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.     

Tissue-specific expression of the chicken alpha A-crystallin gene in cultured lens epithelia and transgenic mice

The present experiments show that the single gene for the lens-specific protein alpha A-crystallin of chickens and mice uses a different subset of cis- and trans-acting regulatory elements for expression in transfected embryonic chicken lens epithelial cells. A chicken alpha A-crystallin-chloramphenicol acetyltransferase (CAT) fusion gene required 162 base pairs whereas the murine alpha A-crystallin-CAT fusion gene required only 111 base pairs of 5'-flanking sequences for efficient tissue-specific expression in the transfected chicken lens cells. Gel retardation and competition experiments were performed using embryonic chicken lens nuclear extract and oligodeoxynucleotides identical to the 5'-flanking region of the chicken (-170/-111) and murine (-111/-88 and -88/-55) alpha A-crystallin gene. The results indicated that these homologous promoters use different nuclear factors for function. Methylation interference analysis identified a dyad of symmetry (CTGGTTCCCACCAG) at position -153 to -140 in the chicken alpha A-crystallin promoter which binds one or more lens nuclear factors. Gel mobility shift experiments using nuclear extracts of brain, reticulocytes, and muscle of embryonic chickens or HeLa cells suggested that the factor(s) binding to the chicken alpha A-crystallin gene promoter sequences are not lens specific. Despite differences in the functional and protein-binding properties of the alpha A-crystallin gene promoter of chickens and mice, expression of the chicken alpha A-crystallin-CAT fusion gene in transgenic mice was lens specific, consistent with a common underlying mechanism for expression of the alpha A-crystallin gene in chickens and mice.     

Transgenic Xenopus laevis tadpoles: a transient in vivo model system for the manipulation of lens function and lens development.

Rodent gamma-crystallin promoters were recognized as lens-specific promoters in micro-injected Xenopus laevis tadpoles and targeted the expression of the chloramphenicol acetyl transferase (CAT) reporter gene to the tadpole lens. The onset of expression coincided with lens cell formation. The level of expression continued to increase up to 9 days of development (stage 47), stayed at that level till at least day 13 and dropped by only 57% at day 21. In contrast, the level of expression of a non-tissue-specific promoter, the SV40 early promoter, decreased rapidly in the eye during development and was only detectable up to stage 44 (day 5). The stability of the CAT activity in the lens was assessed by delivering a pulse of activity from a heat shock promoter-CAT fusion gene. The half-life of the CAT activity in the eye was the same as that in the tail. The increase in CAT activity in the lens thus depends upon continued activity of the injected gamma-crystallin promoters. Our data demonstrate that mammalian promoters can be used to target gene expression to specific tissues during Xenopus laevis development.     

Tissue- and species-specific promoter elements of rat gamma-crystallin genes.

The 5' flanking regions of the six rat gamma-crystallin genes (gamma A-gamma F) are all capable of conferring lens-specific expression to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene in either transdifferentiating chicken neural retina cells or mouse lens epithelial cells. Deletion mapping of the most active gamma-crystallin promoter region, the gamma D region, showed that at least three elements are required for maximal expression in mouse lens epithelial cells: element(s) located between -200 and -106, a conserved CG rich region around position -75, and a CG stretch around -15. The region between -200 and -106 was dispensable in transdifferentiating chicken neural retina cells, which instead required the region between -106 and -78. The maximal activity of the gamma E and gamma F promoters was also dependent upon the integrity of the conserved CG region located around -75. A synthetic oligonucleotide containing this sequence was capable of lens-specific enhancement of the activity of the tk promoter in transdifferentiating chicken neural retina cells but not in mouse lens epithelial cells. Our results further show that this region may contain a silencer element, active in non-lens tissues, as well.     

Insulin-like growth factors, IGF-1, IGF-2 and somatomedin C trigger cell proliferation in mammalian epithelial cells cultured in a serum-free medium

A single insulin-like growth factor which constitutes part of a defined serum-free medium is sufficient to stimulate DNA synthesis and mitosis in mammalian lens epithelial cells. Rabbit lenses were cultured in KEI-4, a medium which mimics rabbit aqueous humor, or in KEI-4 containing insulin growth factor I (IGF I), insulin growth factor II (IGF II) or somatomedin C. The magnitude of DNA synthesis and mitosis was evaluated on whole mount preparations of the epithelium at various times of culture. IGF I and II, the most highly purified of the insulin-like growth factors, and somatomedin C were equipotent lens mitogens, were active at the ng level, were more mitogenic toward lens epithelial cells than insulin, and initiated cell proliferation throughout the normally amitotic central region of the lens epithelium. The time course of the mitotic response elicited by the insulin-like growth factors was identical to that noted in lenses cultured in medium supplemented with serum or insulin. The present results, coupled with those of other investigators, suggest that insulin-like factors may regulate cell division in the mammalian lens in vivo.     

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM D-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 microM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123+/-10 microgram/10(6) cells (n=5) to 3.9+/-1.9 microgram/10(6) cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.     

The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis)

Since aldose reductase is localized primarily in lens epithelial cells, osmotic insults induced by the accumulation of sugar alcohols occur first in these cells. To determine whether the accumulation of sugar alcohols can induce lens epithelial cell death, galactose-induced apoptosis has been investigated in dog lens epithelial cells. Dog lens epithelial cells were cultured in Dulbecco's modified Eagle's mimimum essential medium (DMEM) supplemented with 20% fetal calf serum (FCS). After reaching confluence at fifth passage, the medium was replaced with the same DMEM medium containing 50 mM d-galactose and the cells were cultured for an additional 2 weeks. Almost all of the cells cultured in galactose medium were stained positively for apoptosis with the terminal deoxynucleotidyl transferance-mediated biotin-dUTP nick end labeling (TUNEL) technique. Agarose gel electrophoresis of these cells displayed obvious DNA fragmentation, known as a ladder formation. All of these apoptotic changes were absent in similar cells cultured in galactose medium containing 1 μM of the aldose reductase inhibitor AL 1576. Addition of AL 1576 also reduced the cellular galactitol levels from 123±10 μg/10⁶ cells (n=5) to 3.9±1.9 μg/10⁶ cells (n=5). These observations confirm that galactose induced apoptosis occurs in dog lens epithelial cells. Furthermore, the prevention of apoptosis by an aldose reductase inhibitor suggests that this apoptosis is linked to the accumulation of sugar alcohols.     

Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.     

Pyruvate modulates antioxidant status of cultured human lens epithelial cells under hypergalactosemic conditions

Lens epithelial cells are the metabolic unit of the lens and antioxidant enzymes are mainly concentrated here. The purpose of this study was to maintain human lens epithelial cells (HLEC) in culture and examine the status of antioxidant enzymes (glutathione peroxidase (GSHPx), catalase (CAT), glutathione-S-transferase (GST)), lipid peroxidation product malondialdehyde (MDA) and glutathione (GSH) levels in these cells under normal as well as hypergalactosemic (30 mM galactose) conditions. Further, effect of pyruvate, a physiological antioxidant has also been evaluated on these parameters. For conducting experiments, anterior capsule specimens obtained from fresh cadaver eyes from eye bank were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% fetal calf serum. Upon confluency, the cells were subcultured in three separate flasks containing DMEM alone (normal group), DMEM + 30 mM D-galactose (control group), DMEM + 30 mM D-galactose + 5 mM pyruvate (test group) and incubated for 24 or 72 h. These cells were observed under the phase contrast microscope for any morphological changes and harvested for the estimation of various antioxidant parameters. Our results show significant weakened antioxidant defense in HLEC when incubated in the presence of galactose as compared to normal. Addition of pyruvate significantly modulated levels of GSH, MDA, GSHPx, CAT and GST.     

An attempt to assay the state of determination by using transfected genes as probes in transdifferentiation of neural retina into lens

Hybrid genes coding for chloramphenicol acetyltransferase (CAT) with a non-specific retroviral, lens-specific delta-crystallin or lens-specific alpha-crystallin promoters were constructed to transfect the transdifferentiating (lentoidogenic) and non-transdifferentiating (non-lentoidogenic) cultures of chicken embryonic neural retina for assaying the state of determination towards lens differentiation. The expression occurred only when CAT genes with lens-specific promoters were transfected to the cultures maintained in the conditions permissive to lentoidogenesis. The expression of these exogenous, lens-specific CAT genes began at stages of culturing that were earlier than the expression of endogenous crystallin. Presumably, there are two steps in the transdifferentiation of neural retina into lens; acquisition of capacity to express crystallin genes and derepression of the endogenous crystallin genes.