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J. Biol. Chem., Vol. 280, Issue 38, 32634-32639, September 23, 2005

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Maintenance of the Keratocyte Phenotype during Cell Proliferation Stimulated by Insulin^*

Kurt Musselmann, Bridgette Alexandrou, Bradley Kane, and John R. Hassell1

From the Department of Biochemistry and Molecular Biology, University of South Florida College of Medicine and Shriners Hospitals for Children Tampa, Tampa, Florida, 33612

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/cm²) and high (20,000 cells/cm²) density in serum-free medium. Keratocyte proliferation was measured by [³H]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.

Received for publication, April 29, 2005 , and in revised form, July 25, 2005.

^* This work was supported by a Grant EY08104 from the National Institutes of Health (to J. R. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Shriners Hospital for Children, Research Molecular Biology, 12502 Pine Dr., Tampa, FL 33612. Tel.: 813-975-7144; Fax: 813-975-7127; E-mail: jhassell{at}shctampa.usf.edu.

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