RESUMO
BACKGROUND: Numerous studies have reported on the health benefits of sesamin, a major lignin found in sesame (S. indicum) seeds. Recently, sesamin was shown to have the ability to promote chondroitin sulfate proteoglycan synthesis in normal human chondrocytes. This study assesses the anti-inflammatory effect of sesamin on proteoglycans production in 3D chondrocyte cultures. METHODS: To evaluate the effects of sesamin on IL-1ß-treated human articular chondrocytes (HAC) pellets, the pellets were pre-treated with IL-1ß then cultured in the presence of various concentrations of sesamin for 21 days. During that period, the expression of IL-1ß, glycosaminoglycans (GAGs) content and Chondroitin sulfate proteoglycans (CSPGs) synthesis genes (ACAN, XT-1, XT-2, CHSY1 and ChPF) was measured. The GAGs accumulation in the extracellular matrix was determined on day 21 by histological analysis. RESULTS: There was clear evidence that sesamin upregulated expression of all the CSPGs synthesis genes, in contrast to the down-regulation of IL-1ß expression both in genes and in protein levels. The level of release and matrix accumulation of GAGs in IL-1ß pre-treated HAC pellets in the presence of sesamin was recovered. These results correlate with the histological examination which showed that sesamin enhanced matrix CSPGs accumulation. CONCLUSIONS: Sesamin enhances CSPGs synthesis, suppresses IL-1ß expression and ameliorates IL-1ß induced inflammation in human chondrocytes. Sesamin could have therapeutic benefits for treating inflammation in osteoarthritis.
Assuntos
Condrócitos/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Dioxóis/farmacologia , Interleucina-1beta/metabolismo , Lignanas/farmacologia , Adulto , Agrecanas/genética , Agrecanas/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Feminino , Glucuronosiltransferase , Humanos , Masculino , Pessoa de Meia-Idade , Enzimas Multifuncionais , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Adulto JovemRESUMO
To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH(4)OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
Assuntos
Ácido Ascórbico/farmacologia , Técnicas de Cultura de Células , Córnea/efeitos dos fármacos , Ceratócitos da Córnea/efeitos dos fármacos , Engenharia Tecidual/métodos , Ácido Valproico/farmacologia , Animais , Apoptose , Bovinos , Proliferação de Células , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Córnea/metabolismo , Citometria de Fluxo/métodos , Sulfato de Queratano/biossíntese , Lumicana , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Proteoglicanas/biossíntese , Coelhos , Vimentina/biossínteseRESUMO
Our previous study has reported that electroacupuncture (EA) promotes survival, differentiation of bone marrow mesenchymal stem cells (MSCs), and functional improvement in spinal cord-transected rats. In this study, we further investigated the structural bases of this functional improvement and the potential mechanisms of axonal regeneration in injured spinal cord after MSCs and EA treatment. Five experimental groups, 1) sham control (Sham-control); 2) operated control (Op-control); 3) electroacupuncture treatment (EA); 4) MSCs transplantation (MSCs), and 5) MSCs transplantation combined with electroacupuncture (MSCs + EA), were designed for this study. Western blots and immunohistochemical staining were used to assess the fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs) proteins expression. Basso, Beattie, Bresnahan (BBB) locomotion test, cortical motor evoked potentials (MEPs), and anterograde and retrograde tracing were utilized to assess cortical-spinal neuronal projection regeneration and functional recovery. In the MSCs + EA group, increased labeling descending corticospinal tract (CST) projections into the lesion site showed significantly improved BBB scales and enhanced motor evoked potentials after 10 weeks of MSCs transplant and EA treatment. The structural and functional recovery after MSCs + EA treatment may be due to downregulated GFAP and CSPGs protein expression, which prevented axonal degeneration as well as improved axonal regeneration.
Assuntos
Axônios/fisiologia , Eletroacupuntura/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos da Medula Espinal/terapia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Proteína Glial Fibrilar Ácida/biossíntese , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/metabolismo , Análise de SobrevidaRESUMO
Axonal injury is a major hallmark of traumatic brain injury (TBI), and it seems likely that therapies directed toward enhancing axon repair could potentially improve functional outcomes. One potential target is chondroitin sulfate proteoglycans (CSPGs), which are major axon growth inhibitory molecules that are generally, but not always, up-regulated after central nervous system injury. The current study was designed to determine temporal changes in cerebral cortical mRNA or protein expression levels of CSPGs and to determine their regional localization and cellular association by using immunohistochemistry in a controlled cortical impact model of TBI. The results showed significant increases in versican mRNA at 4 and 14 days after TBI but no change in neurocan, aggrecan, or phosphacan. Semiquantitative Western blot (WB) analysis of cortical CSPG protein expression revealed a significant ipsilateral decrease of all CSPGs at 1 day after TBI. Lower CSPG protein levels were sustained until at least 14 days, after which the levels began to normalize. Immunohistochemistry data confirm previous reports of regional increases in CSPG proteins after CNS injury, seen primarily within the developing glial scar after TBI, but also corroborate the WB data by revealing wide areas of pericontusional tissue that are deficient in both extracellular and perineuronal net-associated CSPGs. Given the evidence that CSPGs are largely inhibitory to axonal growth, we interpret these data to indicate a potential for regional spontaneous plasticity after TBI. If this were the case, the gradual normalization of CSPG proteins over time postinjury would suggest that this may be temporally as well as regionally limited.
Assuntos
Lesões Encefálicas/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Regulação da Expressão Gênica , Proteínas do Tecido Nervoso/biossíntese , Animais , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Proteoglicanas de Sulfatos de Condroitina/genética , Cicatriz/etiologia , Cicatriz/genética , Cicatriz/metabolismo , Cicatriz/patologia , Gliose/etiologia , Gliose/genética , Gliose/metabolismo , Gliose/patologia , Masculino , Córtex Motor/lesões , Córtex Motor/metabolismo , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/lesões , Córtex Somatossensorial/metabolismo , Fatores de Tempo , Versicanas/biossíntese , Versicanas/genética , CicatrizaçãoRESUMO
BEHAB (brain-enriched hyaluronan-binding protein)/brevican is the most abundant chondroitin sulfate proteoglycan in the extracellular matrix of the adult rat brain. BEHAB/brevican expression is up-regulated coincident with glial cell proliferation and/or motility, including during early central nervous system development and in invasive glioma. An understanding of the molecular interactions that mediate BEHAB/brevican function is still in its infancy because of the existence of several BEHAB/brevican isoforms, each of which may mediate different functions. Here, we describe a novel BEHAB/brevican isoform, B/b130, and demonstrate that it is neither the glycosylphosphatidylinositol-linked splice variant of BEHAB/brevican nor a cleavage product of the full-length protein (B/b150). B/b130 is an underglycosylated isoform of BEHAB/brevican, lacking glycosaminoglycan chains as well as most of the sugars that invest B/b150. B/b130 localizes exclusively to the particulate fraction of rat brain and associates with the cell membrane by a previously undescribed calcium-independent mechanism. In addition, B/b130 is the major isoform of BEHAB/brevican that is up-regulated in a rat model of invasive glioma and may therefore contribute to the invasive ability of glioma cells. Further understanding of BEHAB/brevican isoforms will advance our knowledge of the function of this ECM component and may help identify new potential therapeutic targets for primary brain tumors.
Assuntos
Encéfalo/metabolismo , Proteínas de Transporte/biossíntese , Proteínas de Transporte/química , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/química , Glioma/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Regulação para Cima , Animais , Western Blotting , Encéfalo/embriologia , Brevicam , Linhagem Celular , Membrana Celular/metabolismo , DNA Complementar/metabolismo , Matriz Extracelular/metabolismo , Feminino , Glicosídeo Hidrolases/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Imunoquímica , Imuno-Histoquímica , Lectinas Tipo C , Camundongos , Microscopia de Fluorescência , Invasividade Neoplásica , Ligação Proteica , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Frações Subcelulares/metabolismo , TransfecçãoRESUMO
The differentiation and morphogenesis of neural tissues involves a diversity of interactions between neural cells and their environment. Many potentially important interactions occur with the extracellular matrix (ECM), a complex association of extracellular molecules organised into aggregates and polymers. The large modular glycoprotein, Tenascin-C, and the chondroitin sulphate proteoglycan, DSD-1-PG/Phosphacan, have complex and frequently overlapping expression patterns in the developing CNS. Their presence in zones of cell proliferation, migration, and differentiation, as well as in boundary structures, suggest that they may be involved in the modulation of an extensive range of cellular processes. They are both strongly up-regulated in a range of CNS lesions and pathologies, being components of the glial scar, and expressed by gliomas. Functional roles in many cellular processes are possible through their extensive molecular interaction sites, both with each other, and with many of the same cell surface receptors, adhesion molecules, growth factors and other matrix proteins. These multiple interactions involve sites on both their protein domains and on the heterogeneous carbohydrate groups with which they are post-translationally modified. In vitro assays demonstrate cell-type specific effects on adhesion, migration and the formation and extension of cellular processes, including neurites and axons.
Assuntos
Sulfatos de Condroitina/metabolismo , Matriz Extracelular/química , Matriz Extracelular/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Tenascina/fisiologia , Animais , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/química , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Proteoglicanas de Sulfatos de Condroitina/fisiologia , Sulfatos de Condroitina/biossíntese , Sulfatos de Condroitina/química , Sulfatos de Condroitina/fisiologia , Matriz Extracelular/metabolismo , Humanos , Ligantes , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Tenascina/biossíntese , Tenascina/químicaRESUMO
Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10(5) cell/cm2 and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and beta-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro.
Assuntos
Calcificação Fisiológica , Condrócitos/citologia , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Proteoglicanas , Agrecanas , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Calcificação Fisiológica/genética , Diferenciação Celular/genética , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Proteoglicanas de Sulfatos de Condroitina/genética , Colágeno/biossíntese , Feto , Glicoproteínas/biossíntese , Glicoproteínas/genética , Lectinas Tipo C , Osteocalcina/biossíntese , Osteocalcina/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
We used immunohistochemistry to examine possible molecular interactions between the subplate and growing thalamocortical axons in rat fetuses. In the cortical anlage of embryonic day 16 (E16), the subplate first appeared below the cortical plate. Among chondroitin sulfate proteoglycans, phosphacan was uniformly distributed throughout the cortical wall, whereas neurocan was localized only in the subplate at E16. Neural cell adhesion molecules, NCAM-H, TAG-1, and L1, were detected in the cortical anlage. Both cortical neurons and growing axons were diffusely immunopositive for NCAM-H, and TAG-1 immunoreactivity was found on immature neurons and cortical efferent axons but not on thalamocortical axons. L1 immunoreactivity was specifically localized on the growing thalamocortical axons. When the locations of neurocan and L1 were compared in the developing cortex, L1-bearing axons were found to extend to neurocan-immunopositive regions; neurocan immunoreactivity was intense in the subplate at E16, when small numbers of L1-immunoreactive thalamocortical axons began to invade the cortex. At E17, many L1-positive axons were observed in the subplate that expressed neurocan specifically. Double immunostaining showed that L1-positive axons and neurocan immunoreactivity overlapped in the subplate at E17. After E18, neurocan expression gradually extended to the lower part of the cortical plate; it extended to the entire cortex by E21, 1 day before birth. By E21, L1-bearing axons had invaded the lower part of the cortical plate. The present study demonstrated that the neurocan expression precedes growth of L1-bearing thalamocortical afferent fibers. Because neurocan can bind to L1 molecule in vitro, these results suggest that neurocan and L1 play some important roles in pathfinding of the thalamocortical afferent fibers during rat corticogenesis.
Assuntos
Moléculas de Adesão Celular Neuronais , Córtex Cerebral/embriologia , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Neurônios/fisiologia , Tálamo/embriologia , Animais , Axônios/fisiologia , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Proteoglicanas de Sulfatos de Condroitina/análise , Contactina 2 , Desenvolvimento Embrionário e Fetal , Imuno-Histoquímica/métodos , Lectinas Tipo C , Complexo Antígeno L1 Leucocitário , Glicoproteínas de Membrana/análise , Proteínas do Tecido Nervoso/análise , Moléculas de Adesão de Célula Nervosa/análise , Moléculas de Adesão de Célula Nervosa/biossíntese , Neurocam , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Tálamo/citologia , Tálamo/metabolismoRESUMO
This study examines the early organization of glial cells, together with the expression of chondroitin sulfate proteoglycans in the developing thalamus of ferrets. Glia were identified with antibodies against vimentin and glial fibrillary acidic protein and the chondroitin sulfate proteoglycans were identified by using an antibody against chondroitin sulfate side chains. Our results reveal three striking features of early thalamic development. First, there is a distinct population of glial fibrillary acidic protein-immunoreactive astrocytes (first seen at E30) that resides in the perireticular thalamic nucleus of the primordial internal capsule. These glial fibrillary acidic protein-immunoreactive astrocytes of the perireticular nucleus are transient and form a conspicuous feature of the early developing forebrain. They are first apparent well before any glial fibrillary acidic protein-immunoreactive astrocytes are seen in other regions of the thalamus (at about P8). Further, unlike in other thalamic regions, these peculiar perireticular astrocytes do not express vimentin before they express glial fibrillary acidic protein. Second, in the reticular thalamic nucleus, the radial glial cells express glial fibrillary acidic protein; they are the only ones to do so in the thalamus during development. The glial fibrillary acidic protein-immunoreactive radial glial cells of the reticular nucleus form a rather distinct band across the developing thalamus at these early stages (E30-P1). Finally, and preceding the expression of glial fibrillary acidic protein, the radial glial cells of the reticular nucleus, unlike those in other thalamic regions, are associated closely with the expression of chondroitin sulfate proteoglycans (E20-E30). Later (after E30), the expression of the chondroitin sulfate proteoglycans in the reticular nucleus declines sharply. The significance of this finding is related to the early organization of the cortico-fugal and cortico-petal pathways.
Assuntos
Envelhecimento/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Desenvolvimento Embrionário e Fetal , Regulação da Expressão Gênica no Desenvolvimento , Neuroglia/fisiologia , Tálamo/fisiologia , Animais , Biomarcadores , Proteoglicanas de Sulfatos de Condroitina/análise , Furões , Proteína Glial Fibrilar Ácida/análise , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , Núcleos Talâmicos/embriologia , Núcleos Talâmicos/crescimento & desenvolvimento , Núcleos Talâmicos/fisiologia , Tálamo/embriologia , Tálamo/crescimento & desenvolvimento , Vimentina/análiseRESUMO
The presence of proteoglycans bearing galactosaminoglycan chains has been reported, but none has been identified previously in the matrix of the Engelbreth-Holm-Swarm tumor, which is a source of several basement membrane components. This tumor matrix contains perlecan, a large, low buoyant density heparan sulfate proteoglycan, widespread in many basement membranes and connective tissues. We now identify two distinct proteoglycan species from this tumor source, which are substituted with galactosaminoglycans and which show basement membrane localization by immunohistochemistry. One species is perlecan but, in addition to being present as a heparan sulfate proteoglycan, it is also present as a hybrid molecule, with dermatan sulfate chains. A minor population of perlecan apparently lacks heparan sulfate chains totally, and some of this is substituted with chondroitin sulfate. The second species is immunologically related to basement membrane-chondroitin sulfate proteoglycan (BM-CSPG) and bears chondroitin sulfate chains. No BM-CSPG was detectable which was substituted with heparan sulfate chains. A combination of immunological and molecular approaches, including cDNA cloning, showed that perlecan and BM-CSPG are distinct in core protein structure. Both are, however, basement membrane components, although there are tissue-specific differences in their distribution.
Assuntos
Membrana Basal/química , Proteoglicanas de Sulfatos de Condroitina/análise , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/análise , Neoplasias Experimentais/química , Proteoglicanas/análise , Animais , Anticorpos , Anticorpos Monoclonais , Western Blotting , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Clonagem Molecular , DNA Complementar , Técnica Indireta de Fluorescência para Anticorpo , Heparitina Sulfato/biossíntese , Humanos , Imuno-Histoquímica , Córtex Renal/citologia , Camundongos , Proteoglicanas/biossíntese , Coelhos , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Pele/citologiaRESUMO
Multipotential hemopoietic progenitor cells (FDCP-mix) proliferate in culture medium supplemented with horse serum. When transferred to a medium without serum, cells do not proliferate and enter a quiescent state. Both proliferative and quiescent cells synthesize only chondroitin sulfate proteoglycan (CS-PG) which is associated to the cell membrane. Incorporation of 35SO4 into CS-PG was 4-fold higher in quiescent than in proliferative cells. Flow cytometric studies using monoclonal antibodies which recognize the core protein or the CS chains, showed that the increased uptake of sulfate was not the consequence of an increase in the abundance of CS-PG. Further characterization demonstrated that CS-PG isolated from quiescent cells exhibited a slightly higher hydrodynamic size than CS-PG from proliferative cells. However, the glycosaminoglycan chains from PG derived from proliferative and quiescent cells have the same hydrodynamic size. Through ion-exchange chromatography we observed that the mean charge density of PG from quiescent cells was higher than in proliferative cells, suggesting a higher sulfation degree from PG synthesized by quiescent cells. This was confirmed by flow cytometric studies using monoclonal antibody 2B6, which recognizes the unsaturated terminal disaccharide of chondroitin-4-O-sulfate.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/biossíntese , Células-Tronco Hematopoéticas/metabolismo , Animais , Compartimento Celular , Divisão Celular , Linhagem Celular , Proteoglicanas de Sulfatos de Condroitina/isolamento & purificação , Cromatografia em Gel , Células Clonais , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Membranas/metabolismo , Camundongos , Tamanho da Partícula , Sulfatos/metabolismoRESUMO
Proteoglycans metabolically labelled with [35S]sulphate and [3H]glucosamine or [3H]leucine were isolated from the incubation medium and cell layer of human adult mesangial cells and glomerular visceral epithelial cells using sequential DEAE chromatography purification steps followed by gel-filtration chromatography. The proteoglycan composition of each peak was analysed by treatment with HNO2, chondroitinase ABC or chondroitinase AC followed by chromatography on Sephadex G-50 columns. Heparan sulphate proteoglycan (HSPG) and dermatan sulphate proteoglycan were detected in both the culture medium and cell layer of mesangial cells. Culture medium of glomerular visceral epithelial cells contained HSPG and a second proteoglycan with the properties of a hybrid molecule containing HS and chondroitin sulphate (CS). The cell layer contained HSPG and CSPG. Detailed analysis of the hybrid molecule revealed that it had an apparent molecular mass of 400 kDa. SDS/PAGE of hybrid molecules, after treatment with heparitinase and chondroitinase ABC, revealed a core protein of 80 kDa. Using 1.8% polyacrylamide/0.6% agarose-gel electrophoresis, we deduced that the HS and CS were independently attached to one core protein. Because glomerular-basement-membrane HSPG is thought to be derived from mesangial cells and glomerular visceral epithelial cells and this molecule is involved in several kidney diseases, we investigated its synthesis in more detail. Anti-(rat glomerular-basement-membrane HSPG) monoclonal antibodies (JM403) and anti-(human glomerular-basement-membrane HSPG) polyclonal antibodies (both antibodies known to react with the large basement-membrane HSPG, perlecan) reacted strongly with HSPG obtained from both mesangial cells and glomerular visceral epithelial cells. However, the hybrid molecule did not react with these antibodies, suggesting that the HS side chain and the core protein were different from glomerular-basement-membrane HSPG. To quantify HS we performed an inhibition ELISA using mouse antibodies specific for glomerular-basement-membrane HS glycosaminoglycan side chains. Glomerular visceral epithelial cells produced significantly higher levels of HS (between 197.56 and 269.40 micrograms/72 h per 10(6) cells) than mesangial cells (between 29.8 and 45.5 micrograms/72 h per 10(6) cells) (three different cell lines; n = 3; P < 0.001). HS production by these cells was inhibited by cycloheximide, revealing that it was synthesized de novo. Expression of perlecan mRNA, demonstrated using reverse transcriptase PCR, was different in the two cell types. We conclude that glomerular visceral epithelial cells and mesangial cells have characteristic patterns of proteoglycan production. Glomerular visceral epithelial cells produced a hybrid proteoglycan containing CS and HS independently attached to its core protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Mesângio Glomerular/metabolismo , Glomérulos Renais/metabolismo , Proteoglicanas/biossíntese , Adolescente , Adulto , Idoso , Sequência de Bases , Células Cultivadas , Criança , Pré-Escolar , Proteoglicanas de Sulfatos de Condroitina/análise , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Cromatografia Líquida de Alta Pressão , DNA Complementar/biossíntese , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Epitélio/metabolismo , Glucosamina/metabolismo , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/biossíntese , Heparitina Sulfato/genética , Humanos , Lactente , Glomérulos Renais/citologia , Leucina/metabolismo , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteoglicanas/genética , RNA/genética , RNA/isolamento & purificação , RNA/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Sulfatos/metabolismo , Radioisótopos de Enxofre , Transcrição Gênica , TrítioRESUMO
We have previously described the cloning of phosphacan, a chondroitin sulfate proteoglycan of nervous tissue which interacts with neurons, glia, neural cell adhesion molecules, and tenascin, and represents the extracellular domain of a receptor-type protein tyrosine phosphatase. We now report the complete cDNA and deduced amino acid sequences of the rat transmembrane phosphatase, and demonstrate that the phosphatase and the extracellular proteoglycan have different 3'-untranslated regions. Northern analysis showed three probable splice variants, comprising the extracellular proteoglycan (phosphacan) and long and short forms of the transmembrane phosphatase. PCR studies of rat genomic DNA indicated that there are no introns at the putative 5' and 3' splice sites or in the 2.6 kb segment which is deleted in the short transmembrane protein. Using variant-specific riboprobes corresponding to sequences in the 3'-untranslated region of phosphacan and in the first or second phosphatase domains of the transmembrane protein, in situ hybridization histochemistry of embryonic rat brain and spinal cord and early postnatal cerebellum demonstrated identical localizations of phosphacan and phosphatase mRNAs.
Assuntos
Processamento Alternativo , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Fosfatases/genética , Ratos/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Primers do DNA , DNA Complementar , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Hibridização In Situ , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/química , Sondas RNA , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores , Receptores de Superfície Celular/biossíntese , Medula Espinal/metabolismo , Medula Espinal/ultraestruturaRESUMO
Proliferating cultures of bovine vascular smooth muscle cells synthesized a variety of proteoglycans corresponding closely to those reported previously for monkey smooth muscle cells. These included a chondroitin sulfate proteoglycan (CSPG) (47%), a dermatan sulfate proteoglycan (DSPG) (22%), and a heparan sulfate proteoglycan (HSPG) (6%) which were secreted into the medium. Heparan sulfate proteoglycan (6%) and a second dermatan sulfate proteoglycan (14%) were also present in the cell layer. Confluent cultures synthesized a similar spectrum of proteoglycans although the medium CSPG and DSPG were of smaller hydrodynamic size. The cell layer HSPG was much reduced relative to DSPG in early proliferating cultures. Previous reports have shown that heparin inhibits vascular smooth muscle cell proliferation. Heparin had two effects on proteoglycan synthesis. In control cultures, 35S-Labeled proteoglycan synthesis doubled during the first 12 h after releasing cells from growth arrest, decreasing during the following 12 h during which time cell division occurred. Treatment with heparin delayed the onset of proliferation by 24 h and this was accompanied by a corresponding delay in the increase in 35S-labeled proteoglycan synthesis associated with the early phase of the cell cycle. Secondly, heparin treatment resulted in an increase in the anionic properties of heparan sulfate proteoglycan synthesized by the cells. This was independent of the proliferative state of the cultures. Pentosan polysulfate, semi-synthetic heparin, and a highly sulfated heparan sulfate modulated both cell proliferation and heparan sulfate proteoglycan synthesis in the same way as heparin.
Assuntos
Heparina/farmacologia , Músculo Liso Vascular/metabolismo , Proteoglicanas/biossíntese , Animais , Aorta , Bovinos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Dermatan Sulfato/biossíntese , Proteoglicanas de Heparan Sulfato , Heparitina Sulfato/biossíntese , Heparitina Sulfato/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Poliéster Sulfúrico de Pentosana/farmacologia , Polissacarídeos/farmacologiaRESUMO
Vitamin D appears to be required for mineralization of skeletal elements. There is also evidence that cartilage proteoglycans may be involved in the regulation of mineralization. Previous studies have shown an alteration in the structure of the proteoglycans of the epiphyseal growth cartilage as a result of the decrease in serum calcium related to deficiency of dietary vitamin D. Vitamin D deficiency also induces a thickening of the epiphyseal growth plate presumably because of the inhibition of maturation of the growth plate chondrocytes. In order to compare the effect on proteoglycan structure with that on growth plate morphology, the proteoglycans of healing epiphyseal cartilage were characterized. The results indicate that, consistent with previous data, in vitamin D-deficient hatching chicks, the proteoglycans of the growth cartilage, but not of the articular cartilage, are smaller in monomer size with slightly smaller chondroitin sulfate chains whose sulfation pattern is unaltered. Sternal cartilage proteoglycans are unaffected. During recovery from vitamin D deficiency, the proteoglycans isolated from the growth cartilage are still not completely normal one day after supplementation with vitamin D, but are indistinguishable from normal by four days. In addition, the results conflict with those of a previous study in which only growth cartilage of hatchling chicks, not sternal or articular cartilage, was reported to synthesize large proteoglycans. Instead, all of these cartilages in the normal chicken have been found in this study to produce large proteoglycans of a size typical for mammalian cartilage and embryonic chick cartilage.
Assuntos
Cartilagem Articular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Glicosaminoglicanos/biossíntese , Lâmina de Crescimento/metabolismo , Proteoglicanas/biossíntese , Raquitismo/tratamento farmacológico , 24,25-Di-Hidroxivitamina D 3/sangue , Animais , Calcifediol/sangue , Calcitriol/sangue , Galinhas , Colecalciferol/administração & dosagem , Esquema de Medicação , Raquitismo/sangueRESUMO
Early steps in the biosynthesis of chondroitin sulfate proteoglycan (CSPG) and collagenous cartilage matrix molecules were examined by the comparison of products translated in mRNA-directed cell-free reactions and those synthesized by intact cartilage cells. RNA isolated from embryonic chicken sterna was used to direct cell-free translation reactions. Chicken sternal chondrocytes in culture were pulse-labeled with [35S]-methionine. The CSPG core protein was identified by immunoprecipitation. The Mr of the cartilage cell-synthetized core protein was determined to be 370K, approximately 10-15K greater than that of the comparable cell-free translation product. Experimental results strongly support the view that the observed difference in Mr reflects the cotranslational addition of mannose-rich, N-asparagine-linked oligosaccharides to the cell-synthesized core protein: 1) the cell-synthesized product was labeled with [3H]-mannose and precipitated by concanavalin A-sepharose beads; 2) the incorporated [3H]-mannose could be subsequently removed by digestion with endoglycosidase H (Endo H); 3) the Mr of the cell-synthesized core protein was reduced by Endo H digestion to that of the comparable cell-free translation product; 4) the core protein synthesized by tunicamycin-treated chondrocytes (inhibited in their ability to add N-asparagine-linked mannose-rich oligosaccharides to proteins) was comparable in electrophoretic mobility to that of the core protein cell-free translation product; and 5) the core protein translated in microsome-coupled cell-free reactions had an Mr 8-10K greater than that of the core protein translated in the absence of microsomes. For the purpose of examining biosynthetic intermediates, chondrocytes were labeled continuously or pulse-chase labeled for varying times. No biosynthetic CSPG intermediates migrating between the core protein and the CSPG monomer were detected. However, a band of 355Kdal appeared to share certain characteristics with the 307Kdal core protein (including its immunoprecipitability with CSPG antibodies), and a 340Kdal band was noted. Type II procollagen and other collagenase-sensitive products of 205Kdal and 110Kdal were observed among translation and chondrocyte-synthesized products. In chondrocytes, all three products exhibited labeling or chase time-dependent increases in Mr which were accelerated by ascorbate supplements and inhibited by the addition of alpha, alpha'-dipyridyl. These results suggest that the observed time-dependent increases in Mr are a consequence of collagen hydroxylation. The 110Kdal and 205Kdal collagenous proteins may be related to the minor collagens recently described in cartilage.