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R.B. Hough, J. Piatigorsky; Cornea-preferred Transcription of Rabbit ALDH1A1: Importance of Cis-elements Mediating a Hypoxic Response . Invest. Ophthalmol. Vis. Sci. 2003;44(13):852.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In the rabbit cornea, aldehyde dehydrogenase 1 (ALDH1A1) is present in an unusually high concentration for a metabolic enzyme. Our goals are to identify the distribution of ALDH1A1 in the rabbit cornea and the cis-regulatory elements that govern its cornea-preferred expression. Methods: Transgenic mice with rabbit ALDH1A1 promoter fragments driving luciferase expression were analyzed. Expression of ALDH1A1 in rabbit tissues was analyzed by Northern and Western blot. Promoter fragments driving expression of luciferase were examined in primary cultures of rabbit cornea cells and hepatoma cell lines (Hep3B, HepG2, and Hepa1c1c7). Results: Northern and Western blots showed rabbit ALDH1A1 is expressed at least 6-7 times higher in the cornea than other tissues. Rabbit ALDH1A1 protein was expressed in the epithelial, stromal, and endothelial cell layers of the cornea at levels of 3.3%, 16.1%, and 10.9% of the total soluble protein, respectively. Transgenic mice with a –3519/+43 promoter fragment of the rabbit ALDH1A1 gene fused to the luciferase reporter gene had more than 4-fold higher luciferase expression in the cornea, iris, sclera, and muscle than in the retina, lung, and liver. A -1054/+43 promoter fragment drove luciferase gene expression primarily in the retina but also to a lesser extent in the cornea of transgenic mice. In transfection experiments, constructs containing the –3519/+43 promoter fragment of the rabbit ALDH1A1 gave 4-fold higher levels of luciferase expression in primary cultures of rabbit cornea stromal and epithelial cells than in hepatoma cell lines. Upstream of the rabbit ALDH1A1 gene are 4 binding sites of the aryl hydrocarbon nuclear translocator (ARNT), a basic helix-loop-helix transcription factor containing a Per-ARNT-Sim domain (bHLH-PAS). The rabbit –3519/+43 ALDH1A1 promoter was stimulated 2.5-fold by dioxin and repressed 3-fold by hypoxia in transfected HepG2 and rabbit corneal cells, respectively. Mutagenesis of the ARNT binding sites in the promoter constructs eliminated stimulation by dioxin and inhibition by hypoxia and reduced the activity in cultured cornea cells. DNA mobility shift assays indicated the binding of proteins in nuclear extracts from rabbit cornea stromal cells to the ARNT binding sites. Conclusions: The rabbit ALDH1A1 –3519/+43 promoter fragment shows cornea-preferred activity in transgenic mice and transfected cells. The presence of ARNT binding sites and the responses to dioxin and hypoxia suggest transcriptional regulation of rabbit ALDH1A1 by members of the bHLH-PAS family.
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