Keratan sulfate synthesis by corneal stromal cells within three-dimensional collagen gel cultures

The corneal stromal cells from 2-day-old chicks were cultured on plastic dishes or within three-dimensional collagen gel in the presence or absence of growth factor (EGF, bFGF, PDGF, TGF-beta 1, or their combinations). The cells were labeled with [35S] sulfate and [3H]-glucosamine, and the radio-labeled proteoglycans were examined. Keratan sulfate was synthesized to some extent (15.4-16.9% of total synthesis for medium fraction; 8.0% for cell layer fraction) in a primary culture even when the cells were cultured on plastic dishes, although the values were very much lower than that (42.7%) in the stromal fraction of organ culture of corneal explants. The primary culture in collagen gel showed some increase in the proportion of keratan sulfate synthesis as compared with the culture on plastic. Among growth factors, addition of EGF to the culture in gel caused a further increase in the proportion of keratan sulfate synthesis. bFGF and TGF-beta 1 increased proteoglycan synthesis as a whole to some extent, but chondroitin sulfate/dermatan sulfate synthesis was increased preferentially and, consequently, the proportion of keratan sulfate synthesis to total synthesis was decreased. PDGF also caused some decrease in the proportion. In the culture after one passage (secondary culture), the keratan sulfate synthesis decreased markedly (8.6-8.3% of total synthesis for medium fraction; 2.7% for cell layer or gel fraction) and a large chondroitin sulfate/dermatan sulfate proteoglycan appeared whether the cells were cultured on plastic or in collagen gel. But, when the medium was changed to CG medium (serum-free medium) in the middle of either primary or secondary cultures, the keratan sulfate synthesis (27.8% for medium fraction; 15.6% for gel fraction) was maintained at the level of that of the primary culture in gel. EGF and bFGF were not additive to the effect of CG medium on the keratan sulfate synthesis in the secondary culture. Instead, EGF and bFGF stimulated hyaluronic acid synthesis in the culture. The mechanism of these changes in the expression type of proteoglycan and their significance remain to be clarified.