Keratocan, a cornea-specific keratan sulfate proteoglycan, is regulated by lumican

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.

Fig. 1. Schematic of Kera-Lum minigene used for transgenic mouse generation

Lum cDNA containing the c-Myc tag was ligated to the 3.2-kb keratocan promoter followed by a BGH polyadenylation signal with pBSK vector (A). Shown is PCR genotyping of three lines of Kera-Lum transgenic mice, a nontransgenic littermate (NTG), and a positive plasmid DNA control. The presence of the 1,500-bp fragment is positive for the transgenic construct (B). Shown is RT-PCR of Kera-Lum5, -25, and -38 of the Kera-Lum transgenic mice and a nontransgenic control. The presence of the 1,016-bp DNA fragment is positive for the transcript from the transgene (C). Shown are Western blots using anti-lumican and c-Myc antibodies from partially purified corneal extracts from three lines of transgenic animals compared with a nontransgenic control probing for lumican. The transgenic animals show a 3–4-fold increase in lumican (about 45 kDa) expression as compared with the nontransgenic control. c-Myc Western blotting revealed a positive signal in all three transgenic lines but no signal in the nontransgenic mice (D).

Fig. 2. Transmission electron micrographs showing similar stromal architecture in stromas from nontransgenic and transgenic mouse corneas

The collagen fibril diameters in corneas of wild type and transgenic mice are virtually identical. In addition, fibril packing and spacing are comparable in normal and overexpressing lines. Identical results were observed in both the anterior and posterior regions of the stroma. Bar, 300 nm.

Fig. 3

Western blots probing for keratocan in the three transgenic lines (A) and in Lum^+/+, Lum^+/–, and Lum^–/– mouse corneas (B). Keratocan is up-regulated ~5-fold in the Kera-Lum lines as compared with the nontransgenic control. Keratocan levels decrease in the heterozygous (Lum^+/–) and homozygous (Lum^–/–) knockout corneas. Northern blotting hybridization of total RNA (10 μg) from two corneas of Lum^–/–, Lum^+/–, wild-type Lum^+/+, nontransgenic littermate (Lum^+/+), and Kera-Lum transgenic mice. Keratocan mRNA is down-regulated in Lum^–/– mouse corneas and up-regulated in the Kera-Lum transgenic mouse (C).

Fig. 4. Histological analyses of lumican-null mouse corneas 5 days after intrastromal injection of pSecLum or empty vector control plasmid DNA

Hematoxylin and eosin (H&E) staining revealed no significant morphological changes between empty vector- and pSecLum-injected corneas. Immunohistochemistry using anti-lumican antibody detects the presence of lumican in the pSecLum-injected cornea.

Fig. 5. Western blot for lumican (bottom) and keratocan (top) of corneal extracts from Lum^–/– mice 5 days after intrastromal injection of pSecLum or empty vector control plasmid DNA

Lumican levels increase as expected following intrastromal injection of pSecLum plasmid DNA. Keratocan levels also increase 2-fold after injection of pSecLum plasmid DNA, as compared with the empty vector control.

Fig. 6. Down-regulated expression of keratocan by bovine keratocytes treated with lumican siRNA

A, lumican was detected by Western blotting (as described under “Experimental Procedures”) in culture medium (lanes 2 and 4) or cell lysates (lanes 1 and 3) of primary cultures of bovine keratocytes that had been transfected (lanes 3 and 4) with siRNA to bovine lumican or mock-transfected controls (lanes 1 and 2) 96 h after transfection. Transfection of siRNA significantly suppresses the synthesis of lumican. B, quantitative real time RT-PCR was used to determine relative mRNA pools in primary keratocytes 72 h after lumican siRNA transfection (solid bars) or mock transfection (patterned bars) using primer/probes for keratocan, aldehyde dehydrogenase (ALDH), and biglycan as previously described (39).

Fig. 7. Keratocan promoter activity assay performed in Lum^–/– animals measuring the ability of the keratocan promoter to drive β-GEO expression in the presence or absence of lumican

The ability of an empty vector (Vector) or a lumican-expressing plasmid DNA construct (Lumican) to influence the expression of a β-GEO reporter gene by the keratocan promoter was measured 4 days after intrastromal injection. The presence of lumican significantly increases the activity of the keratocan promoter as shown in β-galactosidase 10^–6 units/cornea as compared with vector control.