RESUMEN
The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal-epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal-epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal-epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.
Asunto(s)
Membrana Basal/metabolismo , Colágeno/metabolismo , Dermis/metabolismo , Epidermis/metabolismo , Matriz Extracelular/metabolismo , Laminina/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Colágeno/efectos de los fármacos , Colágeno Tipo IV/efectos de los fármacos , Colágeno Tipo IV/metabolismo , Colágeno Tipo VII/efectos de los fármacos , Colágeno Tipo VII/metabolismo , Dermis/efectos de los fármacos , Dermis/ultraestructura , Inhibidores Enzimáticos/farmacología , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/ultraestructura , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/ultraestructura , Laminina/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Microscopía Electrónica , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
To investigate the pathophysiological role of matrix metalloproteinase (MMP)-9 in the skin, we analyzed MMP-9 expression from human keratinocytes in culture. MMP-9 and the terminal differentiation marker involucrin were co-localized in the same keratinocytes with a high concentration of Ca(2+), a potent stimulator of differentiation. We identified the novel KRE-M9 element, further downstream to the previously reported TPA responsive element in the MMP-9 promoter, and both of these two elements were shown to be important for MMP-9 transcription and Ca(2+) induction. The concomitant upregulation of MMP-9 and involucrin transcripts was probably due to the very similar gene regulatory elements, KRE-M9 and KRE-4, in their respective promoters. These results indicate a novel mechanism of transcriptional regulation for MMP-9 in the process of keratinization, implying the probable association of apoptosis and differentiation of keratinocytes in epidermal skin tissue.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Queratinocitos/enzimología , Queratinas/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Calcio/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Genes Reporteros , Humanos , Inmunohistoquímica , Hibridación in Situ , Queratinocitos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusión , Piel/citología , Piel/metabolismoRESUMEN
Laminins (Ln), together with Type IV collagen and nidogen-1, form the structural integrity of the basement membranes (BM). In this study we used immunohistochemistry to show the distribution of laminin chains alpha1, alpha3, alpha5, beta1, beta2, beta3, gamma1, gamma2, as well as Type IV collagen, in various types of carcinomas and in normal tissues. Except for diffuse gastric carcinomas and infiltrative breast carcinomas, the malignant epithelial tumor clusters were surrounded by quite a continuous BM in most tumors. These BMs comprised most abundantly Ln alpha5, beta1, and gamma1 chains. Conversely, the Ln alpha1 chain, a component of laminins-1 and -3, showed the most restricted distribution in BMs of both normal tissues and malignancies, being moderately present in carcinomas of thyroid gland and ovary and in intraductal carcinomas of breast. In other types of carcinomas, immunoreactivity for Ln alpha1 chain was found more randomly and was practically negative in carcinomas of tongue, stomach, and colon. These findings were comparable to those observed by in situ hybridization, which showed that carcinomas of thyroid gland and intraductal carcinomas of breast constitutively expressed Ln alpha1 mRNA and that the epithelial tumor cells were the main producers of it. The results suggest that epithelial malignancies, except for infiltrative breast and diffuse gastric carcinomas, produce more notable amounts of BM macromolecules in their growth substratum than has previously been anticipated. Corroborating their widespread distribution in normal epithelial tissues, the chains of Lns-5 and -10 are the most abundant Ln molecules in the corresponding carcinomas.
Asunto(s)
Carcinoma/química , Laminina/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Membrana Basal/química , Carcinoma/genética , Carcinoma/patología , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Neoplasias Gastrointestinales/química , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/patología , Humanos , Inmunohistoquímica , Hibridación in Situ , Laminina/genética , Neoplasias Pulmonares/química , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Neoplasias Mamarias Animales/química , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Neoplasias Ováricas/química , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Neoplasias de la Tiroides/química , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Neoplasias de la Lengua/química , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patologíaRESUMEN
The basement membrane is considered to act as a barrier which hinders cancer cells from invading the surrounding stroma. In order to assess changes in essential components during neoplasia in the lung, we immunohistochemically studied distribution patterns of laminins alpha 3 and alpha 5 in 40 adenocarcinomas and 8 squamous cell carcinomas. The a 5 chain was generally preserved at the periphery, frequently disrupted in foci with alveolar collapse and absent in foci of fibroblastic proliferation within adenocarcinomas. Fragmentation and absence of laminin alpha 3 chain were more prominent than for alpha 5 chain. Laminin alpha 3 chain was partially fragmented or absent in peripheral areas of adenocarcinomas, being significantly different from alpha 5 chain. Non-small cell lung cancers with reduced alpha 5 chain showed a tendency for greater lymph node metastasis. In cultured normal air way epithelial cells, both laminin alpha 3 and alpha 5 chains were found to be expressed by northern analysis. Eleven of the twelve cultured lung cancer cell lines did not express alpha 3 chain and expression of alpha 5 chain was reduced in three. Quantitative RT-PCR analysis also demonstrated expression of laminin alpha 3 chain in adenocarcinoma tissues to be significantly lower than in normal lung tissues. These results suggest that expression of laminin alpha chains is often reduced in lung cancer cells and this might contribute to basement membrane fragmentation and subsequent proliferation of stromal elements, as well as play some role in the process of cancer cell invasion.
Asunto(s)
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Laminina/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/patología , Secuencia de Aminoácidos , Northern Blotting , Carcinoma de Células Escamosas/patología , División Celular , Línea Celular , Humanos , Inmunohistoquímica , Laminina/análisis , Neoplasias Pulmonares/patología , Datos de Secuencia Molecular , Estadificación de Neoplasias , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , ARN Mensajero/análisis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Chick cDNA clones for a new member of the FACIT (fibril-associated collagens with interrupted triple helices) subfamily have been isolated and sequenced. The collagen chain encoded by these cDNAs was assigned the next consecutive number, making it the alpha1(XX) collagen chain. Assignment of type XX collagen to the FACIT family was based on sequence similarities to types XII and XIV collagen. Type XX collagen mRNA is not abundant in the chick embryo. It is most prevalent in corneal epithelium. It is also detectable by reverse transcription polymerase chain reaction in embryonic skin, sternal cartilage, and tendon, but is barely detectable in calvaria, notochord, or neural retina at select stages of development, suggesting that it is not expressed in these tissues. The cDNA predicts that the alpha1(XX) collagen polypeptide is smaller than the short forms of collagen XII and XIV. A polyclonal antibody against a synthetic alpha1(XX) peptide reacts with polypeptide bands of 185, 170, and 135 kDa by Western blot analysis. From its similarity to types XII and XIV collagen, type XX is expected to bind to collagen fibrils, projecting the amino-terminal domains away from the fibrillar surface. The projecting NC 3 domains are predicted to be about half the length of those of collagen XIV.
Asunto(s)
Colágeno/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Colágeno/química , Cartilla de ADN , ADN Complementario , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Laminins are heterotrimeric extracellular matrix molecules, present in a wide range of basement membranes within human tissues. They consist of a combination of different alpha, beta, and gamma subunits. Three different gamma subunits have been described to date. Two of them, the gamma1 and gamma2 chains are constituents of basement membrane related laminins, while the gamma3 chain was detected in skin, heart, lung, reproductive tract, brain, and in the retina. Unlike other laminins, the expression of the gamma3 chain was localized to peripheral nerves and to the apical surface of ciliated epithelial cells and in the retina. To further investigate the function and the possible pathogenic role of laminin gamma3 in human disease, we elucidated the structure of the corresponding LAMC3 gene which encodes this polypeptide. Here we report the genomic organization of the LAMC3 gene and a mutation detection strategy for use in genetic studies.
Asunto(s)
Laminina/genética , Mutación , Polimorfismo Genético , Sustitución de Aminoácidos , Secuencia de Bases , ADN/genética , Análisis Mutacional de ADN , Cartilla de ADN , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Mutación Missense , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
We have studied the synthesis of laminins (Ln) and determined the specific integrins mediating the adhesion of immortalized human corneal epithelial cells to mouse Ln-1, and human Lns-5 and -10. Immunofluorescence microscopy of the cells demonstrated integrin alpha(2), alpha(3), alpha(6), beta(1)and beta(4)subunits, integrins alpha(6)and beta(4)being found in a typical 'leopard-skin' like manner. Immunoprecipitation studies showed that the cells produced alpha 3, beta 3 and gamma 2 chains of Ln-5, but not Lns-1 or -10. In culture Ln-5 was found as small plaques beneath the adhering cells within 1 hr, while in 4 hr widely spread Ln-5 plaques were observed in colocalization with beta(4)integrin subunit. By using a quantitative cell adhesion assay and function-blocking monoclonal antibodies we showed that integrin beta(1)subunit plays a role in mediating corneal epithelial cell adhesion to mouse Ln-1. However, none of the available function-blocking antibodies to integrin alpha-subunits inhibited the adhesion. Integrin alpha(3)beta(1)complex mediated the adhesion of corneal epithelial cells to human Lns-5 and -10. Integrin complex alpha(3)beta(1), as well as laminin alpha(3)chain, was also shown to mediate cell adhesion to newly produced endogenous Ln-5. The present results show that integrin alpha(3)beta(1)complex mediates the adhesion of corneal epithelial cells to Lns-5 and -10, while a yet unknown integrin alpha subunit appears to play a role in the adhesion to Ln-1. The results also show that among corneal basement membrane laminins, Ln-5 is synthetized by epithelial cells while Ln-10 may be a product of keratocytes.
Asunto(s)
Epitelio Corneal/fisiología , Laminina/fisiología , Animales , Anticuerpos Monoclonales , Western Blotting , Adhesión Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Epitelio Corneal/citología , Humanos , Integrinas/metabolismo , Ratones , Pruebas de Precipitina , Isoformas de ProteínasRESUMEN
Sciellin is a precursor of the cornified envelope of mammalian stratified epithelia characterized by a central core of nonidentical repeats. We characterized the genomic structure of human sciellin and showed that each homologous repeat was encoded by one exon. We also characterized mouse sciellin and showed that mouse sciellin and human sciellin (HGMW-approved symbol SCEL) share a similar gene organization and protein expression pattern. This one exon/one repeat organization is unique among other cornified envelope precursors characterized by homologous repeats. We identified an alternatively spliced isoform of human sciellin, absent in mouse, characterized by an additional repeat at the beginning of the core domain. During embryonic development, the accumulation of sciellin transcript and the accumulation of sciellin protein in the epidermis correlated with the activation of markers of terminal differentiation in epidermis. Mouse sciellin was also identified in simple epithelia with barrier properties, lending further support to its importance in epithelial function.
Asunto(s)
Proteínas Portadoras/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Epitelio/metabolismo , Exones , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Piel/metabolismoRESUMEN
The netrins are a family of laminin-related molecules. Here, we characterize a new member of the family, beta-netrin. beta-Netrin is homologous to the NH(2) terminus of laminin chain short arms; it contains a laminin-like domain VI and 3.5 laminin EGF repeats and a netrin C domain. Unlike other netrins, this new netrin is more related to the laminin beta chains, thus, its name beta-netrin. An initial analysis of the tissue distribution revealed that kidney, heart, ovary, retina, and the olfactory bulb were tissues of high expression. We have expressed the molecule in a eukaryotic cell expression system and made antibodies to the expressed product. Both in situ hybridization and immunohistochemistry were used to describe the cellular source of beta-netrin and where beta-netrin is deposited. beta-Netrin is a basement membrane component; it is present in the basement membranes of the vasculature, kidney, and ovaries. In addition, beta-netrin is expressed in a limited set of fiber tracts within the brain, including the lateral olfactory tract and the vomeronasal nerve. Functional studies were performed and show that beta-netrin promotes neurite elongation from olfactory bulb explants. Together, these data suggest that beta-netrin is important in neural, kidney, and vascular development.
Asunto(s)
Laminina , Proteínas de la Membrana/aislamiento & purificación , Familia de Multigenes , Proteínas del Tejido Nervioso/aislamiento & purificación , Bulbo Olfatorio/efectos de los fármacos , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Membrana Basal/química , Femenino , Genitales Femeninos/química , Humanos , Técnicas In Vitro , Riñón/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/farmacología , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/farmacología , Netrinas , Neuritas , Bulbo Olfatorio/química , Ratas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Distribución TisularRESUMEN
Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.
Asunto(s)
Laminina , Placenta/química , Procesamiento Proteico-Postraduccional/fisiología , Western Blotting , Coriocarcinoma , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Isomerismo , Laminina/química , Laminina/genética , Laminina/aislamiento & purificación , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/aislamiento & purificación , Pruebas de Precipitina , Radioisótopos de Azufre , Células Tumorales Cultivadas , Neoplasias UterinasRESUMEN
Components of the extracellular matrix exert myriad effects on tissues throughout the body. In particular, the laminins, a family of heterotrimeric extracellular glycoproteins, have been shown to affect tissue development and integrity in such diverse organs as the kidney, lung, skin, and nervous system. Of these, we have focused on the roles that laminins play in the differentiation and maintenance of the nervous system. Here, we examine the expression of all known laminin chains within one component of the CNS, the retina. We find seven laminin chains-alpha3, alpha4, alpha5, beta2, beta3, gamma2, and gamma3-outside the retinal basement membranes. Anatomically, these chains are coexpressed in one or both of two locations: the matrix surrounding photoreceptors and the first synaptic layer where photoreceptors synapse with retinal interneurons. Biochemically, four of these chains are coisolated from retinal extracts in two independent complexes, confirming that two novel heterotrimers-alpha4beta2gamma3 and alpha5beta2gamma3-are present in the retinal matrix. During development, all four of these chains, along with components of laminin 5 (the alpha3, beta3, and gamma2 chains) are also expressed at sites at which they could exert important effects on photoreceptor development. Together, these data suggest the existence of two novel laminin heterotrimers in the CNS, which we term here laminin 14 (composed of the alpha4, beta2, and gamma3 chains) and laminin 15 (composed of the alpha5, beta2, and gamma3 chains), and lead us to hypothesize that these laminins, along with laminin 5, may play roles in photoreceptor production, stability, and synaptic organization.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Laminina/genética , Retina/metabolismo , Adulto , Envejecimiento , Secuencia de Aminoácidos , Animales , Membrana Basal/metabolismo , Bovinos , Matriz Extracelular/metabolismo , Humanos , Hibridación in Situ , Técnicas In Vitro , Laminina/análisis , Laminina/química , Datos de Secuencia Molecular , Neuronas/metabolismo , Fragmentos de Péptidos/química , Ratas , Retina/citología , Retina/crecimiento & desarrollo , Alineación de Secuencia , Sinapsis/metabolismo , Transcripción GenéticaRESUMEN
We have generated transgenic mice expressing green fluorescent protein (GFP) driven by 2.453-kb (-2,362 to +91) of the 5'-upstream region of the human vascular endothelial growth factor (VEGF) promoter to monitor changes of VEGF gene transcription in situ. Neonatal transgenic mice exhibited GFP-derived fluorescence in tissues that have been previously reported to express VEGF mRNA expression, including lung, cartilage, and brain. In normal skin during postnatal development, moderate fluorescence was observed in the upper epidermis and, more prominently, in the outer root sheath keratinocytes of hair follicles. Strong up-regulation of GFP fluorescence was observed in the hyperplastic epidermis of the wound edge at 48 hours after wounding, whereas little GFP fluorescence was detected in the dermis. In situ hybridization confirmed an identical expression pattern of VEGF mRNA in these wounds. Topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA) induced strong VEGF-GFP expression in suprabasal epidermis. Little or no fibroblast-derived fluorescence was seen both in the wound model and after TPA application. By confocal laser microscopy, increased GFP fluorescence was detectable in the epidermis of intact mouse ear skin as early as 6 hours after topical TPA treatment. Importantly, GFP fluorescence was also measurable in the skin of living transgenic mice. These results resolve the present controversy regarding the ability of VEGF-GFP transgenic mouse models to correctly reflect established patterns of VEGF expression, and show the model to be a powerful tool for the in vivo monitoring of VEGF gene expression.
Asunto(s)
Factores de Crecimiento Endotelial/genética , Linfocinas/genética , Regiones Promotoras Genéticas/genética , Piel/metabolismo , Animales , Células Cultivadas , Dermis/citología , Dermis/metabolismo , Células Epidérmicas , Epidermis/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes , Humanos , Queratinocitos/citología , Queratinocitos/metabolismo , Proteínas Luminiscentes/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Piel/citología , Acetato de Tetradecanoilforbol/farmacología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Cicatrización de HeridasRESUMEN
Extensive remodeling of the extracellular matrix (ECM) occurs in inflammatory tissues. The celiac lesion in the small intestine is characterized by inflammation accompanied by profound morphological alterations. We used immunohistochemistry to determine the distribution of laminin, fibronectin, and tenascin isoforms in small intestinal biopsies of untreated patients with celiac disease. In normal mucosa, the distribution of laminin isoforms defines three epithelial basement membrane (BM) zones. We found that the organization of these zones was maintained in the celiac mucosa. Thus, components of laminin-5 (alpha3 and beta3) were found in the surface epithelial BM, laminin alpha2 chain was found selectively at crypt bottoms, and laminin alpha5 chain was the sole alpha-type chain in middle crypt BMs. Likewise, the distribution of fibronectin and tenascin resembled that of the normal gut. The organization of pericryptal fibroblasts and lamina propria smooth muscle strands, as defined by immunostaining for alpha-smooth muscle actin, also remained unchanged in the celiac mucosa. Unexpectedly, major ECM changes were not detected in the celiac lesion.
Asunto(s)
Enfermedad Celíaca/metabolismo , Fibronectinas/metabolismo , Mucosa Intestinal/metabolismo , Laminina/metabolismo , Tenascina/metabolismo , Adolescente , Adulto , Membrana Basal/metabolismo , Niño , Preescolar , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Lactante , Mucosa Intestinal/irrigación sanguínea , Microscopía Fluorescente , Músculo Liso/metabolismoRESUMEN
Epithelial cells maintained in culture medium containing low calcium proteolytically process laminin 5 (alpha3beta3gamma2) within the alpha3 and gamma2 chains (). Experiments were designed to identify the enzyme(s) responsible for the laminin 5 processing and the sites of proteolytic cleavage. To characterize the nature of laminin 5 processing, we determined the N-terminal amino acid sequences of the proteolytic fragments produced by the processing events. The results indicate that the first alpha3 chain cleavage (200-l65 kDa alpha3) occurs within subdomain G4 of the G domain. The second cleavage (l65-l45 kDa alpha3) occurs within the lIla domain, 11 residues N-terminal to the start of domain II. The gamma chain is cleaved within the second epidermal growth factor-like repeat of domain Ill. The sequence cleaved within the gamma2 chain matches the consensus sequence for the cleavage of type I, II, and III procollagens by bone morphogenetic protein-1 (BMP-1), also known as type I procollagen C-proteinase (). Recombinant BMP-1 cleaves gamma2 in vitro, both within intact laminin 5 and at the predicted site of a recombinant gamma2 short arm. alpha3 is also cleaved by BMP-1 in vitro, but the cleavage site is yet to be determined. These results show the laminin alpha3 and gamma2 chains to be substrates for BMP-1 in vitro. We speculate that gamma2 cleavage is required for formation of the laminin 5-6 complex and that this complex is directly involved in assembly of the interhemidesmosomal basement membrane. This further suggests that BMP-1 activity facilitates basement membrane assembly, but not hemidesmosome assembly, in the laminin 5-rich dermal-epidermal junction basement membrane in vivo.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Laminina/metabolismo , Metaloendopeptidasas/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 1 , Bovinos , Células Cultivadas , Cartilla de ADN , Humanos , Hidrólisis , Queratinocitos/citología , Queratinocitos/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
We have investigated re-epithelialization following induction of suction blisters in humans in intact blisters, open wounds, i.e. blister roofs removed immediately after blister induction, and calcipotriol-pretreated open wounds. Intact blisters simulate blister healing in bullous disease, while open wounds simulate re-epithelialization during wound healing. Re-epithelialization was clearly faster in open wounds than in intact blisters, and was not affected by calcipotriol pretreatment. Bullous pemphigoid antigen 2 (BP180), bullous pemphigoid antigen 1 (BP230), plectin/hemidesmosomal 1 protein (HD1), laminin 5, laminin alpha5, laminin beta1, type VII collagen, tenascin-C, beta4, alphavbeta5, alpha5 and alpha9 integrins were studied in intact blisters and open wounds by immunohistochemistry. Hemidesmosomal plaque proteins BP230 and plectin/HD1, which connect the keratin cytoskeleton to the hemidesmosome, appeared earlier at the leading edge in intact blisters than in open wounds. Band-like immunostaining in the basement membrane for laminin 5, alpha5 and beta1 chains was continuous in blister bases, but partially discontinuous in open wound bases. The other antigens studied showed similar expression in intact blisters and open wounds. BP180, BP230, plectin/HD1, beta4 integrin, laminin 5 and tenascin-C expression were further studied in calcipotriol-pretreated open wounds. Calcipotriol did not affect the expression of these antigens. The immunohistochemical results suggest that the keratin cytoskeleton is linked to the basal plasma membrane of migrating basal cells via BP230 and plectin/HD1 earlier in the more slowly re-epithelializing blisters than in open wounds. An intact laminin sheath may inhibit keratinocyte migration in intact blisters.
Asunto(s)
Vesícula/fisiopatología , Calcitriol/análogos & derivados , Agonistas de los Canales de Calcio/uso terapéutico , Proteínas Portadoras , Proteínas del Citoesqueleto , Fármacos Dermatológicos/uso terapéutico , Proteínas del Tejido Nervioso , Colágenos no Fibrilares , Adulto , Autoantígenos/inmunología , Vesícula/tratamiento farmacológico , Vesícula/metabolismo , Calcitriol/uso terapéutico , División Celular/efectos de los fármacos , Colágeno/inmunología , Colágeno/metabolismo , Desmosomas/efectos de los fármacos , Desmosomas/metabolismo , Método Doble Ciego , Distonina , Eosina Amarillenta-(YS) , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hematoxilina , Humanos , Integrinas/efectos de los fármacos , Integrinas/inmunología , Integrinas/metabolismo , Proteínas de Filamentos Intermediarios/inmunología , Queratinocitos/citología , Laminina/efectos de los fármacos , Laminina/inmunología , Laminina/metabolismo , Masculino , Plectina , Piel/inmunología , Coloración y Etiquetado , Tenascina/inmunología , Cicatrización de Heridas/fisiología , Colágeno Tipo XVIIRESUMEN
The formation of the hair follicle and its cyclical growth, quiescence, and regeneration depend on reciprocal signaling between its epidermal and dermal components. The dermal organizing center, the dermal papilla (DP), regulates development of the epidermal follicle and is dependent on signals from the epidermis for its development and maintenance. GFP specifically expressed in DP cells of a transgenic mouse was used to purify this population and study the signals required to maintain it. We demonstrate that specific Wnts, but not Sonic hedgehog (Shh), maintain anagen-phase gene expression in vitro and hair inductive activity in a skin reconstitution assay.
Asunto(s)
Proteínas Aviares , Folículo Piloso/crecimiento & desarrollo , Cabello/crecimiento & desarrollo , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Transactivadores , Proteínas de Pez Cebra , Animales , Animales Recién Nacidos , División Celular , Células Cultivadas , Embrión de Pollo , Técnicas de Cocultivo , Fibroblastos/citología , Fibroblastos/metabolismo , Genes Reporteros/genética , Cabello/citología , Cabello/metabolismo , Folículo Piloso/citología , Folículo Piloso/metabolismo , Proteínas Hedgehog , Queratinocitos/citología , Ratones , Ratones Desnudos , Ratones Transgénicos , Proteínas/genética , Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Transgenes/genética , Proteínas Wnt , Proteína Wnt3RESUMEN
The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. Epidermal-dermal communication through the basement membrane is important for skin homeostasis. The basement membrane contains specialized structures, called the anchoring complex, which ensure the stability of connection and communication between these two tissue compartments. The proteins within the anchoring complex provide links to both the intracellular cytoskeletal keratins in keratinocytes and connective tissue proteins of the dermis. One of the key components of the complex is laminin 5, which is essential to epidermal cell attachment. The biological function of laminin 5 has been investigated by using a skin equivalent model in vitro and during keratinocyte sheet grafting in vivo. As a major link between the epidermal basal cells and the papillary dermis, laminin 5 initiates hemidesmosome formation and provides stable attachment of the epidermis to the dermis. Laminin 5 also accelerates the assembly of basement membranes and may enhance the recovery of damaged skin. An intact basement membrane at the epidermal-dermal junction is essential to stability of the skin.
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Membrana Basal/fisiología , Dermis/citología , Dermis/fisiología , Células Epidérmicas , Epidermis/fisiología , Laminina/fisiología , Animales , Niño , Humanos , Fenómenos Fisiológicos de la PielRESUMEN
Interplay between laminin-5 (Ln-5) and its integrin (Int) receptors alpha2beta1, alpha3beta1 and alpha6beta4 has been implicated in the progression and invasion of carcinomas. In this study we found abundant immunoreactivity for chains of Ln-5 (alpha3-beta3-gamma2) and Ln-10 (alpha5-beta1-gamma1), as well as for type VII collagen, in basement membranes (BM) of colorectal adenomas. In carcinomas of all differentiation grades, Lns were seen in tumor BMs, whereas type VII collagen was almost absent. Ln-5 appeared to accumulate along the invading edges of carcinomas, while Ln-10 was mostly absent. Immunoreactivity for Ln al chain, a component of Lns-1 and -3, was not seen in adenomas or carcinomas. Immunoreactivity for alpha2, alpha6, beta1 and beta4 Ints was found in all tumors and that for alpha3 Int in all adenomas and most of the carcinomas, often in colocalization with Ln-5. Immunoblotting of carcinoma tissues showed that the gamma2 chain of Ln-5 was present as typical Mr 105000 and 155000 isoforms. Immunoprecipitation experiments showed production of Ln-5 by cultured colon carcinoma cells. In quantitative cell adhesion experiments, function-blocking MAbs to alpha3 and beta1 Int subunits, but not those to Int alpha2 or alpha6 subunits, significantly inhibited the adhesion of cells to Ln-5. Our results suggest that BM composition in colorectal adenomas reflects the properties of surface epithelial BM of colorectal mucosa. In invading carcinomas, trimeric Ln-5, produced by carcinoma cells, is a major BM component and the cells use the alpha3beta1 Int complex for adhesion to Ln-5.
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Adenoma/metabolismo , Carcinoma/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neoplasias Colorrectales/metabolismo , Integrinas/metabolismo , Adenoma/patología , Membrana Basal/metabolismo , Carcinoma/patología , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/biosíntesis , Neoplasias Colorrectales/patología , Células HT29 , Humanos , Integrina alfa3beta1 , Ligandos , Células Tumorales Cultivadas , KalininaRESUMEN
Laminin 5 is essential in epithelial attachment to stromal tissues, suggesting that it might improve keratinocyte attachment in a variety of clinical situations. In this study, we examined the effect of exogenous laminin 5 upon the efficiency of transplantation of keratinocyte sheets in animal models. Keratinocyte sheets were prepared according to the method of Rheinwald & Green (1975). Purified laminin 5 was added to the sheets of group 1 (1.0 microg per cm2), Dulbecco's modified Eagle's medium alone was added to group 2. The sheets were grafted to the panniculus carnosus of nude mice (BALB/C nu/nu) (n = 12) and nude rats (Fisher 344) (n = 15). The take rate was assessed by measurement of the area of surviving epithelium at 7 d postgrafting. Laminin 5 bound the keratinocyte sheets of group 1. At 7 d postgrafting, the area of epithelialization of group 1 was significantly larger than that of group 2. Immunohistochemistry staining showed that collagen IV, laminin 5, and collagen VII stained more strongly at the dermal-epidermal junction in group 1 than in group 2. Integrin chains alpha6 and beta4 were similar in both groups. Electron microscopy at day 3 after grafting, showed the lamina densa of group 1 to be more continuous than in group 2. Pretreatment of cultured human keratinocyte sheets with laminin 5 improved the extent of epithelial coverage and increased the rate of neobasement membrane formation. The results suggest that laminin 5 promotes epithelial attachment by increasing the rate of basement membrane assembly.
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Moléculas de Adhesión Celular/farmacología , Supervivencia de Injerto/efectos de los fármacos , Queratinocitos/trasplante , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/ultraestructura , Moléculas de Adhesión Celular/análisis , Trasplante de Células , Colágeno/análisis , Humanos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratas , Ratas Endogámicas F344 , Ratas Desnudas , Piel/química , Piel/efectos de los fármacos , Piel/ultraestructura , Trasplante de Piel/métodos , Factores de Tiempo , Trasplante Heterólogo , KalininaRESUMEN
Recent BP230-knockout experiments with subsequent blistering and recently identified plectin/HD1 mutations in epidermolysis bullosa simplex patients suggest that defective expression of BP230 and plectin/HD1 may predispose to blister formation in human skin. We have studied the expression of the epithelial adhesion complex as well as the basement membrane and anchoring fibril antigens in uninvolved dermatitis herpetiformis skin to find out if alterations can be detected in these structures predisposing to the blister formation typical of the disease. Ten uninvolved dermatitis herpetiformis skin specimens, which all showed clear granular deposits of IgA under the basement membrane in direct immunofluorescence and five normal skin specimens, were studied by indirect immunofluorescence technique. Six uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for BP230 and four uninvolved dermatitis herpetiformis skin specimens showed distinctly decreased immunoreaction for plectin/HD1. All five skin controls showed strong immunoreactions for BP230 and plectin/HD1. Other hemidesmosomal proteins including BP180 and integrin alpha6beta4, as well as basement membrane proteins laminin-5, laminin-1, nidogen and type IV collagen, and the anchoring fibril protein type VII collagen showed a normal strong expression. Our results suggest that alterations in BP230 and plectin/HD1 may contribute or predispose to blister formation in dermatitis herpetiformis skin.