Lumican Modulates Keratocan Gene Expression

Abstract

Abstract: : Purpose: Keratan sulfate proteoglycans (KSPG) contribute to corneal transparency by regulation of collagen fibrillogenesis producing small, uniform fibril diameters and regular interfibrillar spacing. Lumican null mice (Lum^–/–) exhibit corneal opacity and misarranged collagen matrices, whereas keratocan null mice (Kera^–/–) have a mild phenotype of thin but transparent cornea. Here, we examine the hypothesis that lumican serves to mediate keratocan expression in addition to serving as a regulator of collagen fibrillogenesis. Methods: Keratocan expression in Lum^–/–, Lum^+/+, and Kera–Lum/Lum^+/+ mice (transgenics with lumican expressed under the Kera promoter) was determined by northern hybridization, and western blot analysis and immunohistochemistry with anti–Lum and anti–Kera antibodies. The coupling of keratocan expression with lumican was also examined by intrastromal injection of a Lum minigene and by co–injection of the Lum minigene with a Kera–ßGeo reporter gene into the corneal stroma of Lum^–/– mice, and in vitro using siRNA knockdown of lumican expression in primary keratocyte cultures. Results:Transgenic mice that overexpress lumican in the cornea also have an increased expression of keratocan, whereas the lumican–null corneas show a decrease in keratocan expression at both the protein and mRNA level. Co–injection of a Lum minigene caused increased ß–galactosidase activity driven by the keratocan promoter. Knock down of Lum expression by transfecting cultured bovine keratocytes with siRNA also lead to a down regulated Kera expression.Conclusions:Our results reveal the ability of lumican to modulate gene expression of another KSPG family member in the adult cornea, indicating a novel regulatory interaction of these two closely related KSPGs. It also provides an explanation for the differences in severity of corneal manifestation found in Lum^–/– and Kera^–/– mice. The former exemplifies a dramatic reduction of corneal KSPGs that are necessary for the formation of an organized stroma collagen matrix found in transparent corneas.

Keywords: cornea: stroma and keratocytes • gene/expression • transgenics/knock-outs