Keratocan, a Cornea-specific Keratan Sulfate Proteoglycan, Is Regulatedby Lumican

Eric C Carlson,Chia-Yang Liu,Tai-Ichiro Chikama,Yasuhito Hayashi,Candace W.-C Kao,David E Birk,James L Funderburgh,James V Jester,Winston W.-Y Kao

doi:10.1074/jbc.m500249200

Abstract

Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, keratocan, carries the remaining keratan sulfate of the cornea, but keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased keratocan. This coupling of keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum-/- mice. Small interfering RNA knockdown of lumican in vitro reduced keratocan expression, whereas co-injection of a lumican-expressing minigene with a beta-galactosidase reporter driven by the keratocan promoter demonstrated an increase of keratocan transcriptional activity in response to lumican expression in Lum-/- corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum-/- and Kera-/- mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.

Highlights

D Recipient of an Olga Weiss Scholarship from Research To Prevent Blindness (RPB). g A Julius and Doris Stein RPB Professor of Ophthalmology. i Recipients of the RPB Senior Scientific Investigator Award. j To whom correspondence should be addressed: Dept. of Ophthalmology, University of Cincinnati, 3223 Eden Ave., Cincinnati, OH 4582670527
lumican-expressing plasmid DNA construct (Lumican) plays a role in several biological processes such as wound healing, epithelialmesenchymal transition, and tumorigenesis [12, 18, 19, 21, 21, 41]
The results reported here indicate a novel biological role of lumican as a modulator of keratocan gene expression by keratocytes

Summary

Introduction

D Recipient of an Olga Weiss Scholarship from RPB. g A Julius and Doris Stein RPB Professor of Ophthalmology. i Recipients of the RPB Senior Scientific Investigator Award. j To whom correspondence should be addressed: Dept. of Ophthalmology, University of Cincinnati, 3223 Eden Ave., Cincinnati, OH 4582670527. The delayed epithelial wound healing phenotype in LumϪ/Ϫ mice is potentially due to the involvement of lumican in cellular migration, adhesion, and/or proliferation [12, 16]. Lumican-null (LumϪ/Ϫ) mice manifest corneal opacity, skin fragility, and impaired collagen fibrillogenesis [1, 12]; ablation of the keratocan gene (Kera) only results in a subtle manifestation of thin but transparent cornea in which little changes of the collagen matrix can be detected [26, 27]. Using a number of different experimental approaches, lumican expression was found to exert a direct effect on the expression of keratocan These findings provide a new explanation for the differences of clinical manifestations in corneas of LumϪ/Ϫ and KeraϪ/Ϫ mice and document a novel cellular regulatory function of lumican

Objectives

Results

Conclusion