Transketolase gene expression in the cornea is influenced by environmental factors and developmentally controlled events.

PURPOSE: Transketolase (TKT) has been proposed to be a corneal crystallin, and its gene and protein are abundantly expressed in the corneal epithelium of several mammals. A marked up-regulation of TKT gene expression coincides with the time of eyelid opening in the mouse. Here, we examined whether exposure to incident light contributes to the up-regulation of TKT gene expression during cornea maturation. METHODS: Mice were raised in either standard light/dark cycling conditions or total darkness. In some cases, subcutaneous injections of epidermal growth factor (EGF) were given beginning on the day of birth to induce early eyelid opening. RNA was prepared from the corneas of mothers and pups and subjected to Northern blot analyses. In addition, the relative levels of TKT mRNA and/or enzyme activity were examined in the corneas of human, bovine, rat, chicken, and zebrafish. RESULTS: TKT mRNA levels were 2.1-fold higher in the corneas of 25-day-old mouse pups ( 12 days after eyelid opening) that had been born and raised in light/dark conditions compared to pups born and raised in total darkness. By contrast, the level of TKT mRNA in the mature corneas of adult mice maintained in the dark for 2-8 weeks did not vary greatly from those of mice maintained in light/dark conditions. Interestingly, TKT mRNA levels in the corneas of dark-raised mice, although reduced, did exhibit the increase characteristically observed before and after eyelid opening. In addition, TKT mRNA levels were elevated fivefold in the corneas of 28-day-old mice raised in darkness and injected with EGF compared to uninjected mice also deprived of light. The EGF-injected mice opened their eyes 3 days early, and their corneal epithelium did not grossly differ from that of control mice. TKT mRNA and/or enzyme activity was found to be much higher in the corneas than in other tissues of humans, bovines, and rats but was extremely low in the corneas of chicken and zebrafish. CONCLUSION: Our studies suggest that both exposure to incident light and events surrounding the process of eyelid opening play a role in the up-regulation of TKT gene expression observed during corneal maturation in mice. Light appears to play a less important role in the mature cornea in maintaining high levels of TKT gene expression. The low levels of TKT in the cornea of chicken and zebrafish support the notion that TKT acts as a taxon-specific enzyme-crystallin in mammals. The involvement of environmental signals for this putative, mammalian cornea crystallin contrasts with the purely developmental signals involved in the up-regulation of the crystallin genes of the lens.

Enhancer-independent promoter activity of the mouse alphaB-crystallin/small heat shock protein gene in the lens and cornea of transgenic mice.

The alphaB-crystallin/small heat shock protein gene is expressed very highly in the mouse eye lens and to a lesser extent in many other nonocular tissues, including the heart, skeletal muscle and brain. Previously we showed in transgenic mice that lens-specific alphaB-crystallin promoter activity is directed by a proximal promoter fragment (-164/+44) and that non-lens promoter activity depends on an upstream enhancer (-427/-259) composed of at least 5 cis-control elements. Here we have used truncated alphaB-crystallin promoter-CAT transgenes to test by biphasic CAT assays and/or histochemistry for specific expression in the cornea and lens. Deletion either of 87 bp (-427/-340) from the 5' end of the alphaB-crystallin enhancer or of the whole enhancer (-427/-258) abolished alphaB-crystallin promoter activity in all tissues except the lens and corneal epithelium when examined by the biphasic CAT assay in 4-5-week-old transgenic mice. These truncations also lowered promoter strength in the lens. The -426/+44-CAT, -339/+44-CAT and -164/+44-CAT (previously thought to be lens-specific in transgenic mice) transgenes were all expressed in the 4-6-week-old corneal epithelium when examined histochemically. Immunohistochemical staining confirmed the presence of endogenous alphaB-crystallin in the mature corneal epithelial cells. CAT gene expression driven by the alphaB-crystallin promoter with or without the enhancer was evident in the embryonic and 4-6-week-old lens. By contrast, activity of the alphaB-crystallin promoter/enhancer-CAT transgene was not detectable in the corneal epithelium before birth. Taken together, these results indicate that the intact enhancer of the alphaB-crystallin/small heat shock protein gene is required for promoter activity in all tissues tested except the lens and cornea.

The cellular basis of corneal transparency: evidence for 'corneal crystallins'.

In vivo corneal light scattering measurements using a novel confocal microscope demonstrated greatly increased backscatter from corneal stromal fibrocytes (keratocytes) in opaque compared to transparent corneal tissue in both humans and rabbits. Additionally, two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1 (ALDH1), isolated from rabbit keratocytes showed unexpectedly abundant expression ( approximately 30% of the soluble protein) in transparent corneas and markedly reduced levels in opaque scleral fibroblasts or keratocytes from hazy, freeze injured regions of the cornea. Together these data suggest that the relatively high expressions of TKT and ALDH1 contribute to corneal transparency in the rabbit at the cellular level, reminiscent of enzyme-crystallins in the lens. We also note that ALDH1 accumulates in the rabbit corneal epithelial cells, rather than ALDH3 as seen in other mammals, consistent with the taxon-specificity observed among lens enzyme-crystallins. Our results suggest that corneal cells, like lens cells, may preferentially express water-soluble proteins, often enzymes, for controlling their optical properties.

Aldehyde dehydrogenase class 3 expression: identification of a cornea-preferred gene promoter in transgenic mice.

Aldehyde dehydrogenase class 3 (ALDH3) constitutes 20-40% of the total water-soluble proteins in the mammalian cornea. Here, we show by Northern blot analysis that ALDH3 expression in the mouse is at least 500-fold higher in the cornea than in any other tissue examined, with very low levels of expression detected in the stomach, urinary bladder, ocular lens, and lung. Histochemical localization reveals that this exceptional level of expression in the mouse cornea occurs in the anterior epithelial cells and that little ALDH3 is present in the keratocytes or corneal endothelial cells. A 13-kbp mouse ALDH3 promoter fragment containing >12 kbp of the 5' flanking sequence, the 40-bp untranslated first exon, and 29 bp of intron 1 directed cat reporter gene expression to tissues that express the endogenous ALDH3 gene, except that transgene promoter activity was higher in the stomach and bladder than in the cornea. By contrast, when driven by a 4.4-kbp mouse ALDH3 promoter fragment [1,050-bp 5' flanking region, exon 1, intron 1 (3.4 kbp), and 7 bp of exon 2] expression of the cat reporter gene was confined to the corneal epithelial cells, except for very low levels in the liver, effectively reproducing the corneal expression pattern of the endogenous ALDH3 gene. These results indicate that tissue-specific expression of ALDH3 is determined by positive and negative elements in the 5' flanking region of the gene and suggests putative silencers located in intron 1. We demonstrate regulatory sequences capable of directing cornea-specific gene expression, affording the opportunity for genetic engineering in this transparent tissue.

Transketolase is a major protein in the mouse cornea.

Earlier experiments in this laboratory identified a highly expressed 65-68-kDa protein in both mouse and human corneas (Cuthbertson, R. A. , Tomarev, S. I., and Piatigorsky J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4004-4008). Here, we demonstrate that this protein is transketolase (TKT; EC 2.2.1.1), an enzyme in the nonoxidative branch of the pentose-phosphate pathway, based on peptide and cDNA isolation and sequence analysis of mouse cornea protein and RNA samples, respectively. While expressed at low levels in a number of tissues, the 2.1-kilobase TKT mRNA was expressed at a 50-fold higher level in the adult mouse cornea. The area of most abundant expression was localized to the cornea epithelial cell layer by in situ hybridization. Western blot analysis confirmed TKT protein abundance in the cornea and indicated that TKT may comprise as much as 10% of the total soluble protein of the adult mouse cornea. Soluble cornea extracts exhibited a correspondingly high level of TKT enzymatic activity. TKT expression increased progressively through cornea maturation, as shown by Northern blot, in situ hybridization, Western blot, and enzymatic analyses. TKT mRNA and protein were expressed at low levels in the cornea prior to eye opening, while markedly increased levels were observed after eye opening. Taken together, these observations suggest that TKT may be a cornea enzyme-crystallin, and suggest that the crystallin paradigm and concept of gene sharing, once thought to be restricted to the lens, apply to other transparent ocular tissues.