Activated IGF-1R inhibits hyperglycemia-induced DNA damage and promotes DNA repair by homologous recombination
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《美国生理学杂志》
Division of Nephrology and Hypertension, Department of Medicine, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey
Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania
ABSTRACT
The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.
genotoxicity; DNA double-strand breaks; Rad51; reactive oxygen species; danger signal
HYPERGLYCEMIA AND DIABETES mellitus are associated with an exponential increase in reactive oxygen species (ROS) production at the cellular level (9, 14), which play an important role in the development of diabetic complications (2, 22). ROS-dependent signals have also been linked to defects in genomic maintenance systems and the aging process (5). In a recent communication (9), we documented a strong IGF-1R oxidant-resistant phenotype in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at a high ambient glucose concentration. This novel IGF-1R antioxidant function was closely coupled with expression of the survival phenotype and inhibition of the apoptosis program. These observations are in keeping with genetic experiments, in which longevity in mice was directly linked to increased resistance to oxidant stress (6, 13). Interestingly, the IGF-1R (6) and the adaptor protein p66Shc, a key IGF-1R signaling molecule, have both emerged as major genetic determinants of longevity and oxidative stress, in mammals.
As recently reviewed (5), cell survival and longevity are closely linked with the maintenance of genomic integrity. The DNA double helix is a target for ROS-dependent signals, which inflict more than 100 different types of DNA lesions, ranging from base modifications to single-strand breaks (SSB) and potentially lethal double-strand breaks (DSB) (5, 13). DNA repair is a fundamental mechanism by which cells protect themselves from oxidative stress. Failure to repair DSB commits a cell to a death sentence or malignant transformation (1, 23, 24). The two major repair mechanisms for DSB are homologous recombination (HRR) and nonhomologous end joining (NHEJ) (24). HRR is dependent on the availability of a template, synthesized during the S-phase of the cell cycle. The breast cancer susceptibility gene (BRCA2) and Rad51, a structural and functional homolog of bacterial RecA recombinase, are essential for the error-free repair of DSB by HRR (1, 23). Following detection of DSB, BRCA2 recruits Rad51 to the junction of DSBs. The first step in DSB repair by HRR involves the processing of the DNA break to produce a single-strand region with a 3' overhang, via ATM-activated 5'-3' endonuclease complex. Replication protein A initially binds to the 3' overhang and is subsequently replaced by Rad51, which searches for a homologous donor sequence and catalyzes strand exchange with the donor DNA (10). Alternatively, NHEJ, which is rapid and error prone, proceeds without a template by using the end binding Ku70/Ku80 complex and DNA protein kinase (DNA-PK). Repair is completed by ligation with the enzyme XRCC4-ligase (12).
The activated IGF-1R transmits a powerful survival signal in several cell lines and is a critical determinant of growth and development. The insulin receptor substrate (IRS) family is a major cytoplasmic substrate for the activated IGF-1R (15). Recent investigations suggest a novel function of the IGF-1R/IRS-1 pathway is the intracellular trafficking of Rad51 to the nucleus (24). In the proposed model, Rad51 is sequestered in the cytoplasm, bound to the NH2-terminal domain of IRS-1. Ligand activation of the IGF-1R results in phosphorylation at IRS-1 tyrosine residues, which in turn, attenuates this the protein-protein interaction, facilitating translocation of Rad51 to foci of damaged DNA. Accordingly, we set out to determine whether IGF-1 will protect genomic DNA of MMC and NHMC from the hyperglycemic superoxide (O2–) danger signal and whether the activated IGF-1R enhances the repair of DSB by HRR, thereby preventing growth arrest and cellular senescence. Our results document IGF-1R-dependent signals prevent genotoxic stress by suppressing hyperglycemic O2– danger signals and enhancing the repair of DSB by HRR.
METHODS
Reagents. IGF-1, NAC, penicillin, streptomycin, and D-glucose were purchased from Sigma. All culture media were purchased from GIBCO-BRL and Bio-Whittaker.
MMC culture. SV40 MMC were obtained from the American Type Culture Collection. MMC exhibit phenotypic characteristics of mesangial cells in primary culture (25). A limited number of studies were also performed with NHMC to ensure that results were not influenced by transformation. MMC cultures were maintained under conditions previously established in our laboratory (8). For experimental studies, 80% confluent MMC were plated in serum-free medium (SFM; 0.2% BSA) and incubated for 12 h and divided into different experimental groups as described below.
To determine whether hyperglycemia induces DNA damage, serum-starved MMC were maintained at 5 or 25 mM glucose for 16 h in the presence and absence of IGF-1 (100 ng/ml) or the cell-permeable free radical scavenger N-acetylcysteine (NAC; 50 μM). DNA damage was determined by single-gel electrophoresis (comet assay).
NHMC culture. An identical protocol to that described immediately above was performed with NHMC obtained from Bio-Whittaker. Culture conditions were as follows; NHMC were maintained in mesangial cell basal medium (Bio-Whittaker), supplemented with 5% FBS, 30 mg/ml of gentamycin, and 15 μg/l amphotericin B, in a humidified incubator at 37°C and 5% CO2-95% air (8, 9). For experimental studies, 70% confluent primary NHMC cultures were incubated in SFM (0.2% BSA) for 16 h. All experiments were performed using NHMC from passages 5-6.
Comet assay. Overall DNA damage was analyzed by alkaline single cell gel electrophoresis (comet assay) (20) with some modifications. Briefly, an aliquot of 1 x 105 cells was suspended in 0.75% LMP agarose and spread on microscopic slides precoated with 0.5% NMP agarose (Sigma). The cells were lysed for 1 h at 4°C in a buffer containing 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10. The slides were placed in an electrophoresis unit, and DNA was allowed to unwind for 40 min in the running buffer (300 mM NaOH, 1 mM EDTA, pH >13). Electrophoresis was conducted for 30 min at 0.73 V/cm. The slides were neutralized with 0.4 M Tris, pH 7.5, stained with 2 mg/ml 4',6'-diamidino-2-phenylindole and covered with coverslips. Olive tail moment was calculated from 100 images randomly selected from each sample, using Comet 5.0 image analysis system (Kinetic Imaging, Liverpool, UK).
Homologous recombination-directed DNA repair. Plasmid pDR-GFP (generously provided by M. Jasin, Sloan-Kettering Cancer Center, New York, NY) (16) was stably transfected by using a calcium phosphate reagent (Promega, Madison, WI) into MMC and NHMC. Stable clones were selected in puromycin (2 μg/ml). pDRGFP contains a nonactive green fluorescent protein (GFP) gene (SceGFP) as a recombination reporter and a fragment of the GFP gene as a donor for homologous repair. The SceGFP cassette has an inactivating insertion, which consists of two stop codons and a restriction site for the rare cutting endonuclease I-SceI. When I-SceI is expressed in DR-GFP expressing clones, it inflicts DSBs within the SceGFP fragment, providing a signal for homologous recombination and reconstruction of functional GFP. To analyze the effectiveness of HRR, cells were transiently transfected with 3 μg of pCA-Sce and 1 μg of pDsRed1-Mito (Clontech, Palo Alto, CA) by using Fungene 6 reagent (Roche, Indianapolis, IN). PCA-Sce contains I-SceI cDNA to generate DSBs in SceGFP cDNA (16), and pDsRed1-Mito contains red fluorescent protein with a mitochondrial localization signal to control for the efficiency of transfection. DNA repair by HRR was evaluated by counting cells with both green nuclear fluorescence and red mitochondrial fluorescence vs. all positively transfected cells (red and green vs. only red cells at 72 h after transfection).
NHEJ. The cell free NHEJ assay (11) was used with some modifications (24). An aliquot of 107 MMC was washed three times with ice-cold PBS and lysed in hypotonic buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl (pH 7.5), 2 μg/ml aprotinin, 2 μg/ml leupeptin, 0.5 mM PMSF, 0.5 mM dithiothreitol, 25 mM NaF, 0.2 mM NaVO3] for 10 min on ice. Following centrifugation at 6,000 g for 3 min, nuclear pellets were resuspended in buffer B [20 mM HEPES, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 7.5), and protease inhibitors as in buffer A], and samples were frozen in liquid nitrogen. Following centrifugation (at 30,000 g for 30 min), supernatants were dialyzed overnight against buffer C [25 mM Tris·HCl (pH 7.5), 1 mM EDTA, 10% glycerol and proteinase inhibitors as in buffer A], and aliquots were stored at –70°C. NHEJ reactions were performed under the following conditions: 10 μg of nuclear lysate, 1 mM ATP, 0.25 mM deoxynucleoside triphosphates, 25 mM Tris acetate (pH 7.5), 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM diothiothreitol. After 5 min of preincubation at 37°C, the reaction mixture was supplemented with the substrate [200 ng of XhoI-XbaI-linearized pBluescript KS(+)]. The reaction mixture was incubated for 1 h at 37°C to ligate the plasmid and was treated with proteinase K (1 μg/reaction at 65°C for 30 min) to digest DNA bound proteins. Products of NHEJ reactions were resolved on 0.5% agarose gel containing 0.5 μg/ml ethidium bromide. For each experiment, the sensitivity of the assay was evaluated by running control samples in which increasing amounts of the substrate (0–500 ng) and increasing amounts of the nuclear extract (0–20 μg) were evaluated.
Immunofluorescent detection of hyperglycemic-oxidant stress. The trafficking of 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H2TMRos or Redox Sensor Red CC-1; Molecular Probes, Eugene, OR) was used to detect reactive oxygen intermediates in MMC and NHMC, as previously described (8, 9). Redox Senosr Red CC-1 is oxidized in the presence of O2– and H2O2. Briefly, cells were loaded at 37°C for 20 min with Redox Sensor Red CC-1 (1 μM) and a mitochondria-specific dye, MitoTeracker green FM (50 nM; Molecular Probes). Culture slides were washed and mounted with PBS and visualized with Nikon fluorescence microscope (Nikon Eclipse E800) equipped with triple filter cube and charge-coupled device (CCD) camera (Nikon DXM1200). The staining was performed in quadruplicate for each group and 30 random fields (average 500 cells) were studied in replicate. Images were captured using Nikon ACT-1 (Version 1.12) software and combined for publishing format using Adobe Photoshop 6.0 software.
Immunoblotting. To evaluate levels of selected DNA repair proteins, MMC were lysed on ice with 400 μl of lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% EGTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 0.2 mM sodium orthovanante, 10 μg/ml aprotinin]. DNase was added to lysis buffer to improve recovery of DNA bound proteins. Proteins (50 μg) were separated on 4 to 15% SDS polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membranes. Blots were probed with the following rabbit polyclonal antibodies; anti-Rad51 (Ab-1; Oncogene) anti-Ku70 (Serotec, Oxford, UK), anti-Ku80 (Serotec). An anti-Grb-2 antibody (Transduction Laboratories) was used as a control to monitor equal loading conditions.
Immunocytofluorescence. Serum-starved NHMC and MMC were cultured on poly-D-lysine coated Lab-Tek culture slides. Before immunostaining, cells were maintained at 5 or 25 mM glucose for 16 h, in the presence and absence of IGF-1. For immunostaining, cells were fixed and permeabilized with a buffer containing 0.02% Triton X-100 and 4% formaldehyde in PBS. Fixed cells were washed three times in PBS and blocked in 1% BSA for 30 min at 37°C. Rad51 was detected by mouse anti-Rad51 monoclonal antibody (UBI) followed by a FITC-conjugated goat anti-mouse secondary antibody (Molecular Probes). Phospho-histone H2AX (H2AX) was detected by a mouse monoclonal antibody that recognizes phosphorylated serine within the amino acid sequence 134–142 of human histone H2A.X (UBI) and rhodamine-conjugated goat anti-mouse secondary antibody (Molecular Probes). Negative controls were performed in the presence of irrelevant, anti-bromodeoxyuridine antibody in place of primary antibody. In all cases, DNA was counterstained with 4'6'-diamidino-2-phenylindole (DAPI). Specific staining was visualized with an inverted Olympus 1 x 70 fluorescence microscope equipped with a Cook Sensicom ER camera (Olympus America, Melville, NY). Final images were prepared with Adobe Photoshop to demonstrate subcellular localization and colocalization of Rad51 and H2A.X.
Quantification of Rad51/H2AX colocalization. The percentage of colocalization between Rad51 and H2AX was calculated from the entire volume of the nucleus by utilizing SlideBook 4 software (Intelligent Imaging Innovations, Denver, CO), according to the manufacturer’s instructions. Images were captured by an inverted fluorescent microscope equipped with a motorized z-axis.
Rad51 siRNA. MMC were transfected with Dharmacon-designed smart pool siRNA directed against target sequences of mouse Rad51 delivered by Lipofectamine 2000. To control for specificity of Rad51 siRNA, irrelevant siRNAs against target sequences of nuclear lamin were also purchased from Dharmacon and delivered into MMC by Lipofectamine. The final concentration for both sets of oligonucleotides was 100 nM.
Cell cycle analysis. Aliquots of MMC were fixed in 70% ethanol for 30 min at 4°C, cells were centrifuged at 390 g for 5 min, and the resulting pellets were resuspended in 1 ml of freshly prepared propidium iodide-RNaseA solution for 30 min at 37°C. Cell cycle distribution was analyzed by FACS caliber using the Cell Quest program (21).
Statistical analysis. Data are expressed as means ± SD. Comparisons between two values were performed by unpaired Student’s t-test. For multiple comparisons among different groups of data, the significant differences were determined by the Bonferroni method. Significance was defined at P 0.05.
RESULTS
Hyperglycemia-induced DNA damage. To determine whether mesangial cells exposed to hyperglycemia exhibit DNA damage and whether IGF-1 prevents or attenuates genotoxic stress, NHMC and MMC were plated in SFM containing 5 mM (normal glucose; NG) or 25 mM glucose (high glucose; HG) for 16 h. As shown in Fig. 1, comet assay detected baseline level of DNA damage in serum-starved NHMC and MMC at 5 mM glucose. A striking increase in this parameter was observed at 25 mM glucose, in both NHMC and MMC. Taken together, hyperglycemia activates a danger signal that inflicts damage to genomic DNA.
Activated IGF-1R inhibits O2– stress signal and DNA damage. We next set out to determine whether ROS production, triggered by hyperglycemia, inflicted the marked increase in genomic damage detected by comet assay. We previously documented the antioxidant function of the IGF-1R in NHMC and MMC maintained under hyperglycemic conditions (8, 9). The addition of IGF-1 or NAC suppressed hyperglycemia-induced DNA damage (Fig. 2A). To document the inhibition of ROS production in this system, MMC were loaded with the oxidant-sensitive dye Redox Sensor red CC-1 and the mitochondrial-specific dye mitotracker green FM. IGF-1 and NAC prevented high-glucose-induced oxidation of red CC-1, as indicated by the marked reduction of bright yellow orange fluorescence (Fig. 2B). Taken together, hyperglycemic ROS production is sufficient to induce genomic damage, which is interrupted by IGF-1R-dependent signals.
Effect of IGF-1 on DNA repair mechanisms. To determine whether the activated IGF-1R promotes genomic stability by enhancing DNA repair, we examined the effect of IGF-1 on HRR and NHEJ. The assay to evaluate HRR is based on the reconstruction of wt GFP from two nonfunctional heteroallelic fragments of GFP cDNA delivered into MMC by the pDRGFP expression vector (16). MMC carrying the integrated DR-GFP construct were transiently transfected with two additional expression vectors: endonuclease I-SceI, to generate DSB in GFP cDNA and red fluorescent protein with a mitochondrial localization signal, to measure efficiency of transfection. IGF-1 induced a 1.8-fold increase in the percentage of MMC at high glucose repairing SceI-induced DNA breaks (Fig. 3). Taken together, IGF-1 promotes the reconstruction of wt GFP by HRR in MMC maintained under hyperglycemic conditions. The results indicate the presence of an IGF-1R-mediated component of HRR, operative under hyperglycemic conditions.
We next confirmed serum-starved MMC at high glucose (16 h) have a population of cells, which replicate DNA. Serum starvation and high glucose had only marginal effects on cell cycle distribution (Fig. 4). A small percentage of MMC progressed to apoptosis as previously reported (8, 9). As expected, at the 16-h interval, IGF-1 induced a detectable shift in the cell distribution from G1 to G2/M. The distribution of cells in the S phase was similar among the three groups examined, indicating the IGF-1 group did not have an overrepresentation of cells in S phase. These data indicate IGF-1R-dependent repair of DSBs by HRR was not affected by cell cycle kinetics.
To determine whether IGF-1 also enhances the repair of DSBs by NHEJ, we performed a cell-free NHEJ assay. As shown in Fig. 5, NHEJ was detected in MMC under control and experimental conditions, but IGF-1 did not increase the level of in vitro ligation of pBluescript KS (+) measured in nuclear extracts. R503 fibroblasts were used as a positive control for NHEJ assay (24). Taken together, NHEJ and HRR participate in the repair of hyperglycemia-induced DSB. IGF-1 promotes repair by HRR, but not NHEJ.
Effect of IGF-1 on the expression of DNA repair proteins. Rad51 is a key enzyme in the repair of DNA by HRR (24). Immunoblot analysis performed on lysates of MMC maintained at 25 mM glucose, in the presence and absence of IGF-1, did not detect a difference in the expression of Rad51 (Fig. 6). Similarly, expression of DNA repair proteins for NHEJ, Ku70 and Ku80, were not affected by IGF-1, suggesting IGF-1 repair is not at the level of DNA repair enzyme protein expression.
Effect of IGF-1 on Rad51 nuclear foci formation. Based on the above results, we considered the possibility that IGF-1 enhances HRR by facilitating the colocalization of Rad51 with foci of nuclear damage. Because functional complexes of DNA repair proteins form detectable nuclear foci at sites of DNA lesions, which can be detected by immunofluorescent labeling techniques (24), we performed immunocytofluorescent labeling to determine whether Rad51 colocalizes with DNA/protein complexes. As shown in Fig. 7, A and B, MMC and NHMC maintained at high glucose concentration exhibit an increase in Rad51-positive nuclear foci, which increased by 1.6- and 1.7-fold, respectively, in the presence of IGF-1. Conversely, MMC and NHMC maintained at normal glucose concentration exhibit a comparatively small percent of Rad51-positive nuclear foci.
To confirm Rad51 colocalizes at sites of DNA strand breaks, we labeled cells with antibodies to H2AX and Rad51. This immunolabeling was performed because H2AX is phosphorylated within mega bp surrounding DNA strand breaks (17). As shown in Fig. 8, at high glucose concentration in the presence of IGF-1, foci demonstrating colocalization of Rad51 and H2AX were detected in nuclei of MMC and NHMC, indicative of repair by HRR. We next asked what percent of H2AX foci were labeled by Rad51. The values of 24.7 and 14% for MMC and NHMC, respectively, were derived from the entire volume of the nucleus by utilizing a computerized image-analysis system. Taken together, the activated IGF-1R promotes the subcellular trafficking of Rad51 to foci of nuclear damage, a pivotal event in DSB repair by HRR.
Effect of knockdown of Rad51 by siRNA on hyperglycemia-induced DNA damage. To assess the functional role of Rad51 translocation in the repair of hyperglycemia-induced DSBs, MMC were transiently transfected with Rad51 siRNA or nuclear lamin siRNA. Immunoblot analysis (Fig. 9A) showed barely detectable levels of Rad51 expression in Rad51 siRNA cells, whereas lamin siRNA did not affect Rad51 protein levels but did silence nuclear lamin gene expression. The percentage of MMC positive for Rad51 nuclear foci was markedly attenuated in MMC expressing Rad51 siRNA oligonucleotides under euglycemic and hyperglycemic conditions (Fig. 9B). We next asked whether knockdown of Rad51 expression and translocation increases hyperglycemia-induced DNA damage. As shown in Fig. 9C, olive tail moment detected by comet assay was increased in Rad51 siRNA cells. Collectively, these data confirm the importance of HRR in the repair of hyperglycemia-induced DSB.
DISCUSSION
The present study identifies novel mechanism(s) by which the activated IGF-1R protects mesangial cells from hyperglycemia-induced danger signals, which threaten genomic integrity and cell viability. We showed here that hyperglycemia triggers ROS-dependent signals that target DNA and that activated IGF-1R interrupts this sequence to protect against genotoxic stress. IGF-1 also facilitates the repair of DSB by specifically enhancing HRR via the translocation of Rad51 to foci of DNA damage, a pivotal event in the repair process. Finally, the activated IGF-1R did not alter the expression of Rad51, Ku70 or Ku80, suggesting DNA repair was not regulated on the level of gene expression or protein stability, but was driven by a unique IGF-1R/IRS-1/Rad51 signaling pathway (24).
Hyperglycemia-induced DNA damage. This is the first report documenting that IGF-1 protects genomic stability of a resident glomerular cell maintained under hyperglycemic conditions by suppressing O2– generation and enhancing the repair of damaged DNA. The novel IGF-1R antioxidant function was highly effective in shutting down the exponential increase in ROS production triggered by hyperglycemia. ROS-dependent signals induce multiple DNA lesions, ranging from base modifications to SSBs and DSBs. In mammals, two major cellular responses to genotoxic stress are apoptosis (18) and cellular senescence (3). Apoptosis eliminates severely damaged cells, whereas senescent cells growth arrest without dying and may acquire altered functions that can in principle disrupt tissue homeostasis (5). An important and relevant question concerns the adaptation of resident glomerular cells to the hyperglycemic O2– stress signal. We reported that IGF-1 rescues cells from the hyperglycemia-induced apoptosis program (9). Here, under conditions of high ambient glucose concentration, by comet assay we identify populations of mesangial cells at risk for progression to apoptosis or senescence, which were rescued by IGF-1. The data suggest that IGF-IR’s antioxidant function plays a key role in protecting genomic DNA from the O2– danger signal, maintaining cell viability and genomic integrity. The molecular basis for IGF-1R’s antioxidant function has not been identified; however, the activated IGF-1R induces a strong oxidant-resistant phenotype that reflects the inhibition of ROS production in cytosolic and mitochondrial compartments (9). Among the downstream targets of the IGF-1R signaling pathway are the Bcl-2 proteins, Bcl-2, and BclXL, which function as free radical scavengers (4) and also inhibit the mitochondria permeability transition pore (4). IGF-1R-dependent signals increase the availability of Bcl-2 and BclXL by downregulation of the proapoptosis Bax and phosphorylation/inactivation of Bad (9, 15). Taken together, in MMC and NHMC maintained at high ambient glucose concentration, IGF-1R’s antioxidant function is linked to the inhibition of genomic danger signals, preserving cell function and viability.
Activated IGF-1R and DNA repair. A fundamental mechanism by which cells protect themselves against oxidative stress involves the detection and repair of DSB (5). As expected, hyperglycemia-induced DNA damage triggered the activation of the two main pathways for DSB repair, NHEJ and HRR (5, 9). Hyperglycemia and SFM did not affect population of cells in the S phase, a prerequisite for repair by HRR. Flow cytometry detected a small population of cycling cells progressing to apoptosis in our system. Nonetheless, the proapoptotic effect of high glucose and the prosurvival properties of IGF-1 are consistent with data obtained by TUNEL and ELISA cell death assay. Interestingly, the error-prone NHEJ has been suggested as the major pathway for the repair of DSB in mammalian cells (7, 19). The results of the present study are consistent with such an analysis. In our system, Rad51 the key enzyme for HRR did not colocalize with the preponderance of H2AX foci, implying HRR was not the predominant pathway for DSB repair. Alternatively, the activated IGF-1R was shown to promote DSB repair by HRR but had no effect on NHEJ. We hypothesize that IGF-1R-dependent trafficking of Rad51 to foci of nuclear damage was a pivotal event in promoting repair by HRR. To test this hypothesis, we examined the effect of inhibiting Rad51 expression. At high glucose, MMC expressing Rad51 siRNA exhibit a striking reduction in Rad51 nuclear foci and increased olive tail moment by comet assay. Taken together, these data document the importance of HRR in the repair of hyperglycemia-induced DSB.
Activated IGF-1R and genomic maintenance. We speculate that the antioxidant function of IGF-1R is critical for preventing DSBs and progression to apoptosis, whereas HRR restores the genomic integrity of damaged DNA. The activated IGF-1R induces a strong oxidant-resistant phenotype that provides protection from the exponential increase in O2– production, triggered by hyperglycemia. As shown here, IGF-1R’s antioxidant function was translated into inhibition of DNA damage. Moreover, in the absence of the antioxidant function of the IGF-1R, ROS-induced DNA damage would likely overwhelm the contribution of HRR to genomic maintenance. This contention is supported by our recent work documenting oxidant stress as the proximate signal in the hyperglycemia-induced apoptosis program (8), which is completely inhibited by IGF-1 (9). Interestingly, IGF-1R cytoprotection was dependent on recruitment of both Akt/PKB and the ERK subfamily of MAPKs. Based on the results of the present study, it seems reasonable to infer that DNA repair mechanisms are not sufficient to prevent progression to apoptosis in the absence of the IGF-1R antioxidant and prosurvival gene program. We hypothesize the activated IGF-1R coordinates cell rescue via multiple signaling pathways at the level of IRS-1, promoting the repair of DSB by HRR, via trafficking of Rad51 to foci of nuclear damage. Taken together, we identified an IGF-1R survival pathway, which, on the one hand, shuts down hyperglycemia-induced danger signals that target the DNA double helix, while, on the other, enhances DNA repair by HRR, thereby maintaining genomic integrity and cell viability.
In summary, the activated IGF-1R protects MMC and NHMC from hyperglycemia-induced DNA damage. In keeping with evolving concepts in which ROS have been shown to be genotoxic, hyperglycemia-mediated ROS production was shown to inflict DSBs, the most lethal of DNA strand breaks. The activated IGF-1R induced a strong oxidant-resistant phenotype, inhibiting ROS production in MMC and NHMC maintained at high glucose and detection of DNA lesions by comet assay. Moreover, we document IGF-1R-dependent repair of DSB by HRR at a high glucose concentration. A key role for the major HRR enzyme Rad51 was documented at the foci of nuclear damage. This fundamental mechanism of cytoprotection links IGF-1R’s antioxidant function with the maintenance of genomic integrity and may have wider implications for disease modification in the diabetic glomerulus.
GRANTS
This work was supported in part by National Institutes of Health Grant 1R01-CA/NS-95518 (to K. Reiss). The authors also gratefully acknowledge the support and generosity of the Wildwood Foundation.
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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Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania
ABSTRACT
The IGF-1R is a genetic determinant of oxidative stress and longevity. Hyperglycemia induces an exponential increase in the production of a key danger signal, reactive oxygen intermediates, which target genomic DNA. Here, we report for the first time that ligand activation of the IGF-1R prevents hyperglycemia-induced genotoxic stress and enhances DNA repair, maintaining genomic integrity and cell viability. We performed single gel electrophoresis (comet assay) to evaluate DNA damage in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at high ambient glucose concentration. Hyperglycemia inflicted an impressive array of DNA damage in the form of single-strand breaks (SSBs) and double-strand breaks (DSBs). The inclusion of IGF-1 to culture media of MMC and NHMC prevented hyperglycemia-induced DNA damage. To determine whether DNA damage was mediated by reactive oxygen species (ROS), ROS generation was evaluated, in the presence of IGF-1, or the free radical scavenger n-acetyl-cysteine (NAC). IGF-1 and NAC inhibited hyperglycemic-induced ROS production and hyperglycemia-induced DNA damage. We next asked whether IGF-1 promotes the repair of DSB under hyperglycemic conditions, by homologous recombination (HRR) or nonhomologous end joining (NHEJ). Repair of DSB by NHEJ and HRR was operative in MMC maintained under hyperglycemic conditions. IGF-1 increased HRR by nearly twofold, whereas IGF-1 did not affect DNA repair by NHEJ. IGF-1R enhancement of HRR correlated with the translocation of Rad51 to foci of DNA damage. Inhibition of Rad51 expression by short interfering RNA experiments markedly decreased percentage of MMC positive for Rad51 nuclear foci and increased hyperglycemic DNA damage. We conclude that the activated IGF-1R rescues mesangial cells from hyperglycemia-induced danger signals that target genomic DNA by suppressing ROS and enhancing DNA repair by HRR.
genotoxicity; DNA double-strand breaks; Rad51; reactive oxygen species; danger signal
HYPERGLYCEMIA AND DIABETES mellitus are associated with an exponential increase in reactive oxygen species (ROS) production at the cellular level (9, 14), which play an important role in the development of diabetic complications (2, 22). ROS-dependent signals have also been linked to defects in genomic maintenance systems and the aging process (5). In a recent communication (9), we documented a strong IGF-1R oxidant-resistant phenotype in serum-starved SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC), maintained at a high ambient glucose concentration. This novel IGF-1R antioxidant function was closely coupled with expression of the survival phenotype and inhibition of the apoptosis program. These observations are in keeping with genetic experiments, in which longevity in mice was directly linked to increased resistance to oxidant stress (6, 13). Interestingly, the IGF-1R (6) and the adaptor protein p66Shc, a key IGF-1R signaling molecule, have both emerged as major genetic determinants of longevity and oxidative stress, in mammals.
As recently reviewed (5), cell survival and longevity are closely linked with the maintenance of genomic integrity. The DNA double helix is a target for ROS-dependent signals, which inflict more than 100 different types of DNA lesions, ranging from base modifications to single-strand breaks (SSB) and potentially lethal double-strand breaks (DSB) (5, 13). DNA repair is a fundamental mechanism by which cells protect themselves from oxidative stress. Failure to repair DSB commits a cell to a death sentence or malignant transformation (1, 23, 24). The two major repair mechanisms for DSB are homologous recombination (HRR) and nonhomologous end joining (NHEJ) (24). HRR is dependent on the availability of a template, synthesized during the S-phase of the cell cycle. The breast cancer susceptibility gene (BRCA2) and Rad51, a structural and functional homolog of bacterial RecA recombinase, are essential for the error-free repair of DSB by HRR (1, 23). Following detection of DSB, BRCA2 recruits Rad51 to the junction of DSBs. The first step in DSB repair by HRR involves the processing of the DNA break to produce a single-strand region with a 3' overhang, via ATM-activated 5'-3' endonuclease complex. Replication protein A initially binds to the 3' overhang and is subsequently replaced by Rad51, which searches for a homologous donor sequence and catalyzes strand exchange with the donor DNA (10). Alternatively, NHEJ, which is rapid and error prone, proceeds without a template by using the end binding Ku70/Ku80 complex and DNA protein kinase (DNA-PK). Repair is completed by ligation with the enzyme XRCC4-ligase (12).
The activated IGF-1R transmits a powerful survival signal in several cell lines and is a critical determinant of growth and development. The insulin receptor substrate (IRS) family is a major cytoplasmic substrate for the activated IGF-1R (15). Recent investigations suggest a novel function of the IGF-1R/IRS-1 pathway is the intracellular trafficking of Rad51 to the nucleus (24). In the proposed model, Rad51 is sequestered in the cytoplasm, bound to the NH2-terminal domain of IRS-1. Ligand activation of the IGF-1R results in phosphorylation at IRS-1 tyrosine residues, which in turn, attenuates this the protein-protein interaction, facilitating translocation of Rad51 to foci of damaged DNA. Accordingly, we set out to determine whether IGF-1 will protect genomic DNA of MMC and NHMC from the hyperglycemic superoxide (O2–) danger signal and whether the activated IGF-1R enhances the repair of DSB by HRR, thereby preventing growth arrest and cellular senescence. Our results document IGF-1R-dependent signals prevent genotoxic stress by suppressing hyperglycemic O2– danger signals and enhancing the repair of DSB by HRR.
METHODS
Reagents. IGF-1, NAC, penicillin, streptomycin, and D-glucose were purchased from Sigma. All culture media were purchased from GIBCO-BRL and Bio-Whittaker.
MMC culture. SV40 MMC were obtained from the American Type Culture Collection. MMC exhibit phenotypic characteristics of mesangial cells in primary culture (25). A limited number of studies were also performed with NHMC to ensure that results were not influenced by transformation. MMC cultures were maintained under conditions previously established in our laboratory (8). For experimental studies, 80% confluent MMC were plated in serum-free medium (SFM; 0.2% BSA) and incubated for 12 h and divided into different experimental groups as described below.
To determine whether hyperglycemia induces DNA damage, serum-starved MMC were maintained at 5 or 25 mM glucose for 16 h in the presence and absence of IGF-1 (100 ng/ml) or the cell-permeable free radical scavenger N-acetylcysteine (NAC; 50 μM). DNA damage was determined by single-gel electrophoresis (comet assay).
NHMC culture. An identical protocol to that described immediately above was performed with NHMC obtained from Bio-Whittaker. Culture conditions were as follows; NHMC were maintained in mesangial cell basal medium (Bio-Whittaker), supplemented with 5% FBS, 30 mg/ml of gentamycin, and 15 μg/l amphotericin B, in a humidified incubator at 37°C and 5% CO2-95% air (8, 9). For experimental studies, 70% confluent primary NHMC cultures were incubated in SFM (0.2% BSA) for 16 h. All experiments were performed using NHMC from passages 5-6.
Comet assay. Overall DNA damage was analyzed by alkaline single cell gel electrophoresis (comet assay) (20) with some modifications. Briefly, an aliquot of 1 x 105 cells was suspended in 0.75% LMP agarose and spread on microscopic slides precoated with 0.5% NMP agarose (Sigma). The cells were lysed for 1 h at 4°C in a buffer containing 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10 mM Tris, pH 10. The slides were placed in an electrophoresis unit, and DNA was allowed to unwind for 40 min in the running buffer (300 mM NaOH, 1 mM EDTA, pH >13). Electrophoresis was conducted for 30 min at 0.73 V/cm. The slides were neutralized with 0.4 M Tris, pH 7.5, stained with 2 mg/ml 4',6'-diamidino-2-phenylindole and covered with coverslips. Olive tail moment was calculated from 100 images randomly selected from each sample, using Comet 5.0 image analysis system (Kinetic Imaging, Liverpool, UK).
Homologous recombination-directed DNA repair. Plasmid pDR-GFP (generously provided by M. Jasin, Sloan-Kettering Cancer Center, New York, NY) (16) was stably transfected by using a calcium phosphate reagent (Promega, Madison, WI) into MMC and NHMC. Stable clones were selected in puromycin (2 μg/ml). pDRGFP contains a nonactive green fluorescent protein (GFP) gene (SceGFP) as a recombination reporter and a fragment of the GFP gene as a donor for homologous repair. The SceGFP cassette has an inactivating insertion, which consists of two stop codons and a restriction site for the rare cutting endonuclease I-SceI. When I-SceI is expressed in DR-GFP expressing clones, it inflicts DSBs within the SceGFP fragment, providing a signal for homologous recombination and reconstruction of functional GFP. To analyze the effectiveness of HRR, cells were transiently transfected with 3 μg of pCA-Sce and 1 μg of pDsRed1-Mito (Clontech, Palo Alto, CA) by using Fungene 6 reagent (Roche, Indianapolis, IN). PCA-Sce contains I-SceI cDNA to generate DSBs in SceGFP cDNA (16), and pDsRed1-Mito contains red fluorescent protein with a mitochondrial localization signal to control for the efficiency of transfection. DNA repair by HRR was evaluated by counting cells with both green nuclear fluorescence and red mitochondrial fluorescence vs. all positively transfected cells (red and green vs. only red cells at 72 h after transfection).
NHEJ. The cell free NHEJ assay (11) was used with some modifications (24). An aliquot of 107 MMC was washed three times with ice-cold PBS and lysed in hypotonic buffer A [10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl (pH 7.5), 2 μg/ml aprotinin, 2 μg/ml leupeptin, 0.5 mM PMSF, 0.5 mM dithiothreitol, 25 mM NaF, 0.2 mM NaVO3] for 10 min on ice. Following centrifugation at 6,000 g for 3 min, nuclear pellets were resuspended in buffer B [20 mM HEPES, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 7.5), and protease inhibitors as in buffer A], and samples were frozen in liquid nitrogen. Following centrifugation (at 30,000 g for 30 min), supernatants were dialyzed overnight against buffer C [25 mM Tris·HCl (pH 7.5), 1 mM EDTA, 10% glycerol and proteinase inhibitors as in buffer A], and aliquots were stored at –70°C. NHEJ reactions were performed under the following conditions: 10 μg of nuclear lysate, 1 mM ATP, 0.25 mM deoxynucleoside triphosphates, 25 mM Tris acetate (pH 7.5), 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM diothiothreitol. After 5 min of preincubation at 37°C, the reaction mixture was supplemented with the substrate [200 ng of XhoI-XbaI-linearized pBluescript KS(+)]. The reaction mixture was incubated for 1 h at 37°C to ligate the plasmid and was treated with proteinase K (1 μg/reaction at 65°C for 30 min) to digest DNA bound proteins. Products of NHEJ reactions were resolved on 0.5% agarose gel containing 0.5 μg/ml ethidium bromide. For each experiment, the sensitivity of the assay was evaluated by running control samples in which increasing amounts of the substrate (0–500 ng) and increasing amounts of the nuclear extract (0–20 μg) were evaluated.
Immunofluorescent detection of hyperglycemic-oxidant stress. The trafficking of 2,3,4,5,6-pentafluorodihydrotetramethylrosamine (PF-H2TMRos or Redox Sensor Red CC-1; Molecular Probes, Eugene, OR) was used to detect reactive oxygen intermediates in MMC and NHMC, as previously described (8, 9). Redox Senosr Red CC-1 is oxidized in the presence of O2– and H2O2. Briefly, cells were loaded at 37°C for 20 min with Redox Sensor Red CC-1 (1 μM) and a mitochondria-specific dye, MitoTeracker green FM (50 nM; Molecular Probes). Culture slides were washed and mounted with PBS and visualized with Nikon fluorescence microscope (Nikon Eclipse E800) equipped with triple filter cube and charge-coupled device (CCD) camera (Nikon DXM1200). The staining was performed in quadruplicate for each group and 30 random fields (average 500 cells) were studied in replicate. Images were captured using Nikon ACT-1 (Version 1.12) software and combined for publishing format using Adobe Photoshop 6.0 software.
Immunoblotting. To evaluate levels of selected DNA repair proteins, MMC were lysed on ice with 400 μl of lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 10% EGTA, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 0.2 mM sodium orthovanante, 10 μg/ml aprotinin]. DNase was added to lysis buffer to improve recovery of DNA bound proteins. Proteins (50 μg) were separated on 4 to 15% SDS polyacrylamide gel (Bio-Rad) and transferred to nitrocellulose membranes. Blots were probed with the following rabbit polyclonal antibodies; anti-Rad51 (Ab-1; Oncogene) anti-Ku70 (Serotec, Oxford, UK), anti-Ku80 (Serotec). An anti-Grb-2 antibody (Transduction Laboratories) was used as a control to monitor equal loading conditions.
Immunocytofluorescence. Serum-starved NHMC and MMC were cultured on poly-D-lysine coated Lab-Tek culture slides. Before immunostaining, cells were maintained at 5 or 25 mM glucose for 16 h, in the presence and absence of IGF-1. For immunostaining, cells were fixed and permeabilized with a buffer containing 0.02% Triton X-100 and 4% formaldehyde in PBS. Fixed cells were washed three times in PBS and blocked in 1% BSA for 30 min at 37°C. Rad51 was detected by mouse anti-Rad51 monoclonal antibody (UBI) followed by a FITC-conjugated goat anti-mouse secondary antibody (Molecular Probes). Phospho-histone H2AX (H2AX) was detected by a mouse monoclonal antibody that recognizes phosphorylated serine within the amino acid sequence 134–142 of human histone H2A.X (UBI) and rhodamine-conjugated goat anti-mouse secondary antibody (Molecular Probes). Negative controls were performed in the presence of irrelevant, anti-bromodeoxyuridine antibody in place of primary antibody. In all cases, DNA was counterstained with 4'6'-diamidino-2-phenylindole (DAPI). Specific staining was visualized with an inverted Olympus 1 x 70 fluorescence microscope equipped with a Cook Sensicom ER camera (Olympus America, Melville, NY). Final images were prepared with Adobe Photoshop to demonstrate subcellular localization and colocalization of Rad51 and H2A.X.
Quantification of Rad51/H2AX colocalization. The percentage of colocalization between Rad51 and H2AX was calculated from the entire volume of the nucleus by utilizing SlideBook 4 software (Intelligent Imaging Innovations, Denver, CO), according to the manufacturer’s instructions. Images were captured by an inverted fluorescent microscope equipped with a motorized z-axis.
Rad51 siRNA. MMC were transfected with Dharmacon-designed smart pool siRNA directed against target sequences of mouse Rad51 delivered by Lipofectamine 2000. To control for specificity of Rad51 siRNA, irrelevant siRNAs against target sequences of nuclear lamin were also purchased from Dharmacon and delivered into MMC by Lipofectamine. The final concentration for both sets of oligonucleotides was 100 nM.
Cell cycle analysis. Aliquots of MMC were fixed in 70% ethanol for 30 min at 4°C, cells were centrifuged at 390 g for 5 min, and the resulting pellets were resuspended in 1 ml of freshly prepared propidium iodide-RNaseA solution for 30 min at 37°C. Cell cycle distribution was analyzed by FACS caliber using the Cell Quest program (21).
Statistical analysis. Data are expressed as means ± SD. Comparisons between two values were performed by unpaired Student’s t-test. For multiple comparisons among different groups of data, the significant differences were determined by the Bonferroni method. Significance was defined at P 0.05.
RESULTS
Hyperglycemia-induced DNA damage. To determine whether mesangial cells exposed to hyperglycemia exhibit DNA damage and whether IGF-1 prevents or attenuates genotoxic stress, NHMC and MMC were plated in SFM containing 5 mM (normal glucose; NG) or 25 mM glucose (high glucose; HG) for 16 h. As shown in Fig. 1, comet assay detected baseline level of DNA damage in serum-starved NHMC and MMC at 5 mM glucose. A striking increase in this parameter was observed at 25 mM glucose, in both NHMC and MMC. Taken together, hyperglycemia activates a danger signal that inflicts damage to genomic DNA.
Activated IGF-1R inhibits O2– stress signal and DNA damage. We next set out to determine whether ROS production, triggered by hyperglycemia, inflicted the marked increase in genomic damage detected by comet assay. We previously documented the antioxidant function of the IGF-1R in NHMC and MMC maintained under hyperglycemic conditions (8, 9). The addition of IGF-1 or NAC suppressed hyperglycemia-induced DNA damage (Fig. 2A). To document the inhibition of ROS production in this system, MMC were loaded with the oxidant-sensitive dye Redox Sensor red CC-1 and the mitochondrial-specific dye mitotracker green FM. IGF-1 and NAC prevented high-glucose-induced oxidation of red CC-1, as indicated by the marked reduction of bright yellow orange fluorescence (Fig. 2B). Taken together, hyperglycemic ROS production is sufficient to induce genomic damage, which is interrupted by IGF-1R-dependent signals.
Effect of IGF-1 on DNA repair mechanisms. To determine whether the activated IGF-1R promotes genomic stability by enhancing DNA repair, we examined the effect of IGF-1 on HRR and NHEJ. The assay to evaluate HRR is based on the reconstruction of wt GFP from two nonfunctional heteroallelic fragments of GFP cDNA delivered into MMC by the pDRGFP expression vector (16). MMC carrying the integrated DR-GFP construct were transiently transfected with two additional expression vectors: endonuclease I-SceI, to generate DSB in GFP cDNA and red fluorescent protein with a mitochondrial localization signal, to measure efficiency of transfection. IGF-1 induced a 1.8-fold increase in the percentage of MMC at high glucose repairing SceI-induced DNA breaks (Fig. 3). Taken together, IGF-1 promotes the reconstruction of wt GFP by HRR in MMC maintained under hyperglycemic conditions. The results indicate the presence of an IGF-1R-mediated component of HRR, operative under hyperglycemic conditions.
We next confirmed serum-starved MMC at high glucose (16 h) have a population of cells, which replicate DNA. Serum starvation and high glucose had only marginal effects on cell cycle distribution (Fig. 4). A small percentage of MMC progressed to apoptosis as previously reported (8, 9). As expected, at the 16-h interval, IGF-1 induced a detectable shift in the cell distribution from G1 to G2/M. The distribution of cells in the S phase was similar among the three groups examined, indicating the IGF-1 group did not have an overrepresentation of cells in S phase. These data indicate IGF-1R-dependent repair of DSBs by HRR was not affected by cell cycle kinetics.
To determine whether IGF-1 also enhances the repair of DSBs by NHEJ, we performed a cell-free NHEJ assay. As shown in Fig. 5, NHEJ was detected in MMC under control and experimental conditions, but IGF-1 did not increase the level of in vitro ligation of pBluescript KS (+) measured in nuclear extracts. R503 fibroblasts were used as a positive control for NHEJ assay (24). Taken together, NHEJ and HRR participate in the repair of hyperglycemia-induced DSB. IGF-1 promotes repair by HRR, but not NHEJ.
Effect of IGF-1 on the expression of DNA repair proteins. Rad51 is a key enzyme in the repair of DNA by HRR (24). Immunoblot analysis performed on lysates of MMC maintained at 25 mM glucose, in the presence and absence of IGF-1, did not detect a difference in the expression of Rad51 (Fig. 6). Similarly, expression of DNA repair proteins for NHEJ, Ku70 and Ku80, were not affected by IGF-1, suggesting IGF-1 repair is not at the level of DNA repair enzyme protein expression.
Effect of IGF-1 on Rad51 nuclear foci formation. Based on the above results, we considered the possibility that IGF-1 enhances HRR by facilitating the colocalization of Rad51 with foci of nuclear damage. Because functional complexes of DNA repair proteins form detectable nuclear foci at sites of DNA lesions, which can be detected by immunofluorescent labeling techniques (24), we performed immunocytofluorescent labeling to determine whether Rad51 colocalizes with DNA/protein complexes. As shown in Fig. 7, A and B, MMC and NHMC maintained at high glucose concentration exhibit an increase in Rad51-positive nuclear foci, which increased by 1.6- and 1.7-fold, respectively, in the presence of IGF-1. Conversely, MMC and NHMC maintained at normal glucose concentration exhibit a comparatively small percent of Rad51-positive nuclear foci.
To confirm Rad51 colocalizes at sites of DNA strand breaks, we labeled cells with antibodies to H2AX and Rad51. This immunolabeling was performed because H2AX is phosphorylated within mega bp surrounding DNA strand breaks (17). As shown in Fig. 8, at high glucose concentration in the presence of IGF-1, foci demonstrating colocalization of Rad51 and H2AX were detected in nuclei of MMC and NHMC, indicative of repair by HRR. We next asked what percent of H2AX foci were labeled by Rad51. The values of 24.7 and 14% for MMC and NHMC, respectively, were derived from the entire volume of the nucleus by utilizing a computerized image-analysis system. Taken together, the activated IGF-1R promotes the subcellular trafficking of Rad51 to foci of nuclear damage, a pivotal event in DSB repair by HRR.
Effect of knockdown of Rad51 by siRNA on hyperglycemia-induced DNA damage. To assess the functional role of Rad51 translocation in the repair of hyperglycemia-induced DSBs, MMC were transiently transfected with Rad51 siRNA or nuclear lamin siRNA. Immunoblot analysis (Fig. 9A) showed barely detectable levels of Rad51 expression in Rad51 siRNA cells, whereas lamin siRNA did not affect Rad51 protein levels but did silence nuclear lamin gene expression. The percentage of MMC positive for Rad51 nuclear foci was markedly attenuated in MMC expressing Rad51 siRNA oligonucleotides under euglycemic and hyperglycemic conditions (Fig. 9B). We next asked whether knockdown of Rad51 expression and translocation increases hyperglycemia-induced DNA damage. As shown in Fig. 9C, olive tail moment detected by comet assay was increased in Rad51 siRNA cells. Collectively, these data confirm the importance of HRR in the repair of hyperglycemia-induced DSB.
DISCUSSION
The present study identifies novel mechanism(s) by which the activated IGF-1R protects mesangial cells from hyperglycemia-induced danger signals, which threaten genomic integrity and cell viability. We showed here that hyperglycemia triggers ROS-dependent signals that target DNA and that activated IGF-1R interrupts this sequence to protect against genotoxic stress. IGF-1 also facilitates the repair of DSB by specifically enhancing HRR via the translocation of Rad51 to foci of DNA damage, a pivotal event in the repair process. Finally, the activated IGF-1R did not alter the expression of Rad51, Ku70 or Ku80, suggesting DNA repair was not regulated on the level of gene expression or protein stability, but was driven by a unique IGF-1R/IRS-1/Rad51 signaling pathway (24).
Hyperglycemia-induced DNA damage. This is the first report documenting that IGF-1 protects genomic stability of a resident glomerular cell maintained under hyperglycemic conditions by suppressing O2– generation and enhancing the repair of damaged DNA. The novel IGF-1R antioxidant function was highly effective in shutting down the exponential increase in ROS production triggered by hyperglycemia. ROS-dependent signals induce multiple DNA lesions, ranging from base modifications to SSBs and DSBs. In mammals, two major cellular responses to genotoxic stress are apoptosis (18) and cellular senescence (3). Apoptosis eliminates severely damaged cells, whereas senescent cells growth arrest without dying and may acquire altered functions that can in principle disrupt tissue homeostasis (5). An important and relevant question concerns the adaptation of resident glomerular cells to the hyperglycemic O2– stress signal. We reported that IGF-1 rescues cells from the hyperglycemia-induced apoptosis program (9). Here, under conditions of high ambient glucose concentration, by comet assay we identify populations of mesangial cells at risk for progression to apoptosis or senescence, which were rescued by IGF-1. The data suggest that IGF-IR’s antioxidant function plays a key role in protecting genomic DNA from the O2– danger signal, maintaining cell viability and genomic integrity. The molecular basis for IGF-1R’s antioxidant function has not been identified; however, the activated IGF-1R induces a strong oxidant-resistant phenotype that reflects the inhibition of ROS production in cytosolic and mitochondrial compartments (9). Among the downstream targets of the IGF-1R signaling pathway are the Bcl-2 proteins, Bcl-2, and BclXL, which function as free radical scavengers (4) and also inhibit the mitochondria permeability transition pore (4). IGF-1R-dependent signals increase the availability of Bcl-2 and BclXL by downregulation of the proapoptosis Bax and phosphorylation/inactivation of Bad (9, 15). Taken together, in MMC and NHMC maintained at high ambient glucose concentration, IGF-1R’s antioxidant function is linked to the inhibition of genomic danger signals, preserving cell function and viability.
Activated IGF-1R and DNA repair. A fundamental mechanism by which cells protect themselves against oxidative stress involves the detection and repair of DSB (5). As expected, hyperglycemia-induced DNA damage triggered the activation of the two main pathways for DSB repair, NHEJ and HRR (5, 9). Hyperglycemia and SFM did not affect population of cells in the S phase, a prerequisite for repair by HRR. Flow cytometry detected a small population of cycling cells progressing to apoptosis in our system. Nonetheless, the proapoptotic effect of high glucose and the prosurvival properties of IGF-1 are consistent with data obtained by TUNEL and ELISA cell death assay. Interestingly, the error-prone NHEJ has been suggested as the major pathway for the repair of DSB in mammalian cells (7, 19). The results of the present study are consistent with such an analysis. In our system, Rad51 the key enzyme for HRR did not colocalize with the preponderance of H2AX foci, implying HRR was not the predominant pathway for DSB repair. Alternatively, the activated IGF-1R was shown to promote DSB repair by HRR but had no effect on NHEJ. We hypothesize that IGF-1R-dependent trafficking of Rad51 to foci of nuclear damage was a pivotal event in promoting repair by HRR. To test this hypothesis, we examined the effect of inhibiting Rad51 expression. At high glucose, MMC expressing Rad51 siRNA exhibit a striking reduction in Rad51 nuclear foci and increased olive tail moment by comet assay. Taken together, these data document the importance of HRR in the repair of hyperglycemia-induced DSB.
Activated IGF-1R and genomic maintenance. We speculate that the antioxidant function of IGF-1R is critical for preventing DSBs and progression to apoptosis, whereas HRR restores the genomic integrity of damaged DNA. The activated IGF-1R induces a strong oxidant-resistant phenotype that provides protection from the exponential increase in O2– production, triggered by hyperglycemia. As shown here, IGF-1R’s antioxidant function was translated into inhibition of DNA damage. Moreover, in the absence of the antioxidant function of the IGF-1R, ROS-induced DNA damage would likely overwhelm the contribution of HRR to genomic maintenance. This contention is supported by our recent work documenting oxidant stress as the proximate signal in the hyperglycemia-induced apoptosis program (8), which is completely inhibited by IGF-1 (9). Interestingly, IGF-1R cytoprotection was dependent on recruitment of both Akt/PKB and the ERK subfamily of MAPKs. Based on the results of the present study, it seems reasonable to infer that DNA repair mechanisms are not sufficient to prevent progression to apoptosis in the absence of the IGF-1R antioxidant and prosurvival gene program. We hypothesize the activated IGF-1R coordinates cell rescue via multiple signaling pathways at the level of IRS-1, promoting the repair of DSB by HRR, via trafficking of Rad51 to foci of nuclear damage. Taken together, we identified an IGF-1R survival pathway, which, on the one hand, shuts down hyperglycemia-induced danger signals that target the DNA double helix, while, on the other, enhances DNA repair by HRR, thereby maintaining genomic integrity and cell viability.
In summary, the activated IGF-1R protects MMC and NHMC from hyperglycemia-induced DNA damage. In keeping with evolving concepts in which ROS have been shown to be genotoxic, hyperglycemia-mediated ROS production was shown to inflict DSBs, the most lethal of DNA strand breaks. The activated IGF-1R induced a strong oxidant-resistant phenotype, inhibiting ROS production in MMC and NHMC maintained at high glucose and detection of DNA lesions by comet assay. Moreover, we document IGF-1R-dependent repair of DSB by HRR at a high glucose concentration. A key role for the major HRR enzyme Rad51 was documented at the foci of nuclear damage. This fundamental mechanism of cytoprotection links IGF-1R’s antioxidant function with the maintenance of genomic integrity and may have wider implications for disease modification in the diabetic glomerulus.
GRANTS
This work was supported in part by National Institutes of Health Grant 1R01-CA/NS-95518 (to K. Reiss). The authors also gratefully acknowledge the support and generosity of the Wildwood Foundation.
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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