Abstract
Purpose: :
The core proteins of corneal keratan sulfate proteoglycans have been shown to regulate collagen fibril assembly and growth. The hydroxylation of lysine and proline residues in collagen is necessary for stable triple helix formation and subsequent fibril assembly. Ascorbate is a co–factor required for the hydroxylation of these amino acids. Here we determine the importance of ascorbic acid for keratan sulfate proteoglycan synthesis in a chemically defined culture medium that stimulates proliferation and maintains keratocyte phenotype (JBC, 2005, 280: 32634–9).
Methods: :
Keratocytes were isolated by collagenase digestion and plated out at 20,000 cells/cm2 in DMEM/F12. The medium was changed on day 1 to DMEM/F12 with or without 1 mM 2–phospho–l–ascorbic acid (ASC) or DMEM/F12 supplemented with 10 ug/ml insulin with or without ASC. The cells were radiolabeled on day 1 for 72 h with either 35SO4 and digested with endo–ß–galactosidase (eßg) to determine keratan sulfate synthesis or 3H–glycine and digested with collagenase to determine collagen synthesis using or on day 4 for 30 minutes with 35S–express for immunoprecipitation of lumican, keratocan and prostaglandin D synthase (PGDS) precursor proteins. Media was collected on day 4, digested with eßg and core proteins were detected by Western blot. DNA content was measured on days 1 and 4 using Cyquant. Ethyl–3,4–dihydroxy benzoate (EDB) was used to inhibit proline hydroxylase. All measurements were expressed per DNA.
Results: :
ASC alone had no effect on collagen synthesis, but insulin and insulin+ASC increased collagen synthesis 4– and 8–fold, respectively. ASC or insulin alone did not increase keratocan or lumican precursor protein synthesis. Insulin+ASC, however, increased both 6–fold but had no effect on PDGS. Keratocytes cultured ASC accumulated 4– and 3–fold more keratocan and lumican, respectively, than keratocytes in DMEM/F12. Culture in medium containing insulin alone did not increase the accumulation of any of these components. Culture in insulin+ASC, however, stimulated keratocan and lumican accumulation 9–fold and keratan sulfate synthesis 5–fold when compared to control. EDB prevented this stimulation.
Conclusions: :
These data show that although insulin alone stimulates collagen synthesis, insulin did not stimulate the synthesis and accumulation of keratan sulfate proteoglycans unless ASC was also present. This suggests that the synthesis and secretion of keratocan and lumican may be linked with the synthesis and hydroxylation of collagen.
Keywords: cornea: stroma and keratocytes • cornea: basic science • extracellular matrix