Maintenance of the Keratocyte Phenotype during Cell Proliferation Stimulated by Insulin

Abstract

Keratocytes normally express high levels of aldehyde dehydrogenase and keratocan. They proliferate and lose their keratocyte markers when they become fibroblastic during corneal wound healing. Keratocytes cultured in fetal bovine serum also become fibroblastic, proliferate, and lose these markers. In this report, we studied the effects of three serum growth factors, fibroblast growth factor-2, insulin, and platelet-derived growth factor-BB, on keratocyte proliferation and the maintenance of the keratocyte markers in 7-day cultures in cells plated at low (5,000 cells/cm 2 ) and high (20,000 cells/cm 2 ) density in serum-free medium. Keratocyte proliferation was measured by [ 3 H]thymidine incorporation and by DNA content of the cultures. Cytosolic aldehyde dehydrogenase and keratocan accumulated in the medium were quantified by Western blot. The results showed that all the growth factors stimulated proliferation, but insulin stimulated proliferation more consistently. The keratocyte markers aldehyde dehydrogenase and keratocan were maintained after 7 days in culture in all growth factors, but keratocyte cell morphology was only maintained in medium containing insulin. Most of the proteoglycans were degraded in cultures of keratocytes plated at low density and cultured in the absence of growth factors. This degradation was prevented when keratocytes were cultured in the presence of the growth factors or when keratocytes were plated at high density. The results of this study show that insulin can expand keratocytes in vitro, maintain their phenotype, and prevent proteoglycan degradation.Keratocytes, the cells of the mature corneal stroma, have a dendritic morphology and are responsible for the maintenance of the extracellular matrix of the stroma. These cells produce lumican and keratocan, two keratan sulfate proteoglycans (1, 2) that are necessary to maintain the transparency and shape of the stroma. Lumican and keratocan are members of the small leucine-rich protein family and are the most abundant keratan sulfate proteoglycans in the corneal stroma (3). Lumican is also present in many tissues (1) as a glycosylated protein. Keratocan is exclusively found as a proteoglycan in the cornea (1, 4). Both keratocan and lumican (3, 5, 6) interact with collagen fibrils and modulate their size and spacing (3,5,7,8). The collagen fibrils of both lumican (3,5,7,9) and keratocan (3, 10) knock-out mice are larger and less densely packaged than the collagen heterofibrils in the normal stroma, and in the lumican null mouse the corneas lose transparency (3, 7). The corneas in the keratocan null mouse were reduced in thickness, most likely because of reduced hydration of the corneal stroma. Mutations in the human keratocan gene have been identified as a cause of cornea plana (10 -12). This suggests that keratocan plays a fundamental role in maintenance of the corneal structure and that its function cannot be rescued by other members of the small leucine-rich protein family (13).Keratocytes normally exhibit a dendritic morphology b...

IGF-II is present in bovine corneal stroma and activates keratocytes to proliferate in vitro

“…This indicates that keratocytes cultured in IGF-II can proliferate and maintain their native phenotype similar to that shown for keratocytes cultured in 10 micrograms of insulin/ml (Musselmann et al 2005;Musselmann et al 2006;Guerriero et al 2007). IGF-II and high concentrations of insulin can bind IGF-IR, the receptor for IGF-I, to activate cell proliferation [for review see (Lelbach et al 2005)].…”

“…DNA synthesis was determined in cultures that were radiolabeled with 740,000 Bq 3 H-thymidine/ml of medium for 72 hours beginning on day 4. The incorporation of the radiolabel into DNA was determined as previously described (Musselmann et al 2005).…”

“…In this way it was possible to directly compare the amount of a particular protein produced by the same number of cells. SDS/PAGE of samples from the pooled media and cell layer extracts were run reduced as previously described (Musselmann et al 2005) and Western blots were performed using the ECL Western blotting analysis system (GE Healthcare, Piscataway, NJ). Primary antibodies included: mouse monoclonal anti-α-SM actin (clone asm-1, Roche Molecular Biochemicals), rabbit anti-ALDH (provided by R. Lindahl, University of South Dakota), mouse monoclonal anti-fibronectin (clone TV-1, Millipore), rabbit anti-SPARC (provided by L. Fisher, NIDCR, NIH), rabbit anti-IGF-I (PAA1, Cell Sciences), rabbit anti-IGF-II (PA0382, Cell Sciences), rabbit anti-IGFBP-2 (US Biologicals).…”

“…Keratocytes isolated from the stroma by collagenase digestion and cultured in serum free media retain their dendritic morphology and their quiescence (Jester et al 1996;Beales et al 1999) but can be activated to proliferate by a number of growth factors including, FGF(fibroblast growth factor)-2, PDGF(platelet derived growth factor), TGF(transforming growth factor)-β, IL(interleukin)-1-α, IGF(insulin like growth factor)-I and insulin (Long et al 2000;Jester et al 2002;Jester and Ho-Chang 2003;Musselmann et al 2005). Antibodies to TGF-β have been shown to reduce keratocyte activation in corneal wounds (Jester et al 1997) and this suggests that TGF-β is present in wounds and activates keratocytes in vivo as well.…”

The effects of riboflavin and ultraviolet light on keratocytes cultured in vitro

“…The corneal stroma consists of collagen fibers, extracellular matrix (ECM), and keratocytes, which exhibit dendritic morphology and function in the synthesis of collagen fibrils. The uniform arrangement of these collagen fibers allows corneal transparency (4,5) . A number of diseases have been shown to affect the arrangement and resistance of corneal collagen fibers.…”