Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways

Abstract

Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (؊3519 to ؉43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5 flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl 2 , which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl 2 treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3␣/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways.Most mammalian corneas accumulate (5 to 40% depending on species) aldehyde dehydrogenase 3 (ALDH3A1) (unified nomenclature for the ALDH gene family [http://www.uchsc .edu/sp/sp/alcdbase/alcdbase.html]) mostly, but not exclusively, in their epithelium (47). While the function of the abundant ALDH3A1 in mammalian corneal epithelial cells is not established, detoxification of lipid peroxide radicals induced by UV light (26), absorption of UV (1), and a suspected structural role (47) are among the suggested possibilities. The fact that elimination of mouse ALDH3A1 (50% of corneal water-soluble protein) by homologous recombination in mice fails to show a corneal phenotype has increased the mystery of its function (44). Deficiencies in ALDH have also been implicated in keratoconus, which affects the corneal epithelium (18).Rabbits are exceptional in that they accumulate ALDH1A1 rather than ALDH3A1 in the cornea (29). ALDH1A1 comprises about 3% and ALDH3A1 comprises about 5% of the total soluble protein in human cornea (31). In contrast to the situation with ALDH3A1 in other mammals, rabbit ALDH1A1 is more abundant in the stromal cells of the cornea than in the epithelium. Wound healing experiments after freeze-induced injury suggested that the prevalence of ALDH1A1 may contribute to transparency of rabbit stromal cells (29), analogous to the role of crystallins in the lens. Indeed, -crystallin (ALDH1A8) is a lens crystallin, comprising 25% of the watersoluble protein of the elephant shrew lens (20). A protein equally similar to ALDH1A1 and ALDH2...

Identification of Candidate Klf4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of Klf4 in the Mouse Cornea

“…9,10 Different transcription factors influencing corneal morphogenesis and their target genes are known. [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] Despite these advances, our knowledge of the genetic network of transcription factors regulating embryonic morphogenesis, postnatal maturation, and maintenance of cornea remains incomplete.…”

Conditional Deletion of the Mouse Klf4 Gene Results in Corneal Epithelial Fragility, Stromal Edema, and Loss of Conjunctival Goblet Cells

“…Relatively little is known about the genetic network of transcription factors required for embryonic morphogenesis, postnatal maturation, and maintenance of cornea (1,2,10,12,20,23,26,32,39,46,47,55,59,60,65). Serial analysis of gene expression identified KLF4, a member of the Krüppel-like transcription factor (KLF) subfamily of Cys2-His2 zinc finger proteins, as one of the most highly expressed transcription factors in both 9-day-old and 6-week-old mouse cornea (50).…”

Rabbit Models of Contact Lens–Associated Corneal Hypoxia: A Review of the Literature

“…Numerous structural and biochemical changes occurring in the rabbit cornea during postnatal development would complicate the use of adolescent animals. 3,16 …”

“…The cornea is an avascular tissue that experiences a dramatic (i.e., approximately 75%) decrease in oxygen tension at the corneal surface when the eyelids are closed. 3 The rabbit and human corneas differ in their thickness. 4,5 Human corneal thickness is approximately 500 m, whereas the thickness of the rabbit cornea is approximately 400 m. 6 The thickness of the epithelium of the cornea is slightly greater in human corneas (50 m) than in rabbit corneas (40 m).…”

“…A 4% thickening of the cornea occurs from eyelid closure alone (e.g., during sleep). 3 Contact lens-associated corneal edema correlates with the frequency of infectious keratitis in a rabbit model 26 Desquamation of corneal epithelial cells can be characterized in vivo through the use of tandem scanning confocal microscopy. 28 This structural change was observed after 1 day of wear of RGP lenses with low oxygen transmissibility.…”