Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-depende - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:46 |只看该作者 |倒序浏览 |打印

作者：Janaki Chintapalli,, Shuo Yang,, David Opawumi, Sunita Ray Goyal, Nazia Shamsuddin, Ashwani Malhotra, Krzysztof Reiss, and Leonard G. Meggs作者单位：2 Division of Nephrology and Hypertension, Department of Medicine, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey; and 1 Center for Neurovirology and Cancer Biology, Temple University, School of Medicine, Philadelphia, Pennsylvania ( y; x8 ?9 h! B' O1 c$ v
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      【摘要】. i% i) u* C9 B5 w
   Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.
      【关键词】 reactive oxygen species DNA damage redox function' x( O' n0 {$ E6 s+ t; s3 Q
   P 66S HC A PROTEIN IS ONE of three isoforms encoded by the mammalian ShcA locus. The three overlapping Shc proteins, p66ShcA, p52ShcA, and p46ShcA, all share a COOH-terminal Src homology 2 (SH2) domain, central collagen-homologous (CH) region, and NH 2 -terminal phosphotyrosine binding domain. P46ShcA and P52ShcA are the products of alternative translation initiation sites within the same transcript, whereas p66ShcA is distinguished by a unique NH 2 -terminal region (CH2), generated from alternative splicing ( 6 ). The ShcA family of proteins are cytoplasmic substrates for the tyrosine kinase family of receptors ( 5 ), which participate in mitogenesis via the recruitment of the adaptor protein Grb2 ( 19 ) and subsequent activation of the Ras signaling pathway. Several lines of evidence support a dominant role for wild-type (WT) p66ShcA protein in the intracellular pathways that convert oxidative stress to apoptosis ( 3, 26, 27 ), whereas homozygous mutation of the WTp66Shc gene in mice confers increased resistance to oxidative stress, aging, and apoptosis ( 22, 24 ). Recently, a clearer understanding of the WTp66ShcA gene has emerged, based on evidence WTp66ShcA protein functionally interacts with the mammalian Forkhead homolog FKHRL1 (FOXO3a) ( 26 ). In the proposed scheme, reactive oxygen species (ROS) induce phosphorylation of WTp66ShcA protein at a critical CH2 Ser-36 residue, a modification that serves to promote the intracellular generation of ROS ( 26, 27 ) and the recruitment of Akt/PKB, which directly phosphorylates and inactivates members of the FOXO family ( 35 ).

Hyperglycemia and diabetes mellitus are associated with an exponential increase in ROS production at the cellular level ( 10, 12, 13 ), which may play a causal role in the development of diabetic complications ( 2, 33 ). We have recently identified a novel IGF-1R antioxidant function that protects SV40 murine mesangial cells (MMC) and normal human mesangial cells (NHMC) from high glucose (HG)-induced DNA strand breaks (DSB) ( 39 ) and apoptosis ( 13 ). IGF-1 induces a strong oxidant-resistant phenotype, which reflects inhibition of ROS production in the cytosolic and mitochondrial compartments ( 13, 39 ). The divergent phenotypes induced by the activated IGF-1 receptor and WTp66ShcA protein have important implications for the survival and function of resident glomerular cells. Although we have shown IGF-1 to be a potent antioxidant and prosurvival factor ( 13, 39 ), concerns have been raised about the therapeutic application of IGF-1-based therapy, driven by the association of IGF-1 with diabetic end-organ complications ( 7, 29 ). We hypothesized that gene-based strategies targeting specific redox-sensitive component(s) of the IGF-1 signaling pathway may induce the oxidant-resistant phenotype independently of IGF-1 receptor activation.

Accordingly, we set out to silence expression of the WTp66ShcA gene by transfecting MMC and NHMC with either mutant 36 p66ShcA or p66ShcA short-intervening RNA (siRNA) to test the hypothesis that p66ShcA-deficient cell lines will be more resistant to HG-induced ROS generation, DNA damage, and cell death. Our results demonstrate p66ShcA-deficient cells maintained at HG exhibit the oxidant-resistant phenotype and protection from HG-induced DNA damage.

METHODS
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Reagents. Penicillin, streptomycin, and D -glucose were purchased from Sigma (St. Louis, MO). All culture media were purchased from GIBCO-BRL (Grand Island, NY) and Bio-Whittaker (Walkersville, MD).4 K1 j0 D& R/ B8 H  v: l

MMC and NHMC culture. MMC and NHMC were obtained from the American Type Culture Collection (San Diego, CA). MMC exhibit phenotypic characteristics of mesangial cells in primary culture ( 38 ). A limited number of studies were also performed with NHMC to ensure that results were not influenced by MMC transformation ( 12, 13, 39 ). MMC and NHMC cultures were maintained under conditions previously established in our laboratory ( 12, 13, 39 ). For experimental studies, at 80% confluent growth, cells were plated in serum-free medium (SFM; 0.2% BSA), incubated for 12 h, and then divided into different experimental groups as described below.

Constructs and antibodies. Mutant 36p66ShcA was the kind gift of Toren Finkel (Cardiovascular Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD). The epitope-tagged HA-FOXO3a construct was generously provided by Michael Greenberg (Harvard Medical School, Boston, MA). Phosphorylated FOXO3a was detected using an anti-phospho-FOXO3a (Thr-32) antibody obtained from Cell Signaling Technology (Beverly, MA). ShcA protein levels were determined by immunoblot analysis using an antibody recognizing all ShcA isoforms (Cell Signaling). Phosphorylation at Ser-36 of WTp66ShcA protein was detected by a mouse monoclonal anti-phospho-serine antibody that recognizes the 66-kDa form of Shc phosphorylated at Ser-36 (Calbiochem, La Jolla, CA). Phospho-Akt levels were determined with an antibody to pSer473-Akt (Cell Signaling).

Transfection. MMC and NHMC were transfected with pcDNA/p66ShcA SA expression vector by nucleoporation (Amaxa Nucleofector I, Amaxa, Cologne, Germany). In separate studies, isoform-specific p66ShcA siRNA ( 16 ) was delivered to MMC and NHMC by Lipofectamine 2000 (Invitrogen, Carlsbad, CA). To control for specificity of p66ShcA siRNA, irrelevant siRNA against target sequences of nuclear lamin were purchased from Dharmacon (Lafayette, CO). The final concentration for both sets of oligonucleotides was 100 nM.
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Detection of HG-induced oxidant stress. The trafficking of 2,3,4,5,6-pentafluorodihydrotetramethylfosamine (PF-H2TMRos or Redox Sensor Red CC-1; Molecular Probes-Invitrogen, Carlsbad, CA) was used to detect reactive oxygen intermediates, as previously described ( 12, 13, 39 ). Redox Sensor Red CC-1 is oxidized in the presence of O 2 - and H 2 O 2. Briefly, cells were loaded at 37°C for 20 min with Redox Sensor Red CC-1 (1 µM) and a mitochondria-specific dye, MitoTracker green FM (50 nM; Molecular Probes). Culture slides were washed with PBS and visualized with a Nikon fluorescence microscope (Nikon Eclipse E800) equipped with a triple-filter cube and charge-coupled device 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 using Adobe Photoshop 6.0 software.
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Determination of ROS kinetics at HG. The kinetics of ROS metabolism at HG was determined by measuring the intensity of the fluorescent signal from the redox-sensitive fluoroprobe 2',7'-dichlorofluorescein diacetate (DCFDA) at multiple time points. DCFDA is converted by intracellular esterases to 2'7'-dichlorodihydrofluorescein, which, in turn, is oxidized by H 2 O 2 to the highly fluorescent 2'7'-dichlorohydrofluorescein (DCF). Briefly, cells were loaded with 10 mM DCFDA in phenol red-free DMEM containing either 5 or 25 mM glucose. Cells were incubated in a 24-well plate at 37°C for 30 min and washed with phenol red-free DMEM. DCF fluorescence was detected by a Fluorescence Multi-Well Plate Reader CytoFluor 4000 (PerSeptive Biosystems) set for excitation of 485 nm and emission of 530 nm. The intensity of the fluorescent signal was calculated with Microsoft Excel using the equation {[(F t -F 0 )/F 0 ] x 100} ( 37 ).

Immunoblotting. Cells were lysed on ice with 400 µl of lysis buffer (in mM: 50 HEPES, pH 7.5, 150 NaCl, 1.5 MgCl 2, 1 EDTA, 1 PMSF, and 0.2 sodium orthovanate as well as 10% EGTA, 10% glycerol, 1% Triton X-100, and 10 µg/ml aprotinin). Proteins were separated on 4-15% SDS polyacrylamide gels (Bio-Rad, Hercules, CA) and then transferred to nitrocellulose membranes.
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Immunofluorescent staining. Parental and mutant p66ShcA cells were cultured on poly- D -lysine-coated Lab-Tek slides. The plasmid HA-FOXO3a was delivered by nucleofection. At 24 h posttransfection, cells were maintained in SFM at 5 or 25 mM glucose. For immunostaining, cells were fixed and permeabilized with a buffer containing 0.02% Triton X-100 and 4% formaldehyde in PBS, washed three times in PBS, and blocked in 1% BSA for 30 min at 37°C. The coverslips were then incubated with anti-HA antibody diluted in PBS-BSA for 1 h and washed five times with PBS. Cells were then incubated for 1 h with a secondary antibody (Alexa Fluor 568 goat anti-mouse IgG, Molecular Probes) diluted in PBS-BSA containing 4'6 diamidino-2-phenylindole (DAPI). Specific staining was visualized with an inverted Olympus 1X70 fluorescence microscope equipped with a Cook Sensicom ER camera (Olympus America, Melville, NY). An average of 500 cells/dish were counted in three separate experiments. Final images were prepared with Adobe Photoshop.! X" n% \& z( t* R$ a( W

Single-cell gel electrophoresis (comet assay). DNA damage was analyzed by comet assay ( 39 ) with some modifications. Briefly, an aliquot of 1 x 10 5 cells was suspended in 0.75% LMP-agarose and spread on microscope slides precoated with 0.5% NMP-agarose (Sigma). The cells were lysed for 1 h at 4°C in a buffer containing 2.5M NaCl, 100 mM EDTA, 1% Triton X-100, and 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 DAPI, and mounted using coverslips. Olive tail moment was calculated from 100 images randomly selected form each sample, using the Comet 5.0 image-analysis system (Kinetic Imaging, Liverpool, UK).
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Analysis of DNA fragmentation by ELISA. Histone-associated DNA fragments were quantified by Cell Death Detection ELISA (Roche Diagnostic, Branchburg, NJ) as previously described ( 12, 13, 39 ).

Statistical analysis. Data are expressed as means ± SD. 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

Generation of p66ShcA-deficient cell lines. Two approaches were employed to knock down WTp66ShcA function in MMC and NHMC: stable transfection with a mutant 36 p66ShcA expression vector and transient transfection with isoform-specific p66ShcA siRNA. Immunoblot analysis ( Fig. 1 A ) shows the position of mutant 36 ( top band) and WTp66ShcA protein. Figure 1 B shows immunoblot analysis of lysates from cells transfected with isoform-specific p66ShcA siRNA ( 16 ). Nuclear lamin siRNA was used as a control. p66ShcA siRNA cells show barely detectable levels of WTp66ShcA protein, whereas in control cells WTp66ShcA expression was not affected by lamin siRNA.
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Fig. 1. Representative immunoblot analysis of ShcA isoforms. A : murine mesangial cells (MMC) transfected with empty vector (EV) and MMC stably transfected with mutant 36 p66ShcA (arrow). B : MMC transfected with lamin short-intervening (si) RNA and MMC transfected with isoform-specific p66ShcA siRNA.
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WTp66ShcA-dependent regulation of HG-induced oxidant stress. HG alters the redox status of cells through the overproduction of ROS by the mitochondrial electron transport chain and NADPH oxidase ( 12, 13, 31 ). Accordingly, we next asked whether p66ShcA-deficient cells exhibit resistance to HG-induced intracellular ROS generation. Serum-starved parental cells and mutant 36 p66ShcA cells were maintained at 5 or 25 mM glucose for 16 h. To document the presence of O 2 - and H 2 O 2, cells were loaded with the redox-sensitive probe Redox Sensor Red CC-1 and the mitochondria-specific dye MitoTracker green FM. As shown in Fig. 2 B, parental cells at HG exhibit bright yellow/orange fluorescence in mitochondria due to the colocalization of oxidized Red CC-1 and Mito Tracker green, whereas oxidation of Red CC-1 in cytoplasm shows red/orange fluorescence, indicative of increased ROS production in the mitochondria and cytosolic compartments. Conversely, as shown in Fig. 2 D, HG-induced ROS production is inhibited in p66ShcA-deficient cells. To rule out confounding effects of SV40 transformation, we repeated this protocol with serum-starved parental NHMC ( Fig. 2, E and F ) and NHMC transfected with A mutant 36 p66ShcA construct ( Fig. 2, G and H ). As expected, the mutant construct inhibited HG-induced ROS production.6 v1 J% a5 K! r% @5 o* \; `- p8 \

Fig. 2. Glucose-induced reactive oxygen species (ROS) generation. Serum-starved parental and p66ShcA-deficient cells were maintained at 5 (normal glucose; NG) or 25 mM (high glucose; HG) for 16 h. Cells were loaded with the redox-sensitive dye Redox Sensor Red CC-1 and the mitochondrial specific-dye MitoTracker green FM. A and B : parental MMC at NG ( A ) and HG ( B ). Parental MMC at HG show yellow/orange fluorescence (arrow) due to the colocalization of Red Sensor CC-1 (red fluorescence) and Mito Tracker FM (green fluorescence) at mitochondria. Red/orange fluorescence is due to oxidation of Redox Sensor CC-1 in cytosolic compartment. C and D : mutant 36 p66ShcA MMC at NG ( C ) and HG ( D ). Note absence of fluorescent signal in mitochondria or cytosol in C and D. E and F : parental normal human mesangial cells (NHMC) at NG ( E ) and HG ( F ). G and H : mutant 36 p66ShcA NHMC at NG ( G ) and HG ( F ). Note intensity of yellow/orange fluorescent signal in parental NHMC at HG ( F, arrow) and barely detectable signal in mutant 36 p66ShcA NHMC at HG ( H )." ~; a5 G( b% N4 q, g4 O  I7 k

To determine whether inhibition of the HG-induced ROS production in p66ShcA-deficient cells was due to a shift in the kinetics of ROS metabolism, cells were loaded with the redox-sensitive fluoroprobe DCFDA, and the intensity of the DCF fluorescent signal was measured at multiple time points. As shown in Fig. 3, p66ShcA-deficient cells exhibit attenuation in the amplitude of the kinetic curves for ROS metabolism at HG, indicative of the oxidant-resistant phenotype. The striking inhibitory effect was more sustained in MMC stably transfected with mutant 36 p66ShcA, but it was also detected in MMC transiently transfected with isoform-specific p66ShcA siRNA. Taken together, silencing the WTp66ShcA gene with mutant 36 p66ShcA or p66ShcA siRNA induces a strong oxidant-resistant phenotype.3 c/ }( f9 S1 x4 {

Fig. 3. Effect of silencing wild-type (WT) p66ShcA on kinetics of ROS metabolism at HG. Parental and p66ShcA-deficient cells were studied after 16 h at NG or HG. Cells were loaded with the redox-sensitive probe 2',7'-dichlorofluorescein diacetate (DCFDA). The intensity of the 2'7'-dichlorohydrofluorescein (DCF) fluorescent signal was determined at indicated intervals. Data are presented as means ± SD and represent 5 independent experiments. * P 0.05.
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Mutant 36 p66Shc inhibits phosphorylation of WTp66ShcA, Akt/PKB, and FOXO3a. WTp66ShcA redox function is activated by ROS-dependent phosphorylation at a Ser-36 residue position ( 26 ). We hypothesized that HG phosphorylation of WTp66ShcA protein at Ser-36 will be attenuated in mutant 36 p66ShcA cells. To test this hypothesis, we performed immunoblot analysis with an anti-ShcA/p66 (pSer-36) mouse monoclonal antibody. This antibody recognizes the 66-kDa isoform of ShcA phosphorylated at Ser-36 and does not cross react with nonphosphorylated p66ShcA, mutant 36 p66ShcA, or with unrelated phosphorylation sites ( 27 ). As shown in Fig. 4 A, at HG mutant 36 p66ShcA cells exhibit no change in the phosphorylation status of Ser-36, whereas parental cells show an upregulation in phosphorylation at Ser-36 of WTp66ShcA protein.

Fig. 4. Mutant 36 p66ShcA attenuates glucose-induced phosphorylation of FOXO3a. Parental and p66ShcA-deficient cells were maintained for 16 h at NG or HG. A : protein extracts were isolated and separated by PAGE, and nitrocellulose filters were probed with phospho-antibodies for Ser-36 of p66ShcA, Ser-473 of Akt/PKB, and Thr-32 of FOXO3a. B : effect of N -acetylcysteine (NAC) on HG-induced phosphorylation of Akt/PKB. Results shown are representative of 3-4 experiments.
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WTp66ShcA interacts with FOXO3a by facilitating ROS-dependent activation of Akt/PKB, which, in turn, via an evolutionarily conserved pathway, phosphorylates and inactivates FOXO3a ( 26, 35 ). To determine whether this signaling pathway is dormant in mutant 36 p66ShcA cells, the phosphorylation status of Akt/PKB was examined. Immunoblot analysis shows an upregulation of phospho-Akt/PKB levels in parental cells at HG, whereas mutant 36 p66ShcA cells exhibit no detectable alteration in the phosphorylation status of Akt/PKB ( Fig. 4 A ). Serum starvation is known to increase intracellular oxidants ( 26 ) and, under euglycemic conditions, the level of phospho-Akt/PKB was increased in parental cells vs. p66ShcA-deficient cells, indicative of the potent inhibitory effect on WTp66ShcA redox function. To confirm that phosphorylation of Akt/PKB was redox dependent, immunoblots were repeated at HG and normal glucose ( Fig. 4 B ), in the presence and absence of N -acetylcysteine (NAC; 50 µM).' Z7 `' F0 T6 ?8 P* H- ^+ E3 y

The phosphorylation status of FOXO3a and its subcellular localization are critical for its transcriptional activity ( 26, 35 ). The sites of FOXO3a phosphorylation by Akt/PKB have been mapped to three key regulatory residues ( 35 ). In our system, we tested Thr-32, a site on FOXO3a known to be phosphorylated by Akt/PKB ( 26, 35 ). We hypothesized the HG will increase phosphorylation of FOXO3a in parental cells, whereas this posttranslational modification will be attenuated in mutant 36 p66ShcA cells. To test this hypothesis, lysates from parental and p66ShcA-deficient cells were probed with a phospho-Thr-32 FOXO3a antibody. This polyclonal antibody detects endogenous levels of FOXO1/FOXO3a, only when these transcription factors are phosphorylated on Thr-24 of FOXO1 or Thr-32 of FOXO3a (Cell Signaling Technology). Immunoblot analysis shows upregulation in the phosphorylation of FOXO3a protein at Thr-32 ( top band) in parental cells at HG ( Fig. 4 A ) but no change in the phosphorylation status of FOXO3a in mutant 36 p66ShcA cells.% _( ^3 U& `5 \' J' L- r
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To evaluate the subcellular localization of FOXO3a at HG, parental and mutant 36 p66ShcA cells were cotransfected with HA-FOXO3a and indirect immunofluorescent staining was performed with an antibody against the HA-epitope ( Fig. 5 A ). In parental cells at HG, HA-FOXO3a translocates from nucleus to cytoplasm, as indicated by the absence of green fluorescence in the nuclear compartment, counterstained with DAPI (blue fluorescence). Figure 5 B shows the percent cells positive for nuclear HA-FOXO3a at HG and normal glucose. Parental cells at HG show a marked reduction in nuclear-positive HA-FOXO3a cells. An identical analysis performed with mutant 36 p66ShcA cells detected a nearly identical percentage of cells positive for nuclear HA-FOXO3a at HG and normal glucose. Taken together, inhibition of WTp66ShcA redox function prevents hyperglycemia-induced phosphorylation and redistribution of FOXO3a, the stress response regulator.* e. |, Y7 D* P  u6 j' i

Fig. 5. Mutant 36 p66ShcA attenuates glucose-induced nuclear export of HA-FOXO3a. Parental and p66ShcA-deficient cells were transfected with an expression plasmid encoding HA-tagged WT FOXO3a. At 24 h posttransfection, cells were maintained for 16 h at NG or HG. A : HA-FOXO3a subcellular localization detected by immunofluorescent staining (green) with anti-HA antibody. Nuclear DNA (blue) was stained with 4'6 diamidino-2-phenylindole (DAPI). Images were merged to detect nuclear localization or export of HA-FOXO3a. B : quantification of means ± SD of 3-5 experiments. * P 0.05. C : representative images of HA-FOXO3a immunostaining.( q! r* c. t1 f+ f6 O
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Mutant 36 p66ShcA inhibits HG-induced DNA damage. Cell survival and longevity are closely linked with the maintenance of genomic stability ( 9 ). The DNA double helix is a target for ROS-dependent signals ( 9 ). We hypothesized that p66ShcA-deficient cells will be resistant to HG-induced oxidative DNA damage. As shown in Fig. 6 A, single-cell gel electrophoresis detected baseline level of DSB (olive tail moment) in serum-starved parental cells maintained at normal glucose concentration. This parameter increased 2.5-fold at HG, indicative that hyperglycemic ROS production is sufficient to inflict genomic damage. Conversely, p66ShcA-deficient cells at HG exhibit no change from control olive tail moment. In agreement with previous reports from our laboratory ( 39 ), NAC-inhibited hyperglycemia induced increases in olive tail moment. Similarly, HG-induced apoptosis was attenuated ( Fig. 6 B ) in p66ShcA-deficient cells, whereas parental cells exhibit a 50% increase in apoptotic cell death. Taken together, mutant 36 p66ShcA induces a dominant interfering phenotype, which prevents HG-induced oxidative DNA damage and apoptosis.

Fig. 6. Mutant 36 p66ShcA attenuates HG-induced oxidative DNA damage. Parental and p66ShcA-deficient cells were maintained for 16 h at NG or HG. A : effect of NAC on HG-induced DNA strand breaks (olive tail moment) determined by single-cell gel electrophoresis (comet assay). B : apoptosis as determined by the ELISA cell death assay. Histone-associated DNA fragments are presented as optical density at 405 nm relative to control value. For each assay, 20 µl of lysate (2.0 mg/ml) were used. Data are presented as means ± SD and represent 3-5 independent experiments. OD, optical density. * P 0.05.7 y/ M' p. o2 K& R  j

DISCUSSION2 H( q! M, Y+ \( q- Y# ?0 ~8 h
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The present study documents a pivotal role for WTp66ShcA protein in the generation of hyperglycemic oxidant stress. We have shown that transfection of mesangial cell lines with either a mutant 36 p66ShcA construct or an isoform-specific p66ShcA siRNA induces a strong oxidant-resistant phenotype which suppresses HG-induced ROS production. Moreover, our data indicate WTp66ShcA may function as a potentially harmful regulatory gene, in response to the stress of hyperglycemia. Inhibition of WTp66ShcA redox function prevented glycooxidant-dependent regulation of FOXO3a and nuclear export of this potent stress response regulator. Finally, oxidative DNA damage is a by-product of hyperglycemic stress, which results in irreversible cell injury. We demonstrate here that p66ShcA-deficient cells maintained at HG exhibit resistance to DSB and apoptotic cell death.
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This is the first report in a resident glomerular cell line documenting resistance to HG-induced apoptosis by silencing the redox-sensitive WTp66ShcA gene. Importantly, the survival program was closely coupled with the attenuation of HG-induced ROS production. These results are in accord with genetic experiments in invertebrates ( 15, 18, 23, 28, 32, 35 ), in which several genes have been identified that promote cell survival, in part by increasing resistance to oxidative stress. We employed two strategies to silence the WTp66ShcA gene. Stable transfection with mutant 36 p66ShcA was found to confer a powerful antioxidant effect at HG, shifting the kinetic curve for ROS metabolism downward, indicative of the oxidant-resistant phenotype. This pervasive inhibition of glycooxidant stress was also observed with acute loss of WTp66ShcA redox function, induced by siRNA knockdown experiments. Based on the low levels of intracellular oxidants detected in p66ShcA-deficient cells, we speculate that WTp66ShcA may act as a transducer to amplify HG-induced ROS generation by NADPH oxidase and mitochondria. Taken together, we have identified a pivotal role for WTp66ShcA redox function, as a precursor to free radical injury ( 1, 25 ), in mesangial cell lines.
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The mammalian Forkhead homolog FOXO3a is a potent stress response regulator and downstream target of WTp66Shc protein. FOXO family members participate in various cellular functions, including apoptosis, cell cycle progression, and antioxidant defense ( 35 ). In our system, we elected to study FOXO3a, which has been shown to regulate the expression of antioxidant enzymes e.g., MgSOD, catalase, and stress-related gene products ( 26, 35 ). WTp66ShcA transduces ROS-dependent signals phosphorylating Ser-473 of Akt/PKB protein ( 26 ), a key intermediary in the phosphorylation and inactivation of FOXO3a ( 35 ). In parental cells at HG, cross talk between WTp66ShcA and Akt/PKB was attenuated by NAC, indicative of redox-dependent posttranslational modification of Akt/PKB protein ( 4, 14, 36 ). Our findings also document the pivotal role of WTp66ShcA in this redox signaling pathway, as p66ShcA-deficient cells at HG also exhibit marked attenuation of Ser-473 phosphorylation. Furthermore, the absence of serum or growth factors in these protocols strongly suggests changes in the phosphorylation status of Akt/PKB at HG, reflects cell redox status, and is not mediated through phosphoinositide 3- kinase. In agreement with previous reports, the phosphorylation status of FOXO3a was a key determinant of subcellular localization ( 26, 35 ). The phosphorylated form of FOXO3a was increased in parental cells at HG, resulting in translocation of HA-FOXO3a to the cytoplasm. Conversely, at HG p66ShcA-deficient cells exhibit no detectable alteration in the phosphorylation status of FOXO3a or in the nuclear export of HA-FOXO3a, conditions associated with the transcriptionally active state of FOXO3a ( 35 ).' v) ?4 r5 U2 H

ROS-dependent signals are known to induce multiple DNA lesions, ranging from single-base modifications to single-strand breaks and double-strand breaks ( 9 ). In our system, we determined the functional significance of inhibiting cross talk between WTp66ShcA and FOXO3a by evaluating oxidant-induced DSB and apoptosis ( 12, 39 ). Our data show unequivocally that inhibition of WTp66ShcA redox function prevents oxidative DNA damage and apoptotic cell death, implicating FOXO3a in the maintenance of genomic integrity. An unresolved issue concerns how FOXO3a uncouples the redox stimulus of high ambient glucose from oxidative-induced DSB ( 39 ) and apoptosis ( 12, 13 ). In this regard, the signaling pathway from phosphoinositide 3-kinase to Akt/PKB has been reported to control lifespan in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of the FOXO family of transcription factors ( 9, 23, 28, 35 ). Our findings are in agreement with a growing body of evidence, suggesting an important role for FOXO3a in stress resistance and the aging process in mammals ( 15 - 17 ). A fundamental mechanism by which cells protect themselves against oxidative stress and the aging process involves DNA repair mechanisms ( 9, 35 ). The growth arrest and DNA damage response gene (Gadd45) is a direct target of FOXO3a that promotes FOXO3a-dependent DNA repair ( 34 ). We speculate that FOXO3a, under conditions of HG-induced oxidant stress, promotes DNA repair and cell rescue, whereas more severe environmental stress, e.g., radiation, may activate FOXO3a-dependent apoptotic programs. Taken together, the biological consequences of Akt/PKB and FOXO3a signaling, may be cell type specific and stress type dependent. Alternatively, the proapoptosis redox-sensitive transcription factor p53 is also activated in mesangial cells at HG ( 12, 13 ) but requires WTp66ShcA to execute the apoptotic program ( 24 ). Interruption of this protein-protein interaction in p66ShcA-deficient cells may have contributed to expression of the survival phenotype.

The present study has certain limitations, including the necessity to maintain cells under serum-free conditions to eliminate the confounding effects of serum and contained growth factors on signaling pathways linked to cell survival and oxidant stress ( 13, 26, 39 ). Second, we must acknowledge the shortcomings of an in vitro cell culture system in simulating a complex metabolic disorder, such as diabetes mellitus. Finally, one can only speculate as to whether our data can be extrapolated to the insulin-resistant state of type 2 diabetes mellitus.9 t8 j$ z! s5 N4 f8 I& v3 J

Taking into account the above limitations, we believe our work may have implications for disease modification in the diabetic glomerulus. The present study clearly shows silencing WTp66ShcA in mesangial cells increases resistance to HG-induced oxidative stress and promotes the survival phenotype. Overproduction of ROS is the key danger signal in the development and progression of diabetic glomerulopathy ( 14 ). At the cellular level, recent work has linked ROS to induction of the apoptotic program in visceral epithelial cells (podocytes) of Akita and db/db genetic models of diabetes mellitus ( 31 ), an event that precedes the onset of proteinuria ( 31 ). These results confirm earlier work in mesangial cells ( 12, 13, 39 ) along with cardiac myocytes ( 11, 20 ), neurons ( 30 ), and retinal pericytes ( 8 ), indicating that oxidant-induced apoptosis may account for significant rates of cell death in kidney and other organ systems in diabetes. The application of a gene-based strategy to inhibit apoptosis, attenuate or prevent pathological remodeling, and preserve organ function ( 21 ) may represent an exciting new avenue for therapeutic intervention in the kidney. Whether strategies that incorporate siRNA to silence disease-causing genes ( 40 ), such as WTp66ShcA, can be applied in vivo to the diabetic glomerulus remains to be determined.$ b# r- P8 e9 s2 n. M+ f  o) ~
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GRANTS
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This work was supported by National Institutes of Health Grants 1R01-CA/NS-95518 (K. Reiss) and 1RO1-HL072852 (A. Malhotra) and a grant from the Wildwood Foundation (L. G. Meggs).
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ACKNOWLEDGMENTS
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We thank M. Greenberg and T. Finkel for reagents.
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