1Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska 68198; 2Department of Ophthalmology, Fukui Medical University, Fukui 910 1193, Japan; and 3Center for Ophthalmic Research, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
Submitted 9 February 2004 ; accepted in final form 14 April 2004
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ABSTRACT |
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lens epithelium-derived growth factor; gene promoters; transcription regulation; stress and heat shock elements
Lens epithelium-derived growth factor (LEDGF) is a novel growth and survival factor and a transcriptional activator (10, 11, 32, 33). Cells expressing higher levels of LEDGF survive remarkably well against a wide variety of stress factors (8, 19, 21, 25, 33). Notably, LEDGF mRNA and protein expression is significantly upregulated in conditions of thermal and oxidative stress (28). Our in vitro and in vivo analyses (1, 19, 21, 25, 28, 33) collectively indicate that overexpression of LEDGF provides a selective survival advantage in growing cells by blocking death pathways. In view of the adverse effects of ethanol on cells and tissues and its role in the induction of cataractogenesis (20), we were interested in determining whether LEDGF protects cells from ethanol stress. Because ADH and ALDH are involved in RA synthesis as well as detoxification (5, 9), the levels of these genes were monitored in lens epithelial cells (LECs) overexpressing LEDGF. We additionally assayed the potential of LEDGF to enhance the survival of cells exposed to ethanol and its role in RA production.
A study on the tissue distribution of mouse ADH1 and ADH4 mRNA revealed that ADH1 is primarily expressed in eye, liver, small intestine, kidney, ovary, and uterus (40), whereas class IV mRNA is primarily expressed in eye, stomach, ovary, thymus, and skin. Tissues expressing these two ADH classes possess large numbers of epithelial cells. These enzymes oxidize retinol to RA, and this process is required to regulate epithelial cell differentiation (2). There are five ADH classes in mammals including mice (14). The regulation of these genes is currently poorly understood. However, a number of binding sites and transcriptional regulatory elements have been identified in the promoter region, and a long purine-pyrimidine sequence in mouse ADH1 is suggested to play a role in gene expression (18, 38).
LEDGF, a stress-inducible transcriptional survival factor (28, 34), binds and transactivates heat shock (HSE; nGAAn) and stress response (STRE; A/TGGGGGA/T) elements present in the promoters of stress response genes (8, 30). Cells overexpressing LEDGF exhibit increased levels of ADH1, ADH4, and RALDH2 mRNA. In earlier reports (8, 30), we predicted that the DNA binding sites of LEDGF are located in the promoter regions of ADH and ALDH. Computer-based bioinformatic analysis of the 5'-flanking region of mouse ADH and ALDH gene promoters disclosed the presence of potential LEDGF binding sites, STRE, and/or HSE (see Fig. 5). Our present findings additionally confirm that LEDGF is induced by ethanol stress. Moreover, cells overexpressing LEDGF produce higher levels of RA and resist the cytotoxic effects of ethanol.
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MATERIALS AND METHODS |
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Plasmid construction, expression, and purification of glutathione S-transferase-LEDGF.
A fusion protein of LEDGF and glutathione S-transferase (GST) was generated by inserting the entire coding sequence of LEDGF cDNA into the BamHI and EcoRI sites of a pGEX-2T vector (Pharmacia Biotech, Piscataway, NJ). The construct was used to transform Escherichia coli (BL21) (33). Expression of the GST-LEDGF fusion protein was induced with isopropyl -D-1-thiogalactopyranoside (IPTG) (7). Protein was purified with glutathione-Sepharose 4B beads (Pharmacia Biotech) following the manufacturer's protocol. The protein concentration was determined by the Bradford method (3).
Construction of LEDGF antisense. We subcloned LEDGF cDNA into a pcDNA3 vector in the reverse orientation to generate a full-length LEDGF antisense construct.
Cell culture and transfection. Mouse LECs and COS-7 cells were cultured in DMEM with 10% FCS at 37°C in an atmosphere of air-CO2 (19:1). All transfections were performed by the lipofectamine (GIBCO-BRL, Bethesda, MD) method according to the manufacturer's protocol. Cells were plated at a density of 1 x 106 cells per 100-mm culture dish. After 1012 h, cells were transfected with pEGFP-LEDGF, pEGFP-vector, ADH, or RALDH2-chloramphenicol acetyltransferase (CAT) constructs.
Real-time PCR and semiquantitative reverse transcription-PCR. To monitor the level of LEDGF in LECs after ethanol treatment, total RNA was isolated with a single-step guanidine thiocyanate-phenol-chloroform extraction method (TRIzol reagent, Invitrogen) and converted to cDNA with Superscript II RNase H reverse transcriptase (28). Quantitative real-time PCR was performed with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) in the ABI 7000 sequence detector system (Applied Biosystems). The sequences of LEDGF primers and probe used were as follows: LEDGF primers, 5'-CAGCTCGAGTAGATGAAGTTCCT-3' and 5'GGGTTCCAAAAAAGAAAATGGGTAGTT-3'; LEDGF TaqMan MGB probe, 5'-ATGGAGCTGTAAAACCA-3' (FAM dye labeled). The relative quantity of LEDGF mRNA was assessed by the comparative CT method and normalized with predeveloped TaqMan rodent GAPDH as an endogenous control reagent (Applied Biosystems) following the manufacturer's protocol.
To determine the levels of mouse ADH1, ADH4, and RALDH2 in LECs, we synthesized two pairs of primers (Table 1). -Actin primers were used as control (28). LECs (1 x 106) in 100-mm culture dishes were transiently transfected with pEGFP-LEDGF or pEGFP vector, and mRNA was isolated after 48 h of transfection with the Micro-Fast Track kit (Invitrogen, Carlsbad, CA). cDNA synthesis was performed with a kit (Invitrogen) and used in PCR with specific primers. The following conditions were used: 94°C for 32 min, 15, 25, or 35 cycles of 94°C for 1 min, 55°C for 2 min, 72°C for 3 min, and a final extension step at 72°C for 7 min. The resultant PCR products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining.
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Construction of ADH1, ADH4, and RALDH2-CAT reporter vectors. The 5'-flanking regions of mouse ADH1, ADH4, and RALDH2 gene promoters were isolated with a genomic PCR kit (Clontech Genomics) with specific primers containing restriction sites (Table 2, underlined). The resulting promoter fragments were cloned into the EcoRI sites of the TA vector (Invitrogen) and sequenced after amplification. Constructs were digested with the appropriate enzymes and ligated to pCAT BasicVector (Promega, Madison, WI). The resulting plasmid was amplified and used for the CAT assay.
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CAT assay. The CAT assay was performed with a CAT-ELISA kit (Roche Diagnostics). Mouse LECs were transfected with pEGFP-LEDGF, pEGFP-vector, and the ADH1, ADH4, and RALDH2-CAT constructs. After 72 h, cells were harvested and extracts were prepared and normalized to the soluble protein level. The CAT-ELISA procedure was performed according to the manufacturer's protocol, and absorbance was measured at 405 nm with a microtiter plate ELISA reader. To monitor LEDGF promoter activity, cells were transfected with either the LEDGF-promoter-CAT construct or empty CAT vector and treated with ethanol (25, 50, and 100 mM) for 48 h or left untreated and the CAT assay was performed.
Cell viability assay (MTS assay). Cells overexpressing LEDGF were treated with different concentrations of ethanol (25, 50, and 100 mM), and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay was performed as described previously (7, 8).
Statistical analysis. The unpaired Student's t-test was used to assess the statistical significance of the differences between the groups.
Western analysis. Lysates from ethanol-treated or untreated cells or those transfected with EGFP-LEDGF or EGFP vector were prepared in ice-cold RIPA buffer (1% Igepal CA-630; Sigma, St Louis, MO), 0.5% (wt/vol) sodium deoxycholate, 0.1% (wt/vol) sodium dodecyl sulfate (SDS) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma), and protease inhibitor (Complete-Mini, Roche Diagnostics). The protein concentration was determined by the Bradford method. Protein samples were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, blots were incubated overnight at 4°C with rabbit polyclonal anti-LEDGF COOH-terminal antibody (1:10,000 dilution) or GFP polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed with PBS-Tween 20, incubated with anti-rabbit IgG labeled with horseradish peroxidase (Santa Cruz Biotechnology), and visualized by the enhanced chemiluminescence method according to the manufacturer's protocol.
Monitoring of endogenous RA levels in cells overexpressing LEDGF. We used the RA response elements (RARE)-secreted alkaline phosphate (SEAP) reporter system (Clontech) to monitor the levels of RA in cells overexpressing LEDGF. The SEAP vector contains RA response elements. COS-7 cells were transfected with pEGFP-LEDGF, pEGFP-vector, and SEAP expression vector. Cells were additionally transfected with negative controls (SEAP vector lacking enhancer elements) to determine background levels of reporter gene activity. The supernatant was collected after 48 and 72 h of transfection. A fluorescent SEAP assay was performed following the manufacturer's protocol. Placental alkaline phosphatase was used as a positive control to calculate the values.
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RESULTS |
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We next determined the changes in promoter activity of LEDGF after ethanol stress. LECs were transfected with a LEDGF promoter linked to the CAT reporter gene (Fig. 1Ca) or empty CAT vector, and the resulting CAT values were quantified (Fig. 1Cb). CAT values were significantly high (P < 0.001) in cells exposed to various concentrations of ethanol (25, 50, and 100 mM) compared with unexposed cells (Fig. 1Cc). No detectable changes in the CAT value were observed when cells containing empty vector were treated with ethanol (Fig. 1Cc). Our results strongly suggest that ethanol exposure activates the expression and function of the ledgf gene.
LECs overexpressing LEDGF display protection against ethanol stress.
To determine whether LEDGF has a protective effect against ethanol stress, LECs were overexpressed with pEGFP-LEDGF or with empty vector. Overexpression was confirmed by Western blot using GFP antibody (Fig. 2A) and anti-LEDGF antibody (Fig. 2B). Transfected cells harbored higher levels of LEDGF, including both the native protein (60 kDa; Fig. 2B, lanes 3 and 4) and EGFP-LEDGF (90 kDa; Fig. 2A, lane 2). To monitor the survival effect of LEDGF against ethanol stress, cells were exposed to 25, 50, and 100 mM ethanol for 24 h and the MTS assay was performed after a recovery period. Significant differences in the number of live cells were observed between cells overexpressing LEDGF and controls after exposure to different concentrations of ethanol (Fig. 2C). Viability was significantly higher (P < 0.001) in cells overexpressing LEDGF that were exposed to ethanol. The results suggest that LEDGF protects cells from ethanol toxicity.
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DISCUSSION |
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Mouse ALDH1 and RALDH2, which are expressed predominantly in retina, oxidize retinal (15). These enzymes are additionally involved in the oxidation of a wide variety of exogenous and endogenous aldehydes, which are among the most toxic of the metabolites of alcohols (15). In this report, we show that ADH1, ADH4, and RALDH2 (enzymes that function as retinol dehydrogenases in vitro) transcripts are present in significant amounts in mouse LECs overexpressing LEDGF (Figs. 3 and 4). ADH1 and ADH4 are additionally detected in a wide variety of epithelial tissues that convert retinal to RA (5, 6, 17).
Using a transfection system, we showed LEDGF-dependent elevation of RA in cells overexpressing LEDGF compared with control (Fig. 8) and LEDGF antisense-transfected cells as evidenced by the downregulated SEAP values (P < 0.001). These results suggest that LECs generate RA in the presence of LEDGF. It is reported that RA synthesis occurs locally in many epithelial cell types throughout the body (13). Genetic studies provide evidence that adh1-, adh3-, and adh4-null mice have defects in ethanol clearance, formaldehyde toxicity, and metabolism of retinol to RA (4). A 10-fold decrease in RA production is reported in adh1/ knockout and adh1/4/ double-knockout mice (23, 24). One of the important findings of the present study is that cells overexpressing LEDGF confer resistance to ethanol stress (Fig. 2C) that is correlated with the elevated expression of adh1, adh4, and raldh2 genes by LEDGF. Similar earlier reports show that these genes are involved in alcohol detoxification and RA production (26).
Recent findings indicate that ethanol has an acute effect on lens cation homeostasis. Clinical and epidemiologic studies over the last 20 years have suggested that moderate to heavy consumption of alcohol is a risk factor for cataract (20). Excess alcohol causes a loss in lens calcium homeostasis, which may be one of the cellular mechanisms that contribute to cataract development. In our experiments, 150 and 200 mM ethanol were cytotoxic to LECs in vitro and resulted in reduced LEDGF expression (data not shown). The diminution of LEDGF expression at a higher concentration of ethanol might be a critical event in destabilizing the homeostasis of cells, resulting in loss of cellular resistance. Other studies have demonstrated that ethanol exposure causes modifications in the membrane permeability of lens that in turn induce increased calcium permeability of lens lipid membranes, resulting in pathological effects (12, 39). The fetal alcohol syndrome involves ethanol inhibition of ADH-catalyzed RA synthesis. RA additionally regulates the fibronectin gene in LECs, thus playing an important role in the functional adhesion of epithelium to the lens capsule (27). Moreover, RA inhibits the formation of mesenchyme cells, which may be activated in pathological transformations, i.e., in anterior capsular cataract from lens epithelium (22). We reported previously (29, 34) that loss of LEDGF from cells is one of the prime events of cell death during stress, whereas cells overexpressing this protein survive well. A decrease in LEDGF in LECs or other epithelial cells may affect homeostasis, thus impairing the physiological function(s) of these cells. Further studies are required to clarify the mechanism(s) of regulation of these physiologically important genes to determine their function(s). We have shown in the present study that LEDGF is one of the transcriptional regulators of ADH and ALDH genes, which play a major role in ethanol detoxification and production of RA in cells.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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