Association of superoxide anions with retinal pigment epithelial cell apoptosis induced by mononuclear phagocytes.

PURPOSE: Oxidative stress of the retinal pigment epithelium by reactive oxygen species and monocytic infiltration have been implicated in age-related macular degeneration. The purpose of this study was to determine the role of superoxide anions (O(2)(-)) in mononuclear phagocyte-induced RPE apoptosis. METHODS: Mouse RPE cell cultures were established from wild-type and heterozygous superoxide dismutase 2-knockout (Sod2(+/-)) mice. The intracellular reactive oxygen species, O(2)(-) and hydrogen peroxide, were measured by using dihydroethidium assay and 5-(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate, acetyl ester assay, respectively. RPE apoptosis was evaluated by Hoechst staining and terminal deoxynucleotidyltransferase dUTP nick-end labeling assay. Changes in mitochondrial membrane potential were detected by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide dye. Activated caspases and caspase-3 were detected in situ by FITC-VAD-fmk staining and caspase-3 substrate, respectively. RESULTS: Mononuclear phagocytes and interferon-gamma-activated mononuclear phagocytes induced the production of intracellular RPE O(2)(-), a decrease in RPE mitochondrial membrane potential, caspase activation, and apoptosis of mouse RPE cells. All theses changes were significantly enhanced in the Sod2(+/-) RPE cells. Activated mononuclear phagocytes induced more of these oxidative and apoptotic changes in RPE cells than did unstimulated mononuclear phagocytes. CONCLUSIONS: The authors have shown that the decreased expression of SOD2 and increased superoxide production correlate with RPE apoptosis induced by unstimulated and activated mononuclear phagocytes. The authors suggest that elevated O(2)(-) levels due to genetic abnormalities of SOD2 or immunologic activation of mononuclear phagocytes lead to greater levels of RPE apoptosis. The present study could serve as a useful model to characterize RPE/phagocyte interaction in AMD and other retinal diseases.

The effects of GPX-1 knockout on membrane transport and intracellular homeostasis in the lens.

Glutathione peroxidase-1 (GPX-1) is an enzyme that protects the lens against H2O2-mediated oxidative damage. The purpose of the present study was to determine the effects of GPX-1 knockout (KO) on lens transport and intracellular homeostasis. To investigate these lenses we used (1) whole lens impedance studies to measure membrane conductance, resting voltage and fiber cell gap junction coupling conductance; (2) osmotic swelling of fiber cell membrane vesicles to determine water permeability; and (3) injection of Fura2 and Na+-binding benzofuran isophthalate (SBFI) into fiber cells to measure [Ca2+]i and [Na+]i, respectively, in intact lenses. These approaches were used to compare wild-type (WT) and GPX-1 KO lenses from mice around 2 months of age. There were no significant differences in clarity, size, resting voltage, membrane conductance or fiber cell membrane water permeability between WT and GPX-1 KO lenses. However, in GPX-1 KO lenses, coupling conductance was 72% of normal in the outer shell of differentiating fibers and 45% of normal in the inner core of mature fibers. Quantitative Western blots showed that GPX-1 KO lenses had about 50% as much labeled Cx46 and Cx50 protein as WT, whereas they had equivalent labeled AQP0 protein as WT. Both Ca2+ and Na+ accumulated significantly in the core of GPX-1 KO lenses. In summary, the major effect on lens transport of GPX-1 KO was a reduction in gap junction coupling conductance. This reduction affected the lens normal circulation by causing [Na+]i and [Ca2+]i to increase, which could increase cataract susceptibility in GPX-1 KO lenses.

Proteomic and transcriptomic analyses of retinal pigment epithelial cells exposed to REF-1/TFPI-2.

PURPOSE: The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2. METHODS: Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions. RESULTS: Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11. CONCLUSIONS: The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions.

Sigma receptor ligands protect human retinal cells against oxidative stress.

This study was undertaken to investigate the role of sigma receptors during the oxidative damage on human retinal pigment epithelial cells, and to assess whether sigma receptor ligands enhance survival and protect DNA of cells challenged by oxidative stress. Pretreatment with PRE-084, a sigma1 receptor agonist, resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with PRE-084. The effects of PRE-084 were antagonized by pretreatment with sigma1 receptor antagonists (NE-100 and BD1047), but interestingly were synergized by cotreatment with BD1047 that also presented an affinity for the sigma2 receptor. The results suggest that sigma1 receptors play an important role against retinal damage, even though sigma2 receptor involvement cannot be excluded.

SOD2 protects against oxidation-induced apoptosis in mouse retinal pigment epithelium: implications for age-related macular degeneration.

PURPOSE: Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. Because manganese superoxide dismutase (SOD2), localized in mitochondria, is known to be a key enzyme that protects the cells against oxidative stress, this study was undertaken to examine oxidation-induced apoptosis in cultured RPE cells with various levels of SOD2. METHODS: Primary cultures of RPE cells were established from wild-type (WT), heterozygous Sod2-knockout mouse (HET) and hemizygous Sod2 mice with overexpression of the enzyme (HEMI). Purity of the RPE cell cultures was verified by immunostaining with antibody to RPE65 and quantified by flow cytometry. Oxidative stress was induced in RPE cells by exposing them to H(2)O(2) (0-500 muM) for 1 hour and reculturing them in normal medium for various times (0-24 hours). Apoptosis in the RPE was examined by TUNEL staining and quantified by cell-death-detection ELISA. Mitochondrial transmembrane potential (MTP) was measured by a cationic dye, and cytochrome c leakage from mitochondria was analyzed by Western blot analysis. RESULTS: More than 95% of the cells in each culture were RPE65 positive, and the relative SOD2 levels in HET, WT, and HEMI cells were 0.6, 1.0, and 3.4, respectively. H(2)O(2)-induced apoptotic cell death was both dose and time dependent, and apoptosis in these cells was related to the cellular SOD2 level. Disruption of MTP and release of cytochrome c were observed to occur before apoptotic cell death, and they correlated with cellular SOD2. CONCLUSIONS: The results demonstrate a critical role of SOD2 in protection against oxidative challenge. Cells from HET mice showed greater apoptotic cell death, whereas in those from HEMI mice, cell death induced by oxidative injury was suppressed.

Neuroactive steroids protect retinal pigment epithelium against oxidative stress.

This study was undertaken to assess whether neuroactive steroids, 17beta-estradiol and dehydroepiandrosterone-sulfate, enhance survival and protect DNA of human retinal pigment epithelial cells challenged by oxidative stress, and to investigate the role of sigma1 receptors in the effects of neuroactive steroids. Retinal pigment epithelial cells were treated with various concentrations of neuroactive steroids and then exposed to hydrogen peroxide. Pretreatment with steroids resulted in significant increased viability in a dose-related manner. DNA damage induced by oxidative insult was significantly lower with steroid pretreatment. The effects of 17beta-estradiol and dehydroepiandrosterone-sulfate were antagonized by pretreatment with a sigma1 receptor antagonist. The results suggest that neuroactive steroids protect retinal cells from oxidative stress, and that this effect is mediated by sigma1 receptors.

Inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in a human lens epithelial cell line.

Posterior capsule opacification (PCO) after cataract surgery is caused by growth of residual human lens epithelial (HLE) cells on the posterior capsule. We have shown that extracellular matrix (ECM) is an essential factor for HLE cell attachment and migration. The purpose of this study was to examine the inhibitory effects of Arg-Gly-Asp (RGD) peptide on cell attachment and migration in an HLE cell line. HLE cell line cells (SRA 01/04) that were obtained by transfection of large T antigen of SV40 were cultured in the absence of serum. The culture dishes were coated with type IV collagen, laminin or fibronectin, and Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) RGD peptide (0.1, 0.3, 1.0, 2.0 mg/ml) was added to the medium. The number of attached cells was counted after 90 min of incubation, and the inhibitory effects of GRGDSP RGD peptide on cell attachment were calculated. Cell attachment on the fibronectin-coated dishes was inhibited by GRGDSP RGD peptide at concentrations higher than 0.3 mg/ml; the inhibitory rate was 80% at a concentration of 2.0 mg/ml. The inhibition of cell attachment by GRGDSP RGD peptide on laminin-coated dishes appeared only at a concentration of 2.0 mg/ml, whereas no effects were observed on the type IV collagen-coated dishes. The inhibitory effects of GRGDSP RGD peptide on cell migration were measured in medium containing 2.0 mg/ml of GRGDSP RGD peptide after 1, 3, 5 and 7 days of culture. Cell migration was inhibited by GRGDSP RGD peptide from 1 day of culture on the fibronectin-coated dishes and from 5 days of culture on the laminin-coated dishes, whereas no effects were observed on the type IV collagen-coated dishes. GRGDSP RGD peptide inhibited cell attachment and migration on laminin and fibronectin that have RGD sequences. These data suggested that RGD peptide may have the potential to prevent PCO.

Ubiquitin-dependent lysosomal degradation of the HNE-modified proteins in lens epithelial cells.

4-hydroxynonenal (HNE), a highly reactive lipid peroxidation product, may adversely modify proteins. Accumulation of HNE-modified proteins may be responsible for pathological lesions associated with oxidative stress. The objective of this work was to determine how HNE-modified proteins are removed from cells. The data showed that alphaB-crystallin modified by HNE was ubiquitinated at a faster rate than that of native alphaB-crystallin in a cell-free system. However, its susceptibility to proteasome-dependent degradation in the cell-free system did not increase. When delivered into cultured lens epithelial cells, HNE-modified alphaB-crystallin was degraded at a faster rate than that of unmodified alphaB-crystallin. Inhibition of the lysosomal activity stabilized HNE-modified alphaB-crystallin, but inhibition of the proteasome activity alone had little effect. To determine if other HNE-modified proteins are also degraded in a ubiquitin-dependent lysosomal pathway, lens epithelial cells were treated with HNE and the removal of HNE-modified proteins in the cells was monitored. The levels of HNE-modified proteins in the cell decreased rapidly upon removal of HNE from the medium. Depletion of ATP or the presence of MG132, a proteasome/lysosome inhibitor, resulted in stabilization of HNE-modified proteins. However, proteasome-specific inhibitors, lactacystin-beta-lactone and epoxomicin, could not stabilize HNE-modified proteins in the cells. In contrast, chloroquine, a lysosome inhibitor, stabilized HNE-modified proteins. The enrichment of HNE-modified proteins in the fraction of ubiquitin conjugates suggests that HNE-modified proteins are preferentially ubiquitinated. Taken together, these findings show that HNE-modified proteins are degraded via a novel ubiquitin and lysosomal-dependent but proteasome-independent pathway.

Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress.

Age-related cataract, an opacity of the eye lens, is the leading cause of visual impairment in the elderly, the etiology of which is related to oxidative stress damage. Oxidation of methionine to methionine sulfoxide is a major oxidative stress product that reaches levels as high as 60% in cataract while being essentially absent from clear lenses. Methionine oxidation results in loss of protein function that can be reversed through the action of methionine sulfoxide reductase A (MsrA), which is implicated in oxidative stress protection and is an essential regulator of longevity in species ranging from Escherichia coli to mice. To establish a role for MsrA in lens protection against oxidative stress, we have examined the levels and spatial expression patterns of MsrA in the human lens and have tested the ability of MsrA to protect lens cells directly against oxidative stress. In the present report, we establish that MsrA is present throughout the human lens, where it is likely to defend lens cells and their components against methionine oxidation. We demonstrate that overexpression of MsrA protects lens cells against oxidative stress damage, whereas silencing of the MsrA gene renders lens cells more sensitive to oxidative stress damage. We also provide evidence that MsrA is important for lens cell function in the absence of exogenous stress. Collectively, these data implicate MsrA as a key player in lens cell viability and resistance to oxidative stress, a major factor in the etiology of age-related cataract.

Huntingtin Interacting Protein 1 mutations lead to abnormal hematopoiesis, spinal defects and cataracts.

Huntingtin Interacting Protein 1 (HIP1) binds clathrin and AP2, is overexpressed in multiple human tumors, and transforms fibroblasts. The function of HIP1 is unknown although it is thought to play a fundamental role in clathrin trafficking. Gene-targeted Hip1-/- mice develop premature testicular degeneration and severe spinal deformities. Yet, although HIP1 is expressed in many tissues including the spleen and bone marrow and was part of a leukemogenic translocation, its role in hematopoiesis has not been examined. In this study we report that three different mutations of murine Hip1 lead to hematopoietic abnormalities reflected by diminished early progenitor frequencies and resistance to 5-FU-induced bone marrow toxicity. Two of the Hip1 mutant lines also display the previously described spinal defects. These observations indicate that, in addition to being required for the survival/proliferation of cancer cells and germline progenitors, HIP1 is also required for the survival/proliferation of diverse types of somatic cells, including hematopoietic progenitors.

Purification, molecular cloning, and expression of a novel growth-promoting factor for retinal pigment epithelial cells, REF-1/TFPI-2.

PURPOSE: Retinal pigment epithelial (RPE) cells are known to play important roles in maintaining the homeostasis of the retina and in controlling choroidal neovascularization. The purpose of this study was to identify a factor or factors that would stimulate RPE cells to proliferate. METHODS: To isolate such a factor, 100 L of human-fibroblast-conditioned medium underwent ion-exchange, hydrophobic, and reverse-phase chromatographies followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The growth-promoting activity of the factor was examined in a human K-1034 RPE cell line and human primary RPE cells. RESULTS: The different chromatographic processes isolated a 31-kDa factor that had RPE cell growth-promoting properties. This factor, which we have named RPE cell factor (REF)-1, promotes growth of RPE cells but not of human umbilical vein endothelial cells (HUVECs). The amino-terminal sequence and molecular cloned cDNA of REF-1 were identical with those of tissue-factor pathway inhibitor (TFPI)-2, a family of TFPIs, and placental protein (PP)-5, a serine protease inhibitor. The cDNA expression of REF-1/TFPI-2 with pcDL-pSRalpha vector in Chinese hamster ovary (CHO) cells confirmed the growth-promoting activity for RPE cells. The major component of the recombinant REF-1/TFPI-2 expressed in CHO cells had a molecular mass of 31 kDa and exerted growth-promoting activity in RPE cells but not in human endothelial cells and fibroblasts in vitro. REF-1/TFPI-2 also had protease inhibitory activity. The other family factor, TFPI-1, did not promote RPE cell growth. CONCLUSIONS: REF-1/TFPI-2 is a novel growth-promoting factor for RPE cells but not for endothelial cells and fibroblasts. Its properties make it potentially beneficial for intraocular therapy for the repair and maintenance of RPE cells.

Growth factor induced activation of Rho and Rac GTPases and actin cytoskeletal reorganization in human lens epithelial cells.

PURPOSE: To determine the involvement of the Rho GTPases-mediated signaling pathway in growth factor-stimulated actomyosin cytoskeletal organization and focal adhesion formation in lens epithelial cells. METHODS: Serum starved human lens epithelial cells (SRA01/04) were treated with different growth factors including epidermal growth factor (EGF), basic-fibroblast growth factor (b-FGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF-beta), insulin-like growth factor 1 (IGF-1), lysophosphatidic acid (LPA), and thrombin. Growth factor stimulated activation of Rho and Rac GTPases were evaluated by GTP-loading pull-down assays. Changes in actin cytoskeletal organization and focal adhesions were determined by fluorescence staining using FITC-phalloidin and anti-vinculin antibody/rhodamine-conjugated secondary antibody, respectively. Fluorescence images were recorded using either confocal or fluorescence microscopy. RESULTS: Rho GTPase activity was significantly augmented in human lens epithelial cells treated with EGF, b-FGF, TGF-beta, IGF-1, and LPA. Rac GTPase activation, in contrast, was significantly enhanced in response to only EGF or b-FGF. Serum starved human lens epithelial cells exhibited a strong increase in cortical actin stress fibers and integrin-mediated focal adhesions in response to b-FGF, PDGF, TGF-beta, thrombin, and LPA. While EGF induced a striking increase in membrane ruffling and a marginal increase on focal adhesion formation, IGF-1 had no effect on either. Pretreatment of lens epithelial cells with C3-exoenzyme (an irreversible inhibitor of Rho-GTPase), lovastatin (an isoprenylation inhibitor), or the Rho kinase inhibitor Y-27632 abolished the ability of the different growth factors to elicit actin stress fiber and focal adhesion formation. EGF induced membrane ruffling, however, was not suppressed by Y-27632 and C3-exoenzyme. CONCLUSIONS: These results demonstrate that different growth factors induce actin cytoskeleton reorganization and formation of cell-ECM interactions in lens epithelial cells and this response of growth factors appears to be mediated, at least in part, through the Rho/Rho kinase-mediated signaling pathway. The ability of growth factors to trigger activation of Rho and Rac GTPases along with actomyosin cytoskeletal reorganization and formation of focal adhesions might well play a crucial role in lens epithelial cell proliferation, migration, elongation and survival.

The effect of up- and downregulation of MnSOD enzyme on oxidative stress in human lens epithelial cells.

PURPOSE: Gene knockouts serve as useful experimental models to investigate the role of antioxidant enzymes in protection against oxidative stress in the lens. In the absence of gene knockout animals for Mn-containing superoxide dismutase (MnSOD), the effect of this enzyme on oxidative stress was investigated in a human lens epithelial cell line (SRA 01/04) in which the enzyme was up- or downregulated by transfection with sense and antisense expression vectors for MnSOD. METHODS: Human lens epithelial cells (SRA 01/04) were transfected with plasmids containing sense and antisense human cDNA for MnSOD. The enzyme levels were determined by Western blot analysis and immunocytochemistry. The protective effect of the enzyme against the cytotoxicity of H(2)O(2) was evaluated by cell viability, DNA strand breaks, and morphologic changes observed by transmission electron microscopy. In addition, the protective effect of this enzyme against photochemically induced stress and UVB irradiation in cells was assessed by measuring the damage of cellular DNA. RESULTS: The MnSOD level in upregulated cells was three times higher than in downregulated cells, and the enzyme surrounded the nucleus. Cells with elevated enzyme levels were more resistant to the cytotoxic effect of H(2)O(2) with greater cell viability. MnSOD-deficient cells showed dramatic mitochondrial damage when exposed to 50 micro M H(2)O(2) for 1 hour. Similarly, oxidative challenge by H(2)O(2), photochemically generated reactive oxygen species, or UVB irradiation produced greater DNA strand breaks in MnSOD-deficient cells than in those in which the enzyme was upregulated. CONCLUSIONS: These findings demonstrate the protective effect of MnSOD in antioxidant defense of cultured lens epithelial cells. This approach to modulating the enzyme level in cultured cells provides a new experimental model for study of the role of antioxidant enzymes in the lens.

Cellular distribution of lens epithelium-derived growth factor (LEDGF) in the rat eye: loss of LEDGF from nuclei of differentiating cells.

Lens epithelium-derived growth factor (LEDGF) enhances the survival and growth of cells. To understand LEDGF's spatial localization and its putative function(s) during proliferation and differentiation, we localized LEDGF during terminal differentiation in whole rat lenses, lens epithelial cell (LEC) explants stimulated with FGF-2, and insulin, iris, human LECs with lentoids. In addition, intracellular localization of LEDGF was performed in other ocular tissues: ciliary body, retina, and cornea. We found the immunopositivity of nuclear LEDGF decreased in LECs of the equatorial region. In contrast, immunopositivity of LEDGF was detected in the cytoplasm of LECs and superficial fiber cells. After treating LEC explants with FGF-2 and insulin, which are known to be differentiating factors for LECs, the nuclei of these cells showed no LEDGF immunopositivity, but explants did express p57(kip2), a differentiation marker protein. Also, immunopositive LEDGF was not detected in the nuclei of differentiated cells, lentoid body, and corneal epithelial cells. This demonstrated that the loss of LEDGF from the nucleus may be associated with the process of terminal differentiation that might be in some way common with the biochemical mechanisms of apoptosis. The spatial and temporal distribution of LEDGF in the present study also provides a vision for further investigation as to how this protein is involved in cell fate determination.

Spectrum and range of oxidative stress responses of human lens epithelial cells to H2O2 insult.

PURPOSE: Oxidative stress (OS) is believed to be a major contributor to age-related cataract and other age-related diseases. METHODS: cDNA microarrays were used to identify the spectrum and range of genes with transcript levels that are altered in response to acute H(2)O(2)-induced OS in human lens epithelial (HLE) cells. HLE cells were treated with 50 microM H(2)O(2) for 1 hour in the absence of serum, followed by a return to complete medium. RNAs were prepared from treated and untreated cells at 0, 1, 2, and 8 hours after H(2)O(2) treatment. RESULTS: The data showed 1171 genes that were significantly up- and downregulated in response to H(2)O(2) treatment. Several functional subcategories of genes were identified, including those encoding DNA repair proteins, antioxidant defense enzymes, molecular chaperones, protein biosynthesis enzymes, and trafficking and degradation proteins. Differential expression of selected genes was confirmed at the level of RNA and/or protein. Many of the identified genes (e.g., glutathione S-transferase [MGST2], thioredoxin reductase beta, and peroxiredoxin 2) have been identified as participants in OS responses in the lens and other systems. Some genes induced by OS in the current study (e.g., oxygen regulated protein [ORP150] and heat shock protein [HSP40]) are better known to respond to other forms of stress. Two genes (receptor tyrosine kinase [AXL/ARK] and protein phosphatase 2A) are known to be differentially expressed in cataract. Most of the genes point to a novel pathways associated with OS. CONCLUSIONS: The present data provide a global perspective on those genes that respond to acute OS, point to novel genes and pathways associated with OS, and set the groundwork for understanding the functions of OS-related genes in lens protection and disease.

Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses.

PURPOSE: To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses. METHODS: Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy. RESULTS: Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses. CONCLUSIONS: Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.

UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects.

The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5 mWcm(-2), 340-410 nm wavelength, peak at 365 nm) for 4-5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70-90% of 340-400 nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30-35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein-thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, alpha- and beta- tubulin, vimentin and alpha-actinin (60-100%). The results indicate that a 4-5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O(2) present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.

Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway.

Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERT-transfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerase-negative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.