Regulation of heavy subunit chain of γ-glutamylcysteine synthetase by tumor necrosis factor-α in lens epithelial cells: role of LEDGF/p75
Abstract
TNF-α induces oxidative stress by generating reactive oxygen species (ROS). This molecule elevates the expression of γ-glutamylcysteine synthetase heavy subunit (γ-GCS-HS). Lens epithelium-derived growth factor (LEDGF)/p75, a transcriptional protein, is inducible by oxidative stress and protects cells from various stresses by upregulating stress-responsive genes. This paper presents evidence that TNF-α elevates the expression of LEDGF and that LEDGF is one of the transactivators of γ-GCS-HS gene. An analysis of the γ-GCS-HS promoter sequence (−819 to +518 nt) revealed the presence of putative sites for LEDGF binding. Gel mobility assay confirmed the binding of LEDGF to the heat shock element (nGAAn) and the stress response element (A/TGGGGA/T) present in γ-GCS-HS promoter. Transactivation experiments showed activation of γ-GCS-HS promoter in cells overexpressing LEDGF or treated with a sublethal dose of TNF-α (20 ng/ml). Downregulation of γ-GCS-HS promoter activity in cells transfected with LEDGF small interfering RNA validated the finding. Notably, cells treated with TNF-α (20 ng/ml) for 24 h had an increased abundance of LEDGF and γ-GCS-HS mRNA and protein. In contrast, cells treated with TNF-α for longer periods or with higher concentrations of TNF-α showed reduced expression of LEDGF and γ-GCS-HS and increased cellular death with higher ROS levels. Cells overexpressing LEDGF revealed elevated GSH levels (10–15%), a condition that may potentially eliminate the insult to cells induced by TNF-α. Thus TNF-α regulation of LEDGF may be physiologically important, as elevated expression of LEDGF increases the expression of endogenous γ-GCS-HS gene, the catalytic subunit of the regulating enzyme in GSH biosynthesis that may constitute a protective mechanism in limiting oxidative stress induced by inflammatory cytokines.
reactive oxygen species (ROS), a source of oxidative stress, play a role in cellular signaling (3, 15). Exposure to low levels of oxidant can enhance the expression of numerous enzymes involved in antioxidant defense, including γ-glutamylcysteine synthetase (γ-GCS) (40). Induction of this gene has been found to engender resistance to subsequent exposure to higher oxidant concentrations (50). Likewise, the acquisition of stable resistance to oxidant injury is associated with the increased expression of genes involved in antioxidant defense (45, 46). Induction of γ-GCS heavy subunit (HS) by TNF-α may also afford protection from oxidative stress (23).
TNF-α is one of the ROS producers. Stimulation of TNF-α receptors rapidly raises the levels of intracellular ROS (53) that are potent mediators of the killing activity of the cytokines (9). Cells can be made resistant to TNF-α challenge by prior exposure to sublethal doses of TNF-α. This finding suggests that lower concentrations of TNF-α lead to the induction of protective genes that engender protective effects in cells. Several reports have described the expression of TNF-α-induced survival genes, including plasminogen activator inhibitor type 2, the zinc finger protein A20, Bcl-2, and γ-GCS-HS (4, 14, 23). Biomolecules, such as TNF-α, that regulate the levels of intracellular redox GSH may have a direct role in activation of the transcription factor(s) and thereby may regulate GSH synthesis.
GSH is a ubiquitous cellular nonprotein sulfhydryl that plays a prominent role in maintenance of intracellular redox balance and in cellular defense against oxidative stress (21, 30). GSH is synthesized from its constituent amino acids in two sequential enzymatic reactions catalyzed by γ-GCS and GSH synthetase (37). γ-GCS is the regulatory and rate-limiting enzyme and is composed of two subunits, heavy (GCS-HS, 73 kDa) and light (GCS-LS, 28 kDa) chains (37). The heavy subunit possesses all of the catalytic activity (12). However, γ-GCS-HS promoter has been shown to contain a large number of potential consensus elements for transcriptional proteins such as activator protein (AP)-1 and NF-κB sites, an antioxidant-responsive element and an AP-2 site, and metal response elements (MREs) (5, 48). Earlier studies demonstrated that TNF-α plays a regulatory role in gene transcription (1, 2, 11, 19, 23, 35, 36), and some TNF-α-regulated genes contain lens epithelium-derived growth factor (LEDGF) regulatory elements, heat shock element (HSE; nGAAn) and stress response element (STRE; A/TGGGGA/T) (6, 8, 16, 38, 43).
LEDGF/p75 is a novel transcriptional factor (8, 42) and is identical with p75, a known coactivator of transcription (10, 44). LEDGF is upregulated when cells are exposed to thermal and oxidative stresses (39), and it protects cells from stresses by binding to HSE and STRE present in stress-responsive genes (6, 8, 16, 38). LEDGF is present in lens epithelial cells (LECs) (17), and the presence of TNF-α has been documented in lens cells as well as in the aqueous and vitreous humor. Because various levels of activity of TNF-α have been reported in the eye lens of human and other species (27, 28, 41, 55), the lens would be expected to exhibit TNF-α-related LEDGF dynamics. We have shown (6, 16, 38) that the upregulation of stress-responsive genes and their proteins by LEDGF is one of the mechanisms by which LEDGF prolongs cellular survival.
Moreover, the generation of ROS by TNF-α may be associated with the activation/deactivation and expression of several antideath transcription factors such as LEDGF (19, 32, 33, 39). In the present study, we demonstrated that the modulation of γ-GCS-HS expression in LECs treated with TNF-α was associated with LEDGF expression level. Also, in LECs overexpressing LEDGF, the expression of γ-GCS-HS increased. On the basis of that evidence, we hypothesized that 1) TNF-α generates oxidative stress by accelerating ROS production in LECs, which in turn upregulates the level of LEDGF expression and 2) increased expression of γ-GCS-HS is a consequence of elevated expression of LEDGF. To determine the potential role of LEDGF, we analyzed the 5′-flanking region of human γ-GCS-HS (−819 to +518 nt) (24). This promoter fragment does not contain known regulatory factors like antioxidant response element (ARE) and AP-1. Most striking is the presence of LEDGF binding sites, HSE and STRE (see Fig. 6A), which may be explained by expression pattern changes due to the TNF-α regulation of LEDGF.
In the study described here, we analyzed the cis-acting LEDGF binding elements in the human γ-GCS-HS gene promoter that are responsible for promoter induction by TNF-α and showed evidence of TNF-α-mediated LEDGF induction of γ-GCS-HS protein and mRNA expression. We also demonstrated that TNF-α-induced intracellular ROS level is critical for the regulation of LEDGF, which subsequently influences cellular GSH content by regulating transcription of γ-GCS-HS.
MATERIALS AND METHODS
Cell culture and transfection.
Human (h) and mouse (m) LECs (6–8) were cultured at 37°C in DMEM (GIBCO-BRL) with 10% FBS. Cells were maintained by methods described elsewhere (43). To examine the effect of TNF-α on LECs, cells were plated (5 × 105/well) in six-well plates (Falcon) for 24 h, washed twice, and then replaced in DMEM with 0.2% bovine serum albumin and human recombinant TNF-α (Pepro Tech) at different concentrations (0, 5, 20, and 100 ng/ml). A construct containing a green fluorescent protein (GFP) and LEDGF cDNA was generated with the “living color system” (Clontech), using the plasmid vector pEGFP-c1 for eukaryotic expression. The construct was used to generate stable eukaryotic cells that overexpressed LEDGF [enhanced GFP (EGFP)-LEDGF]. Cells transfected with the empty vector served as control.
Cell survival assay.
A colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay (Promega) was performed. MTS, when added to medium containing viable cells, is reduced to a water-soluble formazan salt. Absorbance at 490 nm was measured.
2′,7′-Dichlorofluorescin diacetate assay.
Cells (1.0 × 104/well) were cultured in 96-well plates for 24 h and then replaced in DMEM with 0.2% BSA in the presence or absence of TNF-α (20 ng/ml). Before the cells were assayed, they were washed once with HBSS and incubated in the same buffer containing 10 μg/ml of 2′,7′-dichlorofluorescin diacetate (DCFH-DA) at 37°C for 30 min. Intracellular fluorescence was detected with excitation at 485 nm and emission at 530 nm by using Spectra Max (Gemini EM; Molecular Devices).
Western blot analysis.
Cells with or without TNF-α treatment were washed, harvested, and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS). An equal amount of protein was resolved onto 10% SDS-PAGE and blotted onto polyvinylidene difluoride membrane (Millipore). Membranes were probed with mouse monoclonal or full anti-LEDGF antibody at 1:1,000 dilution or with anti-γ-GCS-HS at 1:200 dilution (Lab Vision). After being washed with PBS-Tween-20 (PBS-T), membranes were incubated with horseradish peroxidase-anti-mouse IgG or -anti-rabbit IgG as a second antibody. The specific band was visualized with luminol reagent (Santa Cruz Biotechnology). The membranes were stripped and reprobed with anti-actin antibody (Sigma). The densities in each band were analyzed with Scion imaging software.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and 4′,6-diamidino-2-phenylindole staining.
hLECs were treated with or without TNF-α (20 ng/ml) for 72 h. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was carried out with the In Situ Cell Death Detection Kit, Fluorescein (Roche), following the manufacturer's protocol. Samples were directly photomicrographed under a fluorescence microscope and analyzed. For 4′,6-diamidino-2-phenylindole (DAPI) staining, cells were fixed with paraformaldehyde solution (4% in PBS, pH 7.4), washed with PBS, incubated in DAPI solution for 30 min at room temperature, and again washed and mounted. After photomicrography, apoptotic nuclei were counted.
RT-PCR and real-time PCR.
To assess the level of the LEDGF and γ-GCS-HS transcript in LECs, we synthesized a pair of sense (5′-AACACACAGAGATGATTACTACAC-3′) and antisense (5′-TTTCAACATCAAACCTATCCTTAT-3′) primers for LEDGF and a pair of sense (5′-GATCCTCCAGTTCCTGCACATC-3′) and antisense (5′-GGAGATGGTGTATTCTTG-TCC-3′) primers for γ-GCS-HS. RNA was isolated with the single-step guanidine thiocyanate-phenol-chloroform extraction method (TRIzol reagent; Invitrogen). A cDNA synthesis kit for RT-PCR was used to synthesize cDNA. The resulting cDNA was amplified with the specific primers mentioned earlier. The following cycling conditions for the PCR were used: 94°C for 3 min, 25 or 35 cycles of 94°C for 1 min, 56°C for 2 min, 72°C for 3 min, and 7 min for final extension. 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 relative quantities of LEDGF and γ-GCS-HS mRNA were assessed by the comparative cycle threshold method and normalized with β-actin/ribosomal mRNA level as an endogenous control.
Electrophoretic mobility shift assay.
EMSA was performed with nuclear extracts from cells treated with or without TNF-α, following the method of Fatma et al. (8). Oligonucleotides with HSE (nGAAn) or STRE [(T/A)GGGG(A/T)] present in the γ-GCS-HS promoter were synthesized chemically (Invitrogen). Sequences were annealed and end-labeled with [γ-32P]ATP, using T4 polynucleotide kinase (New England Biolabs). The binding reaction was performed in 20 μl of binding buffer containing 20 mM Tris·HCl (pH 8.0), 75 mM KCl, 5% glycerol, 50 μg/ml bovine serum albumin, 0.025% Nonidet P-40, 1 mM EDTA, 5 mM dithiothreitol, and 1 μg of poly(dI-dC). Five femtomoles (10,000 cpm) of the end-labeled probe was incubated on ice for 30 min with 5 μg of nuclear extracts. Samples were resolved on 5% polyacrylamide. The gel was dried and autoradiographed. For the supershift assay, 1 μl of anti-LEDGF antibody was added to the reaction mixture.
Construction of γ-GCS-HS-chloramphenicol acetyltransferase reporter vector and chloramphenicol acetyltransferase assay.
γ-GCS-HS promoter spanning from −819 to +518 nt (see Fig. 6A) was isolated from a human lens cDNA library. A fragment was generated with specific primers and was ligated to the pCAT Basic vector (Promega) and amplified. LECs (5 × 105) were placed in 60-mm petri dishes. After 24 h, the cells were washed and cotransfected with γ-GCS-HS-chloramphenicol acetyltransferase (CAT) reporter construct or CAT empty vector and 1 μg of secreted alkaline phosphatase (SEAP) vector (6). Cells were exposed to TNF-α (20 ng/ml) for 24 h. Cell extracts were prepared and normalized to the soluble protein level. CAT-ELISA was performed according to the manufacturer's protocol. All CAT values were normalized with SEAP activity. CAT assays were performed with cells stably transfected with EGFP-LEDGF after cotransfection with the γ-GCS-HS-CAT reporter gene.
Site-directed mutagenesis.
PCR-based site-directed mutagenesis was performed with a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer's protocol. We made mutations in sequences of γ-GCS-HS promoter at LEDGF binding sites. Two pairs of complementary primers were used to mutate the promoter construct: 5′-GTCCGTGCCATCCAGATAACATAGGTCACCAGTTAATC-3′ and 3′-CAGGCACGGTAGGTCTATTGTATCCAGTGGTCAATTAG-5′ (Mut 1) and 5′-CTTCTCGCGAGCTGCTCCTCTCAACTGCGACCCAATC-3′ and 3′-GAAGAGCGCTCGACGAGGAGAGTTGACGCTGGGTTAG-5′ (Mut 2). Bold letters indicate mutated nucleotides (G/C to A/T).
The plasmid was amplified, and mutation was confirmed by sequencing. These mutant plasmids were used for the CAT assay and CAT values were compared with wild-type CAT-γ-GCS-HS. Mut 3 construct was obtained by using Mut 1 product as a template and Mut 2 primers in reaction mixture.
Preparation of small interfering RNAs and transfection.
The LEDGF-specific small interfering (si)RNA expression plasmid was designed according to the method described by Maertens et al. (20). The sequence was selected from location 1340–1360 (5′-AAAGACAGCATGAGGAAGCGA-3′). The sense and antisense oligonucleotides with the internal loop were synthesized by Invitrogen. These were annealed and ligated into the BamHI and HindIII sites of pSilencer 4.1-CMVneo (Ambion). pSilencer 4.1-CMVneo expressing a scrambled siRNA (Ambion) was used as a control. Transient transfection with siRNAs was performed with siPORT XP-1 (Ambion). Cells were harvested, and Western blot was performed.
Determination of intracellular GSH levels.
The intracellular glutathione level was estimated by colorimetric assay for GSH using the BIOXYTECH GSH-400 kit (OAXIS International). EGFP-LEDGF as well as EGFP empty vector stably transfected cells (1 × 107) were harvested in 5% meta-phosphoric acid (MPA; Sigma) and were homogenized and centrifuged at 10,000 rpm and 4°C for 15 min. Reaction products were mixed and incubated at 25°C for 10 min in the dark. The absorbance sample was taken at 400 nm.
Statistical analyses.
Statistical analyses for multiple comparisons of mean values between cell preparations were made by one-way analysis of variance followed by Fisher's test.
RESULTS
TNF-α-induced morphological changes and apoptosis in LECs.
Cellular effects elicited by TNF-α appear to be dependent on its concentration as well as the duration of its exposure to cells (9, 23, 47). The presence of TNF receptors in LECs is well documented (52). Thus, to study the effects of TNF-α on LECs derived from mouse or human lenses, we used variable concentrations of TNF-α (5, 20, and 100 ng/ml) for variable time periods. Cell viability assay revealed the inhibition of cell growth and cell survival with treatment involving a high dose of TNF-α (100 ng/ml) for 24 h, whereas lower doses of TNF-α (5 and 20 ng/ml) did not alter cell survival. However, at 72 h, 5 and 20 ng/ml TNF-α inhibited the cellular survival of LECs (Fig. 1, A and B). Because TNF-α induces the generation of ROS and thereby produces cell injury or apoptosis (1, 32, 34), we monitored ROS levels in these cells with the fluorescent dye H2-DCFH-DA. An increase in ROS levels was observed 3 h later with 20 ng/ml TNF-α treatment (Fig. 1C). Longer exposure to TNF-α led to further increases in ROS levels. To assess whether TNF-α induced apoptotic cell death in hLECs, we performed TUNEL assay with the In Situ Death Detection Kit (Fig. 2A). Treatment with TNF-α (20 ng/ml for 72 h) resulted in a significant increase in the rate of apoptotic cell death (∼30%) in hLECs (Fig. 2B). A typical apoptotic nucleus (the fragmented and disintegrated nucleus) could be observed in TNF-α-treated cells by TUNEL assay (Fig. 2Ab) and DAPI staining (Fig. 2Cb).
Fig. 1.Concentration- and time-dependent effect of TNF-α on the viability of lens epithelial cells (LECs). Cells were cultured with or without TNF-α at different concentrations for variable time periods, and cell viability was assessed by MTS assay. Mouse (mLECs; A) and human (hLECs; B) LECs were treated with different concentrations of TNF-α for 24 (a) and 72 (b) h. C: effect of TNF-α on intracellular reactive oxygen species (ROS) level. hLECs were treated with TNF-α (20 ng/ml) for 3, 24, 48, and 72 h, and intracellular ROS was measured with 2′,7′-dichlorofluorescin (DCFH-DA). OD, optical density. Results are means ± SD of 4 experiments. *P < 0.05, **P < 0.01 vs. control.
Fig. 2.Morphological changes and apoptotic cell death of LECs treated with TNF-α. Cells treated with or without TNF-α (20 ng/ml) for 72 h were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay (A) and 4′,6-diamidino-2-phenylindole (DAPI) staining (C). A: photomicrograph of TUNEL-positive cells treated without (a) or with (b) TNF-α. B: histogram showing % of TUNEL-positive cells in human (h)LECs treated or untreated with TNF-α (20 ng/ml). *P < 0.05 vs. control. C: photomicrograph of DAPI staining of hLECS cultured without (a) and with (b) TMF-2 (20 ng/ml) for 72 h. White arrows in A and C indicate apoptotic cells. D: photomicrograph of hLECs cultured without (a) or with (b) TNF-α (20 ng/ml) for 72 h. Black arrows indicate dead cells.
To determine whether TNF-α induces morphological changes in LECs, cells were exposed to different concentrations of TNF-α as described in materials and methods, and phase-contrast microscopy was performed to analyze the phenotypic changes. Cells treated with TNF-α showed distinguishable changes: they became elongated and fiberlike and detached more frequently (Fig. 2Db). These changes in the treated cells appeared 24 and 48 h after treatment with 100 and 20 ng/ml TNF-α, respectively. We concluded that changes in LECs induced by TNF-α may be related to cellular ROS levels and that the ROS levels may be dependent on TNF-α concentration or exposure.
TNF-α modulates expression of LEDGF and γ-GCS-HS in a concentration- and exposure-dependent fashion.
Because it is recognized that LEDGF is a survival factor and protects cells from physiological and/or environmental stresses, we were interested in the fate of LEDGF in hLECs after TNF-α treatment. We extracted protein from LECs untreated or treated with different concentrations of TNF-α for 24 h and performed Western blot analysis. Exposure of hLECs to TNF-α (5 and 20 ng/ml) led to increased expression of LEDGF protein (Fig. 3A). In contrast, cells exposed to the higher concentration of TNF-α (100 ng/ml) showed reduced expression of LEDGF protein. Next, we monitored the level of γ-GCS-HS, the regulatory and rate-limiting enzyme composed of two subunits, heavy and light chains. As the catalytic activity resides in the heavy subunit, we also determined the effect of TNF-α on the level of γ-GCS-HS protein. Similar to the expression pattern of LEDGF, the expression of γ-GCS-HS protein increased in cells treated with TNF-α at concentrations of 5 and 20 ng/ml, whereas its expression decreased in cells treated with 100 ng/ml TNF-α (Fig. 3B). We also investigated the effect of lower doses of TNF-α (20 ng/ml) for different time periods (24 and 72 h). The results revealed an increased expression of LEDGF (Fig. 4Aa) and γ-GCS-HS (Fig. 4Ab) protein in hLECs at 24 h, whereas expression levels of both were reduced in cells exposed to TNF-α for 72 h. Similarly, in mLECs, the expression levels of LEDGF (Fig. 4Ba) and γ-GCS-HS (Fig. 4Bb) increased at 24 h but decreased at after 72-h treatment with TNF-α compared with untreated control vehicles.
Fig. 3.Modulation of expression of lens epithelium-derived growth factor (LEDGF) and γ-glutamylcysteine synthetase (γ-GCS) heavy subunit (HS) in LECs exposed to variable concentrations of TNF-α. Cells were incubated for 24 h with various concentrations (0, 5, 20, 100 ng/ml) of TNF-α as indicated. Levels of LEDGF (A) and γ-GCS-HS (B) protein were determined by Western blot analysis. Lane 1, untreated control; lane 2, 5 ng/ml TNF-α; lane 3, 20 ng/ml TNF-α; lane 4, 100 ng/ml TNF-α. Percent increase or decrease over control was determined in 4 different cell preparations. *P < 0.05 vs. control.
Fig. 4.Effect of TNF-α on the expression of LEDGF and γ-GCS-HS protein in LECs. hLECs (A) and mLECs (B) were cultured with or without TNF-α (20 ng/ml) for 24 and 72 h and harvested. The levels of LEDGF (a) and γ-GCS-HS (b) protein were determined by Western blot analysis. Cells cultured with TNF-α showed diminution of LEDGF and γ-GCS-HS at 72 h, whereas LEDGF and γ-GCS-HS expression was increased at 24 h. Percent increase or decrease over control was determined in 4 different cell preparations. *P < 0.05 vs. control.
To gain a better understanding of the regulation of LEDGF and/or γ-GCS-HS by TNF-α at the transcriptional level, we isolated mRNA from mLECs at intervals of 0, 1, 3, 24, 48, and 72 h (Fig. 5A) and carried out RT-PCR and quantitative PCR. The results demonstrated that mLECs treated with TNF-α had an abundance of γ-GCS-HS and LEDGF mRNA after 3–24 h, and expression levels declined thereafter. We performed real-time quantitative PCR to validate the results (Fig. 5B).
Fig. 5.Alteration in the expression of LEDGF and γ-GCS-HS mRNA in LECs after TNF-α exposure. A: RT-PCR showing the profiles of LEDGF, γ-GCS-HS, and β-actin mRNA expression. Total RNA was isolated from mLECs cultured with TNF-α (20 ng/ml) for 0–72 h and subjected to RT-PCR. Resulting products were visualized on agarose gel with EtBr staining. B: real-time PCR disclosed elevated levels of LEDGF mRNA in mLECs after 3 h of TNF-α exposure. LECs (1 × 106) were exposed to 20 ng/ml TNF-α for 3 and 72 h. Results are means ± SD of 4 different experiments. *P < 0.05 vs. control.
TNF-α is a transactivator of γ-GCS-HS gene in LECs.
The experiments described above clearly showed that TNF-α regulated γ-GCS-HS and LEDGF expression in a dose- and time-dependent manner. To determine whether the γ-GCS-HS mRNA levels increased by TNF-α merely represented a stabilization of their mRNA or were due to a higher activation of gene transcription, we performed a transactivation assay. As described in materials and methods, we used the 5′-flanking region of the human γ-GCS-HS promoter linked to CAT vector (Fig. 6A) to transfect the LECs and performed CAT assay after TNF-α treatment (Fig. 6B). LECs transfected with this construct showed a significant increase in CAT activity at 24 h after TNF-α treatment compared with control values.
Fig. 6.A: putative LEDGF binding sites in the 5′-flanking region of γ-GCS-HS gene promoter. Bold letters in the DNA sequence indicate heat shock element (HSE; nGAAn) and stress response element [STRE; (T/A)GGGG(A/T)]. Underlining indicates the sequence of probe used for EMSA. Location of transcription start site is indicated by bent arrow and +1. The start codon and coding sequence are shown as the shaded area at the 3′ end of the sequence. CAT, chloramphenicol acetyltransferase. B: CAT activity of γ-GCS-HS promoter construct and empty CAT vector (Vec) with or without TNF-α treatment. mLECs (a) and hLECs (b) were transfected with either γ-GCS-HS promoter construct or empty CAT vector. Cells were treated with TNF-α at 20 ng/ml for 24 h, and cell extracts were obtained and used for CAT ELISA. Empty CAT vector has shown insignificant CAT activity. Results are means ± SD of 4 individual experiments. *P < 0.05 vs. control (CAT-γ-GCS-HS-transfected cells untreated with TNF-α).
TNF-α alters DNA binding affinity of LEDGF to HSE and STRE in γ-GCS-HS promoter.
A correlation was found between the expression levels of γ-GCS-HS and LEDGF mRNA and protein expression in LECs treated with TNF-α (Figs. 3–5). We predicted that LEDGF may be a transcriptional regulator of γ-GCS-HS gene. Because LEDGF exerts its survival function by the activation of transcription of stress-related genes including αB-crystallin and heat shock protein 27 via binding through HSE (nGAAn) and STRE [(T/A)GGGG(A/T)] (8, 39), and because we found these elements in human γ-GCS-HS promoter (Fig. 6A), we commercially synthesized the oligomers having these sites and used them for EMSA (Fig. 7). As described above, nuclear extracts were isolated from treated or untreated LECs and were incubated with 32P-labeled probe. LEDGF in the nuclear extract of LECs treated with the lower dose of TNF-α (20 ng/ml for 24 h) bound to probe (STRE2; Figs. 6A and 7) and formed Cm1 complex with higher affinity (Fig. 7A, lane 4). In contrast, nuclear extract from LECs treated with TNF-α (100 ng/ml for 72 h) displayed reduced or no binding (Fig. 7A, lane 3). Nuclear extract from untreated LECs bound to probe and formed Cm1 complex that was supershifted and formed a band (Ss1) after addition of LEDGF-specific antibody (Fig. 7, A, lane 2, and B, lane 2), demonstrating the specificity of LEDGF binding. Similar results were obtained with HSE probe (HSE2) present in γ-GCS-HS gene (Fig. 7C). These results demonstrate the ability of TNF-α to modify the DNA binding affinity of LEDGF, which may be associated with the cellular microenvironment. Notably, LECs overexpressed with LEDGF could restore the TNF-α-induced diminution of LEDGF's DNA binding affinity (compare Fig. 7, B, lane 1, and D, lane 1).
Fig. 7.EMSA and supershift assays using LEC nuclear extract with or without treatment of TNF-α. Five micrograms of nuclear extract from normal cells (A and C, lanes 1 and 2) or TNF-α-treated cells (A and C, lanes 3 and 4) was incubated with radiolabeled probes having STRE (AGGGGA or AGAA) (Fig. 6A; STRE 2 and HSE2)-formed protein-DNA complex (Cm1 band, lanes 1 and 4). When LEDGF-specific antibody was added, the Cm1 band shifted to a higher molecular weight position shown by the Ss1 band (A and C, lane 2). Nuclear extract isolated from LECs treated with 100 ng/ml TNF-α for 72 h showed reduced or no binding to probe (A and C, lane 3), whereas nuclear extract from LECs treated with 20 ng/ml TNF-α for 24 h showed enhanced binding to the probe (A and C, lane 4). Ns, nonspecific band (represents equal loading). B and D: LECs overexpressing LEDGF treated with 100 ng/ml for 72 h restored the DNA binding activity of LEDGF to STRE and HSE (lane 1).
LEDGF is a transcriptional regulator of γ-GCS-HS gene.
To establish whether cells overexpressing LEDGF could upregulate the promoter activity of γ-GCS-HS, we used hLECs and mLECs stably transfected with LEDGF and cotransfected with γ-GCS-HS promoter constructs. The transcriptional activity of γ-GCS-HS was examined by CAT assay. Overexpression of LEDGF dramatically enhanced the γ-GCS-HS promoter activity to more than fourfold higher than that of empty EGFP vector-transfected cells (Fig. 8). Furthermore, to determine whether overexpression of LEDGF really upregulated the levels of γ-GCS-HS protein and mRNA, we performed Western blot and quantitative RT-PCR. The results showed higher expression of γ-GCS-HS mRNA and protein in hLECs overexpressing LEDGF (Fig. 9, A and B). We validated the result with LEDGF siRNA and found that the reduced expression of γ-GCS-HS (Fig. 9C) was associated with the reduced expression of LEDGF, indicating that LEDGF is a regulator of the γ-GCS-HS gene.
Fig. 8.Transactivation of γ-GCS-HS CAT in LECs overexpressing LEDGF. mLECs (A) and hLECs (B) were transiently transfected with γ-GCS-HS CAT construct or empty pCAT vector. After 72 h, CAT activity was assayed. These cells previously had been stably transfected with a green fluorescent protein (GFP)-LEDGF construct or empty vector. CAT value in cells transfected with the GFP-LEDGF construct was significantly higher than in cells transfected with the enhanced GFP (EGFP) empty vector (*P < 0.05). Results are means ± SD of 4 individual experiments. C: transactivation of γ-GCS-HS promoter in GFP-LEDGF-transfected cells containing mutated LEDGF binding sites. a: Mutations in γ-GCS-HS promoter. Gray portion represents nonmutated sequences of γ-GCS-HS promoter. b: hLECs overexpressing LEDGF were transiently transfected with Mut 1, Mut 2, or Mut 3 constructs, and CAT activity was measured after 72 h. Results indicated that mutation in LEDGF binding sites downregulated transactivation of γ-GCS-HS promoter.
Fig. 9.Cells overexpressing LEDGF showed elevated expression of γ-GCS-HS protein and mRNA in LECs: Western blot analysis (A) and real-time RT-PCR (B) showing upregulation of γ-GCS-HS protein and mRNA in hLECs overexpressing LEDGF, respectively. A significant increase in γ-GCS-HS level (P < 0.03) was observed in LEDGF-overexpressing cells (V+L) compared with empty vector-transfected cells (V). C: Western blot showing diminution of LEDGF as well as γ-GCS-HS levels in hLECs transfected with LEDGF specific small interfering (si)RNAs (siLED) compared with control (siV). Results are means ± SD of 4 individual experiments. *Significant difference between two groups.
Mutation in HSE and STRE reduced LEDGF-dependent transactivation of γ-GCS-HS promoter.
To demonstrate the contribution of LEDGF binding to the function of γ-GCS-HS promoter, we mutated the sequences HSE2 (−621 to −590) and STRE2 (−156 to −120) as shown in Fig. 6A. In Mut 1 we mutated HSE2 (AGAA to ATAA), and in Mut 2 we mutated STRE2 (TCCCCT to TCCTCT). In Mut 3 (Mut 1 + Mut 2), both HSE2 and STRE 2 were mutated. In Mut 1, there was a 14% loss of CAT activity, in Mut 2 a 22% loss, and in Mut 3, with both Mut 1 and Mut 2 present, a 39% loss (Fig. 8). These results suggest that each LEDGF binding site contributed to the transactivation of γ-GCS-HS promoter. The unmutated sites appeared to be responsible for the remaining promoter activity.
Effect of LEDGF overexpression on GSH level in hLECs.
GSH plays a key role in the cellular defense system against oxidative stress, and GSH level is maintained by γ-GCS-HS. Because our results showed that overexpression of LEDGF enhanced the transcriptional activity of γ-GCS-HS and its protein and mRNA expression, we considered that the cells overexpressing LEDGF may have been carrying higher levels of GSH. Monitoring the amount of GSH in hLECs overexpressing LEDGF (Fig. 10) showed that GSH levels in LEDGF-transfected cells were 20.3% higher than in vector-transfected cells. This experiment revealed that LEDGF regulated GSH levels in LECs through upregulation of γ-GCS-HS.
Fig. 10.Increased glutathione levels in LECs overexpressing LEDGF. The concentration of GSH was estimated according to the manufacturer's protocol (OAXIS International). hLECs were stably transfected with EGFP-vector (B) or EGFP-LEDGF (A), cultured, and incubated at 37°C. Results are means ± SD of 4 individual experiments. *P < 0.05, A vs. B.
LECs overexpressing LEDGF gain resistance to TNF-α-induced insults.
To determine whether overexpression of LEDGF counteracts TNF-α-mediated adverse effects in cells, we compared hLECs stably transfected with EGFP-LEDGF to those transfected with empty vector. After treatment with TNF-α (20 ng/ml for 72 h), vector-transfected cells showed phenotypic changes similar to those observed in nontransfected hLECs (Fig. 11Ab). Surprisingly, cells overexpressing LEDGF after treatment with TNF-α were indistinguishable from control cells (Fig. 11Ad), suggesting that LEDGF prevented TNF-α-mediated adverse effects. We further investigated cell viability by MTS assay to compare cells stably transfected with EGFP-LEDGF or with empty vector treated with TNF-α. Results revealed that the cell death induced by TNF-α exposure in vector-transfected hLECs was significantly inhibited by the overexpression of LEDGF (Fig. 11B; difference between second and fourth bars is statistically insignificant). We surmised that the overexpression of LEDGF abolished the effect of TNF-α, a finding that could be useful to diminish or postpone TNF-α-induced cytotoxic complications.
Fig. 11.A: photomicrograph of hLECs overexpressing LEDGF with or without treatment of TNF-α (20 ng/ml). Human LECs stably transfected with either EGFP vector (a and b) or EGFP-LEDGF (c and d) were cultured without (a and c) or with (b and d) TNF-α (20 ng/ml) for 72 h. Insets: EGFP-LEDGF (c) or EGFP empty vector (a) expression after stable transfection. B: resistance to TNF-α-induced cell damage in hLECs overexpressing LEDGF: MTS assay showing cell viability of hLECs transfected with empty EGFP-vector (gray bars) and EGFP-LEDGF (filled bars) treated with or without TNF-α (20 ng/ml for 72 h). Results are means ± SD of 4 individual experiments. *P < 0.05 vs. EGFP-LEDGF-transfected cells treated with TNF-α.
DISCUSSION
Results of the present study demonstrated that LECs overexpressing LEDGF expressed enhanced levels of endogenous γ-GCS-HS; thus these LECs contained higher GSH levels and acquired a strong resistance to TNF-α cytotoxicity (Figs. 1, 2, and 11B). Our earlier work (6, 8, 16, 38) showed that LEDGF interacts directly with genes containing STRE and/or HSE to activate their transcription. Further study is warranted to identify the mechanism of LEDGF-mediated cellular protection against various stresses. Elevation of γ-GCS-HS by LEDGF may be one of the mechanisms of LEDGF-mediated cellular protection against oxidative stress induced by various environmental/physiological factors including growth factor/cytokines such as TNF-α. Moreover, the eye is exposed to various cytokines and factors that are released as a result of injury, disease processes, or aging (13, 32, 34, 36), and the lens itself can be exposed to inflammatory factors that are subsequently present in the aqueous humor (49). LEDGF is present in LECs (17), and TNF-α receptor has been reported to be present in LECs (52). Thus TNF-α that is present in aqueous and vitreous humors may alter LEDGF dynamics (28, 41, 55). TNF-α, an excitatory cytokine, induces the generation of intracellular ROS. Higher levels of ROS are known to be deleterious, whereas lower levels are beneficial for cells (7, 49). In the present study using LECs and TNF-α as a model, we showed elevated expression of LEDGF in the presence of sublethal doses of TNF-α (Fig. 3) and found that the elevation of expression of γ-GCS-HS gene is LEDGF dependent (Figs. 8 and 9). Thus we propose a novel mechanism of γ-GCS-HS regulation by LEDGF, in which TNF-α-induced regulation of LEDGF expression in LECs plays a critical role, and which is TNF-α concentration dependent (Fig. 3). Furthermore, the ability of mammalian cells to maintain cellular functions during oxidative stress depends on induction of antioxidant enzymes and ROS levels. Our findings provide evidence that TNF-α upregulates the level of GSH as an adaptive control mechanism that attenuates the extent of ROS generation, suggesting that regulation of γ-GCS-HS by LEDGF before exposure to TNF-α may determine the fate of cells.
Furthermore, TNF-α promotes cellular changes including apoptosis through several mechanisms including the overproduction of ROS (9, 11, 32, 34, 36). We found that TNF-α induced apoptosis and morphological changes in LECs (Fig. 2). Sublethal doses of TNF-α upregulated the expression of LEDGF in LECs (Fig. 3). Conversely, LECs exposed to higher concentrations or longer exposure with lower concentrations of TNF-α showed downregulated expression of LEDGF as well as γ-GCS-HS (Figs. 3–5). In light of our findings, it is likely that the expression of LEDGF is regulated by different regulatory signals in response to diverse stimuli. However, in the present study using both hLECs and mLECs, we found that hLECs were more susceptible to TNF-α-induced cellular damage, whereas the cell types showed similar expression patterns of LEDGF and γ-GCS-HS (Figs. 1 and 4). We think that higher susceptibility of hLECs to TNF-α may be associated with decreased levels of antioxidant. Evidence shows that parts of the organ antioxidant defense system can adapt to oxidative stress. For example, activation of the AP-1 transcription factor leads to upregulation of γ-GCS, the rate-limiting enzyme of GSH, in certain cells (31). We observed that TNF-α (20 ng/ml) enhanced γ-GCS-HS mRNA and protein expression and its promoter activity in LECs (Figs. 3–6). An analysis of γ-GCS-HS gene revealed the presence of LEDGF binding sites (Fig. 6A). EMSA and transactivation with LEDGF siRNA showed that LEDGF physically and functionally bound to either HSE or STRE elements in the γ-GCS-HS promoter and transactivated it (Figs. 7 and 9C). These results are consistent with our previous reports (6, 8, 16). However, multiple putative transcription factors are involved in response to different stimuli in different cells. Activation of the γ-GCS-HS gene undoubtedly involves multiple transcription factors and regulatory elements (25, 29), and we have shown that LEDGF is one of these factors.
Moreover, the overexpression of LEDGF in LECs engendered resistance against TNF-α-induced cell damage, by upregulating γ-GCS-HS gene, a rate-limiting enzyme, and thus controlling cellular glutathione content (Fig. 10). Because lower doses of TNF-α enhance the expression of LEDGF, we believe that TNF-α-mediated elevation of LEDGF and consequently LEDGF activation of γ-GCS-HS gene are physiologically important to maintain normal physiological function of cells. Earlier reports showed that sensitive cells can be made resistant to TNF-α challenge by prior exposure to a sublethal dose of TNF-α (4, 14, 18, 21, 22, 27) and that cells overexpressing several protective genes, including plasminogen activator inhibitor type 2, the zinc finger protein A20, and the Bcl-2-related family member A1, show significant resistance against TNF-α toxicity (4, 14, 18, 21, 22, 27). These findings imply that, under certain conditions, TNF-α leads to the induction of genes that confer protective effects on cells. We did not observe significant cell injury in LECs with high expression of LEDGF (5 and 20 ng/ml TNF-α for 24 h). We have shown that external stimuli (e.g., H2O2, UV, thermal stress and TNF-α; present study) activated the expression of LEDGF in LECs and COS-7 cells (39), which indicates that one mechanism of LEDGF-mediated cell survival is the transcriptional regulation of γ-GCS-HS. Nevertheless, it is unlikely that the overall increase in cell viability with LEDGF overexpression is due to the upregulation of γ-GCS-HS and GSH, because increased cell survival may also be associated with heat shock proteins HSP27 and αB-crystallin, a negative regulator of the death pathway (21). However, in the present study, we observed that LECs exposed to TNF-α in higher concentrations or for longer periods showed reduced expression of γ-GCS-HS, which may be due to the attenuation of LEDGF binding to HSE and STRE. We reported previously (7, 38) that reduced expression or attenuation of LEDGF protein by stress is associated with reduced function of stress response genes. Together, the results suggest that longer exposure of cells to TNF-α may attenuate LEDGF function and result in downregulation of γ-GCS-HS expression.
In summary, TNF-α-mediated increase of cellular GSH levels is associated with transcriptional regulation of the γ-GCS-HS genes by LEDGF. Thus LEDGF is one of the regulators of γ-GCS-HS genes, and upregulation of cellular GSH by LEDGF may serve as an additional protective mechanism for maintaining cellular homeostasis. Findings from this study may provide grounds for formulation of cytokine-based induced inductive/adaptive therapy.
GRANTS
Grants provided by the National Eye Institute (EY-13394 to D. P. Singh) and Foundation for Fighting Blindness are gratefully acknowledged.
FOOTNOTES
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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