RESUMEN
The antidiabetic intestinal L cell hormone glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion and inhibits gastric emptying. GLP-1 secretion is stimulated by luminal oleic acid (OA), which crosses the cell membrane by an unknown mechanism. We hypothesized that L cell fatty acid transport proteins (FATPs) are essential for OA-induced GLP-1 release. Therefore, the murine GLUTag L cell model was used for immunoblotting, [(3)H]OA uptake assay, and GLP-1 secretion assay as determined by radioimmunoassay following treatment with OA ± phloretin, sulfo-N-succinimidyl oleate, or siRNA against FATP4. FATP4(-/-) and cluster-of-differentiation 36 (CD36)(-/-) mice received intraileal OA, and plasma GLP-1 was measured by sandwich immunoassay. GLUTag cells were found to express CD36, FATP1, FATP3, and FATP4. The cells demonstrated specific (3)H[OA] uptake that was dose-dependently inhibited by 500 and 1,000 µM unlabeled OA (P < 0.001). Cell viability was not altered by treatment with OA. Phloretin and sulfo-N-succinimidyl oleate, inhibitors of protein-mediated transport and CD36, respectively, also decreased [(3)H]OA uptake, as did knockdown of FATP4 by siRNA transfection (P < 0.05-0.001). OA dose-dependently increased GLP-1 secretion at 500 and 1,000 µM (P < 0.001), whereas phloretin, sulfo-N-succinimidyl oleate, and FATP4 knockdown decreased this response (P < 0.05-0.01). FATP4(-/-) mice displayed lower plasma GLP-1 at 60 min in response to intraileal OA (P < 0.05), whereas, unexpectedly, CD36(-/-) mice displayed higher basal GLP-1 levels (P < 0.01) but a normal response to intraileal OA. Together, these findings demonstrate a key role for FATP4 in OA-induced GLP-1 secretion from the murine L cell in vitro and in vivo, whereas the precise role of CD36 remains unclear.
Asunto(s)
Células Enteroendocrinas/metabolismo , Proteínas de Transporte de Ácidos Grasos/sangre , Péptido 1 Similar al Glucagón/metabolismo , Ácido Oléico/farmacología , Animales , Antígenos CD36/análisis , Antígenos CD36/genética , Células Cultivadas , Células Enteroendocrinas/efectos de los fármacos , Proteínas de Transporte de Ácidos Grasos/análisis , Proteínas de Transporte de Ácidos Grasos/genética , Femenino , Silenciador del Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ácidos Oléicos/farmacología , Floretina/farmacología , Succinimidas/farmacologíaRESUMEN
In chronic renal disease, the progressive accumulation of collagen and other extracellular matrix proteins in the mesangium results in fibrosis, glomerulosclerosis, and eventual renal failure. Mice deficient in proalpha2(I) collagen are not only a model of osteogenesis imperfecta but also accumulate fibrillar homotrimeric type I collagen in the mesangium. This accumulation spreads to the subendothelial space in the peripheral capillary loops. Picosirius red staining of kidney sections demonstrates that in comparison to wild-type mice, Col1a2-deficient homozygous and heterozygous mice exhibit abnormal glomerular collagen deposition in a gene dosage-dependent manner. The glomerulopathy initiates during the first postnatal week, appears progressive following the pattern of glomerular maturation and results in albuminuria in severely affected animals. In situ hybridization revealed no gross differences in steady-state proalpha1(I) and proalpha2(I) collagen mRNA levels among the three genotypes. Quantitative reverse transcriptase-polymerase chain reaction, however, using whole kidney sections showed a twofold increase in steady-state proalpha1(I) collagen mRNA in 1-month homozygous Col1a2-deficient animals compared with wild-type and heterozygous animals. We suggest that glomerular collagen deposition seen in the osteogenesis imperfecta model mice is, in part, owing to pretranslational mechanisms and may represent an over compensation of wound healing.
Asunto(s)
Animales Recién Nacidos/metabolismo , Colágeno Tipo I/metabolismo , Colágeno/deficiencia , Enfermedades Renales/etiología , Enfermedades Renales/fisiopatología , Glomérulos Renales/crecimiento & desarrollo , Albuminuria/etiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Colágeno/genética , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Heterocigoto , Homeostasis , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Glomérulos Renales/patología , Ratones , Ratones Noqueados , Microscopía Electrónica , ARN Mensajero/metabolismoRESUMEN
Alport syndrome, caused by mutations that interfere with the normal assembly of the alpha3alpha4alpha5(IV) collagen network in the glomerular basement membrane (GBM), is the most common inherited glomerular disease leading to renal failure. A detailed knowledge of the underlying pathogenic mechanisms is necessary for developing new, more specific, and effective therapeutic strategies aimed at delaying the onset and slowing disease progression. Studies of several dog and mouse models of Alport syndrome have significantly enhanced our understanding of the disease mechanisms and provided systems for testing potential therapies. In the most widely used Col4a3-/- mouse models of autosomal-recessive Alport syndrome (ARAS), the genetic background strongly affects renal survival. One contributing factor may be the strong ectopic deposition of alpha5alpha6(IV) collagen in the GBM of Col4a3-/- mice on the C57BL/6J background, which is almost undetectable on the 129/Sv background. This isoform 'switch' has not been observed in human ARAS, although it had been reported in the dog model of ARAS. In human patients as well as dog and mouse models of X-linked Alport syndrome, the alpha3-alpha6(IV) collagen chains are absent from the GBM. These biochemical differences among Alport animal models provide an opportunity to determine how the molecular makeup of the GBM affects the glomerular function. At the same time, potentially confounding influences of characteristics unique to a particular strain or model should be carefully considered in the design of studies aiming to define key events underlying the pathobiology of Alport glomerular disease.
Asunto(s)
Modelos Animales de Enfermedad , Nefritis Hereditaria/genética , Animales , Expresión Génica , RatonesRESUMEN
CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. Here we demonstrate that CD2AP localizes to the slit diaphragm in podocytes using immunoelectron microscopy and that nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm.
Asunto(s)
Glomérulos Renales/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión/genética , Línea Celular , Proteínas del Citoesqueleto , Uniones Intercelulares/metabolismo , Glomérulos Renales/citología , Glomérulos Renales/ultraestructura , Proteínas de la Membrana , Ratones , Ratones Noqueados , Microscopía Inmunoelectrónica , Mutación , Pruebas de Precipitina , Unión Proteica , Proteínas/química , Proteínas/genéticaRESUMEN
Laminins are major components of all basement membranes. One laminin that has garnered particular interest, due to its widespread expression pattern and importance during development, is the laminin alpha5 chain. In vitro studies have suggested that the Lutheran blood group glycoprotein/basal cell adhesion molecule (Lu), an Ig superfamily transmembrane protein, is a receptor for laminins containing the alpha5 chain. However, there are no in vivo studies showing that these proteins are capable of interacting in tissues. We have isolated the mouse ortholog of Lu and characterized its expression and localization in mouse tissues. Lu was primarily found on the basal surface of epithelial cells and on muscle cells adjacent to basement membranes containing laminin alpha5. In addition, there was both a dramatic reduction in the basal concentration of Lu in mice lacking laminin alpha5, and a significant increase in Lu protein in transgenic mice overexpressing laminin alpha5. Together, these data provide the first in vivo evidence for an interaction between Lu and laminin alpha5 and support the hypothesis that Lu is a laminin alpha5 receptor. We propose that laminin alpha5 is involved in concentrating Lu on the basal surface of epithelial cells. This may be one mechanism by which basement membrane signals are transmitted to the cell.
Asunto(s)
Laminina/metabolismo , Receptores de Laminina/metabolismo , Animales , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , ADN Complementario/metabolismo , Inmunoglobulina G/metabolismo , Inmunohistoquímica , Riñón/embriología , Riñón/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculos/embriología , Músculos/metabolismo , Miocardio/citología , Miocardio/metabolismo , Unión Proteica , ARN/metabolismo , Transducción de Señal , Factores de Tiempo , Distribución TisularRESUMEN
The laminins are a family of heterotrimeric extracellular matrix molecules that form suprastructural networks in basement membranes and elsewhere. They interact with integrin receptors, playing key roles in modulating programs of cytodifferentiation and maintaining tissue homeostasis in animals. Earlier studies have demonstrated an extensive laminin network in both the developing and adult cochlea, primarily associated with the basement membranes. These studies, however, did not address the laminin chain composition of these networks. In this study, we used antibodies specific for the known laminin chains to examine the composition of laminins in both the developing and adult murine cochlea. The results illustrate a complex and dynamic postnatal developmental regulation pattern for most of these chains, and suggest that an unusually large number of laminin heterotrimers are present in both the developing and adult cochlea. The laminin composition at postnatal day 2 is relatively simple. By postnatal day 7, however, activation of several laminin chains results in a very complex laminin composition. In the basement membrane underlying the region of the basilar membrane under the developing organ of Corti, eight of the 11 known basement membrane laminins are possible by co-localization inference. Dynamic changes in expression continue through day 14, but simplify by adulthood. Thus, the most dynamic period for laminin expression in the mouse cochlea coincides with terminal cytodifferentiation of the cochlear epithelial structures. Considering the well established role of laminins in regulating both embryonic and organ development in other systems, these data suggest a closer look at the role of the laminins in cochlear development and function may be warranted.
Asunto(s)
Envejecimiento/metabolismo , Membrana Basal/metabolismo , Oído Interno/crecimiento & desarrollo , Oído Interno/metabolismo , Laminina/metabolismo , Ratones/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Técnica del Anticuerpo Fluorescente , Isoformas de Proteínas/metabolismoRESUMEN
Mutant mice lacking the integrin alpha8 subunit exhibit variable defects in kidney development with most mutants missing both kidneys. Several lines of evidence indicate that the known extracellular matrix ligands for integrin alpha8beta1 are either dispensable for or not involved in alpha8beta1 signaling during kidney development. This suggests the presence of an unknown ligand. A novel alpha8beta1 ligand, nephronectin, has now been identified. Nephronectin is a new extracellular matrix protein associated with the Wolffian duct and the ureteric bud, epithelial structures with well-defined roles in kidney development.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Integrinas/metabolismo , Riñón/embriología , Riñón/metabolismo , Animales , Matriz Extracelular/metabolismo , Marcación de Gen , Integrinas/genética , Ligandos , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Ratones Mutantes , Especificidad de Órganos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Uréter/embriología , Uréter/metabolismo , Conductos Mesonéfricos/embriología , Conductos Mesonéfricos/metabolismoRESUMEN
Differential gene expression in tumors often involves growth factors and extracellular matrix/basement membrane components. Here, 11,000- gene microarray was used to identify gene expression profiles in brain tumors including high-grade gliomas [glioblastoma multiforme (GBM) and anaplastic astrocytoma], low-grade astrocytomas, or benign extra-axial brain tumors (meningioma) in comparison with normal brain tissue. Histologically normal tissues adjacent to GBMs were also studied. All GBMs studied overexpressed 14 known genes compared with normal human brain tissue. Overexpressed genes belonged to two broad groups: (a) growth factor-related genes; and (b) structural/extracellular matrix-related genes. For most of these 14 genes, expression levels were lower in low-grade astrocytoma than in GBM and were barely detectable in normal brain. Despite normal-appearing histology, gene expression patterns of tissues immediately adjacent to GBM were similar to those of their respective primary GBMs. Two genes were consistently up-regulated in both high-grade and low-grade gliomas, as well as in histologically normal tissues adjacent to GBMs. These genes coded for the epidermal growth factor receptor (previously reported to be overexpressed in gliomas) and for the alpha4 chain of laminin, a major blood vessel basement membrane component. Changes in expression of this laminin chain have not been previously associated with malignant tumors. Overexpression of laminin alpha4 chain in GBM and astrocytoma grade II by gene microarray analysis was confirmed by semiquantitive reverse transcription-PCR and immunohistochemistry. Importantly, an alpha4 chain-containing laminin isoform, laminin-8 (alpha4beta1gamma1), was expressed mainly in blood vessel walls of GBMs and histologically normal tissues adjacent to GBMs, whereas another alpha4 chain-containing laminin isoform, laminin-9 (alpha4beta2gamma1), was expressed mainly in blood vessel walls of low-grade tumors and normal brain. GBMs that overexpressed laminin-8 had a shorter mean time to tumor recurrence (4.3 months) than GBMs with overexpression of laminin-9 (9.7 months, P = 0.0007). Up-regulation of alpha4 chain-containing laminins could be important for the development of glioma-induced neovascularization and glial tumor progression. Overexpression of laminin-8 may be predictive of glioma recurrence.
Asunto(s)
Neoplasias Encefálicas/genética , Glioma/genética , Laminina/genética , Adulto , Anciano , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Glioma/patología , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.
Asunto(s)
Glomérulos Renales , Laminina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Membrana Basal/química , Membrana Basal/fisiología , Capilares/ultraestructura , Endotelio Vascular/ultraestructura , Epítopos/análisis , Fibroblastos/citología , Fibroblastos/fisiología , Isomerismo , Glomérulos Renales/citología , Glomérulos Renales/fisiología , Glomérulos Renales/trasplante , Trasplante de Riñón/métodos , Operón Lac , Laminina/química , Laminina/inmunología , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Datos de Secuencia Molecular , Nefronas/fisiología , Nefronas/trasplante , Nefronas/ultraestructura , Técnicas de Cultivo de Órganos , Proteínas Tirosina Fosfatasas/análisis , Proteínas/análisis , Codorniz , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial VascularRESUMEN
During the past 3 years there have been significant advances in our understanding of the biology of the glomerular podocyte. In particular, two proteins, CD2-associated protein and nephrin, have been identified as critical podocyte proteins that are both required for normal glomerular filtration. In addition to supporting the idea that the slit diaphragm plays a crucial role in glomerular function, these results suggest novel insights into the pathogenesis of glomerular diseases. The present review addresses these recent advances and discusses the implications of the findings.
Asunto(s)
Riñón/fisiología , Proteínas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas del Citoesqueleto , Humanos , Enfermedades Renales/fisiopatología , Glomérulos Renales/fisiología , Ratones , Ratones Noqueados/genética , Proteínas/genética , Insuficiencia Renal/genética , Insuficiencia Renal/mortalidadRESUMEN
Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.
Asunto(s)
Membrana Basal/metabolismo , Colágeno/genética , Proteínas de Homeodominio/metabolismo , Glomérulos Renales/metabolismo , Síndrome de la Uña-Rótula/complicaciones , Insuficiencia Renal/etiología , Animales , Colágeno/biosíntesis , Matriz Extracelular/metabolismo , Regulación de la Expresión Génica , Proteínas con Homeodominio LIM , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Factores de Transcripción , Transcripción GenéticaRESUMEN
Laminins are trimeric glycoprotein components of basement membranes. Each laminin has three structurally similar chains, designated alpha, beta, and gamma. Five laminin alpha chains are now known. In previous studies using monoclonal antibody 4C7, laminin alpha1 was thought to be present in basement membranes of human lung throughout development and in the adult, but recent expression studies have demonstrated that 4C7 identifies laminin alpha5 rather than alpha1. To determine the temporal and spatial patterns of laminin alpha1 and laminin alpha5 in developing human lung, we prepared complementary DNA probes specific for laminin alpha1 and alpha5 messenger RNAs (mRNAs). By Northern analysis, laminin alpha1 mRNA was prominent in first-trimester fetal lung, but was not detectable at 23 wk or at later times. In contrast, laminin alpha5 mRNA was readily detected in early fetal lung and remained present thereafter. Immunohistochemical staining demonstrated laminin alpha1 only in early fetal lung, whereas laminin alpha5 was persistent from the early fetal period. In situ hybridization localized laminin alpha1 expression to distal epithelium in the first-trimester lung, and laminin alpha5 to all epithelium and developing pulmonary arteries from the first trimester through the perinatal period. These studies indicate that laminin alpha1 expression is restricted to early human lung morphogenesis, whereas the expression of laminin alpha5 in human lung is continuous from early lung development through adult life. It is evident that laminin alpha1 and laminin alpha5 have different roles in the development of the human lung.
Asunto(s)
Perfilación de la Expresión Génica , Laminina/genética , Pulmón/metabolismo , Northern Blotting , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Recién Nacido , Laminina/metabolismo , Pulmón/química , Pulmón/embriología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor Nuclear Tiroideo 1 , Distribución Tisular , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
CD2-associated protein (CD2AP) is an adapter molecule that can bind to the cytoplasmic domain of nephrin, a component of the glomerular slit diaphragm. Mice lacking CD2AP exhibit a congenital nephrotic syndrome characterized by extensive foot process effacement, suggesting that CD2AP-nephrin interactions are critical to maintaining slit diaphragm function. We have examined the patterns of expression of both CD2AP and nephrin in developing mouse and human kidney. Both proteins were first detected in developing podocytes at the capillary loop stage of glomerulogenesis and eventually became concentrated near the glomerular basement membrane. CD2AP was also observed diffusely in collecting duct and apically in many cells of proximal and distal tubule. Kidneys from Cd2ap -/- mice initially exhibited normal nephrin localization, but as the mice aged and foot processes became effaced, nephrin disappeared. In laminin-beta(2) mutant mice exhibiting nephrotic syndrome, CD2AP in glomeruli was aberrantly localized in a primarily punctate pattern. Extensive extrarenal expression of CD2AP was observed in endothelial and epithelial cells, in many cases with a specific subcellular localization. Together, these results suggest that CD2AP is not only involved in maintaining the slit diaphragm but may also have a general role in maintaining specialized subcellular architecture. The severity of kidney disease in Cd2ap mutant mice may have eclipsed manifestation of defects in other tissues.
Asunto(s)
Envejecimiento/metabolismo , Feto/metabolismo , Riñón/embriología , Riñón/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas del Citoesqueleto , Desarrollo Embrionario y Fetal , Feto/fisiología , Humanos , Riñón/citología , Glomérulos Renales/embriología , Laminina/genética , Proteínas de la Membrana , Ratones , Mutación/fisiología , Distribución TisularRESUMEN
Netrins are secreted proteins that serve as potent axon guidance molecules in vertebrates and invertebrates. We report the identification of a novel mammalian member of this family. Netrin-4 is similar in predicted size and secondary structure to the other three netrins; all contain, in order, an amino-terminal signal sequence, a laminin-type globular domain of the 'VI' type, three laminin-type epidermal growth factor (EGF) repeats, and a carboxyl-terminal 'netrin module'. In terms of primary sequence, however, netrin-4 is a distant relative of netrins-1-3, and its globular domain is more closely related to those of laminins than to those of other netrins. Netrin-4 is broadly expressed in both neural and non-neural tissues of embryonic and adult mice. In embryonic spinal cord, it is selectively expressed by cells at the lateral margins of the floor plate. In postnatal brain, it is selectively expressed in subsets of neurons, including cerebellar granule and hippocampal pyramidal cells.
Asunto(s)
Factores de Crecimiento Nervioso/biosíntesis , Factores de Edad , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/embriología , Encéfalo/metabolismo , Cerebelo/metabolismo , ADN Complementario/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Expresión Génica , Biblioteca de Genes , Hipocampo/metabolismo , Humanos , Hibridación in Situ , Riñón/metabolismo , Laminina/metabolismo , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/genética , Netrinas , Filogenia , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Médula Espinal/embriología , Factores de Tiempo , Distribución TisularRESUMEN
Matrix metalloproteinases are matrix degrading enzymes implicated in many biological processes, including development and inflammation. Gelatinase B (gelB; also known as MMP-9) is expressed in the kidney and is hypothesized to be involved in basement membrane remodeling and in preventing pathogenic accumulation of extracellular matrix in the kidney. Inhibition of gelB activity in metanephric organ culture disrupts branching morphogenesis of the ureteric bud, suggesting that gelB plays a role in kidney development in vivo. We studied kidneys of gelB-deficient mice to search for developmental, histological, molecular, ultrastructural, and functional defects. Surprisingly, no differences between gelB-/- and control kidneys were detected, and renal function was normal in gelB mutants. In addition, gelB-/- embryonic kidneys developed normally in organ culture. Gelatinase B-deficient mice were bred with Col4a3-/- mice, a model for Alport syndrome, to determine whether gelB influences the progression of glomerulonephritis. This is an important question, as it has been hypothesized that proteases are involved in damaging Alport glomerular basement membrane. However, the presence or absence of gelB did not affect the rate of progression of renal disease. Thus, gelB does not have a discernible role in the normal kidney and gelB is not involved in the progression of glomerulonephritis in a mouse model of Alport syndrome.
Asunto(s)
Enfermedades Renales/patología , Riñón/embriología , Metaloproteinasa 9 de la Matriz/metabolismo , Nefritis Hereditaria/patología , Animales , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Colágeno/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Genotipo , Riñón/enzimología , Riñón/ultraestructura , Enfermedades Renales/enzimología , Pruebas de Función Renal , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Nefritis Hereditaria/enzimología , Técnicas de Cultivo de Órganos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Nerve regeneration studies at the neuromuscular junction (NMJ) suggest that synaptic basal lamina components tell the returning axon where to locate neurotransmitter release machinery, including synaptic vesicle clusters and active zones. Good candidates for these components are the synaptic laminins (LNs) containing alpha4, alpha5, or beta2 chains. Results from a beta2 laminin knockout mouse have suggested a linkage of this extracellular laminin to cytosolic synaptic vesicle clusters. Here we report such a transmembrane link at the electric organ synapse, which is homologous to the NMJ. We immunopurified electric organ synaptosomes and found on their surface two laminins of 740 and 900 kDa. The 740 kDa laminin has a composition of alpha4beta2gamma1 (laminin-9). Immunostaining reveals that as in the NMJ, alpha4 and beta2 chains are concentrated at the electric organ synapse. Using detergent-solubilized synaptosomes, we immunoprecipitated a complex containing alpha4beta2gamma1 laminin, the voltage-gated calcium channel, and the cytoskeletal protein spectrin. Other presynaptic proteins such as 900 kDa laminin are not found in this complex. We hypothesize that alpha4beta2gamma1 laminin in the synaptic basal lamina attaches to calcium channel, which in turn is attached to cytosolic spectrin. Spectrin could then organize synaptic vesicle clusters by binding vesicle-associated proteins.
Asunto(s)
Canales de Calcio/metabolismo , Laminina/metabolismo , Terminales Presinápticos/metabolismo , Espectrina/metabolismo , Sinapsis/metabolismo , Torpedo/metabolismo , Animales , Órgano Eléctrico/metabolismo , Laminina/química , Laminina/aislamiento & purificación , Peso Molecular , Unión Neuromuscular/metabolismo , Pruebas de Precipitina , Sinaptosomas/metabolismoRESUMEN
Laminins are major components of all basement membranes. They are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta, and three gamma chains. Laminin alpha5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, alpha5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin alpha chain deposition in which alpha5 replaces alpha1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin alpha5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin alpha5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells.
Asunto(s)
Membrana Basal/embriología , Glomérulos Renales/embriología , Laminina/metabolismo , Uréter/embriología , Animales , Endotelio Vascular/embriología , Epitelio/embriología , Mesangio Glomerular/embriología , Ratones , Ratones Mutantes , Técnicas de Cultivo de Órganos , Anomalías Urogenitales/genéticaRESUMEN
Interactions between growing axons and synaptic basal lamina components direct the formation of neuromuscular junctions during nerve regeneration. Isoforms of laminin containing alpha5 or beta2 chains are potential basal lamina ligands for these interactions. The nerve terminal receptors are unknown. Here we show that SV2, a synaptic vesicle transmembrane proteoglycan, is complexed with a 900-kDa laminin on synaptosomes from the electric organ synapse that is similar to the neuromuscular junctions. Although two laminins are present on synaptosomes, only the 900-kDa laminin is associated with SV2. Other nerve terminal components are absent from this complex. The 900-kDa laminin contains an alpha5, a beta1, and a novel gamma chain. To test whether SV2 directly binds the 900-kDa laminin, we looked for interaction between purified SV2 and laminin-1, a laminin isoform with a similar structure. We find SV2 binds with high affinity to purified laminin-1. Our results suggest that a synaptic vesicle component may act as a laminin receptor on the presynaptic plasma membrane; they also suggest a mechanism for activity-dependent adhesion at the synapse.
Asunto(s)
Laminina/aislamiento & purificación , Glicoproteínas de Membrana/aislamiento & purificación , Proteínas del Tejido Nervioso/aislamiento & purificación , Unión Neuromuscular/química , Vesículas Sinápticas/química , Sinaptosomas/química , Animales , Elasmobranquios , Pez Eléctrico , Órgano Eléctrico , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Modelos Neurológicos , Proteínas del Tejido Nervioso/metabolismo , Unión ProteicaRESUMEN
Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) alpha2 chain not present in round mesenchymal cells. LM alpha2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM beta1 and gamma1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM alpha2. Dy/dy mice express very low levels of LM alpha2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM alpha-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM alpha2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.
Asunto(s)
Regulación de la Expresión Génica , Laminina/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Músculo Liso/citología , Músculo Liso/embriología , Actinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Desmina/metabolismo , Sistema Digestivo/citología , Sistema Digestivo/metabolismo , Sistema Digestivo/patología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Eliminación de Gen , Regulación de la Expresión Génica/efectos de los fármacos , Laminina/genética , Laminina/inmunología , Laminina/farmacología , Mesodermo/efectos de los fármacos , Ratones , Ratones Endogámicos , Ratones Mutantes , Modelos Biológicos , Músculo Liso/efectos de los fármacos , Músculo Liso/patología , Distrofia Muscular Animal/congénito , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patología , Miosinas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sistema Respiratorio/citología , Sistema Respiratorio/embriología , Sistema Respiratorio/metabolismo , Sistema Respiratorio/patologíaRESUMEN
Renal basement membrane components. Basement membranes are specialized extracellular matrices found throughout the body. They surround all epithelia, endothelia, peripheral nerves, muscle cells, and fat cells. They play particularly important roles in the kidney, as demonstrated by the fact that defects in renal basement membranes are associated with kidney malfunction. The major components of all basement membranes are laminin, collagen IV, entactin/nidogen, and sulfated proteoglycans. Each of these describes a family of related proteins that assemble with each other in the extracellular space to form the basement membrane. Over the last few years, new basement membrane components that are expressed in the kidney have been discovered. Here, the major components and their localization in mature and developing renal basement membranes are described. In addition, the phenotypes of basement membrane component gene mutations, both naturally occurring and experimental, are discussed, as is the aberrant deposition of basement membrane proteins in the extracellular matrix in several renal diseases.