Production of prostaglandin D synthase as a keratan sulfate proteoglycan by cultured bovine keratocytes Invest Ophthalmol Vis Sci 42(6

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-␤-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 anionexchange chromatography (Resource Q; Amersham Pharmacia) and subjected to amino acid sequencing. RESULTS. Keratanase digestion of proteoglycans produced ϳ50 kDa core proteins that immunoreacted with antisera to lumican, keratocan, and osteoglycin-mimecan. Chondroitinase ABC digestion produced a ϳ55-kDa core protein that immunoreacted with antisera to decorin. A 28-kDa band generated by keratanase or endo-␤-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-␤-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. (Invest Ophthalmol Vis Sci. 2001;42:1201-1207 K eratocytes, the principal cell type of the adult corneal stroma, are responsible for producing the extensive and uniquely transparent extracellular matrix of the corneal stroma. 1,2 The keratocytes in adult corneas are quiescent, but on stromal wounding are activated, proliferate, become fibroblasts and myofibroblasts, migrate to the wound site, 3,4 and produce a disorganized extracellular matrix 5 without keratan sulfate 6 -factors that probably contribute to the formation of an opaque scar in the cornea. Keratocytes that have been isolated from the stroma and cultured under standard conditions exhibit characteristics of the fibroblast and myofibroblast phenotypes, including cell shape, the presence of ␣-smooth muscle actin, low levels of keratan sulfate production, expression of the fibronectin receptor, and extensive cell proliferation. 7-11 These cells are not useful for studying properties of the keratocytes that produce corneal transparency. Recent studies, however, indicate that collagenase-isolated keratocytes plated in media without fetal bovine serum do not become fibroblasts or myofibroblasts in culture. Collagenase-isolated rabbit keratocytes cultured in serum-free media do not proliferate, appear dendritic, and have no ␣-smooth muscle actin. 12 Similarly, isolated and cultured bovine keratocytes do not proliferate, appear dendritic, and synthesize high levels of keratan sulfate proteoglycans (KSPGs). This new serum-free keratocyte culturing method provides an opportunity to more fully characterize the keratocyte phenotype and its transition to other phenotypes. In this report, we identify the major secreted proteoglycans of keratocytes in serum-free cell culture and find that the keratocytes make all four previously identified corneal stroma proteoglycans: decorin, lumican, keratocan, and osteoglycin. In addition, they make a novel small KSPG that has been identified as prostaglandin D synthase (PGDS), a secreted product that has not been previously shown to be made by cultured keratocytes or made as a proteoglycan. MATERIALS AND METHODS Keratocyte Isolation and Culture Bovine keratocytes were isolated using a modification of a collagenase digestion method, 13 using only two sequential digestions. Briefly, corneas were procured from twenty-four freshly harvested, adult bovine eyes (Pel-Freeze Biologicals, Rogers, AR), and three 8-mm disks were removed from the central region of each and processed as described before. Two equivalent groups of quartered discs were subjected to collagenase digestion for 30 to 45 minutes at 37°C with shaking at 142 rpm to remove epithelial and endothelial cells. A second digestion with 36 ml of fresh collagenase solution proceeded for 150 minutes under identical conditions. Cells from the second digestion were pelleted by low-speed centrifugation and resuspended in Dulbecco's modified Eagle medium/F-12 (DMEM/F-12, Gibco-Life Technologies, Grand Island, NY). Cell number and viability were determined using trypan blue exclusion. After a second low-speed centrifugation, cells were resuspended in DMEM/F-12 supplemented with 1% platelet-poor horse serum (PPHS; Sigma, St. Louis, MO), plated into six-well tissue culture dishes (Costar, Cambridge, MA) at high density (400,000 cells/well) and allowed to attach overnight at 37°C in 5% CO 2 . Media (2 ml/well) were changed to DMEM/F-12, and incubation proceeded until day 4