RESUMO
A European case of laminin alpha2 deficiency-associated muscular dystrophy in a 12-month-old, female Maine coon pedigree cat is reported. The history and eventual clinical presentation of this cat differed from those of two cats reported in the USA. In this case, the myopathy was characterised by progressively worsening weakness, muscle atrophy and joint contracture. Tendon reflexes were diminished, and motor nerve conduction velocities were slowed. Muscle biopsy demonstrated a dystrophic phenotype with endomysial fibrosis. Occasional thinly myelinated nerve fibres were present within a peripheral nerve specimen. Poorly myelinated fibres were also found at the root level on necropsy specimens. Immunohistochemical staining revealed the absence of laminin alpha2. The cat's family history did not indicate genetic transmission of the disease.
Assuntos
Doenças do Gato/diagnóstico , Laminina/deficiência , Distrofia Muscular Animal/diagnóstico , Animais , Biópsia , Doenças do Gato/genética , Doenças do Gato/patologia , Gatos , Diagnóstico Diferencial , Feminino , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/veterinária , Distrofia Muscular Animal/complicações , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , LinhagemRESUMO
The sarcoglycanopathies are a group of four autosomal recessive limb girdle muscular dystrophies (LGMD 2D, 2E, 2C, and 2F), caused by mutations of the alpha-, beta-, gamma-, or delta-sarcoglycan genes, respectively. The delta-sarcoglycan-deficient hamster has been the most utilized model for gene delivery to muscle by recombinant adeno-associated virus (AAV) vectors; however, human patients with delta-sarcoglycan deficiency are exceedingly rare, with only two patients described in the United States. Here, we report construction and use of AAV vectors expressing either alpha- or beta-sarcoglycan, the genes responsible for the most common forms of the human sarcoglycanopathies. Both vectors showed successful short-term genetic, biochemical, and histological rescue of both alpha- and beta-sarcoglycan-deficient mouse muscle. However, comparison of persistence of expression in 51 injected mice showed substantial differences between AAV alpha-sarcoglycan (alpha-SG) and beta-sarcoglycan (beta-SG) vectors. AAV-beta-SG showed long-term expression with no decrease in expression for more than 21 months after injection, whereas AAV-alpha-SG showed a dramatic loss of positive fibers between 28 and 41 days post-injection (p = 0.006). Loss of immunopositive myofibers was correlated with significant inflammatory cell infiltrate, primarily macrophages. To determine whether the loss of alpha-sarcoglycan-positive fibers was due to an immune response or cytotoxic effect of alpha-sarcoglycan overexpression, severe combined immunodeficient (SCID) mouse muscle was assayed for cytotoxicity after injection with AAV-alpha-SG, AAV-beta-SG, or phosphate-buffered saline. The results were consistent with overexpression of alpha-sarcoglycan causing significant cytotoxicity. The cytotoxicity of alpha-sarcoglycan, and not beta- or delta-sarcoglycan overexpression, was consistent with biochemical studies of the hierarchical order of assembly of the sarcoglycan complex. Our data suggest that even closely related proteins might require different levels of expression to avoid toxicity and achieve long-term tissue rescue.
Assuntos
Proteínas do Citoesqueleto/genética , Dependovirus , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Glicoproteínas de Membrana/genética , Distrofias Musculares/terapia , Animais , Proteínas do Citoesqueleto/uso terapêutico , Distroglicanas , Glicoproteínas de Membrana/uso terapêutico , Camundongos , Camundongos Knockout , Camundongos SCID , Fibras Musculares Esqueléticas/citologia , Distrofias Musculares/genética , SarcoglicanasRESUMO
Cell growth and differentiation are controlled in many tissues by paracrine factors, which often require proteolytic processing for activation. Metalloproteases of the metzincin family, such as matrix metalloproteases and ADAMs, recently have been shown to be involved in the shedding of growth factors, cytokines, and receptors. In the present study, we show that hydroxamate-based inhibitors of metalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusion of C(2)C(12) myoblasts and provoke myotube hypertrophy. HIMPs did not seem to effect hypertrophy via proteins that have previously been shown to regulate muscle growth in vitro, such as insulin-like growth factor-I, calcineurin, and tumor necrosis factor-alpha. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced in C(2)C(12) cells treated with HIMPs, as suggested by the presence of nonprocessed myostatin precursor only in hypertrophic myotubes. Myostatin is a known negative regulator of skeletal muscle growth, belonging to the transforming growth factor-beta/bone morphogenetic protein superfamily. These results indicate that metalloproteases are involved in the regulation of skeletal muscle growth and differentiation, that the proteolytic maturation of myostatin in C(2)C(12) cells may be directly or indirectly linked to the activity of some unidentified HIMP-sensitive metalloproteases, and that the lack of myostatin processing on HIMP treatment may be a mediator of myotube hypertrophy in this in vitro model.
Assuntos
Metaloendopeptidases/antagonistas & inibidores , Músculo Esquelético/citologia , Inibidores de Proteases/farmacologia , Fator de Crescimento Transformador beta/fisiologia , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Clonais , Humanos , Ácidos Hidroxâmicos/farmacologia , Indicadores e Reagentes , Fator de Crescimento Insulin-Like I/metabolismo , Ligantes , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/ultraestrutura , MiostatinaRESUMO
The most common form of muscular dystrophy in dogs and humans is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome, and, therefore, disease-causing mutations in dystrophin occur most often in males. Therefore, females with dystrophin deficiency or other forms of muscular dystrophy may be undiagnosed or misdiagnosed. Immunohistochemistry was used to analyze dystrophin and a number of other muscle proteins associated with muscular dystrophy in humans, including sarcoglycans and laminin alpha2, in muscle biopsy specimens from 5 female dogs with pathologic changes consistent with muscular dystrophy. The female dogs were presented with a variety of clinical signs including generalized weakness, muscle wasting, tremors, exercise intolerance, gait abnormalities, and limb deformity. Serum creatine kinase activity was variably high. One dog had no detectable dystrophin in the muscle; another was mosaic, with some fibers normal and others partly dystrophin-deficient. A 3rd dog had normal dystrophin but no detectable laminin alpha2. Two dogs could not be classified. This study demonstrates the occurrence of dystrophin- and laminin alpha2-associated muscular dystrophy and the difficulty in clinical diagnosis of these disorders in female dogs.
Assuntos
Doenças do Cão/diagnóstico , Distrofina/análise , Laminina/análise , Distrofia Muscular Animal/diagnóstico , Animais , Cruzamento , Creatina Quinase/sangue , Doenças do Cão/genética , Doenças do Cão/patologia , Cães , Distrofina/deficiência , Feminino , Imuno-Histoquímica , Laminina/deficiência , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologiaRESUMO
Laminins, a family of heterotrimeric proteins with cell adhesive/signaling properties, are characteristic components of basement membranes of vasculature and tissues. In the present study, permeabilized platelets were found to react with a monoclonal antibody to laminin gamma1 chain by immunofluorescence. In Western blot analysis of platelet lysates, several monoclonal antibodies to gamma1 and beta1 laminin chains recognized 220- to 230-kDa polypeptides, under reducing conditions, and a structure with much slower electrophoretic mobility under nonreducing conditions. Immunoaffinity purification on a laminin beta1 antibody-Sepharose column yielded polypeptides of 230, 220, 200, and 180 kDa from platelet lysates. In the purified material, mAbs to beta1 and gamma1 reacted with the two larger polypeptides, while affinity-purified rabbit antibodies to laminin alpha4 chain recognized the smallest polypeptide. Identity of the polypeptides was confirmed by microsequencing. One million platelets contained on average 1 ng of laminin (approximately 700 molecules per cell), of which 20-35% was secreted within minutes after stimulation with either thrombin or phorbol ester. Platelets adhered to plastic surfaces coated with the purified platelet laminin, and this process was largely inhibited by antibodies to beta1 and alpha6 integrin chains. We conclude that platelets contain and, following activation, secrete laminin-8 (alpha4beta1gamma1) and that the cells adhere to the protein by using alpha6beta1 integrin.
Assuntos
Plaquetas/química , Plaquetas/metabolismo , Integrinas/metabolismo , Laminina/metabolismo , Adesividade Plaquetária/fisiologia , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Western Blotting , Citometria de Fluxo , Humanos , Integrina alfa6beta1 , Integrinas/imunologia , Laminina/análise , Dados de Sequência MolecularRESUMO
Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Lectinas Tipo C , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Animais , Bupivacaína/metabolismo , Linhagem Celular , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento/genética , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Camundongos Endogâmicos mdx , Polirribossomos/metabolismo , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , RegeneraçãoRESUMO
The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.
Assuntos
Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Proteína ADAM12 , Sequência de Aminoácidos , Animais , Células COS , Cisteína/química , Desintegrinas/química , Desintegrinas/genética , Humanos , Hidrólise , Proteínas de Membrana/química , Proteínas de Membrana/genética , Metaloendopeptidases/química , Metaloendopeptidases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de AminoácidosRESUMO
The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.
Assuntos
Antígenos CD/genética , Cadeias alfa de Integrinas , Doenças Musculares/congênito , Doenças Musculares/genética , Mutação , Sequência de Bases , Criança , Pré-Escolar , Clonagem Molecular , DNA Complementar , Feminino , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/genéticaRESUMO
The ADAM (A Disintegrin And Metalloprotease) family of cell-surface proteins may have an important role in cellular interactions and in modulating cellular responses. In this report we describe a novel, secreted form of human ADAM 12 (meltrin alpha), designated ADAM 12-S (S for short), and a larger, membrane-bound form designated ADAM 12-L (L for long form). These two forms arise by alternative splicing of a single gene located on chromosome 10q26. Northern blotting demonstrated that mRNAs of both forms are abundant in human term placenta and are also present in some tumor cell lines. The ADAM 12-L transcript can also be detected in normal human adult skeletal, cardiac, and smooth muscle. Human A204 embryonal rhabdomyosarcoma cells that do not differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these transfected cells contained ectopic muscle cells of apparent mouse origin as shown by species-specific markers. These results may have potential applications in the development of muscle-directed gene and cell therapies.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Proteínas ADAM , Proteína ADAM12 , Adulto , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 10 , Clonagem Molecular , DNA Complementar , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas Musculares/genética , Fases de Leitura Aberta , RNA Mensageiro/genéticaRESUMO
Adhalin, or alpha-sarcoglycan, is a 50-kDa glycoprotein that was originally characterized as a muscle membrane protein. The importance of adhalin is suggested by the diseases associated with its absence, notably the limb-girdle muscular dystrophies. However, the function of adhalin is unknown. To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation in vitro. The proper localization of adhalin to the muscle cell membrane was observed only in late stages of myotube maturation, coincident with the re-distribution of caveolin-3 and dystrophin. These data suggest that adhalin is highly specific for striated muscle and that it is linked with the formation of a fully functional muscle fiber.
Assuntos
Caveolinas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Músculo Esquelético/química , Músculo Esquelético/citologia , Sequência de Aminoácidos , Animais , Caveolina 3 , Diferenciação Celular , Clonagem Molecular , DNA Complementar/química , Imunofluorescência , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Distrofias Musculares/genética , RNA Mensageiro/análise , Sarcoglicanas , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Regulação para CimaRESUMO
A combination of congenital central nervous, ocular and muscular abnormalities is characteristic of muscle-eye-brain disease (MEB), of Fukuyama congenital muscular dystrophy (FCMD), and of Walker-Warburg syndrome (WWS). The nosological relationship of these inherited malformative disorders is still unestablished, although the genetic locus for FCMD has been excluded in MEB. We present the first postmortem neuropathological study of MEB based on 2 male patients. Apart from sharply limited occipital agyric areas, their brains showed coarse gyri with an abnormally nodular surface ("cobblestone cortex"). Both the cerebral and cerebellar cortices showed a total disorganization without horizontal lamination. The haphazardly oriented cortical neurons formed irregular clusters or islands, separated by gliovascular strands extending from the pia. The ocular abnormalities included a pronounced glial preretinal membrane. Although MEB shares the cobblestone cortex-type malformation with FCMD and WWS, the cerebral and ocular manifestations are less severe than in WWS. Furthermore, a consistently weak staining for laminin alpha2 chain (merosin) was found in muscle biopsy specimens from 4 MEB patients, while normal immunoreactivity was observed for the laminin beta2 chain, reported to be severely deficient in WWS. These findings support nosological independence of MEB.
Assuntos
Encefalopatias/patologia , Oftalmopatias/patologia , Atrofia Muscular/patologia , Adulto , Criança , Feminino , Humanos , Imuno-Histoquímica , MasculinoRESUMO
Laminin (laminin-1; alpha 1-beta 1-gamma 1) is known to promote myoblast proliferation, fusion, and myotube formation. Merosin (laminin-2 and -4; alpha 2-beta 1/beta 2-gamma 1) is the predominant laminin variant in skeletal muscle basement membranes; genetic defects affecting its structure or expression are the causes of some types of congenital muscular dystrophy. However, the precise nature of the functions of merosin in muscle remain unknown. We have developed an in vitro system that exploits human RD and mouse C2C12 myoblastic cell lines and their clonal variants to study the roles of merosin and laminin in myogenesis. In the parental cells, which fuse efficiently to multinucleated myotubes, merosin expression is upregulated as a function of differentiation while laminin expression is downregulated. Cells from fusion-deficient clones do not express either protein, but laminin or merosin added to the culture medium induced their fusion. Clonal variants which fuse, but form unstable myotubes, express laminin but not merosin. Exogenous merosin converted these myotubes to a stable phenotype, while laminin had no effect. Myotube instability was corrected most efficiently by transfection of the merosin-deficient cells with the merosin alpha 2 chain cDNA. Finally, merosin appears to promote myotube stability by preventing apoptosis. Hence, these studies identify novel biological functions for merosin in myoblast fusion and muscle cell survival; furthermore, these explain some of the pathogenic events observed in congenital muscular dystrophy caused by merosin deficiency and provide in vitro models to further investigate the molecular mechanisms of this disease.
Assuntos
Laminina/análise , Fibras Musculares Esqueléticas/química , Animais , Apoptose/fisiologia , Sequência de Bases , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , DNA Complementar/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Laminina/deficiência , Laminina/genética , Camundongos , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/fisiologia , Distrofia Muscular Animal/congênito , Distrofia Muscular Animal/metabolismo , Rabdomiossarcoma , Transfecção , Células Tumorais Cultivadas/químicaRESUMO
Because recent information suggests that the localized deposition of protease inhibitors is one mechanism by which cells regulate pericellular proteolysis during tissue invasion, the distribution of type 1 plasminogen activator inhibitor (PA1-1) associated with the invasive human glioma cell line U-251 was investigated. Direct and reverse fibrin zymography indicated the presence of urokinase-like plasminogen activator (u-PA) and PAI-1 in U-251 conditioned media and cell lysates. PA1-1 antigen was detected immunologically in cytoplasmic granules present within cellular processes of U-251 cells and these organelles could be isolated on Percoll density gradients in a high density band. In contrast, u-PA activity and another secreted protein, amyloid beta-protein precursor, were only present in the low density region of the gradients. Functional analysis of PAI-1 in the granules contained within the high density fractions revealed the presence of active PAI-1. Incubation of U-251 cells with the secretagogue, 8-bromoadenosine 3':5'-cyclic monophosphate, resulted in a 3-fold increase in the release of PAI-1 in the media conditioned by these cells. These data suggest that the human glioma cell line U-251 contains PAI-1 in a rapidly releasable form, which may provide another mechanism by which these tumors could regulate proteolytic activity in a localized manner.
Assuntos
Fibrinólise , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Fracionamento Celular , Linhagem Celular , Centrifugação com Gradiente de Concentração , Fibrina , Glioma , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Organelas/efeitos dos fármacos , Organelas/metabolismo , Organelas/ultraestrutura , Inibidor 1 de Ativador de Plasminogênio/isolamento & purificação , Povidona , Dióxido de Silício , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/isolamento & purificaçãoRESUMO
Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin beta 2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.
Assuntos
Anormalidades Múltiplas/metabolismo , Proteínas do Citoesqueleto/análise , Anormalidades do Olho/metabolismo , Laminina/análise , Glicoproteínas de Membrana/análise , Músculos/metabolismo , Distrofias Musculares/metabolismo , Membrana Basal/metabolismo , Pré-Escolar , Humanos , Imuno-Histoquímica , Masculino , Sarcoglicanas , SíndromeRESUMO
The classic murine muscular dystrophy strain, dy, was first described almost 40 years ago. We have identified the molecular basis of an allele of dy, called dy2J, by detecting a mutation in the laminin alpha 2 chain gene--the first identified mutation in laminin-2. The G to A mutation in a splice site consensus sequence causes abnormal splicing and expression of multiple mRNAs. One mRNA is translated into an alpha 2 polypeptide with a deletion in domain VI. The truncated protein apparently lacks important qualities of the wild type protein and is unable to provide sufficient muscle stability.
Assuntos
Laminina/genética , Camundongos Mutantes/genética , Proteínas Musculares/genética , Distrofia Muscular Animal/genética , Proteínas do Tecido Nervoso/genética , Mutação Puntual , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Basal/metabolismo , Membrana Basal/patologia , Adesão Celular , DNA Complementar/genética , Feminino , Glicosilação , Laminina/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Músculos/patologia , Distrofia Muscular Animal/patologia , Processamento de Proteína Pós-Traducional , Splicing de RNA , RNA Mensageiro/biossíntese , Deleção de SequênciaRESUMO
BACKGROUND: The laminins are heterotrimeric basement membrane glycoproteins. Eight subunits that can be assembled into laminins have been characterized and are known as: A, B1, B2, S, M, K, B2t, B1k laminin chains. Although many neoplastic cells secrete laminins and some of them even assemble basement membranes, the pattern of production of various laminin subunits remains to be explored. EXPERIMENTAL DESIGN: The expression of laminin was examined in several human carcinoma cells using a panel of specific cDNA probes as well as polyclonal and chain specific monoclonal antibodies. For this purpose a human laminin S chain 2 kb cDNA was isolated and characterized and used together with existing probes for laminin chains. RESULTS: All carcinoma cell lines had a high level of expression of three light chains (B1, S and B2) mRNA. In contrast, the heavy chains of laminin, A and M, were expressed in negligible amounts as detected by Northern blotting and PCR. The only exception was the HU-1 lung adenocarcinoma cell line which expressed significant quantities of laminin M chain mRNA and lower levels of laminin A chain mRNA. The presence in the HU-1 cells of translated polypeptides was demonstrated by immunofluorescence staining. The cells contained both B1 and S chain laminin in the cell layer, but preferentially secreted the B1 chain into the culture supernatant as shown by Western blotting. The 300 to 400 kDa M chain immunoreactive band was found in laminin secreted into the culture medium of HU-1 cells. Immunoprecipitation of biosynthetically labeled proteins showed that the M chain was synthesized as a complex with B chains. Little or no A chain laminin was detected in the culture medium supernatant. HU-1 cells also synthesized the newly described laminin variant, epiligrin which was secreted into the medium. Thus, the HU-1 cells secreted two laminin variants: M-B1-B12 laminin and epiligrin into the culture medium. Immunostaining of HU-1 nude mice tumors showed that tumor basement membranes contained M, B1, and B2 laminin and epiligrin immunoreactivity but apparently no S chain. CONCLUSIONS: All human carcinoma cell lines produced laminin chains B1, B2 and S, but no or little A or M. The only exception was the lung carcinoma cell line HU-1. Human HU-1 carcinoma cells in culture synthesize several homologous laminin chains and regulate the process of assembly, secretion and deposition of laminin variants into tumor basement membranes. These data indicate that the tumor cells vary among themselves with regards to laminin production and that some of them, like HU-1 may produce essentially all laminin chains simultaneously.
Assuntos
Carcinoma/genética , Laminina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Sondas de DNA , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunológicas , Técnicas In Vitro , Queratinócitos/metabolismo , Masculino , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Transplante de Neoplasias , Peptídeos/química , RNA Mensageiro/genética , Transplante Heterólogo , Células Tumorais Cultivadas , CalininaRESUMO
The intestinal tissue is characterized by important morphogenetic movements during development as well as by a continuous dynamic crypt to villus epithelial cell migration leading to differentiation of specialized cells. In this study, we have examined the spatio-temporal distribution of laminin A and M chains as well as of alpha 6 and beta 4 integrin subunits in adult and developing human and mouse intestine by indirect immunofluorescence. Selective expression of the constituent polypeptides of laminin isoforms (A and M chains) was demonstrated. In the mature human intestine, A and M chains were found to be complementary, the M chain being restricted to the base of crypts and the A chain lining the villus basement membrane. In the developing human intestine, M chain expression was delayed as compared to that of A chain; as soon as the M chain was visualized, it exhibited the typical localization in the crypt basement membrane. A somewhat different situation was found in the adult mouse intestine, since both M and A chains were found in the crypts. During mouse intestinal development the delayed expression of the M chain as compared to that of the A chain was also obvious. The absence of M chain expression in mutant dy mouse did not impair intestinal morphogenesis nor cell differentiation. The expression of alpha 6 and beta 4 subunits was not coordinated. In both species the alpha 6 expression preceded that of beta 4. Furthermore, while beta 4 staining in adult mouse intestine was detected at the basal surface of all cells lining the crypt-villus, that of alpha 6 was mainly confined to the crypt cell compartment. An overall similarity of location between alpha 6 integrin subunit and laminin A chain at the epithelial/stromal interface was noted. These data indicate that the spatial and temporal distribution of laminin variants in the developing intestine may be characteristic for each species and that interactions of laminin variants with particular receptors may be important for induction and/or maintenance of differentiated cells.
Assuntos
Integrinas/análise , Intestinos/química , Laminina/análise , Adulto , Animais , Animais Lactentes , Membrana Basal/química , Membrana Basal/metabolismo , Criança , Desenvolvimento Embrionário e Fetal , Imunofluorescência , Humanos , Integrina alfa6 , Integrina beta4 , Integrinas/biossíntese , Mucosa Intestinal/metabolismo , Intestinos/embriologia , Laminina/biossíntese , Masculino , Camundongos , MorfogêneseAssuntos
Laminina , Animais , Membrana Basal/química , Adesão Celular , Células Cultivadas , Matriz Extracelular/química , Feminino , Humanos , Laminina/análise , Laminina/química , Laminina/isolamento & purificação , Laminina/metabolismo , Neoplasias/química , Neoplasias/ultraestrutura , Neuritos/metabolismo , Neurônios/citologia , Placenta/química , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
An assay for the determination of relative concentrations of integrins on clonal cultured cells is described. Unpurified monoclonal antibodies to integrin subunits, present in ascites or hybridoma culture media, are immobilized on a plastic surface via goat antibodies to mouse IgG. Cell attachment to the integrin-coated substrate is quantitated after fixation and staining of the cells. The assay is specific and very sensitive; only cells with the relevant integrins attach to antibody-coated substrates and concentrations of antibody as low as 1-10 ng/ml are sufficient. Titrations of the antibodies on the solid phase allow the estimation of the relative amounts of integrins on different cell types. The results with this assay correlate well with results obtained by flow cytometry.
Assuntos
Anticorpos Monoclonais/metabolismo , Adesão Celular , Integrinas/análise , Linhagem Celular , Coriocarcinoma , Neoplasias do Colo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Citometria de Fluxo/métodos , Glioma , Humanos , Integrinas/imunologia , Integrinas/fisiologia , Leiomiossarcoma , Gravidez , Rabdomiossarcoma , Espectrometria de Fluorescência/métodos , Células Tumorais Cultivadas , Neoplasias UterinasRESUMO
BACKGROUND: Laminins are a family of multifunctional glycoproteins that play a role in various aspects of cell biology. Three different isoforms of laminin have been described, and each comprises a molecule consisting of three subunit polypeptides, the A, B1, B2, M or S chain. EXPERIMENTAL DESIGN: The expression pattern of laminin isoforms was studied by indirect immunofluorescence staining of human peripheral nerve in situ or cell cultures derived from such nerve by using monoclonal antibodies recognizing the subunit epitopes. RESULTS: Selective expression of the subunit polypeptides of laminin isoforms in endoneurium and perineurium was demonstrated. Specifically, an intense immunoreaction for A, B2 and S chain epitopes could be detected in perineurium, whereas endoneurium revealed the presence of B1, B2, M and S chains. Examination of the laminin isoform expression in perineurial cells, Schwann cells, and fibroblasts in cultures derived from normal human nerve indicated, however, that these cells under in vitro conditions were capable of expressing all five laminin chains. Cutaneous neurofibromas, tumors characterized by the presence of mixed cell populations consisting of Schwann cells, perineurial cells, and fibroblasts, demonstrated the expression of B1, B2 and M chain epitopes, whereas only a weak immunostaining could be detected with antibodies recognizing the A and S chains. Similar observations were made on schwannomas, a Schwann cell tumor. CONCLUSIONS: Collectively, the observations of this study attest to the plasticity of neural-derived connective tissue cells with respect to laminin isoform expression. Such plasticity may relate to the cell-cell and cell-matrix interactions during development of peripheral nerves and the potential for neural regeneration.