RESUMEN
Obesity is a global epidemic that contributes to the increasing medical burdens related to type 2 diabetes, cardiovascular disease and cancer. A better understanding of the mechanisms regulating adipose tissue expansion could lead to therapeutics that eliminate or reduce obesity-associated morbidity and mortality. The extracellular matrix (ECM) has been shown to regulate the development and function of numerous tissues and organs. However, there is little understanding of its function in adipose tissue. In this manuscript we describe the role of laminin α4, a specialized ECM protein surrounding adipocytes, on weight gain and adipose tissue function. Adipose tissue accumulation, lipogenesis, and structure were examined in mice with a null mutation of the laminin α4 gene (Lama4-/-) and compared to wild-type (Lama4+/+) control animals. Lama4-/- mice exhibited reduced weight gain in response to both age and high fat diet. Interestingly, the mice had decreased adipose tissue mass and altered lipogenesis in a depot-specific manner. In particular, epididymal adipose tissue mass was specifically decreased in knock-out mice, and there was also a defect in lipogenesis in this depot as well. In contrast, no such differences were observed in subcutaneous adipose tissue at 14 weeks. The results suggest that laminin α4 influences adipose tissue structure and function in a depot-specific manner. Alterations in laminin composition offers insight into the roll the ECM potentially plays in modulating cellular behavior in adipose tissue expansion.
Asunto(s)
Tejido Adiposo/patología , Laminina/deficiencia , Aumento de Peso , Adipocitos/patología , Tejido Adiposo Blanco/patología , Envejecimiento/patología , Animales , Tamaño de la Célula , Dieta Alta en Grasa , Conducta Alimentaria , Laminina/metabolismo , Lipogénesis , Masculino , Ratones Endogámicos C57BL , Obesidad/patología , Grasa Subcutánea/patologíaRESUMEN
We investigated the effects of exendin-4 on neural stem/progenitor cells in the subventricular zone of the adult rodent brain and its functional effects in an animal model of Parkinson's disease. Our results showed expression of GLP-1 receptor mRNA or protein in the subventricular zone and cultured neural stem/progenitor cells isolated from this region. In vitro, exendin-4 increased the number of neural stem/progenitor cells, and the number of cells expressing the neuronal markers microtubule-associated protein 2, beta-III-tubulin, and neuron-specific enolase. When exendin-4 was given intraperitoneally to naïve rodents together with bromodeoxyuridine, a marker for DNA synthesis, both the number of bromodeoxyuridine-positive cells and the number of neuronal precursor cells expressing doublecortin were increased. Exendin-4 was tested in the 6-hydroxydopamine model of Parkinson's disease to investigate its possible functional effects in an animal model with neuronal loss. After unilateral lesion and a 5-week stabilization period, the rats were treated for 3 weeks with exendin-4. We found a reduction of amphetamine-induced rotations in animals receiving exendin-4 that persisted for several weeks after drug administration had been terminated. Histological analysis showed that exendin-4 significantly increased the number of both tyrosine hydroxylase- and vesicular monoamine transporter 2-positive neurons in the substantia nigra. In conclusion, our results show that exendin-4 is able to promote adult neurogenesis in vitro and in vivo, normalize dopamine imbalance, and increase the number of cells positive for markers of dopaminergic neurons in the substantia nigra in a model of Parkinson's disease.
Asunto(s)
Hipoglucemiantes/farmacología , Neuronas/efectos de los fármacos , Trastornos Parkinsonianos/tratamiento farmacológico , Péptidos/farmacología , Recuperación de la Función/efectos de los fármacos , Ponzoñas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Proteína Doblecortina , Exenatida , Receptor del Péptido 1 Similar al Glucagón , Inmunohistoquímica , Ratones , Actividad Motora/efectos de los fármacos , Neuronas/citología , Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Ratas , Receptores de Glucagón/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.
Asunto(s)
Cistinil Aminopeptidasa/metabolismo , Ligandos , Oligopéptidos/metabolismo , Angiotensina II/análogos & derivados , Angiotensina II/síntesis química , Angiotensina II/química , Angiotensina II/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Cricetulus , Cistinil Aminopeptidasa/genética , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Modelos Moleculares , Neuronas/citología , Neuronas/efectos de los fármacos , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Unión Proteica , Conformación Proteica , Ensayo de Unión Radioligante , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/citología , Células Madre/efectos de los fármacos , Relación Estructura-Actividad , Porcinos , TransfecciónRESUMEN
The Schwann cell basal lamina (BL) is required for normal myelination. Loss or mutations of BL constituents, such as laminin-2 (alpha2beta1gamma1), lead to severe neuropathic diseases affecting peripheral nerves. The function of the second known laminin present in Schwann cell BL, laminin-8 (alpha4beta1gamma1), is so far unknown. Here we show that absence of the laminin alpha4 chain, which distinguishes laminin-8 from laminin-2, leads to a disturbance in radial sorting, impaired myelination, and signs of ataxia and proprioceptive disturbances, whereas the axonal regenerative capacity is not influenced. In vitro studies show poor axon growth of spinal motoneurons on laminin-8, whereas it is extensive on laminin-2. Schwann cells, however, extend longer processes on laminin-8 than on laminin-2, and, in contrast to the interaction with laminin-2, solely use the integrin receptor alpha6beta1 in their interaction with laminin-8. Thus, laminin-2 and laminin-8 have different critical functions in peripheral nerves, mediated by different integrin receptors.
Asunto(s)
Axones/fisiología , Laminina/deficiencia , Células de Schwann/citología , Células de Schwann/fisiología , Animales , Animales Recién Nacidos , Axones/efectos de los fármacos , Conducta Animal/fisiología , Movimiento Celular/genética , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/ultraestructura , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/patología , Inmunohistoquímica/métodos , Técnicas In Vitro , Laminina/farmacología , Laminina/fisiología , Ratones , Ratones Noqueados/fisiología , Microscopía Electrónica de Transmisión/métodos , Espasticidad Muscular/genética , Regeneración Nerviosa/genética , Proteínas S100/metabolismo , Células de Schwann/efectos de los fármacos , Nervio Ciático/crecimiento & desarrollo , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patología , Factores de TiempoRESUMEN
During extravasation, neutrophils migrate through the perivascular basement membrane (BM), a specialized extracellular matrix rich in laminins. Laminins 8 (LN-8) (alpha4beta1gamma1) and 10 (LN-10) (alpha5beta1gamma1) are major components of the endothelial BM, but expression, recognition, and use of these laminin isoforms by neutrophils are poorly understood. In the present study, we provide evidence, using a panel of novel monoclonal antibodies against human laminin alpha4 (LNalpha4) chain, that neutrophils contain and secrete LN-8, and that this endogenous laminin contributes to chemoattractant-induced, alphaMbeta2-integrin-dependent neutrophil migration through albumin-coated filters. Phorbol ester-stimulated neutrophils adhered to recombinant human (rh) LN-8, rhLN-10, and mouse LN-1 (mLN-1) (alpha1beta1gamma1) via alphaMbeta2-integrin, and these laminin isoforms strongly promoted chemoattractant-induced neutrophil migration via the same integrin. However, only rhLN-8 enhanced the spontaneous migration. In addition, recruitment of neutrophils into the peritoneum following an inflammatory stimulus was impaired in LNalpha4-deficient mice. rhLN-8 also protected isolated neutrophils from spontaneous apoptosis. This study is the first to identify a specific laminin isoform in neutrophils and provides evidence for the role of LN-8 in the adhesion, migration, extravasation, and survival of these cells.
Asunto(s)
Apoptosis , Movimiento Celular , Laminina/metabolismo , Neutrófilos/citología , Neutrófilos/metabolismo , Aflatoxina B1 , Animales , Anticuerpos Monoclonales/inmunología , Apoptosis/efectos de los fármacos , Caseínas/farmacología , Adhesión Celular/efectos de los fármacos , Células Endoteliales , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Integrinas/metabolismo , Laminina/deficiencia , Laminina/genética , Laminina/farmacología , Ratones , Ratones Noqueados , Neutrófilos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Albúmina SéricaRESUMEN
Laminin-8 (alpha 4 beta 1 gamma 1) is one of the major laminin isoforms expressed in vascular endothelial basement membranes. Here we show that deletion of laminin-8 in mice affects angiogenesis under pathological conditions. Murine tumor models used in laminin alpha 4-deficient mice results in hyperneovascularization and significant promotion of tumor growth and metastasis. The higher tumor growth rates in mutant mice correlate with decreased tumor cell apoptosis. Depletion of laminin alpha 4 chain may alter the structure of vascular basement membranes, leading to increased angiogenesis. Our data suggest that the laminin-8 plays a critical role in the regulation of pathological angiogenesis.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Laminina/deficiencia , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/metabolismo , Animales , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , División Celular/fisiología , Laminina/biosíntesis , Laminina/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Neovascularización Patológica/genética , Neovascularización Patológica/patologíaRESUMEN
Laminins are alphabetagamma heterotrimeric extracellular proteins that regulate cellular functions by adhesion to integrin and nonintegrin receptors. Laminins containing alpha4 and alpha5 chains are expressed in bone marrow, but their interactions with hematopoietic progenitors are unknown. We studied human bone marrow cell adhesion to laminin-10/11 (alpha5beta1gamma1/alpha5beta2gamma1), laminin-8 (alpha4beta1gamma1), laminin-1 (alpha1beta1gamma1), and fibronectin. About 35% to 40% of CD34(+) and CD34(+)CD38(-) stem and progenitor cells adhered to laminin-10/11, and 45% to 50% adhered to fibronectin, whereas they adhered less to laminin-8 and laminin-1. Adhesion of CD34(+)CD38(-) cells to laminin-10/11 was maximal without integrin activation, whereas adhesion to other proteins was dependent on protein kinase C activation by 12-tetradecanoyl phorbol-13-acetate (TPA). Fluorescence-activated cell-sorting (FACS) analysis showed expression of integrin alpha6 chain on most CD34(+) and CD34(+)CD38(-) cells. Integrin alpha6 and beta1 chains were involved in binding of both cell fractions to laminin-10/11 and laminin-8. Laminin-10/11 was highly adhesive to lineage-committed myelomonocytic and erythroid progenitor cells and most lymphoid and myeloid cell lines studied, whereas laminin-8 was less adhesive. In functional assays, both laminin-8 and laminin-10/11 facilitated stromal-derived factor-1alpha (SDF-1alpha)-stimulated transmigration of CD34(+) cells, by an integrin alpha6 receptor-mediated mechanism. In conclusion, we demonstrate laminin isoform-specific adhesive interactions with human bone marrow stem, progenitor, and more differentiated cells. The cell-adhesive laminins affected migration of hematopoietic progenitors, suggesting a physiologic role for laminins during hematopoiesis.
Asunto(s)
Células de la Médula Ósea/citología , Células Madre Hematopoyéticas/citología , Laminina/metabolismo , Antígenos CD34 , Adhesión Celular , Movimiento Celular , Fibronectinas/metabolismo , Hematopoyesis , Humanos , Laminina/fisiología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiología , Receptores de Laminina/metabolismoRESUMEN
We have produced human laminin-8 (alpha4beta1gamma1) using recombinant technology. Approximately half of the recombinant laminin-8 (rLN-8) molecules were found to have a chondroitin sulphate modification in the alpha4 chain. The substituted and non-substituted forms were separated and tested for cell adhesion activity. Lower cell adhesion promoting activity was seen for the substituted form, but the integrin receptor utilization was similar. We also found the human rLN-8 to behave identically in cell adhesion assays compared to a human/mouse hybrid variant of rLN-8.
Asunto(s)
Sulfatos de Condroitina/química , Laminina/biosíntesis , Laminina/química , Animales , Adhesión Celular/fisiología , Línea Celular , Humanos , Integrina alfa6beta1/metabolismo , Laminina/fisiología , Ratones , Multimerización de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Recent studies suggest important functions for laminin-8 (Ln-8; alpha4beta1gamma1) in vascular and blood cell biology, but its distribution in human tissues has remained elusive. We have raised a monoclonal antibody (MAb) FC10, and by enzyme-linked immunoassay (EIA) and Western blotting techniques we show that it recognizes the human Ln alpha4-chain. Immunoreactivity for the Ln alpha4-chain was localized in tissues of mesodermal origin, such as basement membranes (BMs) of endothelia, adipocytes, and skeletal, smooth, and cardiac muscle cells. In addition, the Ln alpha4-chain was found in regions of some epithelial BMs, including epidermis, salivary glands, pancreas, esophageal and gastric glands, intestinal crypts, and some renal medullary tubules. Developmental differences in the distribution of Ln alpha4-chain were detected in skeletal muscle, walls of vessels, and intestinal crypts. Ln alpha4- and Ln alpha2-chains co-localized in BMs of fetal skeletal muscle cells and in some epithelial BMs, e.g., in gastric glands and acini of pancreas. Cultured human pulmonary artery endothelial (HPAE) cells produced Ln alpha4-chain as M(r) 180,000 and 200,000 doublet and rapidly deposited it to the growth substratum. In cell-free extracellular matrices of human kidney and lung, Ln alpha4-chain was found as M(r) 180,000 protein.
Asunto(s)
Laminina/metabolismo , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Membrana Basal/embriología , Membrana Basal/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotelio/embriología , Endotelio/metabolismo , Matriz Extracelular/metabolismo , Humanos , Técnicas para Inmunoenzimas , Inmunohistoquímica , Laminina/inmunología , Especificidad de Órganos , Pruebas de PrecipitinaRESUMEN
The laminin (LN) family of large heterotrimeric extracellular matrix glycoproteins has multiple functions: LNs take part in the regulation of processes such as cell migration, differentiation, and proliferation, in addition to contributing to the structure of basement membranes. LN-10, composed of alpha5, beta1, and gamma1 chains, is widely distributed in most basement membranes of both epithelia and endothelia. We determined the complete human cDNA sequence for the LN alpha5 chain and produced recombinant human LN-10 (rLN-10) in HEK293 cells by triple transfection of full-length cDNAs encoding the human LN alpha5, beta1, and gamma1 chains. The rLN-10 was purified using affinity chromatography and had an apparent molecular mass of approximately 800 kDa in SDS-PAGE and a native domain structure in rotary shadowing electron microscopy. By using function-blocking monoclonal antibodies, integrin alpha(3)beta(1) was found to be a major mediator of adhesion of HT-1080 and human saphenous vein endothelial cells. Human saphenous vein endothelial cells adhered more strongly to rLN-10 than to LN-1 and LN-8 and showed better migration on rLN-10, compared with several other matrices. Considering the cell adhesive and migration-promoting properties of rLN-10 on endothelial cells, this molecule could be useful in improving the biocompatibility and endothelialization of vascular grafts.
Asunto(s)
Laminina/genética , Secuencia de Bases , Adhesión Celular/fisiología , Línea Celular , Movimiento Celular/fisiología , Cromatografía de Afinidad/métodos , Clonación Molecular , Cartilla de ADN , ADN Complementario , Humanos , Laminina/aislamiento & purificación , Laminina/fisiología , Laminina/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin alpha4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin alpha4 chain in microvessel growth and, in the absence of other laminin alpha chains, in the composition of endothelial basement membranes.