Abstract
Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.
Highlights
The corneal stroma is a disc of connective tissue that constitutes about 90% of the mammalian cornea
A recent study has shown that primary bovine keratocytes in low-serum or serum-free media maintain keratan sulfate proteoglycans (KSPG) secretion for several days in culture, whereas growth in 10% fetal bovine serum resulted in decreased KSPG synthesis [23]
We report that keratocyte mitosis is not directly linked to KSPG down-regulation but that subculture after trypsin treatment, in the presence of mitogen-rich fetal bovine serum, results in irreversible loss of KSPG synthesis
Summary
The corneal stroma is a disc of connective tissue that constitutes about 90% of the mammalian cornea. Cells in the healing wound are characterized by secretion of pro-inflammatory cytokines such as interleukin-1␣ and proteolytic enzymes involved in tissue remodeling, collagenase, gelatinase, and stromelysin [3, 4] This remodeling (fibroblastic) phenotype is simulated in vitro when keratocytes are cultured in medium containing fetal bovine serum and subcultured by trypsinization [5]. The high level of expression of these three proteins combined with a specialized glycosylation is a property unique to keratocytes and constitutes an essential feature of the role of the keratocyte in maintenance of corneal transparency This conclusion is supported by the recent demonstration that mice bearing null mutations in the lumican gene lose corneal transparency, whereas mice with a similar mutation in the gene for decorin, the major corneal chondroitin/dermatan sulfate proteoglycan, maintain clear corneas [11, 12]. The presence of FGF-2, supports KSPG synthesis by induction of core protein biosynthesis
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