Structurally Normal Corneas in Aldehyde Dehydrogenase 3a1-Deficient Mice

Nees, David W.; Wawrousek, Eric F.; Robison, W. Gerald; Piatigorsky, Joram

doi:10.1128/mcb.22.3.849-855.2002

Cited by 53 publications

(41 citation statements)

References 44 publications

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“…TKT is concentrated in the corneal epithelium of WT mice (25) enriched in the corneal stroma and endothelium (6) of WT mice. The fact that a 50% reduction in corneal TKT had no detectable effect is analogous to our earlier finding that elimination of ALDH3, the most abundant corneal enzyme and which is also concentrated in the corneal epithelium, had no detectable effect on corneal clarity or structure (16). There was no compensatory protein that accumulated in the corneas of the ALDH3 Ϫ/Ϫ (16) or TKT ϩ/Ϫ (present report; data not shown) mice.…”

Section: Discussionsupporting

confidence: 64%

Transketolase Haploinsufficiency Reduces Adipose Tissue and Female Fertility in Mice

Zhengping

Wawrousek

Piatigorsky

2002

Molecular and Cellular Biology

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Transketolase (TKT) is a ubiquitous enzyme used in multiple metabolic pathways. We show here by gene targeting that TKT-null mouse embryos are not viable and that disruption of one TKT allele can cause growth retardation (Ϸ35%) and preferential reduction of adipose tissue (Ϸ77%). Other TKT ؉/؊ tissues had moderate (Ϸ33%; liver, gonads) or relatively little (Ϸ7 to 18%; eye, kidney, heart, brain) reductions in mass. These mice expressed a normal level of growth hormone and reduced leptin levels. No phenotype was observed in the TKT ؉/؊ cornea, where TKT is especially abundant in wild-type mice. The small female TKT ؉/؊ mice mated infrequently and had few progeny (with a male/female ratio of 1.4:1) when pregnant. Thus, TKT in normal mice appears to be carefully balanced at a threshold level for well-being. Our data suggest that TKT deficiency may have clinical significance in humans and raise the possibility that obesity may be treated by partial inhibition of TKT in adipose tissue.Transketolase (EC 2.2.1.1) (TKT) is a thiamine diphosphate-dependent enzyme linking the nonoxidative branch of the pentose phosphate pathway (PPP) to the glycolytic pathway. The PPP generates sugar phosphates for intermediary biosynthesis and nucleic acid synthesis and NADPH for reductive biosynthesis (26,31). Fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. TKT is expressed in most tissues throughout the animal and plant kingdoms (27). It is prevalent in proliferating tumor cells, and its repression can inhibit tumor cell growth (2).Our interest in TKT was stimulated by its abundance in the cornea of mice (25) and rabbits (9). In mice, TKT comprises approximately 10% (25) and aldehyde dehydrogenase class 3 (ALDH3) comprises approximately 40% (4, 16) of the watersoluble protein of the corneal epithelial cells. By analogy with the use of enzymes as lens crystallins, we have speculated that the abundant metabolic enzymes in the cornea may serve a structural as well as metabolic role (4,21,22).In the present investigation we disrupted the TKT gene by homologous recombination to test whether corneal clarity is dependent on the concentration of this enzyme. We were only able to obtain viable TKT ϩ/Ϫ mice. While their corneas appeared normal, the TKT haploinsufficient mice were often smaller and the small females were relatively infertile. The sizes of the organs were differentially affected, with adipose tissue being markedly reduced in the TKT ϩ/Ϫ mice. It thus appears that in mice, as found earlier in plants (8), a reduction in TKT leads to multiple phenotypes and metabolic disturbances. Moreover, our results suggest that TKT haploinsufficiency or a reduction in TKT activity may have clinical implications in humans. MATERIALS AND METHODS Generation and mating of TKT؉/؊ mice. A 12.5-kb genomic clone containing the mouse TKT gene was isolated from a 129Sv library (Stratagene, La Jolla, Calif.). The targeting construct was created by replacing a 2.8-kb BamHI-BamHI fragment (24) with a 1.6-kb PGK/neo cassette for...

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Section: Discussionsupporting

confidence: 64%

Transketolase Haploinsufficiency Reduces Adipose Tissue and Female Fertility in Mice

Zhengping

Wawrousek

Piatigorsky

2002

Molecular and Cellular Biology

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“…43 and references therein). As mentioned above, ALDH3A1 null mice have transparent cornea (20). However, the lack of any obvious physical-structural functions does not eliminate ALDH3A1 as a corneal crystallin.…”

Section: Discussionmentioning

confidence: 99%

“…To explain the functional significance of such high levels of ALDH3A1 expression, several hypotheses have been postulated, and these include both structural and enzymatic properties (17). It was initially suggested that ALDH3A1 is involved in the transparency and the optical properties of the corneal cells (18), although ALDH3A1 null mice have no apparent corneal abnormalities (19,20). We have previously shown that SWR mice, a strain prone to corneal clouding after exposure to UV radiation (21), lack ALDH3A1 due to mutations in the structural gene (19).…”

Section: Discussionmentioning

confidence: 99%

Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells

Pappa

Brown

Koutalos

et al. 2005

Journal of Biological Chemistry

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Aldehyde dehydrogenase 3A1 (ALDH3A1) is a NAD(P)؉ -dependent enzyme that is highly expressed in mammalian corneal epithelial cells and has been shown to protect against UV-and 4-hydroxynonenal-induced cellular damage, mainly by metabolizing toxic lipid peroxidation aldehydes. Here we report a novel function of ALDH3A1 as a negative cell cycle regulator. We noticed a reduction in ALDH3A1 gene expression in actively proliferating primary human corneal epithelium explant cultures, indicating that ALDH3A1 expression is inversely correlated with replication. To examine this further, a human corneal epithelial cell line (HCE) lacking endogenous ALDH3A1 was stably transfected to express ALDH3A1 at levels similar to those found in vivo. ALDH3A1-transfected cells exhibited an elongated cell cycle, decreased plating efficiency, and reduced DNA synthesis compared with the mock-transfected cells. These effects were associated with reduced cyclin Aand cyclin B-dependent kinase activities and reduced phosphorylation of the retinoblastoma protein (pRb) as well as decreased protein levels of cyclins A, B, and E, the transcription factor E2F1, and the cyclin-dependent kinase inhibitor p21. These observations were further expanded and confirmed on human keratinocyte cells (NCTC-2544) overexpressing ALDH3A1. Consistent with a protective role of an elongated cell cycle, ALDH3A1-transfected cells exhibited increased resistance to the cytotoxic effects of the DNA-damaging agents mitomycin C and Vp-16. Immunohistochemistry and biochemical fractionation revealed that ALDH3A1 is localized both in the nucleus and cytosol of ALDH3A1-transfected cells, implying a possible association between the nuclear localization of the enzyme and its proliferationsuppressive functions. In conclusion, these results suggest that ALDH3A1 may protect corneal epithelial cells against oxidative damage not only through its metabolic function but also by prolonging the cell cycle. The cornea serves as barrier between the external environment and the internal ocular tissues, protecting them against oxidative stimuli that include solar radiation and molecular oxygen. Mammalian corneal epithelial cells express high levels of ALDH3A1 1 (6), an enzyme that is part of a well regulated defense system that protects the eye against oxidative damage (7,8). Because this enzyme represents nearly half of the total soluble protein in the corneal epithelium, levels that exceed those required for normal metabolism, it has been proposed to be a corneal crystallin, mimicking the situation in the lens (6, 9) where metabolic proteins are overexpressed for their intrinsic refractive properties. Additional functions have been hypothesized regarding the role of ALDH3A1 in corneal epithelium. These include direct absorption of UV radiation, scavenging of UV-generated reactive oxygen species (ROS), and a chaperonelike function preventing aggregation of partially denatured proteins (10). We have recently shown that ALDH3A1 protects corneal epithelial cells against oxidative damage indu...

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“…These results indicate an apparent correlation between cellular transparency and the levels of enzyme-crystallins in stromal cells of the cornea. However, ALDH3A1-deficient mice (31) and TKT ϩ/-(32) mice show no corneal phenotype, indicating that the enzyme-crystallins are not required for corneal transparency. Other studies have indicated that the abundant water-soluble enzyme-crystallins protect the cornea against ultraviolet (UV)-induced oxidative injury.…”

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confidence: 99%

Proteomic Analysis of the Soluble Fraction from Human Corneal Fibroblasts with Reference to Ocular Transparency

Karring

Thøgersen

Klintworth

et al. 2004

Molecular & Cellular Proteomics

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The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serumcultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts. Molecular & Cellular Proteomics 3:660 -674, 2004.The cornea, the only transparent connective tissue of the body, is responsible for ϳ70% of the total refractive power of visible light in the eye. The corneal stroma (thickness ϳ450 m) mainly consists of collagen lamellae formed by uniform fibrils composed of type I, III, and V collagen (1, 2). The uniform diameter (ϳ30 nm) and regular spacing of the collagen fibrils (ϳ60 nm between the centers) lead to destructive interference of light except in the forward direction and thus optical transparency of the extracellular matrix of the cornea (3, 4). The stroma also contains multiple layers of quiescent corneal fibroblasts, termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the quiescent keratocytes are transparent except for the nuclei when studied in vivo using scanning slit specular microscopy (5), slit-lamp biomicroscopy, or in vivo confocal microscopy (6). This stealth-like invisibility indicates that the refractive index of the keratocyte cytoplasm and cellular processes is similar to that of the extracellular matrix of the corneal stroma (6).The keratocytes have a dynamic potential for proliferation and transformation. Thus, after injury of the cornea such as excimer laser keratectomy for the treatment of myopia, the keratocytes transform into spindle-shaped fibroblastic cells (7,8). The corneal fibroblasts repopulate the d...

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Structurally Normal Corneas in Aldehyde Dehydrogenase 3a1-Deficient Mice

Cited by 53 publications

References 44 publications

Transketolase Haploinsufficiency Reduces Adipose Tissue and Female Fertility in Mice

Transketolase Haploinsufficiency Reduces Adipose Tissue and Female Fertility in Mice

Human Aldehyde Dehydrogenase 3A1 Inhibits Proliferation and Promotes Survival of Human Corneal Epithelial Cells

Proteomic Analysis of the Soluble Fraction from Human Corneal Fibroblasts with Reference to Ocular Transparency

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