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1.
J Biol Chem ; 288(30): 21850-60, 2013 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-23766508

RESUMEN

SLC35A3 is considered the main UDP-N-acetylglucosamine transporter (NGT) in mammals. Detailed analysis of NGT is restricted because mammalian mutant cells defective in this activity have not been isolated. Therefore, using the siRNA approach, we developed and characterized several NGT-deficient mammalian cell lines. CHO, CHO-Lec8, and HeLa cells deficient in NGT activity displayed a decrease in the amount of highly branched tri- and tetraantennary N-glycans, whereas monoantennary and diantennary ones remained unchanged or even were accumulated. Silencing the expression of NGT in Madin-Darby canine kidney II cells resulted in a dramatic decrease in the keratan sulfate content, whereas no changes in biosynthesis of heparan sulfate were observed. We also demonstrated for the first time close proximity between NGT and mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetylglucosaminyltransferase (Mgat5) in the Golgi membrane. We conclude that NGT may be important for the biosynthesis of highly branched, multiantennary complex N-glycans and keratan sulfate. We hypothesize that NGT may specifically supply ß-1,3-N-acetylglucosaminyl-transferase 7 (ß3GnT7), Mgat5, and possibly mannosyl (α-1,3-)-glycoprotein ß-1,4-N-acetylglucosaminyltransferase (Mgat4) with UDP-GlcNAc.


Asunto(s)
Sulfato de Queratano/biosíntesis , Proteínas de Transporte de Membrana/metabolismo , Polisacáridos/biosíntesis , Interferencia de ARN , Animales , Secuencia de Bases , Transporte Biológico , Células CHO , Línea Celular , Línea Celular Tumoral , Cricetinae , Cricetulus , Perros , Transferencia Resonante de Energía de Fluorescencia , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Microscopía Confocal , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Análisis de Secuencia de ADN , Azúcares de Uridina Difosfato/metabolismo
2.
Exp Cell Res ; 319(7): 967-81, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23399832

RESUMEN

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Sulfato de Queratano/biosíntesis , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Proteoglicanos Tipo Condroitín Sulfato/deficiencia , Humanos , Integrina beta1/metabolismo , Sulfato de Queratano/deficiencia , Lumican , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Próstata/patología , Regulación hacia Arriba
3.
Cell Tissue Bank ; 15(1): 25-34, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23292197

RESUMEN

The in vivo quiescent corneal stroma keratocytes need to be transformed to activated state in order to obtain sufficient number of cells either for monolayer evaluation or corneal stroma reconstruction. This study aimed to investigate the phenotypic characterization of corneal stromal cells during culture expansion from the limbal region of the cornea. Isolated corneal keratocytes from limbal tissue of New Zealand White Strain rabbits' corneas (n = 6) were culture expanded until three passages. Keratocytes morphology was examined daily with viability, growth rate, number of cell doubling and population doubling time were recorded at each passage. The expression of collagen type 1, aldehyde dehydrogenase (ALDH), lumican and alpha smooth muscle actin (α-SMA) were detected by RT-PCR. Immunocytochemistry was also used to detect ALDH, α-SMA, collagen type I and Cytokeratin-3 (CK3). Growth kinetic study revealed that the growth rate was low at the initial passage but increase to about two folds with concomitant reduction in population doubling time in later passages. Freshly isolated and cultured keratocytes expressed collagen type 1, ALDH and lumican but α-SMA expression was absent. However, α-SMA was expressed along with the other genes during culture expansion. Keratocytes at P1 expressed all the proteins except CK3. These results suggest that cultured keratocytes maintained most of the gene expression profile of native keratocytes while the emergence of α-SMA in serial passages showed a mix population of various phenotypes. The phenotypic characterization of monolayer keratocytes provides useful information before reconstruction of bioengineered tissue or in vitro pharmaceutical applications.


Asunto(s)
Queratocitos de la Córnea/citología , Sustancia Propia/citología , Actinas/biosíntesis , Aldehído Deshidrogenasa/biosíntesis , Animales , Bioingeniería , Tratamiento Basado en Trasplante de Células y Tejidos , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Colágeno Tipo I/biosíntesis , Queratocitos de la Córnea/trasplante , Fibroblastos , Expresión Génica , Sulfato de Queratano/biosíntesis , Queratina-3/biosíntesis , Lumican , Fenotipo , Conejos
4.
Mol Genet Metab ; 106(2): 214-20, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22546219

RESUMEN

MPS disorders result from a deficiency or absence of glycosaminoglycan (GAG) degrading enzymes leading to an imbalance between the synthesis and degradation of GAGs and their subsequent accumulation in a range of cells. The inhibition of GAG synthesis using small chemical inhibitors has been proposed as a novel therapeutic approach to treatment. Several inhibitors have been shown to decrease heparan sulphate GAG synthesis and in this study we evaluated a novel fluorinated analog of N-acetylglucosamine (2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-D-glucopyranose (F-GlcNAc)) and rhodamine B for their ability to also inhibit the synthesis of chondroitin/dermatan and keratan sulphate GAGs present in bovine cartilage. Both inhibitors decreased GAG synthesis in chondrocyte monolayer culture and in cartilage chip explant culture in a dose dependent manner. Both inhibitors decreased the size of newly synthesised proteoglycans and in the case of F-GlcNAc this was due to a decrease in newly synthesised GAG chain size. Rhodamine B, however, did not affect GAG chain size, while both inhibitors decreased the amount of chondroitin/dermatan and keratan sulphate GAG equally. The expression of genes responsible for the initiation and elongation of chondroitin/dermatan sulphate and keratan sulphate GAGs were downregulated in the presence of rhodamine B but not in the presence of F-GlcNAc. Thus the 2 inhibitors appear to have differing effects on GAG synthesis, with F-GlcNAc inhibiting the epimerisation of UDP-GlcNAc to UDP-GalNAc thus decreasing the availability of monosaccharides for addition to the growing GAG chain, whereas rhodamine B is more likely to reduce the number of GAG chains. Together with previous data these 2 inhibitors are capable of non-specific inhibition of GAG synthesis, reducing the production of chondroitin/dermatan sulphate, keratan sulphate and heparan sulphate GAGs. As such they would be applicable to therapy in a range of MPS disorders.


Asunto(s)
Acetilglucosamina/análogos & derivados , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Glicosaminoglicanos/biosíntesis , Rodaminas/farmacología , Acetilglucosamina/farmacología , Animales , Cartílago/metabolismo , Bovinos , Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/biosíntesis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Sulfato de Queratano/biosíntesis , Mucopolisacaridosis/genética , Mucopolisacaridosis/metabolismo , Proteoglicanos/metabolismo
5.
Reprod Biol Endocrinol ; 9: 22, 2011 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-21294898

RESUMEN

BACKGROUND: We have previously demonstrated that four members of the family of small leucine-rich-proteoglycans (SLRPs) of the extracellular matrix (ECM), named decorin, biglycan, lumican and fibromodulin, are deeply remodeled in mouse uterine tissues along the estrous cycle and early pregnancy. It is known that the combined action of estrogen (E2) and progesterone (P4) orchestrates the estrous cycle and prepares the endometrium for pregnancy, modulating synthesis, deposition and degradation of various molecules. Indeed, we showed that versican, another proteoglycan of the ECM, is under hormonal control in the uterine tissues. METHODS: E2 and/or medroxiprogesterone acetate (MPA) were used to demonstrate, by real time PCR and immunoperoxidase staining, respectively, their effects on mRNA expression and protein deposition of these SLRPs, in the uterine tissues. RESULTS: Decorin and lumican were constitutively expressed and deposited in the ECM in the absence of the ovarian hormones, whereas deposition of biglycan and fibromodulin were abolished from the uterine ECM in the non-treated group. Interestingly, ovariectomy promoted an increase in decorin, lumican and fibromodulin mRNA levels, while biglycan mRNA conspicuously decreased. Hormone replacement with E2 and/or MPA differentially modulates their expression and deposition. CONCLUSIONS: The patterns of expression of these SLRPs in the uterine tissues were found to be hormone-dependent and uterine compartment-related. These results reinforce the existence of subpopulations of endometrial fibroblasts, localized into distinct functional uterine compartments, resembling the organization into basal and functional layers of the human endometrium.


Asunto(s)
Biglicano/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Decorina/biosíntesis , Estradiol/farmacología , Proteínas de la Matriz Extracelular/biosíntesis , Sulfato de Queratano/biosíntesis , Acetato de Medroxiprogesterona/farmacología , Proteoglicanos/biosíntesis , Útero/metabolismo , Animales , Matriz Extracelular/metabolismo , Femenino , Fibromodulina , Lumican , Ratones , Útero/efectos de los fármacos
6.
Dev Dyn ; 239(3): 844-54, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20108350

RESUMEN

Fibromodulin regulates collagen fibrillogenesis, but its existence/role(s) in the cornea is controversial. We hypothesize that fibromodulin regulates fibrillogenesis during postnatal development of the anterior eye. Fibromodulin is weakly expressed in the limbus at post-natal day (P) 4, increases and extends into the central cornea at P14, becomes restricted to the limbus at P30, and decreases at P60. This differential spatial and temporal expression of fibromodulin is coordinated with emmetropization; the developmental increase in axial length and globe size. Genetic analysis demonstrated that fibromodulin regulates fibrillogenesis in a region-specific manner. At the limbus, fibromodulin is dominant in regulating fibril growth during postnatal development. In the posterior peripheral cornea, cooperative interactions of fibromodulin and lumican regulate fibrillogenesis. These data indicate that fibromodulin plays important roles in the regulation of region-specific fibrillogenesis required for the integration of the corneal and scleral matrices and sulcus development required for establishment of the visual axis.


Asunto(s)
Colágeno/metabolismo , Córnea/embriología , Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteoglicanos/metabolismo , Animales , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Córnea/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/biosíntesis , Fibromodulina , Sulfato de Queratano/biosíntesis , Lumican , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión/métodos , Modelos Genéticos , Proteoglicanos/biosíntesis , ARN Mensajero/metabolismo , Tendones/metabolismo , Factores de Tiempo , Visión Ocular
7.
J Comp Neurol ; 528(1): 14-31, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31237692

RESUMEN

Induction of keratan sulfate in microglia has been found in several animal models of neurological disorders. However, the significance of keratan sulfate-expressing microglia is not fully understood. To address this issue, we analyzed the characteristics of microglia labeled by the 5D4 epitope, a marker of high-sulfated keratan sulfate, in the mouse hippocampus during the latent period after pilocarpine-induced status epilepticus (SE). Only 5D4-negative (5D4- ) microglia were found in the CA1 region of vehicle-treated controls and pilocarpine-treated mice at 1 day after SE onset. A few 5D4+ microglia appeared in the strata oriens and radiatum at 5 days post-SE, and they were distributed into the stratum pyramidale at 14 days post-SE. The expressions of genes related to both anti- and pro-inflammatory cytokines were higher in 5D4+ cells than in 5D4- cells at 5 but not 14 days post-SE. The expressions of genes related to phagocytosis were higher in 5D4+ cells than in 5D4- cells throughout the latent period. The phagocytic activity of microglia, as measured by engulfment of the zymosan bioparticles, was higher in 5D4+ cells than in 5D4- cells. The contact ratios between excitatory synaptic boutons and microglia were also higher in 5D4+ cells than in 5D4- cells at 5 and 14 days post-SE. The excitatory/inhibitory ratios of synaptic boutons within the microglial domain were lower in 5D4+ cells than in 5D4- cells at 14 days post-SE. Our findings indicate that 5D4+ microglia may play some role in epileptogenesis via pruning of excitatory synapses during the latent period after SE.


Asunto(s)
Hipocampo/metabolismo , Sulfato de Queratano/biosíntesis , Microglía/metabolismo , Pilocarpina/toxicidad , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismo , Animales , Citometría de Flujo/métodos , Hipocampo/citología , Masculino , Ratones , Ratones Endogámicos ICR
8.
Trends Biochem Sci ; 17(9): 340-3, 1992 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-1412708

RESUMEN

Avascular connective tissues (cartilage, discs, cornea) change with maturation and aging, particularly in large animals, where diffusion paths are longest. It is suggested that the changes in such tissues are responses to increasing difficulties in obtaining oxygen. Two almost identical structural polymers are made in these tissues: chondroitin sulphate, which requires large amounts of oxygen for biosynthesis and keratan sulphate, which requires relatively little. The observed balance of these polymers in the tissue is proposed to depend on the control of biosynthesis by the ambient oxygen tension, and/or selective breakdown.


Asunto(s)
Tejido Conectivo/ultraestructura , Consumo de Oxígeno/fisiología , Animales , Secuencia de Carbohidratos , Cartílago/metabolismo , Sulfatos de Condroitina/biosíntesis , Tejido Conectivo/metabolismo , Córnea/metabolismo , Disco Intervertebral/metabolismo , Sulfato de Queratano/biosíntesis , Datos de Secuencia Molecular
9.
Wound Repair Regen ; 16(6): 814-23, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-19128253

RESUMEN

Wound healing in oral mucosa is fast and results in little scar formation as compared with skin. The biological mechanisms underlying this property are poorly understood but may provide valuable information about the factors that promote wound regeneration. Small leucine-rich proteoglycans (SLRPs) decorin, biglycan, fibromodulin and lumican are extracellular matrix molecules that regulate collagen fibrillogenesis, inhibit transforming growth factor-beta (TGF-beta) activity and reduce scarring. In the present study, we analyzed accumulation of SLRPs and TGF-beta during non-scarring human oral mucosal wound healing. Biopsies were collected from healthy volunteers from unwounded tissue and from standardized experimental wounds 3-60 days postwounding. Localization of SLRPs, TGF-beta1 and TGF-beta3 was analyzed by immunohistochemical staining and quantitated by image analysis. Double immunostaining was used to study localization of SLRPs or active TGF-beta in distinct cells. Decorin, biglycan, fibromodulin, and TGF-beta isoforms showed significantly increased accumulation in the wound extracellular matrix and distinct wound cells while the abundance of lumican in the extracellular matrix was strongly reduced during wound healing. Localization and abundance of fibromodulin, lumican, and TGF-beta isoforms was also spatiotemporally regulated in the wound epithelium. The findings suggest that SLRPs regulate wound reepithelialization and connective tissue regeneration during oral mucosal wound healing.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Proteínas de la Matriz Extracelular/análisis , Sulfato de Queratano/análisis , Mucosa Bucal , Proteoglicanos/análisis , Factor de Crecimiento Transformador beta/análisis , Cicatrización de Heridas , Biglicano , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Fibromodulina , Humanos , Sulfato de Queratano/biosíntesis , Lumican , Proteoglicanos/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis
10.
Invest Ophthalmol Vis Sci ; 59(3): 1641-1651, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29625490

RESUMEN

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine-transferring enzyme, ß-1,3-N-acetylglucosaminyltransferase-7 (ß3GnT7). A mouse model deficient in ß3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the ß3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via ß3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without ß3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs.


Asunto(s)
Sustancia Propia/metabolismo , Sulfato de Queratano , N-Acetilglucosaminiltransferasas/deficiencia , Animales , Modelos Animales de Enfermedad , Sulfato de Queratano/biosíntesis , Sulfato de Queratano/fisiología , Ratones , Ratones Noqueados , Fenotipo
11.
J Dermatol Sci ; 47(3): 217-26, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17601707

RESUMEN

BACKGROUND: Collagen biosynthesis and deposition is a complex, multistep process, which is tightly regulated to maintain proper tissue homeostasis. Sex steroid hormones have been implicated in regulating collagen synthesis; however the specific mechanisms regulating the process remain largely unknown. OBJECTIVE: To investigate the role of estrogens and androgens in the regulation of genes involved in collagen synthesis and fibrillogenesis using gonadectomized C57/B6 mice. METHODS: Collagen content was assessed by hydroxyproline measurement and acetic acid extraction of collagen with or without the addition of pepsin. The mRNA levels of fibrillar collagens and enzymes involved in fibrillogenesis were determined by QPCR analysis. The protein expression of decorin, lumican and fibromodulin was confirmed by immunostaining. RESULTS: We have shown that castration resulted in a markedly decreased skin thickness and collagen content without affecting collagen solubility. Furthermore, the mRNA levels of fibrillar collagen genes including types I, III, and V were decreased, suggesting that androgens positively regulate the rate of collagen gene transcription. Conversely, ovariectomy mainly affected collagen solubility. The absence of estrogens resulted in decreased expression levels of several of the small leucine-rich repeat proteins and proteoglycans (SLRPs) including decorin, fibromodulin and lumican. CONCLUSIONS: Estrogens may not be directly involved in the regulation of collagen synthesis; however, they may play a critical role in regulating organization and stability of collagen fibrils. Androgens play a positive role in the regulation of collagen biosynthesis. In summary, our data demonstrate that androgens and estrogens regulate distinct aspects of collagen fibrillogenesis in mouse skin.


Asunto(s)
Colágeno/metabolismo , Piel/metabolismo , Animales , Castración , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Femenino , Fibromodulina , Hormonas Esteroides Gonadales/metabolismo , Sulfato de Queratano/biosíntesis , Lumican , Masculino , Ratones , Ovariectomía , Pepsina A/metabolismo , Proteoglicanos/biosíntesis , ARN Mensajero/metabolismo , Factores Sexuales
12.
Curr Opin Struct Biol ; 13(5): 605-11, 2003 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14568616

RESUMEN

Most of the sulfotransferases participating in glycosaminoglycan biosynthesis have now been identified. Their essential role in generating binding sites for proteins interacting with glycosaminoglycans is apparent. These interactions may influence important biological processes such as growth control, signal transduction, cell adhesion and lipid metabolism. Gene targeting in mice as well as studies in Drosophila melanogaster and Caenorhabditis elegans have shown that dysfunction or lack of glycosaminoglycan sulfotransferases may result in severely disturbed embryonic development.


Asunto(s)
Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/química , Sulfotransferasas/química , Sulfotransferasas/metabolismo , Animales , Sulfatos de Condroitina/biosíntesis , Sulfatos de Condroitina/química , Dermatán Sulfato/biosíntesis , Dermatán Sulfato/química , Heparitina Sulfato/biosíntesis , Heparitina Sulfato/química , Humanos , Sulfato de Queratano/biosíntesis , Sulfato de Queratano/química , Ratones , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo
13.
J Biomech ; 40(12): 2686-93, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17346717

RESUMEN

Although growth plate response to mechanical stress has been increasingly studied, our understanding of mechanical modulation of neonatal growth plate is incomplete, especially concerning biochemical changes. This study was designed to explore the cellular and biochemical responses of the cranial base growth plate (CBGP) explant upon cyclic loading. The growth plate with subchondral bone was aseptically isolated from each of 24 neonatal rabbits and fixated in an organ culture system. Cyclic loading was applied to growth plate explants at 200 mN and 1 Hz for 60 min (N=12), whereas control explants were immersed in organ culture for 60 min without mechanical loading (N=12). Computerized image analysis revealed that cyclic loading induced significantly more proliferating chondrocytes than unloaded controls (p<0.001), as well as significantly higher growth plate height at 856+/-30 microm than the unloaded controls at 830+/-36 microm (p<0.05). Immunoblotting with monoclonal antibodies (mAb) disclosed that the average mAb binding area for chondroitin sulfate was significantly higher in the loaded specimens than the unloaded controls at (p<0.001). The average mAb binding area for keratan sulfate was also significantly higher in the loaded specimens than the unloaded controls (p<0.01). Biochemical analysis showed that the average total hyaluronan content of loaded specimens at 0.25+/-0.06 microg/microg DNA was significantly higher than the unloaded controls at 0.09+/-0.05 microg/microg DNA (p<0.01). Taken together, these data suggest that brief doses of cyclic, intermittent forces activate cellular and molecular responses in the CBGP ex vivo. Whether hyaluronan-mediated pathway is involved in the biological responses of growth plate to mechanical loading warrants additional investigations.


Asunto(s)
Placa de Crecimiento/metabolismo , Ácido Hialurónico/biosíntesis , Mecanotransducción Celular , Base del Cráneo/metabolismo , Animales , Animales Recién Nacidos , Sulfatos de Condroitina/biosíntesis , Placa de Crecimiento/patología , Sulfato de Queratano/biosíntesis , Técnicas de Cultivo de Órganos , Conejos , Base del Cráneo/patología , Estrés Mecánico
14.
Invest Ophthalmol Vis Sci ; 47(12): 5260-6, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17122111

RESUMEN

PURPOSE: Ascorbate is required for the hydroxylation of collagen that is present in the corneal stroma. The keratan sulfate proteoglycans (KSPGs) lumican and keratocan are also present, and they interact with collagen and modulate its assembly into fibrils. In this study, ascorbate was added to a defined medium containing insulin, and its effects on the synthesis of collagen and KSPGs by keratocytes were determined. METHODS: Collagenase-isolated keratocytes were cultured with or without insulin with or without ascorbate. Collagen and glycosaminoglycan synthesis was determined by collagenase digestion of incorporated 3H-glycine and by chondroitinase ABC or endo-beta-galactosidase digestion of incorporated 35SO4. KSPGs were detected by Western blot. Collagen stability was determined by pepsin digestion. Ethyl-3,4-dihydroxybenzoate (EDB) was used to inhibit collagen hydroxylation. RESULTS: Insulin stimulated the synthesis of collagen but did not affect the accumulation of lumican and keratocan. Insulin plus ascorbate, however, stimulated the synthesis of collagen and increased the accumulation of these proteoglycans. The accumulation of PGDS, a KSPG that does not interact with collagen, was not affected by ascorbate. Only the collagen synthesized in the presence of ascorbate was pepsin resistant. EDB overrode the effects of ascorbate on pepsin resistance and proteoglycan accumulation. CONCLUSIONS: The results of this study indicate that the accumulation of lumican and keratocan depends in part on the level of collagen synthesis and its hydroxylation. The interaction of lumican and keratocan with the stably folded triple helix provided by hydroxylation may also serve to stabilize these proteoglycans.


Asunto(s)
Ácido Ascórbico/farmacología , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Colágeno/biosíntesis , Sustancia Propia/citología , Fibroblastos/efectos de los fármacos , Insulina/farmacología , Sulfato de Queratano/biosíntesis , Proteoglicanos/biosíntesis , Animales , Western Blotting , Bovinos , Proliferación Celular , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Fibroblastos/metabolismo , Hidroxilación , Lumican
15.
J Orthop Res ; 24(1): 71-9, 2006 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-16419971

RESUMEN

The effects of growth and differentiation factor-5 (GDF-5) on ligament healing were studied using a gap injury model of the medial collateral ligament in rat knee joints. The administration of GDF-5 once at the time of surgery significantly improved the mechanical properties of the femur-ligament-tibia complex. At 3 weeks after surgery, 30 microg of GDF-5 improved the ultimate tensile strength of the complex by 41%, and the stiffness by 60%, compared with the vehicle control (p < 0.05 for both; Fisher's PLSD test). The observation with a transmission electron microscopy revealed that GDF-5 increased the diameter of collagen fibrils in the repair tissue, which was considered to be a possible mechanism for the positive result in the biomechanical testing. Quantitative PCR and in situ hybridization revealed enhanced type I procollagen expression by GDF-5, and the PCR analysis also revealed that the GDF-5 treatment reduced the expression of type III procollagen relative to type I procollagen. The PCR analysis further showed that the expression of decorin and fibromodulin was relatively reduced against type I procollagen by the growth factor, which was considered to be responsible for the increase of collagen fibril diameter in the repair tissue. No adverse effects were observed, and the use of GDF-5 was considered a promising approach to facilitate ligament healing.


Asunto(s)
Proteínas Morfogenéticas Óseas/farmacología , Ligamentos Articulares/lesiones , Cicatrización de Heridas/efectos de los fármacos , Animales , Fenómenos Biomecánicos , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Decorina , Proteínas de la Matriz Extracelular/biosíntesis , Fibromodulina , Factor 5 de Diferenciación de Crecimiento , Hibridación in Situ , Sulfato de Queratano/biosíntesis , Ligamentos Articulares/efectos de los fármacos , Ligamentos Articulares/ultraestructura , Lumican , Masculino , Procolágeno/análisis , Proteoglicanos/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
Oncol Rep ; 16(6): 1225-30, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17089042

RESUMEN

Lumican is a member of a small leucine-rich proteoglycan family, and it is reportedly overexpressed in human breast cancer. The expression of lumican in the extracellular matrix in breast cancer is associated with a high tumor grade, low estrogen receptor levels and young age. Lumican expression has been previously reported in colorectal cancer, but the role of lumican in the tumor is not well understood. In this study, we examined the expression and role of lumican in advanced colorectal cancer. Immunohistochemical staining was performed on 158 patients who underwent curative surgery for advanced colorectal cancer with lymph node metastasis. In the normal colorectal tissues, lumican immunoreactivity was observed in the fibroblasts and neural cells, but not in the colorectal epithelial cells. Lumican was localized in the cytoplasm of the cancer cells and its overexpression was detected in 99 of the 158 (62.7%) colorectal cancer patients. Clinicopathologically, there was no association of lumican expression with age, sex, histological typing, or venous and lymphatic invasion. However, lumican expression tended to correlate with the spread of lymph node metastasis and the depth of tumor invasion (p=0.136 and 0.135, respectively). Furthermore, the survival rate was significantly lower in patients with a high lumican expression level than in those with a low lumican expression level (p=0.048). These results indicate that lumican expression is a potential prognostic factor in patients with advanced colorectal cancer with nodal metastasis.


Asunto(s)
Adenocarcinoma/metabolismo , Biomarcadores de Tumor/análisis , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Sulfato de Queratano/biosíntesis , Metástasis Linfática/patología , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Inmunohistoquímica , Lumican , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de Supervivencia , Tasa de Supervivencia
17.
Cancer Res ; 58(7): 1348-52, 1998 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-9537227

RESUMEN

Lumican mRNA has been identified as being differentially expressed between different regions of the same human breast tumor. In situ hybridization study of 26 independent breast tumors confirmed the presence of lumican mRNA in fibroblast-like cells within stroma and showed a significant increase of its expression in tumor compared to adjacent normal stroma (P < 0.001). Higher lumican expression was associated with higher tumor grade, lower estrogen receptor levels in the tumor, and younger age of the patients (P < 0.05). Reverse transcription-PCR analysis of total RNA extracted from 19 independent breast tissues exhibiting lesions that are thought to parallel tumor progression also suggests that this proteoglycan is differentially expressed during tumor progression.


Asunto(s)
Neoplasias de la Mama/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Sulfato de Queratano/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Mama/metabolismo , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Hibridación in Situ , Lumican , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Dodecil Sulfato de Sodio , Transcripción Genética , Células Tumorales Cultivadas
18.
Cancer Res ; 59(18): 4715-9, 1999 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-10493530

RESUMEN

Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and they are key components of germ cell tumors (GCTs). They express several high molecular weight glycoprotein antigens that are down-regulated upon differentiation. One of these antigens, defined by monoclonal antibody TRA-1-60, can be detected in the serum of GCT patients and provides a useful complement to the established serum markers human chorionic gonadotropin and alpha-fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or alpha-fetoprotein. To examine the relationship of the TRA-1-60-defined antigen to similar antigens defined by other monoclonal antibodies, we have carried out comparative Western blot and immunoprecipitation analyses of human GCT-derived cell lines with monoclonal antibodies TRA-1-60, TRA-1-81, GCTM2, and K21. The TRA-1-60 antigen was detected by Western blot analysis in extracts of all human EC cell lines and in clinical specimens of GCT tested as a diffuse band with a molecular weight of >200,000. A similar but noticeably fainter band was detected in GCT composed of seminoma only. The antigen was not expressed by GCT-derived lines without an EC phenotype. Affinity bead-purified TRA-1-60, TRA-1-81, GCTM2 and K21 antigens reacted in Western blot analysis with each of the other antibodies tested, indicating that the epitopes recognized by each antibody are carried by the same molecular species. This molecule could be metabolically labeled with inorganic [35S]sulfate and was degraded by keratanase. Glycopeptides produced from affinity-purified TRA-1-60 antigen by extensive digestion with Pronase exhibited a molecular weight in excess of 10,000 and were degraded by keratanase. The TRA-1-60 epitope was destroyed by digestion with neuraminidase, but the epitopes defined by TRA-1-81, GCTM2, and K21 were not. Our results indicate that human EC cells generally express a cell surface sialylated keratan sulfate proteoglycan that is subject to modification to yield a variety of epitopes, one of which is recognized by the monoclonal antibody TRA-1-60. Sensitivity to milk alkaline digestion suggests that the oligosaccharides of this proteoglycan are O-linked to a core polypeptide.


Asunto(s)
Biomarcadores de Tumor/análisis , Proteoglicanos Tipo Condroitín Sulfato/análisis , Glicopéptidos/química , Glicoproteínas/análisis , Sulfato de Queratano/análisis , Ácido N-Acetilneuramínico/análisis , Anticuerpos Monoclonales , Antígenos de Neoplasias/análisis , Antígenos de Superficie , Biomarcadores de Tumor/biosíntesis , Western Blotting , Carcinoma Embrionario , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Proteoglicanos Tipo Condroitín Sulfato/química , Glicopéptidos/aislamiento & purificación , Glicoproteínas/biosíntesis , Glicoproteínas/química , Humanos , Sulfato de Queratano/biosíntesis , Sulfato de Queratano/química , Lumican , Proteoglicanos , Sulfatos/metabolismo , Células Tumorales Cultivadas
19.
J Neurosci ; 22(11): 4611-24, 2002 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-12040068

RESUMEN

Keratan sulfate proteoglycans (KSPGs) are extracellular matrix molecules that appear to establish boundaries for axonal growth in the developing brain and spinal cord. In vitro studies confirm that KSPGs define inhibitory boundaries to extending neurites. The aim of the current study was to investigate whether KSPGs are expressed after spinal cord injury (SCI) and thereby might act as potential inhibitors of axonal growth. Adult Fischer 344 rats were subjected to spinal cord lesions, and the temporal and spatial expression of KSPGs was examined using the 5D4 monoclonal anti-KSPG antibody. In the intact spinal cord, a subpopulation of microglia expressed 5D4-KSPG throughout the white and gray matter. Within 24 hr of injury, 5D4-KSPG immunoreactivity substantially increased and appeared on cellular profiles in close proximity to the spinal cord lesion site, peaking 3 d after injury. Double immunolabeling revealed that 5D4-KSPG expression arose from multiple cell types at the lesion site, including reactive microglia, macrophages, and oligodendrocyte progenitors. Astrocytes were not identified as a source of 5D4-KSPG. The robust and extensive production of 5D4-KSPG at sites of SCI precedes the expression of other putatively inhibitory proteoglycan molecules such as chondroitin sulfate proteoglycans. This is the first demonstration that KSPGs are expressed after SCI in a temporal and spatial relationship that could exert an early and important role in modulating axonal growth after SCI.


Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Sulfato de Queratano/biosíntesis , Macrófagos/metabolismo , Microglía/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Células Madre/metabolismo , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Inmunohistoquímica , Lumican , Macrófagos/patología , Microglía/patología , Oligodendroglía/metabolismo , Oligodendroglía/patología , Ratas , Ratas Endogámicas F344 , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/patología , Traumatismos de la Médula Espinal/patología , Células Madre/patología
20.
Biochim Biophys Acta ; 675(1): 132-9, 1981 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-6789884

RESUMEN

Sulfated glycosaminoglycans of cultured bovine lens epithelial cells grown in the presence and in the absence of a retinal growth factor were investigated comparatively. The newly formed [35S]sulfate-labeled glycosaminoglycans were analysed in the extra-, peri- and intracellular compartments of early (4--5th) and late 17--18 h) subcultures. The following results were obtained: (1) Cultured lens epithelial cells grown in the presence or in the absence of the growth factor synthesize chondroitin 4- and 6-sulfates and dermatan sulfate, with heparan sulfate as the main component, the pericellular compartments were particularly rich in heparan sulfate; (2) The distribution pattern of the glycosaminoglycans changes during successive subcultures; the proportion of heparan sulfate increases in the pericellular compartment, the dermatan sulfate to chondroitin sulfate ratio increases in all three compartments; (3) IN contrast to the drastic decrease in the fibronectin levels in the presence of growth factor in the early subcultures, only minor differences were found between the glycosaminoglycan patterns of the treated and non-treated cells.


Asunto(s)
Glicosaminoglicanos/biosíntesis , Sustancias de Crecimiento/farmacología , Cristalino/metabolismo , Animales , Bovinos , Células Cultivadas , Sulfatos de Condroitina/biosíntesis , Dermatán Sulfato/biosíntesis , Epitelio/metabolismo , Heparitina Sulfato/biosíntesis , Sulfato de Queratano/biosíntesis , Cinética , Retina/fisiología
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