RESUMEN
PURPOSE: To characterize the major proteoglycans produced and secreted by collagenase-isolated bovine keratocytes in culture. METHODS: Freshly isolated keratocytes from mature bovine corneas were cultured in serum-free Dulbecco's modified Eagle's medium/ F12. Secreted proteoglycans were radiolabeled with protein labeling mix ((35)S-Express; Dupont NEN Life Science Products, Boston, MA) and digested with chondroitinase ABC, keratanase, and endo-beta-galactosidase to remove glycosaminoglycan chains, and core proteins were analyzed by autoradiography and Western blot analysis. An unidentified keratan sulfate proteoglycan (KSPG) was purified by gel filtration (Superose 6; Amersham Pharmacia, Piscataway, NJ) and anion-exchange chromatography (Resource Q; Amersham Pharmacia) and subjected to amino acid sequencing. RESULTS: Keratanase digestion of proteoglycans produced approximately 50 kDa core proteins that immunoreacted with antisera to lumican, keratocan, and osteoglycin-mimecan. Chondroitinase ABC digestion produced a approximately 55-kDa core protein that immunoreacted with antisera to decorin. A 28-kDa band generated by keratanase or endo-beta-galactosidase digestion did not react with these antibodies. Chromatographic purification and amino acid sequencing revealed that the protein was prostaglandin D synthase (PGDS). Identity was confirmed by Western blot analysis using antisera to recombinant PGDS. PGDS isolated from corneal extracts was not keratanase sensitive but was susceptible to endo-beta-galactosidase, suggesting that it contains unsulfated polylactosamine chains in native tissue and is therefore present as a glycoprotein. CONCLUSIONS: These results indicate that bovine keratocytes, when cultured under serum-free conditions, produce the four known leucine-rich proteoglycans decorin, keratocan, lumican, and osteoglycin/mimecan and maintain a phenotype that is comparable to that of in situ keratocytes. Additionally, these cells produce PGDS, a known retinoid transporter, as a KSPG.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Sustancia Propia/enzimología , Fibroblastos/enzimología , Oxidorreductasas Intramoleculares/biosíntesis , Sulfato de Queratano/biosíntesis , Animales , Autorradiografía , Western Blotting , Bovinos , Células Cultivadas , Cromatografía en Gel , Sustancia Propia/citología , Medio de Cultivo Libre de Suero , Decorina , Proteínas de la Matriz Extracelular , Glicoproteínas/biosíntesis , Lipocalinas , Lumican , Proteoglicanos/biosíntesisRESUMEN
The leucine-rich proteoglycans (also known as "small, leucine-rich proteoglycans," or SLRPs) lumican and decorin are thought to be involved in the regulation of collagen fibril assembly. Preparation of these proteoglycans in chemical amounts without exposure to denaturants has recently been achieved by infecting HT-1080 cells with vaccinia virus that contains an expression cassette for these molecules. Addition of lumican and decorin to a collagen fibrillogenesis assay based on turbidity demonstrated that lumican accelerated initial fibril formation while decorin retarded initial fibril formation. At the end of fibrillogenesis, both proteoglycans resulted in an overall reduced turbidity, suggesting that fibril diameter was lower. The presence of both proteoglycans had a synergistic effect, retarding fibril formation to a greater degree than either proteoglycan individually. Competitive binding studies showed that lumican did not compete for decorin-binding sites on collagen fibrils. Both proteoglycans increased the stability of fibrils to thermal denaturation to approximately the same degree. These studies show that lumican does not compete for decorin-binding sites on collagen, that decorin and lumican modulate collagen fibrillogenesis, and that, in the process, they also enhance collagen fibril stability.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/farmacología , Colágeno/química , Colágeno/metabolismo , Sulfato de Queratano/farmacología , Proteoglicanos/farmacología , Animales , Sitios de Unión , Unión Competitiva , Bovinos , Línea Celular , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Decorina , Matriz Extracelular/química , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular , Humanos , Técnicas In Vitro , Sulfato de Queratano/metabolismo , Lumican , Desnaturalización Proteica/efectos de los fármacos , Proteoglicanos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence. The expression of each of the three corneal KSPGs in the developing and mature chick cornea was investigated by competitive PCR and immuno-biochemical analysis of corneal extracts. Competitive PCR was used to determine the message levels for chick lumican, keratocan and osteoglycin in embryonic day 9, 12, 15, 18 and adult corneas. Results showed that lumican mRNA fluctuated during development but remained at a relatively high level while keratocan and osteoglycin message levels declined steadily from day 9 to adult. Additionally, lumican mRNA was present at higher levels, during all stages of corneal development, than keratocan and at much higher levels than osteoglycin. Antibodies shown to be specific for each KSPG were used to characterize proteoglycans isolated from embryonic and adult chick corneas. KSPGs from embryonic corneas eluted 1-2 fractions earlier on Q-Sepharose than KSPG from adult corneas. Additionally, Western blot analysis showed that embryonic KSPGs were more keratanase-resistant, endo-beta-galactosidase sensitive than adult KSPGs. The results of this study indicate an alteration in sulfation or the fine structure of the glycosaminoglycan chains occurs during corneal maturation for the 3 KSPGs.
Asunto(s)
Pollos/crecimiento & desarrollo , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Córnea/crecimiento & desarrollo , Sulfato de Queratano/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Embrión de Pollo , Pollos/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/genética , Córnea/metabolismo , ADN Complementario/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Sulfato de Queratano/genética , Lumican , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteoglicanos/metabolismoRESUMEN
The recent increased efficacy of diagnosing prostate carcinoma from needle biopsy can be attributed to the accelerated biopsy rate as a result of cancer screening, the greater number of core samples per set, and the increased ability to identify malignancy in progressively smaller gland foci. This improvement in histological judgement has been facilitated by more sophisticated histological criteria, which in turn depend largely on an increasing knowledge of normal histological features and their abnormal counterparts. The recent discovery of the prostate secretory granule (PSG) as part of the normal secretory mechanism has prompted our study of the PSG as a possible additional criterion for distinction between benign and malignant cells in biopsy samples. The proper delineation of PSG required glutaraldehyde-based fixation, but this change in fixation showed additional diagnostic advantages. We quantitated PSG depletion in 150 sequential core biopsy samples, evaluating benign epithelium, dysplasia (PIN), Gleason grade 3, and grade 4 carcinoma separately. Overall, 80% of carcinomas and 63% of high-grade dysplasias were markedly depleted of PSG such that no granules were seen at low-power magnification with routine haematoxylin and eosin stains. This contrast between benign and malignant epithelium was especially prominent in small carcinoma foci greatly assisting in cancer recognition. Comparison between all groups showed an advantage of glutaraldehyde-based tissue fixation over formalin fixation for prostate needle biopsy specimens, providing clear resolution of cytological detail a well as an additional histologic criterion for cancer diagnosis. HUM PATHOL 31:1515-1519.
Asunto(s)
Adenocarcinoma/metabolismo , Próstata/metabolismo , Neoplasia Intraepitelial Prostática/metabolismo , Neoplasias de la Próstata/metabolismo , Vesículas Secretoras/metabolismo , Adenocarcinoma/patología , Biopsia con Aguja , Formaldehído , Glutaral , Humanos , Masculino , Neoplasia Intraepitelial Prostática/patología , Neoplasias de la Próstata/patología , Fijación del Tejido/métodosRESUMEN
PURPOSE: To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes. METHODS: Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS: Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum. CONCLUSIONS: A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.