RESUMEN
BACKGROUND: The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. RESULTS: Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. CONCLUSIONS: Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.
Asunto(s)
Adenocarcinoma/fisiopatología , Comunicación Celular/fisiología , Neoplasias Colorrectales/fisiopatología , Matriz Extracelular/fisiología , Fibroblastos/fisiología , Células del Estroma/fisiología , Sindecano-2/fisiología , Regulación hacia Arriba/fisiología , Adenocarcinoma/patología , Biomarcadores de Tumor/fisiología , Células CACO-2 , Adhesión Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular , Neoplasias Colorrectales/patología , Matriz Extracelular/patología , Fibroblastos/patología , Fibronectinas/fisiología , Células HCT116 , Humanos , Integrinas/fisiología , Proteoglicanos/fisiología , Células del Estroma/patologíaRESUMEN
In the pregnant mouse endometrium, collagen fibrillogenesis is characterized by the presence of very thick collagen fibrils which are topographically located exclusively within the decidualized stroma. This dynamic biological process is in part regulated by the small leucine-rich proteoglycans decorin and biglycan. In the present study we utilized wild-type (Dcn(+/+)) and decorin-deficient (Dcn(-/-)) time-pregnant mice to investigate the evolution of non-decidualized and decidualized collagen matrix in the uterine wall of these animals. Ultrastructural and morphometric analyses revealed that the organization of collagen fibrils in the pregnant endometrium of both non-decidualized and decidualized stroma showed a great variability of shape and size, regardless of the genotype. However, the decidualized endometrium from Dcn(-/-) mice contained fibrils with larger diameter and more irregular contours as compared to the wild-type littermates. In the Dcn(-/-) animals, the proportion of thin (10-50 nm) fibrils was also higher as compared to Dcn(+/+) animals. On day 7 of pregnancy, biglycan was similarly localized in the decidualized endometrium in both genotypes. Lumican immunostaining was intense both in decidualized and non-decidualized stroma from Dcn(-/-) animals. The present results support previous findings suggesting that decorin participates in uterine collagen fibrillogenesis. In addition, we suggest that the absence of decorin disturbs the process of lateral assembly of thin fibrils, resulting in very thick collagen fibrils with irregular profiles. Our data further suggest that decorin, biglycan and lumican might play an interactive role in collagen fibrillogenesis in the mouse endometrium, a process modulated according to the stage of pregnancy.
Asunto(s)
Endometrio/ultraestructura , Proteínas de la Matriz Extracelular/deficiencia , Colágenos Fibrilares/ultraestructura , Proteoglicanos/deficiencia , Animales , Biglicano , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/fisiología , Decidua/ultraestructura , Decorina , Endometrio/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Femenino , Sulfato de Queratano/metabolismo , Sulfato de Queratano/fisiología , Lumican , Ratones , Ratones Noqueados , Microscopía Electrónica , Embarazo , Proteoglicanos/metabolismo , Proteoglicanos/fisiologíaRESUMEN
The mechanical properties of lung tissue are important determinants of lung physiological functions. The connective tissue is composed mainly of cells and extracellular matrix, where collagen and elastic fibers are the main determinants of lung tissue mechanical properties. These fibers have essentially different elastic properties, form a continuous network along the lungs, and are responsible for passive expiration. In the last decade, many studies analyzed the relationship between tissue composition, microstructure, and macrophysiology, showing that the lung physiological behavior reflects both the mechanical properties of tissue individual components and its complex structural organization. Different lung pathologies such as acute respiratory distress syndrome, fibrosis, inflammation, and emphysema can affect the extracellular matrix. This review focuses on the mechanical properties of lung tissue and how the stress-bearing elements of lung parenchyma can influence its behavior.
Asunto(s)
Enfermedades Pulmonares/fisiopatología , Pulmón/fisiología , Mecánica Respiratoria/fisiología , Animales , Colágeno/fisiología , Tejido Elástico/fisiología , Matriz Extracelular/fisiología , Humanos , Proteoglicanos/fisiologíaRESUMEN
The PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) identified as a mutated gene in patients with X-linked hypophosphatemia (XLH), encodes a protein (PHEX) that shows striking homologies to members of the M13 family of zinc metallopeptidases. In the present work the interaction of glycosaminoglycans with PHEX has been investigated by affinity chromatography, circular dichroism, protein intrinsic fluorescence analysis, hydrolysis of FRET substrates flow cytometry and confocal microscopy. PHEX was eluted from a heparin-Sepharose chromatography column at 0.8 M NaCl showing a strong interaction with heparin. Circular dichroism spectra and intrinsic fluorescence analysis showed that PHEX is protected by glycosaminoglycans against thermal denaturation. Heparin, heparan sulfate and chondroitin sulfate inhibited PHEX catalytic activity, however among them, heparin presented the highest inhibitory activity (Ki=2.5+/-0.2 nM). Flow cytometry analysis showed that PHEX conjugated to Alexa Fluor 488 binds to the cell surface of CHO-K1, but did not bind to glycosaminoglycans defective cells CHO-745. Endogenous PHEX was detected at the cell surface of CHO-K1 colocalized with heparan sulfate proteoglycans, but was not found at the cell surface of glycosaminoglycans defective cells CHO-745. In permeabilized cells, PHEX was detected in endoplasmic reticulum of both cells. In addition, we observed that PHEX colocalizes with heparan sulfate at the cell surface of osteoblasts. This is the first report that the metallopeptidase PHEX is a heparin binding protein and that the interaction with GAGs modulates its enzymatic activity, protein stability and cellular trafficking.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Metaloproteasas/metabolismo , Proteínas/metabolismo , Animales , Células CHO , Cromatografía de Afinidad , Dicroismo Circular , Cricetinae , Cricetulus , Transferencia Resonante de Energía de Fluorescencia , Heparina/metabolismo , Heparina/farmacología , Heparitina Sulfato/metabolismo , Heparitina Sulfato/fisiología , Hidrólisis , Unión Proteica , Proteínas/genética , Proteoglicanos/metabolismo , Proteoglicanos/fisiología , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-beta) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-beta isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-beta1, TGF-beta2, soluble betaglycan, TGF-beta1 plus soluble betaglycan, or TGF-beta2 plus soluble betaglycan, and untreated (control). TGF-beta1 and TGF-beta2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-beta1 and TGF-beta2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-beta1 and TGF-beta2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-beta antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-beta promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-beta affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.
Asunto(s)
Embrión de Pollo/embriología , Ovario/embriología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Recuento de Células , División Celular , Embrión de Pollo/metabolismo , Femenino , Humanos , Inmunohistoquímica , Técnicas de Cultivo de Órganos , Ovario/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoglicanos/metabolismo , Proteoglicanos/fisiología , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Proteínas Recombinantes/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.
Asunto(s)
Adhesión Celular/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Transducción de Señal/fisiología , Animales , Proteínas de la Matriz Extracelular/fisiología , Proteoglicanos de Heparán Sulfato/química , Humanos , Glicoproteínas de Membrana/química , Unión Proteica/fisiología , Proteoglicanos/química , Receptores de Superficie Celular/fisiología , SindecanosRESUMEN
The syndecans, heparan sulfate proteoglycans, are abundant molecules associated with the cell surface and extracellular matrix and consist of a protein core to which heparan sulfate chains are covalently attached. Each of the syndecan core proteins has a short cytoplasmic domain that binds cytosolic regulatory factors. The syndecans also contain highly conserved transmembrane domains and extracellular domains for which important activities are becoming known. These protein domains locate the syndecan on cell surface sites during development and tumor formation where they interact with other receptors to regulate signaling and cytoskeletal organization. The functions of cell surface heparan sulfate proteoglycan have been centered on the role of heparan sulfate chains, located on the outer side of the cell surface, in the binding of a wide array of ligands, including extracellular matrix proteins and soluble growth factors. More recently, the core proteins of the syndecan family transmembrane proteoglycans have also been shown to be involved in cell signaling through interaction with integrins and tyrosine kinase receptors.
Asunto(s)
Animales , Humanos , Adhesión Celular/fisiología , Proteoglicanos de Heparán Sulfato/fisiología , Glicoproteínas de Membrana/fisiología , Proteoglicanos/fisiología , Transducción de Señal/fisiología , Proteínas de la Matriz Extracelular/fisiología , Proteoglicanos de Heparán Sulfato/química , Glicoproteínas de Membrana/química , Unión Proteica/fisiología , Proteoglicanos/química , Receptores de Superficie Celular/fisiología , SindecanosRESUMEN
Experiments on articular cartilage have shown nonlinear stress-strain curves under finite deformations as well as intrinsic viscous effects of the solid phase. The aim of this study was to propose a nonlinear biphasic viscohyperelastic model that combines the intrinsic viscous effects of the proteoglycan matrix with a nonlinear hyperelastic constitutive equation. The proposed equation satisfies objectivity and reduces for uniaxial loading to a solid type viscous model in which the actions of the springs are represented by the hyperelastic function proposed by Holmes and Mow [1990. J. Biomechanics 23, 1145-1156.]. Results of the model, that were efficiently implemented in an updated Lagrangian algorithm, were compared with experimental infinitesimal data reported by DiSilverstro and Suh [2001. J. Biomechanics 34, 519-525.] and showed acceptable fitting for the axial force (R(2)=0.991) and lateral displacement (R(2)=0.914) curves in unconfined compression as well as a good fitting of the axial indentation force curve (R(2)=0.982). In addition, the model showed an excellent fitting of finite-deformation confined compression stress relaxation data reported by Ateshian et al. [1997. J. Biomechanics 30, 1157-1164.] and Huang et al. [2005. J. Biomechanics 38, 799-809.] (R(2)=0.993 and R(2)=0.995, respectively). The constitutive equation may be used to represent the mechanical behavior of the proteoglycan matrix in a fiber reinforced model of articular cartilage.
Asunto(s)
Cartílago Articular/fisiología , Simulación por Computador , Matriz Extracelular/fisiología , Modelos Biológicos , Proteoglicanos/fisiología , Animales , Fuerza Compresiva/fisiología , Análisis de Elementos Finitos , Humanos , Dinámicas no Lineales , ViscosidadRESUMEN
The composition and organization of the extracellular matrix of ostrich articular cartilage was investigated, using samples from the proximal and distal surfaces of the tarsometatarsus. For morphological analysis, sections were stained with toluidine blue and analyzed by polarized light microscopy. For biochemical analysis, extracellular matrix components were extracted with 4 M guanidinium chloride, fractionated on DEAE-Sephacel and analyzed by SDS-PAGE. Glycosaminoglycans were analyzed by electrophoresis in agarose gels. Structural analysis showed that the fibrils were arranged in different directions, especially on the distal surface. The protein and glycosaminoglycan contents of this region were higher than in the other regions.SDS-PAGE showed the presence of proteins with molecular masses ranging from 17 to 121 kDa and polydisperse components of 67, 80-100, and 250-300 kDa in all regions. The analysis of glycosaminoglycans in agarosepropylene diamine gels revealed the presence of only chondroitin-sulfate. The electrophoretic band corresponding to putative decorin was a small proteoglycan containing chondroitin-sufate and not dermatan-sulfate, unlike other cartilages. The higher amounts of proteins and glycosaminoglycans and the multidirectional arrangement of fibrils seen in the distal region may be correlated with the higher compression normally exerted on this region
Asunto(s)
Animales , Cartílago Articular/anatomía & histología , Cartílago Articular/citología , Colágeno/fisiología , Aves/anatomía & histología , Proteoglicanos/fisiologíaRESUMEN
The composition and organization of the extracellular matrix of ostrich articular cartilage was investigated, using samples from the proximal and distal surfaces of the tarsometatarsus. For morphological analysis, sections were stained with toluidine blue and analyzed by polarized light microscopy. For biochemical analysis, extracellular matrix components were extracted with 4 M guanidinium chloride, fractionated on DEAE-Sephacel and analyzed by SDS-PAGE. Glycosaminoglycans were analyzed by electrophoresis in agarose gels. Structural analysis showed that the fibrils were arranged in different directions, especially on the distal surface. The protein and glycosaminoglycan contents of this region were higher than in the other regions.SDS-PAGE showed the presence of proteins with molecular masses ranging from 17 to 121 kDa and polydisperse components of 67, 80-100, and 250-300 kDa in all regions. The analysis of glycosaminoglycans in agarosepropylene diamine gels revealed the presence of only chondroitin-sulfate. The electrophoretic band corresponding to putative decorin was a small proteoglycan containing chondroitin-sufate and not dermatan-sulfate, unlike other cartilages. The higher amounts of proteins and glycosaminoglycans and the multidirectional arrangement of fibrils seen in the distal region may be correlated with the higher compression normally exerted on this region
Asunto(s)
Animales , Aves/anatomía & histología , Cartílago Articular/anatomía & histología , Cartílago Articular/citología , Colágeno/fisiología , Proteoglicanos/fisiologíaRESUMEN
The BMP4 signaling pathway plays key roles during early embryonic development and for maintenance of adult homeostasis. In the extracellular space, BMP4 activity is regulated by a group of interacting molecules including the BMP antagonist Chordin, the metalloproteinase Tolloid and Twisted gastrulation (Tsg). In this study, we identified Biglycan (Bgn), a member of the small leucine-rich proteoglycan family, as a new extracellular modulator of BMP4 signaling. Xenopus Bgn (xBgn) is expressed uniformly in the ectoderm and mesoderm and their derivatives during development. Microinjection of Bgn mRNA induced secondary axes, dorsalized the mesoderm and inhibited BMP4 activity in Xenopus embryos. Biochemical experiments showed that Bgn binds BMP4 and Chordin, interaction that increased binding of BMP4 to Chordin. Bgn was also able to improve the efficiency of Chordin-Tsg complexes to block BMP4 activity. Using antisense morpholinos, we demonstrated that Bgn required Chordin to induce double axes in Xenopus. This work unveiled a new function for Bgn, its ability to regulate BMP4 signaling through modulation of Chordin anti-BMP4 activity.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteoglicanos/metabolismo , Transducción de Señal/fisiología , Xenopus/embriología , Animales , Biglicano , Tipificación del Cuerpo/fisiología , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/fisiología , Cartilla de ADN , Ectodermo/metabolismo , Proteínas de la Matriz Extracelular , Glicoproteínas/fisiología , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/fisiología , Mesodermo/metabolismo , Microinyecciones , Oligonucleótidos , Proteoglicanos/fisiología , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de XenopusRESUMEN
Collagen and elastin are thought to dominate the elasticity of the connective tissue including lung parenchyma. The glycosaminoglycans on the proteoglycans may also play a role because osmolarity of interstitial fluid can alter the repulsive forces on the negatively charged glycosaminoglycans, allowing them to collapse or inflate, which can affect the stretching and folding pattern of the fibers. Hence, we hypothesized that the elasticity of lung tissue arises primarily from 1) the topology of the collagen-elastin network and 2) the mechanical interaction between proteoglycans and fibers. We measured the quasi-static, uniaxial stress-strain curves of lung tissue sheets in hypotonic, normal, and hypertonic solutions. We found that the stress-strain curve was sensitive to osmolarity, but this sensitivity decreased after proteoglycan digestion. Images of immunofluorescently labeled collagen networks showed that the fibers follow the alveolar walls that form a hexagonal-like structure. Despite the large heterogeneity, the aspect ratio of the hexagons at 30% uniaxial strain increased linearly with osmolarity. We developed a two-dimensional hexagonal network model of the alveolar structure incorporating the mechanical properties of the collagen-elastin fibers and their interaction with proteoglycans. The model accounted for the stress-strain curves observed under all experimental conditions. The model also predicted how aspect ratio changed with osmolarity and strain, which allowed us to estimate the Young's modulus of a single alveolar wall and a collagen fiber. We therefore identify a novel and important role for the proteoglycans: they stabilize the collagen-elastin network of connective tissues and contribute to lung elasticity and alveolar stability at low to medium lung volumes.
Asunto(s)
Colágeno/fisiología , Colágeno/ultraestructura , Pulmón/citología , Pulmón/fisiología , Modelos Biológicos , Proteoglicanos/fisiología , Proteoglicanos/ultraestructura , Animales , Fenómenos Biomecánicos/métodos , Simulación por Computador , Elasticidad , Técnicas In Vitro , Rendimiento Pulmonar/fisiología , Masculino , Presión Osmótica , Ratas , Ratas Sprague-Dawley , Estrés MecánicoRESUMEN
A osteoartrose é uma doença crônica das articulações que, uma vez instalada, leva seus portadores a uma incapacidade funcional progressiva. Como os proteocondroitins sulfato são os maiores constituintes das cartilagens, espera-se que com a ingestão de glucosamina e condroitina haja uma melhora das condições biológicas desse tecido. Uma vez que não temos conhecimento de estudo da farmacocinética da administração oral dessa associação em seres humanos, o objetivo deste trabalho foi avaliá-la utilizando a associação entre o sulfato de glucosamina (SG) e o sulfato de condroitina (SC) administrada a dois grupos de doze voluntários sadios do sexo masculino (grupo I uma cápsula de (500 mg SG; 400 mg SC) e grupo II quatro cápsulas). Amostras de sangue foram retiradas a intervalos de tempo pré-definidos até 48 horas pós-dose. O SG e o SC foram dosados no plasma pelo método de DMMB (azul de 1,9,dimetildimetileno). A concentração máxima foi atingida em 2 horas (média ±SE; 0,893±0,093 'g/mL, grupo I e 2,222±0,313 'g/mL, grupo II). As áreas sob a curva até 48 horas foram de 10,803±0,965 'g-hr/mL e 38,776±2,981 'g-hr/mL, respectivamente para os grupos I e II. Os dois grupos apresentaram um segundo pico após 18 horas, indicando circulação êntero-hepática. Os nossos resultados indicam que essa associação é absorvida por via oral por mecanismo saturável, o que pode facilitar o seu uso em tratamentos clínicos.
Asunto(s)
Humanos , Masculino , Adolescente , Adulto , Glucosamina/administración & dosificación , Osteoartritis/fisiopatología , Polisacáridos/fisiología , Proteoglicanos/fisiología , Sulfatos de Condroitina/administración & dosificación , Osteoartritis/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Proteoglycans are abundant in the developing brain and there is much circumstantial evidence for their roles in directional neuronal movements such as cell body migration and axonal growth. We have developed an in vitro model of astrocyte cultures of the lateral and medial sectors of the embryonic mouse midbrain, that differ in their ability to support neuritic growth of young midbrain neurons, and we have searched for the role of interactive proteins and proteoglycans in this model. Neurite production in co-cultures reveals that, irrespective of the previous location of neurons in the midbrain, medial astrocytes exert an inhibitory or nonpermissive effect on neuritic growth that is correlated to a higher content of both heparan and chondroitin sulfates (HS and CS). Treatment of astrocytes with chondroitinase ABC revealed a growth-promoting effect of CS on lateral glia but treatment with exogenous CS-4 indicated a U-shaped dose-response curve for CS. In contrast, the growth-inhibitory action of medial astrocytes was reversed by exogenous CS-4. Treatment of astrocytes with heparitinase indicated that the growth-inhibitory action of medial astrocytes may depend heavily on HS by an as yet unknown mechanism. The results are discussed in terms of available knowledge on the binding of HS proteoglycans to interactive proteins, with emphasis on the importance of unraveling the physiological functions of glial glycoconjugates for a better understanding of neuron-glial interactions.
Asunto(s)
Axones/fisiología , Sulfatos de Condroitina/fisiología , Heparitina Sulfato/fisiología , Mesencéfalo/embriología , Neuronas/fisiología , Agrecanos , Animales , Astrocitos/efectos de los fármacos , Astrocitos/fisiología , División Celular/fisiología , Movimiento Celular , Células Cultivadas , Proteoglicanos de Heparán Sulfato/fisiología , Mesencéfalo/citología , Ratones , Neuroglía/fisiología , Polisacárido Liasas/farmacología , Proteoglicanos/fisiologíaRESUMEN
During limb skeletal muscle formation, committed muscle cells proliferate and differentiate in the presence of extracellular signals that stimulate or repress each process. Proteoglycans are extracellular matrix organizers and modulators of growth factor activities, regulating muscle differentiation in vitro. Previously, we characterized proteoglycan expression during early limb muscle formation and showed a spatiotemporal relation between the onset of myogenesis and the expression of decorin, an important muscle extracellular matrix component and potent regulator of TGF-beta activity. To evaluate decorin's role during in vivo differentiation in committed muscle cells, we grafted wild type and decorin-null myoblasts onto chick limb buds. The absence of decorin enhanced the migration and distribution of myoblasts in the limb, correlating with the inhibition of skeletal muscle differentiation. Both phenotypes were reverted by de novo decorin expression. In vitro, we determined that both decorin core protein and its glycosaminoglycan chain were required to reverse the migration phenotype. Results presented here suggest that the enhanced migration observed in decorin-null myoblasts may not be dependent on chemotactic growth factor signaling nor the differentiation status of the cells. Decorin may be involved in the establishment and/or coordination of a critical myoblast density, through inhibition of migration, that permits normal muscle differentiation during embryonic myogenesis.
Asunto(s)
Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteoglicanos/fisiología , Animales , Diferenciación Celular , Línea Celular , Movimiento Celular , Trasplante de Células , Embrión de Pollo , Técnicas de Cocultivo , Decorina , Proteínas de la Matriz Extracelular , Regulación del Desarrollo de la Expresión Génica , Vectores Genéticos , Esbozos de los Miembros/citología , Esbozos de los Miembros/crecimiento & desarrollo , Ratones , Músculo Esquelético/citología , Mioblastos Esqueléticos/citología , Miogenina/metabolismo , Retroviridae/genética , Factor de Crecimiento Transformador beta/metabolismo , Trasplante Heterólogo , beta-Galactosidasa/metabolismoRESUMEN
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/fisiología , Mielopoyesis/fisiología , Animales , División Celular/fisiología , Línea Celular , Extensiones de la Superficie Celular/ultraestructura , Células Cultivadas , Técnicas de Cocultivo , Células del Tejido Conectivo/fisiología , Ferritinas/análisis , Fibroblastos/citología , Fibroblastos/fisiología , Glicocálix/química , Glicocálix/ultraestructura , Glicosaminoglicanos/análisis , Glicosaminoglicanos/aislamiento & purificación , Glicosaminoglicanos/fisiología , Concentración de Iones de Hidrógeno , Indoles/análisis , Ratones , Ratones Endogámicos C3H , Microscopía Electrónica , Microscopía Electrónica de Rastreo , Microscopía de Contraste de Fase , Células Progenitoras Mieloides/química , Células Progenitoras Mieloides/fisiología , Células Progenitoras Mieloides/ultraestructura , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/metabolismo , Compuestos Organometálicos/análisis , Unión Proteica , Proteoglicanos/análisis , Proteoglicanos/fisiología , Seudópodos/ultraestructura , Rojo de Rutenio/análisis , Rojo de Rutenio/farmacología , Dispersión de Radiación , Células del Estroma/química , Células del Estroma/fisiología , Células del Estroma/ultraestructuraRESUMEN
Primordial germ cells are an embryonic cell line that give rise to gametes in vertebrates. They originate outside the embryo proper and migrate by a well-defined route to the genital ridges. Proteoglycans and glycosaminoglycans have distinctive properties that affect many of the characteristics of the extracellular microenvironment of migratory pathways in a variety of developmental systems. The purpose of this work was to identify the proteoglycans and glycosaminoglycans that are spatially and temporally expressed in the migratory pathway of primordial germ cells. We showed that the expression of proteoglycans and glycosaminoglycans in the primordial germ cells migratory pathway changes according to the different phases of the migratory process. Some molecules such as chondroitin-0-sulfate, decorin, and biglycan are present only in certain phases of the migratory process of primordial germ cells. Heparan sulfate, chondroitin-6-sulfate, versican, perlecan, and syndecan-4, although exhibiting some variation in expression were detected during all phases of the migratory process. Our results indicate that the successive steps of primordial germ cell migration require a coordinated expression of proteoglycans and glycosaminoglycans, that should be present in appropriate levels and in specific areas of the embryo, and that the sequential expression of these extracellular matrix molecules is under a genetic program that appears to be common to a variety of cell types during embryonic development.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Células Germinativas/citología , Células Germinativas/metabolismo , Glicosaminoglicanos/biosíntesis , Proteoglicanos/biosíntesis , Animales , Movimiento Celular , Embrión de Mamíferos , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/fisiología , Glicosaminoglicanos/clasificación , Glicosaminoglicanos/fisiología , Ratones , Proteoglicanos/clasificación , Proteoglicanos/fisiología , Factores de TiempoRESUMEN
Transcription of specific skeletal muscle genes requires the expression of the muscle regulatory factor myogenin. To assess the role of the extracellular matrix (ECM) in skeletal muscle differentiation, the specific inhibitors of proteoglycan synthesis, sodium chlorate and beta-D-xyloside, were used. Treatment of cultured skeletal muscle cells with each inhibitor substantially abolished the expression of creatine kinase and alpha-dystroglycan. This inhibition was totally reversed by the addition of exogenous ECM. Myoblast treatment with each inhibitor affected the deposition and assembly of the ECM constituents glypican, fibronectin, and laminin. These treatments did not affect MyoD, MEF2A, and myogenin expression and nuclear localization. Differentiated myoblast treatment with RGDS peptides completely inhibited myogenesis without affecting the expression or nuclear localization of myogenin. Integrin-mediated signaling of focal adhesion kinase was partially inhibited by chlorate and beta-D-xyloside, an effect reversed by the addition of exogenous ECM gel. These results suggested that the expression of myogenin is not sufficient to successfully drive skeletal muscle formation and that ECM is required to complete the skeletal muscle differentiation process.
Asunto(s)
Matriz Extracelular/fisiología , Músculo Esquelético/citología , Miogenina/fisiología , Animales , Biomarcadores , Diferenciación Celular/fisiología , Células Cultivadas , Matriz Extracelular/efectos de los fármacos , Glicósidos/farmacología , Ratones , Músculo Esquelético/fisiología , Proteoglicanos/fisiología , Factores de Transcripción/metabolismoRESUMEN
Members of the heparan sulfate proteoglycan family, the syndecans have emerged as integrators of extracellular signals, such as ECM components or growth factors, that activate cytoplasmic signaling cascades and regulate cytoskeletal functions. Specifically, syndecan-2 has been implicated in various cellular processes, from differentiation to migration, including its participation in cell-cell and cell-matrix adhesion. Here, we focused on the involvement of syndecan-2 in epithelial versus mesenchymal differentiation. Colorectal cancer-derived HT-29 M6 epithelial cells were stably transfected with full-length syndecan-2 cDNA, and the effect on cell morphology, adhesion, and mobility was evaluated. Characteristic features of migratory cells such as loss of intercellular contacts, flatter shape and multiple membrane projections were observed in syndecan-2 transfectants. Western blot analysis of the major component of epithelial adherens junctions, E-cadherin, revealed decreased expression levels. Furthermore, syndecan-2 induced stronger adhesion to collagen type I, specifically inhibited by heparin. This was correlated with an increased ability for migration, as demonstrated by wound healing experiments and transwell assays, without affecting their growth rate. These results indicate that syndecan-2 expression in mucus-secreting HT-29 M6 cells induces differentiation toward a migratory mesenchymal-like phenotype.