RESUMEN
After the luteinizing hormone surge, the cumulus cell-oocyte complexes (COCs) in the preovulatory follicles produce a viscoelastic extracellular matrix, a process that requires the synthesis of hyaluronan as well as the incorporation of some components of the inter-alpha-trypsin inhibitor (IalphaI) family. In this study we report, that a hyaluronan-binding protein, the translated product of tumor necrosis factor-stimulated gene-6 (TSG-6), is also specifically accumulated in this matrix. TSG-6 mRNA expression is quickly upregulated and peaks at approximately 1500 copies/cell 4 h after the ovulatory stimuli as assessed by quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry reveals the colocalization of the TSG-6 protein and hyaluronan around the cumulus and granulosa cells. The TSG-6 protein exists in two distinct populations in the COC matrix as demonstrated by Western-blot analysis. One population is a monomer that is anchored to the matrix by a noncovalent interaction. The second population is a covalent complex with either of the heavy chains of IalphaI and is bound to hyaluronan through a strong interaction that is resistant to denaturing conditions. The specific incorporation of the TSG-6 protein into the COC matrix suggests a structural role for this molecule.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Matriz Extracelular/metabolismo , Oocitos/metabolismo , alfa-Globulinas/química , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Diferenciación Celular , Células Cultivadas , Matriz Extracelular/química , Femenino , Fase Folicular , Ácido Hialurónico/metabolismo , Inmunohistoquímica , Sustancias Macromoleculares , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Peso Molecular , Oocitos/citología , Oocitos/efectos de los fármacos , Folículo Ovárico/citología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de Proteína , Regulación hacia ArribaRESUMEN
Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately 21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h, a finding in line with a predominantly Has2-dependent hyaluronan synthesis and its transcriptional regulation.
Asunto(s)
Factor de Crecimiento Epidérmico/farmacología , Glucuronosiltransferasa/metabolismo , Ácido Hialurónico/metabolismo , Queratinocitos/efectos de los fármacos , Animales , Secuencia de Bases , Cartilla de ADN , Endocitosis , Activación Enzimática , Glucuronosiltransferasa/genética , Hialuronano Sintasas , Queratinocitos/enzimología , Queratinocitos/metabolismo , Cinética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Proteoglycans are macromolecules formed by a protein backbone to which one or more glycosaminoglycan side chains are co-valently attached. They can be secreted by the cells, retained at the cell surface, or stored in intracellular vacuoles. Hyaluronan is an extremely long glycosaminoglycan which, at variance with other glycosaminoglycans, is released into the extracellular matrix as a free polysaccharide not co-valently linked to a core protein. Both proteoglycans and hyaluronan influence many aspects of cell behaviour by multiple interactions with other molecules. They are involved in matrix formation, cell-cell and cell-matrix adhesion, cell proliferation and migration, and show co-receptor activity for growth factors. Both proteoglycan and hyaluranon synthesis change significantly during ovarian follicle development and atresia. This review describes the structure of these molecules and their possible function in ovarian physiology.
Asunto(s)
Ácido Hialurónico , Ácido Hialurónico/fisiología , Folículo Ovárico/fisiología , Proteoglicanos , Proteoglicanos/fisiología , Animales , Fenómenos Fisiológicos Celulares , Matriz Extracelular/metabolismo , Femenino , Fertilización , Humanos , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/química , Masculino , Folículo Ovárico/química , Ovulación , Proteoglicanos/químicaRESUMEN
Maturation of mammalian cumulus cell-oocyte complex in the preovulatory follicle involves the deposition of a hyaluronan-rich extracellular matrix by the cumulus cells. In this study, we report the complete coding sequence of a novel mouse hyaluronan synthase homologue expressed in cumulus cell-oocyte complexes induced to expand in vivo. The time frame of mRNA expression of this molecule correlates well with previous biochemical and immunohistochemical findings on the initiation of hyaluronan synthesis in maturing preovulatory follicles. Evolutionary comparison of the vertebrate hyaluronan synthase homologues indicates that there are at least two genes that code for these proteins.
Asunto(s)
Glucuronosiltransferasa/genética , Glicosiltransferasas , Proteínas de la Membrana , Oocitos/enzimología , Folículo Ovárico/enzimología , Transferasas , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Molecular , Femenino , Expresión Génica , Glucuronosiltransferasa/biosíntesis , Glucuronosiltransferasa/química , Humanos , Hialuronano Sintasas , Ácido Hialurónico/biosíntesis , Ratones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
Tumor necrosis factor stimulated gene-6 (TSG-6) has been previously shown to be induced in vitro in several cell types by proinflammatory cytokines, and in vivo in pathological conditions such as rheumatoid arthritis. In this study, we report the complete coding sequence for the mouse TSG-6 protein, and the exon intron structure and the chromosomal localization of the gene. We have identified a 1605 nt cDNA sequence from mouse cumulus cell oocyte complexes (COCs) induced to expand in vivo. The sequence contains an open reading frame of 825 nt that codes for the 275 amino acid TSG-6 protein. The gene contains six exons separated by 1.1-5.8 kb introns and has been localized to the murine chromosome 2 by linkage analysis. Comparative reverse transcription-polymerase chain reaction studies have revealed that TSG-6 mRNA is specifically expressed after COC expansion induced in vivo, identifying the first non-pathological process in which TSG-6 may play an important role. Since TSG-6 binds to hyaluronan and interacts with inter-alpha-trypsin inhibitor (IalphaI), molecules that are essential for matrix formation by COCs, this protein may have a structural role in the matrix or may enhance the antiproteolytic effect of IalphaI to protect the matrix from degradation.
Asunto(s)
Moléculas de Adhesión Celular/genética , Mapeo Cromosómico , Exones , Intrones , Oocitos/metabolismo , Ovario/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/química , Matriz Extracelular/metabolismo , Femenino , Sustancias Macromoleculares , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Oocitos/citología , Sistemas de Lectura Abierta , Ovario/citología , ARN Mensajero/biosíntesis , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Aggrecan transcripts contain two alternatively spliced exons that code for two epidermal growth factor-like domains (EGF1 and EGF2). Whereas the EGF2 sequence is expressed at a uniform level among different species, the EGF1 sequence has been detected only in human aggrecan transcripts. In this study we have used the nested primer reverse transcription-PCR (RT-PCR) method to compare the expression of the EGF1 exon in human, bovine and dog aggrecan transcripts. Our results indicate that this exon is expressed in a species-specific manner. In addition to its significant expression level in human transcripts, the EGF1 sequence can be detected in a small portion of bovine aggrecan transcripts as shown with nested primer RT-PCR. In contrast, the same module is not detectable in dog aggrecan transcripts, although an EGF1 sequence is present in the dog aggrecan gene. The expression level of the EGF1 exon in the aggrecan transcripts correlates with the strength of the polypyrimidine tract upstream of the exon. The EGF1 sequence also shows much less conservation between the species than the EGF2 sequence. The species-specific expression and high sequence variation of the EGF1 exon imply that this sequence is likely to code for an aggrecan domain having no cartilage-specific function.
Asunto(s)
Empalme Alternativo , Cartílago Articular/metabolismo , Factor de Crecimiento Epidérmico/química , Proteínas de la Matriz Extracelular , Proteoglicanos/biosíntesis , Proteoglicanos/química , Transcripción Genética , Agrecanos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cartilla de ADN , Perros , Feto , Humanos , Lectinas Tipo C , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Immunization of BALB/c mice with human fetal cartilage proteoglycan (PG) produces progressive polyarthritis, and T cells play key roles in the development of the disease. To gain an understanding of how PG is presented to autoreactive T cells by synovial antigen-presenting cells (APC), we examined the abilities of various syngeneic APC in presenting PG to a specific T cell hybridoma 5/4E8, derived from a mouse with PG-induced arthritis. A20 B lymphoma cells and spleen cells were strong presenters of PG, but synoviocytes and P388D1 macrophages could only present PG effectively after stimulation with interferon-gamma (IFN-gamma). The IFN-gamma exerted its effect by up-regulating both MHC class II and intercellular adhesion molecule-1 (ICAM-1) expression by these cells as neutralizing antibodies to Ia, LFA-1 and ICAM-1 inhibited presentation. Our studies also showed that synoviocytes and spleen cells took up and processed PG more rapidly than the cell lines. Cysteine and serine protease-dependent antigen presentation of PG was blocked at 4 degrees C, 18 degrees C and by chloroquine treatment, indicating that presentation required active uptake and processing in an acidic compartment, probably in lysosomes. Also, keratan sulphate-depleted and cyanogen bromide (CNBr) and 2-nitro-5-thiocyanobenzoic acid (NTCB)-cleaved PG elicited stronger T cell responses, as they were more easily processed than the native molecule. Furthermore, CNBr-generated peptides were presented by fixed APC, indicating that core protein fragments of cartilage PG can be presented directly by APC in context with MHC class II molecules.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Artritis/inmunología , Cartílago/inmunología , Proteoglicanos/inmunología , Linfocitos T/inmunología , Animales , Transporte Biológico , Cloroquina/farmacología , Hibridomas/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-2/biosíntesis , Antígeno-1 Asociado a Función de Linfocito/inmunología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteasas/farmacología , Proteoglicanos/química , Proteoglicanos/metabolismo , Bazo/citología , Bazo/inmunología , Relación Estructura-Actividad , TemperaturaRESUMEN
Immunization of BALB/c mice with chondroitin sulfate-depleted proteoglycan (aggrecan) of fetal human cartilage produces progressive polyarthritis and ankylosing spondylitis. The development of the disease in genetically susceptible BALB/c mice is dependent upon the expression of both cell-mediated and humoral immune responses against the host mouse cartilage proteoglycan (PG). Although cartilage PGs from various species have many biochemical and immunological similarities, only a select group of PGs from fetal and newborn human, fetal pig and canine articular cartilages, human osteophytes and human chondrosarcomas are able to induce arthritis in BALB/c mice. Arthritis develops only in mice that also develop autoantibodies to self-cartilage PGs, although autoantibodies occasionally are present in non-arthritic animals as well. The protease-sensitive auto/arthritogenic epitope(s) is located in, or close to, the chondroitin sulfate (CS) attachment region of the PG molecule. The primary structure of the core protein is responsible for the autoimmune/arthritogenic effect of this select group of PGs, whereas the core protein epitopes are masked by glycosaminoglycan (GAG)-side chains. The CS side chains seem to inhibit antigen recognition in all aggrecans with arthritogenic potential, whereas a similar effect with keratan sulfate (KS) appears only in PGs of aging cartilages.
Asunto(s)
Artritis/inmunología , Autoinmunidad , Cartílago Articular/metabolismo , Epítopos , Proteínas de la Matriz Extracelular , Proteoglicanos/inmunología , Agrecanos , Animales , Bovinos , Femenino , Humanos , Inmunoquímica , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Proteoglicanos/químicaRESUMEN
We have isolated and sequenced overlapping cDNA clones encoding the entire core protein of aggrecan (the large aggregating chondroitin sulfate/keratan sulfate proteoglycan of cartilage) from three chondrocyte cDNA libraries of BALB/c mice and localized the aggrecan gene in mouse chromosome 7. We determined 7386 bp of the cDNA sequence, including 132 and 854 nucleotides of 5' and 3' untranslated regions, respectively. The core protein precursor is 2132 amino acids long (M(r) 222,008), including a 19-residue secretory signal peptide. The overall amino acid sequence of the mouse aggrecan shows 91.6% identity to rat and 72.5% to human aggrecan. Comparison of the amino acid sequences of various domains and subdomain structures of mouse aggrecan to known sequences of other species and related proteins (versican, neurocan, link protein, and lymphocyte homing receptor CD44) revealed high levels of identity of the G1, G2, and G3 globular domains and relatively less conserved structures in the interglobular and glycosaminoglycan-attachment regions. Epidermal growth factor (EGF)-like module was detected in only a minor fraction of aggrecan clones, while the complement regulatory protein (CRP)-like domain was regularly expressed in all samples.
Asunto(s)
Proteínas de la Matriz Extracelular , Ratones/genética , Proteoglicanos/genética , Agrecanos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , ADN Complementario/genética , Expresión Génica , Biblioteca de Genes , Humanos , Lectinas Tipo C , Ratones Endogámicos , Datos de Secuencia Molecular , Precursores de Proteínas/genética , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
The carboxyl-terminal globular domain of human aggrecan has been shown previously to contain an alternatively spliced epidermal growth factor (EGF)-like module. We have used reverse transcription/polymerase chain reactions on cartilage-derived RNAs to investigate the heterogeneity in the EGF-like domain content of aggrecans from five species (mouse, rat, dog, bovine, and human). A novel alternatively spliced EGF-like module was detected in human aggrecan, establishing the presence of two of these domains. Highly homologous domains are present in aggrecans of other species, and the expression of these modules is identical (4-8%). They share significant homology with EGF-like domains of differentiation proteins and coagulation factors and have a putative calcium binding site. In contrast to this novel domain (EGF2), the previously described EGF-like module (EGF1) is expressed at a high level exclusively in human aggrecan. The expression of the two EGF-like domains in human aggrecan appears to be independent. Although the function of these domains is not understood, the uniform expression of the EGF2 domain may indicate a general role of this aggrecan module, while the expression of the EGF1 domain may reflect species specificity.
Asunto(s)
Empalme Alternativo , Factor de Crecimiento Epidérmico/genética , Proteínas de la Matriz Extracelular , Proteoglicanos/genética , Agrecanos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN , Perros , Exones , Humanos , Lectinas Tipo C , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Proteoglicanos/química , Ratas , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Células Tumorales CultivadasRESUMEN
The substrate specificity of elastomucoproteinase (EMP), an enzyme which was first isolated from crude pancreatic elastase and described as a proteoglycan-degrading enzyme, determined on tripeptide-p-nitroanilide substrates indicates the existence of a 'new' chymotrypsin-like enzyme. EMP, however, did not cleave any glycosaminoglycans, i.e., its 'mucolytic' effect has been excluded. Activity of EMP on synthetic or protein substrates (e.g., collagen type-II and aggrecan of cartilage) was completely inhibited by serine proteinase inhibitors, which was also found when using cartilage proteoglycan monomers. EMP cleaves the core protein of proteoglycan monomer (aggrecan) into small peptides, some containing glycosaminoglycan chains resulting in an unusual elution profile on Sepharose CL-6B chromatography when compared to the effects of pancreatic and granulocyte elastases, chymotrypsin, cathepsin G and stromelysin. EMP-like activity also was detected in neutrophil granules of bovine leukocytes and polyclonal antibodies were raised against purified bovine EMP to detect the enzyme in both crude elastase preparations and the granule fraction of bovine leukocytes.
Asunto(s)
Cartílago/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Matriz Extracelular , Proteoglicanos/metabolismo , Adulto , Agrecanos , Secuencia de Aminoácidos , Animales , Western Blotting , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Técnicas In Vitro , Lectinas Tipo C , Persona de Mediana Edad , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Fragments of high density cartilage proteoglycan (aggrecan) are released during either the normal or pathological turnover of cartilage proteoglycans, which fragments diffuse into the synovial fluids and then appear in the serum. The keratan sulphate (KS; a glycosaminoglycan side chain of aggrecan) is resistant to enzymatic degradation, it has a relatively low clearance and has a "standard" serum level indicating the actual level of cartilage (proteoglycan) breakdown. Using anti-KS monoclonal antibody in ELISA (enzyme-linked immunosorbent assay), we measured serum KS levels in patients with different joint diseases. The highest KS content (595 ng/ml) was measured in the sera of patients with articular chondrocalcinosis (calcium pyrophosphate crystal deposition disease/pseudogout). Slightly lower KS levels were determined in osteoarthrosis (OA; 578 ng/ml) and much less in rheumatoid arthritis (RA; 421 ng/ml). All these patient groups (either with degenerative or inflammatory joint diseases) expressed slightly higher KS levels compared to control blood donors (295 ng/ml). However, there were remarkable variations between these diseased groups, i. e., KS levels in patients with RA were significantly lower than in patients with OA (p < 0.001) and this difference was more pronounced in rheumatoid patients with I-II Steinbrocker stage (370 ng/ml) or in those treated with non-steroid anti-inflammatory drugs (NSAIDs) (382 ng/ml). Keratan sulphate levels in RA patients chronically treated with corticosteroid (460 ng/ml) or auro-thiomalat (473 ng/ml) indicate that these drugs may influence the cartilage metabolism more effectively than the NSAIDs.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Artritis/metabolismo , Cartílago Articular/metabolismo , Artropatías/metabolismo , Sulfato de Queratano/metabolismo , Adulto , Anciano , Enfermedades de los Cartílagos/metabolismo , Enfermedades de los Cartílagos/patología , Cartílago Articular/patología , Condrocalcinosis/metabolismo , Condrocalcinosis/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoartritis/metabolismoRESUMEN
An in vitro cell line (HT 58) has been established from a human (B) NHL xenograft. The lymphoma cells in culture retained their lymphoblastic appearance, DNA-content, IgM/lambda monoclonality and many immunophenotypic markers. The clonal chromosomal abnormalities involved the chromosomes 1, 2, 3 and 14. The cells expressed and produced chondroitin sulfate proteoglycans identified with mAbs that were raised against human articular cartilage CSPG. The cells also released IgM into the medium as well as substances that stimulated the proliferation of activated normal peripheral B-cells.
Asunto(s)
Linfocitos B/patología , Linfoma no Hodgkin/patología , Anticuerpos Monoclonales , Formación de Anticuerpos , Antígenos de Superficie/análisis , Linfocitos B/inmunología , Linfocitos B/ultraestructura , División Celular , Aberraciones Cromosómicas , Trastornos de los Cromosomas , Técnicas de Cultivo/métodos , ADN de Neoplasias/análisis , Epítopos/análisis , Humanos , Cariotipificación , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/ultraestructura , Microscopía Electrónica , Células Tumorales CultivadasAsunto(s)
Artritis Experimental/inmunología , Autoanticuerpos/análisis , Artropatías/inmunología , Proteoglicanos/inmunología , Linfocitos T/inmunología , Animales , Formación de Anticuerpos , Artritis Reumatoide/inmunología , Humanos , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Espondilitis Anquilosante/inmunologíaRESUMEN
Glycosaminoglycan and core protein components of proteoglycans (PGs) have been studied in three human non-Hodgkin lymphoma xenografts of B cell origin. Lymphomas showed similar GAG content, but different composition of GAG subtypes. This variability was accompanied by an individual capacity to adhere to extracellular matrix elements. The core proteins identified by monoclonal antibodies raised against human cartilage chondroitin sulfate PG were also distinctly expressed and released. These proteins shared by different cell types may have biological significance.