farmacologia , Encefalomielite Autoimune Experimental/patologia , Endotélio/patologia , Matriz Extracelular/metabolismo , Feminino , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Laminina/isolamento & purificação , Meninges/irrigação sanguínea , Meninges/imunologia , Meninges/metabolismo , Meninges/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Laminina/metabolismo , Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The uptake of transferrin and iron by cultured myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus (tsLA24) was compared with that of normal developing myogenic cells which were proliferating at the same rate as the transformed cells. The mechanism of transferrin and iron uptake was the same in the transformed cells as in normal myogenic cells and involved receptor-mediated endocytosis of transferrin. However, there were differences in transferrin receptor numbers and receptor function. The number of receptors in transformed cells was more than twice as great as in the normal cells largely due to increased surface receptor numbers. Despite this, the rate of iron uptake increased by only 20% in the transformed cells due to less efficient cycling of the transferrin receptors and less efficient release of iron from transferrin to intracellular sites. Some internalized iron was released from the transformed cells still bound to transferrin. A fast and a slow rate of transferrin exocytosis were identified in transformed cells, as in normal cells, indicating that there were at least two intracellular pathways for transferrin. The fast pathway predominated in the transformed cells, compared with an equal importance of the two pathways in the normal cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Ferro/metabolismo , Músculos/metabolismo , Transferrina/metabolismo , Animais , Células Cultivadas , Embrião de Galinha , Endocitose , Exocitose , Receptores da Transferrina/análiseRESUMO
Differential gene expression in tumors often involves growth factors and extracellular matrix/basement membrane components. Here, 11,000- gene microarray was used to identify gene expression profiles in brain tumors including high-grade gliomas [glioblastoma multiforme (GBM) and anaplastic astrocytoma], low-grade astrocytomas, or benign extra-axial brain tumors (meningioma) in comparison with normal brain tissue. Histologically normal tissues adjacent to GBMs were also studied. All GBMs studied overexpressed 14 known genes compared with normal human brain tissue. Overexpressed genes belonged to two broad groups: (a) growth factor-related genes; and (b) structural/extracellular matrix-related genes. For most of these 14 genes, expression levels were lower in low-grade astrocytoma than in GBM and were barely detectable in normal brain. Despite normal-appearing histology, gene expression patterns of tissues immediately adjacent to GBM were similar to those of their respective primary GBMs. Two genes were consistently up-regulated in both high-grade and low-grade gliomas, as well as in histologically normal tissues adjacent to GBMs. These genes coded for the epidermal growth factor receptor (previously reported to be overexpressed in gliomas) and for the alpha4 chain of laminin, a major blood vessel basement membrane component. Changes in expression of this laminin chain have not been previously associated with malignant tumors. Overexpression of laminin alpha4 chain in GBM and astrocytoma grade II by gene microarray analysis was confirmed by semiquantitive reverse transcription-PCR and immunohistochemistry. Importantly, an alpha4 chain-containing laminin isoform, laminin-8 (alpha4beta1gamma1), was expressed mainly in blood vessel walls of GBMs and histologically normal tissues adjacent to GBMs, whereas another alpha4 chain-containing laminin isoform, laminin-9 (alpha4beta2gamma1), was expressed mainly in blood vessel walls of low-grade tumors and normal brain. GBMs that overexpressed laminin-8 had a shorter mean time to tumor recurrence (4.3 months) than GBMs with overexpression of laminin-9 (9.7 months, P = 0.0007). Up-regulation of alpha4 chain-containing laminins could be important for the development of glioma-induced neovascularization and glial tumor progression. Overexpression of laminin-8 may be predictive of glioma recurrence.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Laminina/genética , Adulto , Idoso , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Glioma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
It is not known whether epithelial differentiation patterns are reflected in the composition of gingival basement membranes (BMs). We have investigated the expression of laminin isoforms and associated BM components in the murine dento-epithelial junction by using immunofluorescence microscopy. Our results show that chains of laminins 5/6/7/10/11 are expressed in the BM of outer gingival epithelium. The external BM between junctional epithelium (JE) and connective tissue differs from gingival BM by lacking laminin-7 and -11 chains. The internal basal lamina (IBL) between JE and tooth contains only laminin-5. Collagen chains alpha1,2(IV) and nidogen-1 are present in other BMs except the IBL. The dento-epithelial junction thus has a unique BM composition, suggesting that epithelial cells are able to secrete two extracellular matrices in a polarized manner. The exclusive expression of the non-self-polymerizing laminin-5 indicates that the IBL is not a BM by definition, but rather a simple extracellular matrix lacking network structure.
Assuntos
Membrana Basal/metabolismo , Inserção Epitelial/citologia , Células Epiteliais/metabolismo , Gengiva/citologia , Animais , Diferenciação Celular , Inserção Epitelial/metabolismo , Imunofluorescência , Gengiva/metabolismo , Laminina/biossíntese , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Long-EvansRESUMO
The ability of unlabelled heterologous transferrin to interact with transferrin receptors on developing chick myogenic cells was investigated by measuring their capacity to inhibit the surface-binding and internalization of 125I- and 59Fe-labelled ovotransferrin. Transferrins from rat, rabbit, human, and a species of kangaroo (Macropus fuliginosus) were unable to inhibit either surface-binding or internalization of labelled ovotransferrin even at concentrations ten times the molar concentration of the ovotransferrin. Transferrins isolated from the serum of a toad (Bufo marinus) and a lizard (Teliqua rugosa), when added at high concentrations, were found to reduce surface-binding of 125I-Tf by 20-25% but did not inhibit internalization of either 125I-Tf or 59Fe. This suggests that the effects of toad and lizard transferrins are due to non-specific binding to the myogenic cells. In contrast, inhibition of both surface-binding and internalization of labelled ovotransferrin was found when myogenic cells were incubated in the presence of the homologous transferrin (ovotransferrin). The species-specificity of transferrin binding, endocytosis and iron internalization did not vary with the state of proliferation or differentiation of the myogenic cells. However, the intracellular iron utilization was found to differ between differentiating presumptive and terminally differentiated myotubes. Internalized 59Fe was fractioned by gel filtration. In dividing and non-dividing presumptive myoblasts 59Fe was found to elute in three peaks, two with elution volumes corresponding to ferritin and transferrin and one at greater elution volume than that of myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Endocitose , Ferro/metabolismo , Músculos/citologia , Transferrina/metabolismo , Animais , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Ferro/farmacocinética , Ferro/farmacologia , Músculos/embriologia , Ligação Proteica , Especificidade da EspécieRESUMO
The expression of laminin-1 (previously EHS laminin) and laminin-2 (previously merosin) isoforms by myogenic cells was examined in vitro and in vivo. No laminin alpha 2 chainspecific antibodies react with mouse tissues, 50 rat monoclonal antibodies were raised against the mouse laminin alpha 2 chain: their characterization is described here. Myoblasts and myotubes from myogenic cell lines and primary myogenic cultures express laminin beta 1 and gamma 1 chains and form a complex with a 380 kDa alpha chain identified as laminin alpha 2 by immunofluorescence, immunoprecipitation and PCR. PCR from C2C12 myoblasts and myotubes for the laminin alpha 2 chain gene (LamA2) provided cDNA sequences which were used to investigate the in vivo expression of mouse LamA2 mRNA in embryonic tissues by in situ hybridization. Comparisons were made with specific probes for the laminin alpha 1 chain gene (LamA1). LamA2 but not LamA1 mRNA was expressed in myogenic tissues of 14- and 17-day-old mouse embryos, while the laminin alpha 2 polypeptide was localized in adjacent basement membranes in the muscle fibres. In situ hybridization also revealed strong expression of the LamA2 mRNA in the dermis, indicating that laminin alpha 2 is expressed other than by myogenic cells in vivo. Immunofluorescence studies localized laminin alpha 2 in basement membranes of basal keratinocytes and the epithelial cells of hair follicles, providing new insight into basement membrane assembly during embryogenesis. In vitro cell attachment assays revealed that C2C12 and primary myoblasts adhere to laminin-1 and -2 isoforms in a similar manner except that myoblast spreading was significantly faster on laminin-2. Taken together, the data suggest that laminins 1 and 2 play distinct roles in myogenesis.
Assuntos
Laminina/análise , Proteínas Musculares/análise , Músculos/química , Células-Tronco/química , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Sequência de Bases , Linhagem Celular , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Músculos/citologia , Fragmentos de Peptídeos/análiseRESUMO
The experiments described in this study were designed to investigate receptor-mediated endocytosis of transferrin and its role in iron uptake by cultured chick presumptive myoblasts (dividing and non-dividing) and myotubes. The effects of a variety of inhibitors on the internalization of transferrin and iron were investigated and three main effects were found: (i) sulphydryl reagents and microtubular inhibitors reduced the rate of transferrin and iron internalization to similar degrees, (ii) metabolic inhibitors reduced the rate of iron uptake more than that of transferrin endocytosis, and (iii) lysosomotrophic agents almost completely abolished iron accumulation by the cells without any effect on the rate of transferrin internalization. The results suggest that metabolic energy is required not only for the endocytosis of transferrin but also for subsequent steps in the iron uptake process, and that iron release from transferrin occurs in acidified endosomes. Overall, these experiments show that all or virtually all of the iron taken up by developing muscle cells from transferrin occurs as a consequence of receptor-mediated endocytosis of the protein.
Assuntos
Endocitose , Ferro/metabolismo , Músculos/metabolismo , Transferrina/metabolismo , 2,4-Dinitrofenol , Cloreto de Amônio/farmacologia , Animais , Antimicina A/farmacologia , Azidas/farmacologia , Células Cultivadas , Embrião de Galinha , Cloroquina/farmacologia , Colchicina/farmacologia , Dinitrofenóis/farmacologia , Endocitose/efeitos dos fármacos , Metilaminas/farmacologia , Músculos/citologia , Músculos/embriologia , Podofilotoxina/farmacologia , Azida Sódica , Reagentes de Sulfidrila/farmacologia , Vimblastina/farmacologiaRESUMO
The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors.
Assuntos
Transformação Celular Neoplásica , Ferro/metabolismo , Músculos/metabolismo , Receptores da Transferrina/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Vírus do Sarcoma Aviário/genética , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Conalbumina/metabolismo , Replicação do DNA/efeitos dos fármacos , Cinética , Músculos/citologia , Músculos/efeitos dos fármacos , Forbóis/farmacologia , Receptores da Transferrina/efeitos dos fármacosRESUMO
The mechanism of iron uptake and the changes which occur during cellular development of muscle cells were investigated using primary cultures of chick embryo breast muscle. Replicating presumptive myoblasts were examined in exponential growth and after growth had plateaued. These were compared to the terminally differentiated cell type, the myotube. All cells, regardless of the state of growth or differentiation, had specific receptors for transferrin. Presumptive myoblasts in exponential growth had more transferrin receptors (3.78 +/- 0.24 X 10(10) receptors/micrograms DNA) than when division had ceased (1.70 +/- 0.14 X 10(10) receptors/micrograms DNA), while myotubes had 3.80 +/- 0.26 X 10(10) receptors/micrograms DNA. Iron uptake occurred by receptor-mediated endocytosis of transferrin. While iron was accumulated by the cells, apotransferrin was released in an undegraded form. There was a close correlation between the molar rates of endocytosis of transferrin and iron. Maximum rates of iron uptake were significantly higher in myotubes than in presumptive myoblasts in either exponential growth or after growth had plateaued. There were two rates of exocytosis of transferrin, implying the existence of two intracellular pathways for transferrin. These experiments demonstrate that iron uptake by muscle cells in culture occurs by receptor-mediated endocytosis of transferrin and that transferrin receptor numbers and the kinetics of transferrin and iron uptake vary with development of the cells.
Assuntos
Ferro/metabolismo , Músculos/metabolismo , Receptores da Transferrina/metabolismo , Transferrina/metabolismo , Animais , Transporte Biológico , Diferenciação Celular , Células Cultivadas , Embrião de Galinha , Endocitose , Exocitose , Cinética , Músculos/citologia , Células-Tronco/metabolismoRESUMO
Basement membranes of the developing tooth have been previously shown to contain laminins, but the nature of the laminins have not been described. We here studied the distribution of five different laminin alpha chains during tooth development. We show that both epithelial and mesenchymal cells produce laminin alpha chains. The mRNAs of three laminin alpha chains, alpha1, alpha2, and alpha4, were expressed in the tooth mesenchyme, whereas two, the alpha3 and alpha5 chain mRNAs, were found in epithelium. Drastic changes in the expression patterns of the two epithelial chains were found during development. The alpha5 mRNA was widely expressed in tooth epithelia, and the corresponding protein was evenly distributed along the tooth basement membrane throughout embryonic development. This suggests a role for alpha5 as a major laminin alpha chain in tooth basement membrane during embryonic stages. The subsequent disappearance of alpha5 and the drastic increase in alpha3A mRNA expression during terminal ameloblast differentiation and enamel secretion suggest that alpha3A acts as an important chain in the enamel matrix after degradation of tooth basement membrane. These studies show that laminin networks in tooth epithelia form as a result of epithelial-mesenchymal interactions and that the molecular composition of the laminin networks varies drastically during development of tooth.
Assuntos
Expressão Gênica , Laminina/biossíntese , Odontogênese/genética , Animais , Hibridização In Situ , Laminina/genética , Camundongos , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The nature of the laminin alpha chains in the embryonic and adult kidney is still being debated. The present study attempted to clarify this issue by immunofluorescence study using monoclonal antibodies against mouse alpha1, alpha2, and alpha5 chains and in situ hybridization for the alpha2, alpha3B, alpha4, and alpha5 mRNAs. Novel alpha1 chain-specific monoclonal antibodies against E8 fragment revealed a restricted distribution of alpha1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The alpha2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The alpha3B mRNA was found only in a small subset of endothelial cells. The alpha4 mRNA was found transiently in embryonic mesenchyme, with particularly high levels in condensed mesenchyme, close to the tips of the ureteric tree where tubulogenesis is initiated. The alpha5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the alpha5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the alpha5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the alpha5 chain became ubiquitously expressed in basement membranes. Overall, the alpha5 chain exhibited the broadest pattern of expression, followed by the alpha1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several alpha chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the alpha chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear.
Assuntos
Rim/embriologia , Rim/crescimento & desenvolvimento , Laminina/biossíntese , Animais , Animais Recém-Nascidos , Northern Blotting , Hibridização in Situ Fluorescente , Rim/metabolismo , Glomérulos Renais/embriologia , Glomérulos Renais/metabolismo , Laminina/genética , Camundongos , Néfrons/embriologia , Néfrons/metabolismo , RNA Mensageiro , CoelhosRESUMO
Rat monoclonal antibodies were raised against fragment E3 of the mouse Engelbreth-Holm-Swarm (EHS) tumor laminin and selected according to their exclusive reaction with laminin A chain by immunoblotting and staining pattern in embryonic kidneys by immunofluorescence. Immunochemical studies of nine purified antibodies showed a comparable reaction with unfragmented laminin and fragment E3 but no cross-reaction with several other, unrelated laminin fragments including the major cell-binding fragment E8. Reduction or pepsin digestion of fragment E3 reduced or abolished antibody binding indicating that most of the epitopes involved are conformation dependent and do not include carbohydrates. They are, however, not identical as shown by different reactivities after proteolytic or chemical cleavage of E3. Four of the antibodies were highly active in inhibiting cell adhesion of the teratocarcinoma cell line F9 and the Schwannoma cell line RN22 on fragment E3 (IC50 approximately 1 microgram/ml), while the others were distinctly less active. No inhibition was observed for cell adhesion on unfragmented laminin, consistent with previous findings that this is largely mediated by binding of fragment E8 to alpha 6 beta 1 integrin. A distinct correlation was observed between cell adhesion inhibition and the inhibition of heparansulfate proteoglycan and heparin binding to fragment E3. Since heparin is not very efficient in inhibiting cell adhesion, it indicates that heparin- and cell-binding sites on fragment E3 are in close proximity but not identical. Two of the antibodies also showed partial inhibition of kidney tubule formation in organ culture of embryonic kidney mesenchyme while the other antibodies were inactive. It suggests some but probably minor involvement of the fragment E3 structure of laminin in this developmental process.
Assuntos
Anticorpos Monoclonais/química , Túbulos Renais/efeitos dos fármacos , Laminina/química , Animais , Anticorpos/química , Sítios de Ligação de Anticorpos , Heparina/metabolismo , Túbulos Renais/embriologia , Laminina/antagonistas & inibidores , Laminina/imunologia , Camundongos , Proteoglicanas/metabolismo , RatosRESUMO
A method for efficiently infecting primary myogenic cultures with a temperature-sensitive variant of the Prague strain of Rous sarcoma virus (RSV, tsLA24) to obtain a high yield of transformed myogenic cells is reported. It incorporates the use of an amorphous polymer of polycations, Polybrene, to enhance the absorption of the virus by the muscle cells. In addition, other steps which were shown to be important were a) to allow cell attachment before infection, b) to infect at 35 degrees C in low protein medium, c) to use a density of 1 to 1.5 X 10(6) cells/60-mm dish, d) gentle agitation during infection, and e) to minimize the number of passages after infection. The use of the temperature-sensitive virus provided a means of confirming the presence of myogenic cells in transformed cultures. When infected cells were maintained at 35 degrees C (the permissive temperature for virus activity) they exhibited the characteristics of transformed cells. These characteristics included altered cell morphology, the absence of contact-inhibited growth, growth in semisolid medium, and expression of the src oncogene. In contrast, when infected cells were maintained at 41 degrees C (the nonpermissive temperature for virus activity) they do not express src and showed normal myogenic development and ultimately formed myotubes.
Assuntos
Vírus do Sarcoma Aviário , Transformação Celular Viral , Músculos/microbiologia , Animais , Divisão Celular , Embrião de Galinha , DNA Viral/análise , Regulação da Expressão Gênica , Brometo de Hexadimetrina , Músculos/citologia , Mutação , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , TemperaturaRESUMO
Active sequences from the laminin alpha1 and alpha2 chain carboxyl-terminal globular domains (G domain) have been identified by screening overlapping synthetic peptides in a number of biological assays (Nomizu et al. [1995] J. Biol. Chem. 270:20583-20590; Nomizu et al. [1996] FEBS Lett. 396:37-42). We have tested the activity of these peptides in submandibular gland explants of embryonic day 13 mice to determine the functional sites involved in organ development. The laminin alpha1 chain peptide, RKRLQVQLSIRT (residues 2719-2730 and designated AG-73), significantly inhibited epithelial branching morphogenesis. In contrast, other cell adhesive laminin alpha1 chain peptides including the AASIKVAVSADR and NRWHSIYITRFG failed to inhibit the branching. MG-73, a homologue of AG-73 from the laminin alpha2 chain, did not inhibit the branching. The alpha2 chain peptide had no effect, which may be due to the low levels of this laminin chain in day 13 mice. Laminin alpha2 chain-specific monoclonal antibodies strongly reacted with the basement membranes of developed acini but only weakly stained embryonic day 13 submandibular epithelium. The expression of E-cadherin and alpha6 integrin, as detected by immunofluorescence, were unchanged in both AG-73 and control scramble peptide-treated epithelial cells of the explants. In contrast, immunostaining of nidogen/entactin showed that explants treated with AG-73 for 3 days had a discontinuous basement membrane. Explants treated for 3 days with control peptide showed a normal basement membrane. These results suggest that the region containing the AG-73 sequence of the laminin alpha1 chain is crucial for development of submandibular gland at early embryonic stages. The discontinuous basement membrane in AG-73-treated explants may indicate an important role for this region in basement membrane assembly.
Assuntos
Laminina/metabolismo , Glândula Submandibular/embriologia , Glândula Submandibular/metabolismo , Animais , Membrana Basal , Técnicas de Cultura , Epitélio/metabolismo , Laminina/síntese química , Laminina/farmacologia , Camundongos , Morfogênese , Peptídeos/síntese química , Peptídeos/farmacologia , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/ultraestruturaRESUMO
Regulated adhesion of leukocytes to the extracellular matrix is essential for transmigration of blood vessels and subsequent migration into the stroma of inflamed tissues. Although beta(2)-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta(1)- and beta(3)-integrins but not beta(2)-integrin are essential for the adhesion to and migration on extracellular matrix molecules of the endothelial cell basement membrane and subjacent interstitial matrix. Mouse wild type and beta(2)-integrin null PMN and the progranulocytic cell line 32DC13 were employed in in vitro adhesion and migration assays using extracellular matrix molecules expressed at sites of extravasation in vivo, in particular the endothelial cell laminins 8 and 10. Wild type and beta(2)-integrin null PMN showed the same pattern of ECM binding, indicating that beta(2)-integrins do not mediate specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins alpha(5)beta(1) and alpha(v)beta(3), respectively; to laminin 10 via alpha(6)beta(1); but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin alpha(6)beta(1), a major laminin receptor, demonstrating that expression of alpha(6)beta(1) alone is insufficient for ligand binding and suggesting the involvement of accessory factors. Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating that differential cellular signaling via laminins is independent of the extent of adhesion. The data demonstrate that adhesive and nonadhesive interactions with components of the endothelial cell basement membrane and subjacent interstitium play decisive roles in controlling PMN movement into sites of inflammation and illustrate that beta(2)-integrins are not essential for such interactions.
Assuntos
Antígenos CD18/biossíntese , Matriz Extracelular/metabolismo , Granulócitos/metabolismo , Neutrófilos/metabolismo , Animais , Membrana Basal/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endotélio/metabolismo , Fibronectinas/metabolismo , Citometria de Fluxo , Humanos , Integrina alfa6beta1 , Integrinas/metabolismo , Laminina/metabolismo , Ligantes , Camundongos , Oligopeptídeos/metabolismo , Ligação Proteica , Receptores de Fibronectina/metabolismo , Receptores de Vitronectina/metabolismo , Coloração pela Prata , Células Tumorais Cultivadas , Vitronectina/metabolismoRESUMO
We have previously shown that mouse and bovine endothelial cells express a novel 400-kDa laminin alpha chain complexed to beta1 and gamma1 laminin chains. We describe here purification of this laminin isoform from the conditioned medium of a mouse peripheral lymph node endothelial cell line, SVEC. The laminin alpha chain was isolated from the laminin complex, subjected to Edman digestion, and the amino acid sequences of the resulting peptides were determined. Amino acid sequence revealed 100% identity to the predicted amino acid sequence of the recently reported laminin alpha5 gene. A monoclonal antibody to the laminin alpha5 chain was raised (4G6), allowing investigation of its distribution in embryonic, newborn, and mature mouse tissues. The laminin alpha5 chain was expressed mainly by epithelial, endothelial, and myogenic cells: In both embryonic and mature tissues the laminin alpha5 chain was strongly expressed by epithelial cells, the bronchi of the lungs and the developing kidney tubules being the sites of strongest expression. However, laminin alpha5 was not associated with early stages of epithelial cell development, but rather with epithelial cell maturation. Widespread expression of laminin alpha5 in endothelial cells was apparent only in tissues of mature mice, its appearance correlating approximately with sexual maturity. During embryogenesis and in newborn tissues, laminin alpha5 occurred in basement membranes of larger blood vessels only, excluding a role in angiogenic processes. Smooth muscle and skeletal muscle cells were the only other cell types which showed considerable laminin alpha5 expression, with skeletal muscle exhibiting a developmentally regulated pattern of expression: The laminin alpha5 chain occurred in skeletal muscle fiber basement membranes early in embryogenesis (E13-E15) but decreased with development, remaining strongly expressed only at the neuromuscular junction. The data show that laminin alpha5 expression is associated with epithelial and endothelial cell maturation, implicating a role for this laminin chain in the maintenance of differentiated epithelial and endothelial cell phenotype.
Assuntos
Endotélio Vascular/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Laminina/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Bovinos , Linhagem Celular , Epitélio/embriologia , Rim/embriologia , Laminina/genética , Linfonodos/embriologia , Camundongos , Peso Molecular , Músculos/embriologia , RNA Mensageiro/metabolismo , CoelhosRESUMO
Endothelial cells express a 400-kDa or 240-kDa laminin alpha chain, depending on their tissue of origin or physiological state [1, 2]. Using differential display and subsequent screening of a mouse endothelial cell cDNA library we here identify the gene coding for the 240-kDa laminin chain as the laminin alpha 4 gene. The complete mouse laminin alpha 4 cDNA sequence is reported and compared with other laminin alpha chains. In situ hybridization of embryonic and new born mouse tissues revealed expression of laminin alpha 4 mRNA in a subset of endothelium, in particular aortic endothelium, endocardium and endothelium of blood vessels in the skin and in the brain. Strong laminin alpha 4 expression by aortic endothelia was confirmed by data obtained from cultured bovine aortic endothelial cells (BAEC). Isolation of laminin from BAEC conditioned medium revealed a Y-shaped molecule in rotary shadowing. Subsequent sequencing of BAEC laminin resulted in laminin alpha 4, beta 1 and gamma 1 amino acid sequences, confirming that laminin alpha 4 is one of the major laminin alpha chains expressed by aortic endothelium not only in the mouse. In addition, strong laminin alpha 4 mRNA expression occurred in peripheral nerves, cardiac muscle, fat, the dermis of the skin and lung stroma of mouse tissues. The data demonstrate a cytokine and progesterone-regulated differential expression of laminin alpha 4 mRNA in mouse endothelium, suggesting a distinct functional role for this laminin chain in endothelium.
Assuntos
Endotélio Vascular/metabolismo , Laminina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , Clonagem Molecular , Citocinas/farmacologia , DNA Complementar/metabolismo , Expressão Gênica/efeitos dos fármacos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Progesterona/farmacologia , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: The alpha2 chain of laminin-2 (merosin), encoded by a gene on chromosome 6q22, is deficient in about half the cases of congenital muscular dystrophy. Diagnosis of this condition has relied on immunocytochemical analysis of the alpha2 chain in muscle biopsy specimens. We have observed that normal skin also expresses laminin alpha2 in the basement membrane at the junction of the dermis and epidermis. Here we have investigated laminin alpha2 deficiency in skin biopsy specimens from two patients with congenital muscular dystrophy. PARTICIPANTS: Two patients with severe congenital muscular dystrophy gave informed consent to a skin biopsy. The girl was aged 10 and the boy was aged 7. The specimens were labelled with a commercially available mouse monoclonal antibody and a rat monoclonal antibody (4H8-2), which recognise an 80 and a 380 kDa fragment of the alpha2 chain, respectively. The antibodies were visualised by standard methods. A muscle biopsy specimen was available for each case, and was processed with the skin biopsy samples (from the girl a few months previously, from the boy at age 14 days). Skin biopsies were done on four controls with normal expression of laminin alpha2 on their skeletal muscle fibres. FINDINGS We did not detect laminin alpha2 in skin specimens from either case, although the controls were positive. The muscle biopsy specimens from the girl showed a few fibres, with traces of laminin alpha2; those from the boy showed no laminin alpha2. INTERPRETATION: Skin biopsy specimens will provide a useful alternative to muscle biopsy samples for the assessment of laminin-2 (merosin) status in congenital muscular dystrophy.
Assuntos
Laminina/deficiência , Distrofias Musculares/congênito , Pele/patologia , Biópsia , Criança , Feminino , Humanos , Laminina/análise , Masculino , Distrofias Musculares/diagnóstico , Distrofias Musculares/patologiaRESUMO
Laminin-2 (merosin) is a heterotrimer composed of alpha 2, beta 1 and gamma 1 chains. Approximately half of the cases with the classical form of congenital muscular dystrophy (CMD) have a deficiency of the laminin alpha 2 chain, encoded by the LAMA2 gene on chromosome 6q22. This disorder is often termed merosin-deficient CMD. Skeletal and cardiac muscle, and the peripheral and central nervous systems, all express laminin alpha 2 and can be affected in merosin-deficient CMD. Normal skin also expresses all three chains of laminin-2 at the epidermal/dermal junction, around hair follicles and in the sensory nerves. Skin biopsies can therefore be used to assess merosin status in patients. We show here an absence of laminin alpha 2 in skin from four cases of CMD with a severe phenotype and abnormal magnetic resonance image (MRI) of the brain, in contrast to normal expression in one case of mild CMD with normal MRI, and in five controls. An additional case of CMD had a partial deficiency of laminin alpha 2 in the skin and severe motor disability, but a normal MRI. Sensory nerves in this case showed normal expression of laminin alpha 2, in contrast to its absence in the severe cases. The expression of laminin beta 1 was also reduced in skin from cases of merosin-deficient CMD. In contrast to human fetal muscle, the laminin alpha 2 protein was not detected in fetal skin up to 23 weeks of gestation. The laminin beta 1 and gamma 1 chains, and the mRNA for laminin alpha 2, however, were present. Studies of mRNA of cultured skin cells suggest that fibroblasts are the major source of laminin alpha 2, not keratinocytes. Our data show that skin is useful for the assessment of merosin status in patients with CMD and that skin fibroblasts may be a useful source of tissue-specific RNA. In addition, we show that there is a tissue-specific difference in the developmental expression of the laminin alpha 2 protein.
Assuntos
Laminina/deficiência , Distrofias Musculares/congênito , Pele/química , Adolescente , Adulto , Biomarcadores , Biópsia , Estudos de Casos e Controles , Criança , Pré-Escolar , Cromossomos Humanos Par 6 , Técnicas de Cultura , Feminino , Feto , Ligação Genética , Haplótipos , Humanos , Lactente , Laminina/análise , Masculino , Músculo Esquelético/química , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , RNA/química , Índice de Gravidade de Doença , Pele/patologiaRESUMO
The expression of laminin isoforms and laminin-binding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin alpha2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.