RESUMO
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
Affinity chromatography and immunolocalization techniques were used to investigate the mechanism(s) by which endothelial cells interact with the basement membrane component laminin. Bovine aortic endothelial cells (BAEC) membranes were solubilized and incubated with a laminin-Sepharose affinity column. SDS-PAGE analysis of the eluted proteins identified a 69-kD band as the major binding protein, along with minor components migrating at 125, 110, 92, 85, 75, 55, and 30 kD. Polyclonal antibodies directed against a peptide sequence of the 69-kD laminin-binding protein isolated from human tumor cells identified this protein in BAEC lysates. In frozen sections, these polyclonal antibodies and monoclonal antibodies raised against human tumor 69-kD stained the endothelium of bovine aorta and the medial smooth muscle cells, but not surrounding connective tissue or elastin fibers. When nonpermeabilized BAEC were stained in an in vitro migration assay, there appeared to be apical patches of 69 kD staining in stationary cells. However, when released from contact inhibition, 69 kD was localized to ruffling membranes on cells at the migrating front. Permeabilized BAEC stained for 69 kD diffusely, with a granular perinuclear distribution and in linear arrays throughout the cell. During migration a redistribution from diffuse to predominanately linear arrays that co-distributed with actin microfilaments was noted in double-label experiments. The 69-kD laminin-binding protein colocalized with actin filaments in permeabilized cultured microvascular endothelial cells in a continuous staining pattern at 6 h postplating which redistributed to punctate patches along the length of the filaments at confluence (96 h). In addition, 69 kD co-distribution with laminin could also be demonstrated in cultured subconfluent cells actively synthesizing matrix. Endothelial cells express a 69-kD laminin-binding protein that is membrane associated and appears to colocalize with actin microfilaments. The topological distribution of 69 kD and its cytoskeletal associations can be modulated by the cell during cell migration and growth suggesting that 69 kD may be a candidate for a membrane protein involved in signal transduction from extracellular matrix to cell via cytoskeletal connections.
Assuntos
Endotélio Vascular/análise , Laminina/metabolismo , Receptores Imunológicos/análise , Citoesqueleto de Actina/análise , Citoesqueleto de Actina/metabolismo , Animais , Aorta , Bovinos , Adesão Celular , Movimento Celular , Células Cultivadas , Cromatografia de Afinidade , Endotélio Vascular/citologia , Matriz Extracelular/análise , Matriz Extracelular/metabolismo , Imunofluorescência , Humanos , Imunoensaio , Masculino , Microcirculação , Ratos , Ratos Endogâmicos , Receptores de LamininaRESUMO
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
Tetranectin is a protein shared by the blood and the extracellular matrix. Tetranectin is composed of four identical, noncovalently bound polypeptides each with a molecular mass of approximately 21 kD. There is some evidence that tetranectin may be involved in fibrinolysis and proteolysis during tissue remodeling, but its precise biological function is not known. Tetranectin is enriched in the cartilage of the shark, but the gene expression pattern in the mammalian skeletal system has not been determined. In the present study we have examined the expression pattern and putative function of tetranectin during osteogenesis. In the newborn mouse, strong tetranectin immunoreactivity was found in the newly formed woven bone around the cartilage anlage in the future bone marrow and along the periosteum forming the cortex. No tetranectin immunoreactivity was found in the proliferating and hypertrophic cartilage or in the surrounding skeletal muscle. Using an in vitro mineralizing system, we examined osteoblastic cells at different times during their growth and differentiation. Tetranectin mRNA appeared in the cultured osteoblastic cells in parallel with mineralization, in a pattern similar to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet cells contained approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p < 0.01), supporting the notion that tetranectin may play an important direct and/or indirect role during osteogenesis. In conclusion, we have established a potential role for tetranectin as a bone matrix protein expressed in time and space coincident with mineralization in vivo and in vitro.
Assuntos
Proteínas Sanguíneas/fisiologia , Calcificação Fisiológica/fisiologia , Lectinas Tipo C , Osteogênese/fisiologia , Animais , Sequência de Bases , Biomarcadores , Proteínas Sanguíneas/genética , Osso e Ossos/citologia , Bovinos , Diferenciação Celular , Células Cultivadas , Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Neoplasias Experimentais , Células PC12 , RNA Mensageiro/análise , Ratos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.
Assuntos
Integrina beta1/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Transdução de Sinais/fisiologia , Proteínas ADAM , Proteína ADAM12 , Actinas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Neoplasias da Mama , Adesão Celular/fisiologia , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Embrião de Galinha , Condrócitos/citologia , Condrócitos/metabolismo , Neoplasias do Colo , Cisteína , Citoesqueleto/fisiologia , Humanos , Integrina beta1/genética , Integrina beta1/imunologia , Magnésio/farmacologia , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Mesoderma/citologia , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos , Músculo Esquelético/citologia , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteossarcoma , Estrutura Terciária de Proteína , Proteoglicanas/genética , Receptor Cross-Talk/fisiologia , Rabdomiossarcoma , Transdução de Sinais/efeitos dos fármacos , Estresse Mecânico , Sindecanas , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/metabolismoRESUMO
Humans and mice with deficiency of the alpha2 subunit of the basement membrane protein laminin-2/merosin suffer from merosin-deficient congenital muscular dystrophy (MCMD). We have expressed a human laminin alpha2 chain transgene under the regulation of a muscle-specific creatine kinase promoter in mice with complete or partial deficiency of merosin. The transgene restores the synthesis and localization of merosin in skeletal muscle, and greatly improves muscle morphology and integrity and the health and longevity of the mice. However, the transgenic mice share with the nontransgenic dystrophic mice a progressive lameness of hind legs, suggestive of a nerve defect. These results indicate that the absence of merosin in tissues other than the muscle, such as nervous tissue, is a critical component of MCMD. Future gene therapies of human MCMD, and perhaps of other forms of muscular dystrophy, may require restoration of the defective gene product in multiple tissues.
Assuntos
Terapia Genética/métodos , Laminina/deficiência , Distrofia Muscular Animal/congênito , Distrofia Muscular Animal/terapia , Animais , Creatina Quinase/análise , Creatina Quinase/genética , Modelos Animais de Doenças , Expressão Gênica , Marcação de Genes , Membro Posterior/fisiopatologia , Humanos , Laminina/biossíntese , Laminina/genética , Longevidade , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , TransgenesRESUMO
Mutations in genes coding for dystrophin, for alpha, beta, gamma, and delta-sarcoglycans, or for the alpha2 chain of the basement membrane component merosin (laminin-2/4) cause various forms of muscular dystrophy. Analyses of integrins showed an abnormal expression and localization of alpha7beta1 isoforms in myofibers of merosin-deficient human patients and mice, but not in dystrophin-deficient or sarcoglycan-deficient humans and animals. It was shown previously that skeletal muscle fibers require merosin for survival and function (Vachon, P.H., F. Loechel, H. Xu, U.M. Wewer, and E. Engvall. 1996. J. Cell Biol. 134:1483-1497). Correction of merosin deficiency in vitro through cell transfection with the merosin alpha2 chain restored the normal localization of alpha7beta1D integrins as well as myotube survival. Overexpression of the apoptosis-suppressing molecule Bcl-2 also promoted the survival of merosin-deficient myotubes, but did not restore a normal expression of alpha7beta1D integrins. Blocking of beta1 integrins in normal myotubes induced apoptosis and severely reduced their survival. These findings (a) identify alpha7beta1D integrins as the de facto receptors for merosin in skeletal muscle; (b) indicate a merosin dependence for the accurate expression and membrane localization of alpha7beta1D integrins in myofibers; (c) provide a molecular basis for the critical role of merosin in myofiber survival; and (d) add new insights to the pathogenesis of neuromuscular disorders.
Assuntos
Integrinas/biossíntese , Laminina/deficiência , Músculo Esquelético/fisiologia , Distrofias Musculares/congênito , Animais , Diferenciação Celular , Sobrevivência Celular , Cricetinae , Proteínas do Citoesqueleto/biossíntese , Distrofina/deficiência , Distrofina/genética , Humanos , Imuno-Histoquímica , Integrina beta1/metabolismo , Laminina/genética , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Mutantes , Distrofia Muscular Animal/congênito , Receptores de Laminina/biossíntese , Sarcolema/metabolismo , Distribuição TecidualRESUMO
Insulin-like growth factor II (IGF-II) is a major fetal growth factor. The IGF-II gene generates multiple mRNAs with different 5' untranslated regions (5' UTRs) that are translated in a differential manner during development. We have identified a human family of three IGF-II mRNA-binding proteins (IMPs) that exhibit multiple attachments to the 5' UTR from the translationally regulated IGF-II leader 3 mRNA but are unable to bind to the 5' UTR from the constitutively translated IGF-II leader 4 mRNA. IMPs contain the unique combination of two RNA recognition motifs and four hnRNP K homology domains and are homologous to the Xenopus Vera and chicken zipcode-binding proteins. IMP localizes to subcytoplasmic domains in a growth-dependent and cell-specific manner and causes a dose-dependent translational repression of IGF-II leader 3 -luciferase mRNA. Mouse IMPs are produced in a burst at embryonic day 12.5 followed by a decline towards birth, and, similar to IGF-II, IMPs are especially expressed in developing epithelia, muscle, and placenta in both mouse and human embryos. The results imply that cytoplasmic 5' UTR-binding proteins control IGF-II biosynthesis during late mammalian development.
Assuntos
Fator de Crescimento Insulin-Like II/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Linhagem Celular , Galinhas , Clonagem Molecular , DNA Complementar/genética , Desenvolvimento Embrionário e Fetal/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator de Crescimento Insulin-Like II/biossíntese , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.
Assuntos
Proteínas Sanguíneas/fisiologia , Cifose/etiologia , Lectinas Tipo C , Lectinas/fisiologia , Animais , Proteínas Sanguíneas/genética , Densidade Óssea , Suscetibilidade a Doenças , Feminino , Deleção de Genes , Marcação de Genes/métodos , Cifose/genética , Cifose/patologia , Lectinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoporose/etiologia , Ovariectomia , Vértebras Torácicas/anormalidades , Vértebras Torácicas/patologiaRESUMO
Laminin, a glycoprotein antigenically similar or identical to a component of epithelial basement membranes, was identified as a major component of the abundant extracellular matrix synthesized by an experimentally induced rat yolk sac tumor. Immunocytochemical staining revealed laminin in cultured tumor cells as well as in their extracellular matrix. The presence of soluble laminin in the culture media of the tumor cells was demonstrated using metabolic labeling followed by identification by immunoprecipitation and sodium dodecyl sulfate:polyacrylamide gel electrophoresis. This revealed two polypeptides with molecular weights of approximately 200,000 and 400,000. These comigrated with the polypeptides of mouse laminin isolated previously. The yolk sac tumor tissue grown in vivo contained laminin in the tumor cells and in the extracellular material as evidenced by immunofluorescence and immunoperoxidase staining. Immunization with the tumor matrix resulted in an antiserum that contained antilaminin and natifibronectin and was made specific for laminin by absorption with fibronectin. This antiserum precipitated laminin polypeptides from culture medium of yolk sac tumour cells and stained basement membranes in rat tissues in a manner indistinguishable from antilaminin. The presence of laminin in rat yolk sac cells, the presumed origin of our yolk sac tumor, was studied in some detail. Laminin was found to be present in normal cells of the visceral as well as the parietal yolk sac layer and in their basement membranes suggesting, but not proving, that both types of cells have ability to synthesize laminin. Production of laminin and the presence of laminin-containing basement membrane material may be important for the biological behavior of the yolk sac tumor. This tumor will also be a useful source of laminin for chemical and biological characterization of this basement membrane protein.
Assuntos
Membrana Basal/metabolismo , Glicoproteínas/biossíntese , Mesonefroma/metabolismo , Proteínas de Neoplasias/biossíntese , Animais , Membrana Basal/imunologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Epitélio/metabolismo , Fibronectinas/biossíntese , Imunofluorescência , Glicoproteínas/imunologia , Laminina , Mesonefroma/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , RatosRESUMO
Sulfatides (galactosylceramide-I3-sulfate) but not neutral glycolipids or gangliosides adsorbed on plastic promote adhesion of the human melanoma cell line G361. Direct adhesion of G361 cells requires densities of sulfatide greater than 1 pmol/mm2. In the presence of laminin, however, specific adhesion of G361 cells to sulfatide or seminolipid (galactosylalkylacyl-glycerol-I3-sulfate) but not to other lipids is strongly stimulated and requires only 25 fmol/mm2 of adsorbed lipid. The effects of laminin and sulfatide on adhesion are synergistic, suggesting that laminin is mediating adhesion by cross-linking receptors on the melanoma cell surface to sulfatide adsorbed on the plastic. Although thrombospondin binds to sulfatides and G361 cells, it does not enhance, but rather inhibits direct and laminin-dependent G361 cell adhesion to sulfatide. In contrast, C32 melanoma cells also adhere specifically to sulfatide, but adhesion of these cells is not enhanced by laminin or inhibited by antibodies to laminin that block laminin-dependent adhesion of G361 cells. Thrombospondin is a potent inhibitor of C32 cell adhesion to sulfatide. Fucoidan, which inhibits laminin binding to sulfatide, inhibits laminin-dependent adhesion of G361 cells by 50% at 0.2 micrograms/ml. Several other tumor cell lines also attach directly on sulfatide-coated surfaces. Laminin stimulates adhesion to sulfatide of three of the six cell lines tested. The ability of laminin to promote adhesion of tumor cells to sulfatide suggests that binding to sulfatide could participate in laminin-mediated cell-cell adhesion. Thus, many tumor cell lines can attach on sulfatide substrates using endogenous sulfatide binding proteins, and in some cells laminin but not thrombospondin can promote tumor cell adhesion to sulfatide.
Assuntos
Adesão Celular/efeitos dos fármacos , Laminina/farmacologia , Melanoma/patologia , Sulfoglicoesfingolipídeos/metabolismo , Adsorção , Glicoproteínas/farmacologia , Humanos , Laminina/imunologia , Metabolismo dos Lipídeos , Polissacarídeos/farmacologia , Receptores Imunológicos/fisiologia , Receptores de Laminina , Trombospondinas , Células Tumorais CultivadasRESUMO
The involvement of the alpha 6 beta a integrin, a laminin receptor, in breast carcinoma progression needs to be addressed rigorously. We report that a human breast carcinoma cell line, MDA-MB-435, known to be highly invasive and metastatic, expresses three potential integrin laminin receptors: alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1, but uses only alpha 6 beta 1 to mediate adhesion and migration on laminin matrices. To investigate the contribution of alpha 6 beta 1 to the aggressive behavior of these cells, we developed a dominant-negative strategy for knocking out alpha 6 beta 1 function that involved expression of a cytoplasmic domain deletion mutant of the beta 4 integrin subunit by cDNA transfection. Stable transfectants of MDA-MB-435 cells that expressed this mutant beta 4 subunit were inhibited dramatically in their ability to adhere and migrate on laminin matrices, and their capacity to invade Matrigel was reduced significantly. These findings support the hypothesis that alpha 6 beta 1 is important for breast cancer progression. Moreover, this approach is a powerful method that should be useful in assessing the role of alpha 6 beta 1 in other cells.
Assuntos
Neoplasias da Mama/metabolismo , Integrinas/metabolismo , Neoplasias da Mama/patologia , Adesão Celular , Movimento Celular , DNA Complementar/genética , Feminino , Técnicas de Transferência de Genes , Humanos , Integrina alfa6beta1 , Integrinas/genética , Laminina/metabolismo , Receptores de Laminina/metabolismo , Células Tumorais CultivadasRESUMO
The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity of this antiserum against human laminin was studied using the FL cell line of human epithelial-like cells derived from normal amniotic membrane. The antiserum reacted with these cells in immunoperoxidase staining and precipitated metabolically labeled secreted polypeptides which comigrated with polypeptides with molecular weights of 400,000 and 200,000 of rat laminin in sodium dodecyl sulfate:polyacrylamide gel electrophoresis. The neoplastic cells in malignant breast tissues showed strong cytoplasmic staining for laminin, and a positive reaction was aslo found in lymph node metastases. In some cases in which only micrometastases were present, these cells also stained strongly for laminin. In nonmalignant breast tissues, the epithelial cells of the duct were positive for laminin, but the staining was weaker than in the carcinomas. Pretreatment of the fixed tissue sections with trypsin markedly enhanced the staining of basement membranes for laminin. In trypsin-treated sections of normal breast tissue and benign lesions, the laminin staining delineated continuous basement membranes. In carcinomas representing the more differentiated types, basement membranes presumably produced by the tumor cells could be revealed by laminin staining, but they were thinner and discontinuous. The poorly differentiated carcinomas lacked organized basement membranes detectable by laminin staining. Our studies suggest that staining for laminin may be a useful adjunct test for detection of micrometatases in lymph nodes. The correlation of disintegration of the laminin-containing basement membranes of tumors with increasingly anaplastic appearance supports the notion that basement membranes may play a role in tumor invasion.
Assuntos
Membrana Basal/patologia , Neoplasias da Mama/patologia , Mama/ultraestrutura , Carcinoma/patologia , Glicoproteínas/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Laminina , Proteínas de Membrana/metabolismoRESUMO
Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin but not on fibronectin. Synthetic peptide GRGDS corresponding to the fibronectin cell-binding domain inhibited haptotaxis on fibronectin but not on laminin. Both types of anti-laminin receptor antisera inhibited haptotaxis on laminin but not on fibronectin. Using immunohistochemistry, invading human carcinoma cells in vivo exhibited a marked cytoplasmic immunoreactivity for the receptor antigen. Together these findings indicate a specific role for the laminin receptor in laminin-mediated migration and that the ligand binding of the laminin receptor is encompassed in the COOH-terminal end of the protein.
Assuntos
Neoplasias/patologia , Receptores Imunológicos/fisiologia , Adesão Celular , Movimento Celular , Ensaio de Imunoadsorção Enzimática , Fibronectinas/metabolismo , Humanos , Soros Imunes/imunologia , Laminina/metabolismo , Melanoma/patologia , Invasividade Neoplásica , Receptores Imunológicos/análise , Receptores Imunológicos/imunologia , Receptores de LamininaRESUMO
A cDNA coding for a 37 kDa polypeptide has been identified in several species as both the potential precursor of the 67 kDa laminin receptor (37LRP) and a putative ribosome-associated protein (p40). Interestingly, increased expression of this polypeptide (37LRP/p40) is consistently observed in invasive and metastatic cancer cells and is associated with poor prognosis. Southern-blot analysis of human genomic DNA predicted multiple copies of the 37LRP/p40 gene. In this study, we report that the number of copies of this sequence in the human genome is 26 +/- 2. We have sequenced and analyzed 19 genomic clones corresponding to the 37LRP/p40 gene and found that they were all processed pseudogenes. They all lack intronic sequences and show multiple genetic alterations leading in some cases to the appearance of stop codons. Moreover, they all bear characteristic features of retroposons as the presence of a poly(A)-tail at their 3' end and short direct repeated flanking DNA sequences. None of the pseudogenes analyzed present cis-elements in their 5' flanking region such as TATA or GC boxes. Our date reveal that over 50% of the 37LRP/p40 gene copies are pseudogenes most probably generated by retropositional events. The finding of multiple pseudogenes for the 37LRP/p40 suggests that the accumulation of several copies of this gene might have given a survival advantage to the cell in the course of evolution.
Assuntos
Elementos de DNA Transponíveis , Pseudogenes , Receptores de Laminina/genética , Proteínas Ribossômicas/genética , Sequência de Bases , Evolução Biológica , DNA Complementar , Genoma Humano , Humanos , Dados de Sequência Molecular , Família Multigênica , Processamento Pós-Transcricional do RNA , Homologia de Sequência do Ácido NucleicoRESUMO
This series of three short reviews is an attempt to summarize our current knowledge of the in vivo tests of hypotheses of laminin functions. The structures of the laminins have been thoroughly reviewed recently (P. Ekblom and R. Timpl, in press), and I will not attempt to repeat this information here. Instead, I will focus on the recent evidence gathered from gene knock out experiments in mice and from naturally occurring human and mouse gene mutations. The most obvious lesson from the above studies--other than demonstrating the importance of laminins in general--is that the structural diversity of the laminin family members makes highly specialized functions possible. While all laminins may share many functional properties, the individual chains are involved in interactions which cannot be substituted for by other laminins or by other basement membrane components. While this concept is not new, it is very satisfying to see its validity so dramatically confirmed. It is therefore predictable that additional gene ablation experiments using other known and yet undescribed laminin genes will be equally interesting and informative. To me, one of the most striking lessons from these studies is how strongly the induced mouse mutations mimic human disease. With all the concerns with genetic background differences and species specific effects, manipulation of the laminin genes appears to be a particularly good first approach to identifying the causes of human disease. There is an abundant literature accumulated from biochemical and, more recently, molecular structural analyses, and from in vitro systems, suggesting a role of laminins contributing directly to the stability of the basement membrane. There is an equally vast literature supporting an indirect role in mediating cellular behavior, through interactions with various receptors. It is interesting that the in vivo studies summarized above support both activities. In the case of laminin 5 mutations, the phenotypic consequence appears to be due primarily to the loss of an important structural link between the epithelial cytokeratins and the dermal anchoring fibrils. The ultrastructure of the epithelium appears normal, as does the architecture of the papillary dermis. Only the anchoring complex itself is aberrant. The absence of laminin 5 appears not to compromise the development or viability of the epidermis. The basement membrane appears normal-other than the anchoring complex itself. The pathology observed in the newborn is believed to be due to the frictional trauma of birth, with the expectation that the function of the fetal skin is normal in utero. The Herlitz epidermolysis bullosa phenotype is obvious immediately at birth, and it does not progress postnatally beyond the extent to which the affected individual experiences additional frictional trauma or secondary consequences such as infection or fluid loss. Since laminin 5 is only one of a series of structural links within the anchoring complex, one would predict that a loss of any of these links would result in the same phenotype. Current evidence supports this view, as the absence of integrin alpha 6 beta 4 (Vidal et al., 1995; Dowling et al., 1996; Georges-Labouesse et al., 1996; van der Neut et al., 1996) or of collagen VII (A. M. Christiano and J. Uitto, in press) also results in dramatic neonatal dermal-epidermal fragility. The differences in phenotype, such as the pyloric atresia in the case of loss of integrin alpha 6 beta 4, are presumably due to additional functions of the integrin in other tissues or in other developmental processes. Therefore, the laminin 5 mutations may be unique, in that the in vivo studies suggest that the primary role of the molecule is in the elaboration and stability of the anchoring complex, but not in the basement membrane itself. Of course, since the in vivo phenotype reflects only losses that cannot be compensated, this interpretation may be much too narrow. (ABSTRACT TRUNCATED)
Assuntos
Laminina/química , Laminina/fisiologia , Animais , Epidermólise Bolhosa/genética , Matriz Extracelular/fisiologia , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Humanos , Laminina/genética , Camundongos , Distrofias Musculares/genética , Distrofia Muscular Animal/genética , Mutação , Especificidade de ÓrgãosRESUMO
Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism for ADAM 12 involving both furin cleavage and copper binding.
Assuntos
Cobre/farmacologia , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Proteína ADAM12 , alfa-Globulinas/metabolismo , Western Blotting , Cromatografia em Gel , Ativação Enzimática , Humanos , Proteínas Recombinantes/metabolismoRESUMO
Heparan sulfate proteoglycan from the L2 rat yolk sac carcinoma has been purified and partially characterized. The proteoglycan has an apparent Mr of 750 000, 35% of which represents the core protein. The core protein seems to be homogeneous, whereas the heparan sulfate chains are heterogeneous with an Mr of about 50 000-70 000, with 30% of the glucosamine being N-sulfated. Antibodies raised against the core protein of the heparan sulfate proteoglycan reacted with basement membranes of various rat and human tissue.
Assuntos
Membrana Basal/análise , Proteoglicanas de Sulfatos de Condroitina/análise , Disgerminoma/análise , Glicosaminoglicanos/análise , Heparitina Sulfato/análise , Proteoglicanas/análise , Aminoácidos/análise , Animais , Proteoglicanas de Sulfatos de Condroitina/imunologia , Disgerminoma/patologia , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/imunologia , Hexosaminas/análise , RatosRESUMO
To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 20/genética , Laminina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica , Ligação Genética , Humanos , Hibridização In Situ , Laminina/química , Camundongos , Dados de Sequência Molecular , Mutação , RNA Mensageiro/análise , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
In the present study, we examined the regulation of tetranectin gene expression using a human osteoblastic cell line, SV-HFO, that undergoes mineralization upon treatment with dexamethasone. We found that the expression of tetranectin and alkaline phosphatase mRNA was induced by dexamethasone treatment as evidenced by Northern blotting. When transforming growth factor-beta 1 (TGF-beta 1) was added together with dexamethasone to the SV-HFO cell cultures, the mineralization process was markedly suppressed and the expression of tetra nectin and alkaline phosphatase was downregulated in a dose-dependent manner. These results demonstrate that the expression of tetranectin in these osteoblastic cells is regulated by dexamethasone and TGF-beta 1 and that tetranectin expression is tightly linked to the process of mineralization.