Sonia C. Flores and J. Andres MelendezE. Aplin, Yu-Tzu Tai, Julio Aguirre-Ghiso, Mazurkiewicz, Peter J. Bartholomew, AndrewRegan, Kristin K. Nelson, Joseph E. Kip M. Connor, Sita Subbaram, Kevin J. 

OxidationAngiogenic Phenotype via PTEN Regulates the2O2Mitochondrial H

Mechanisms of Signal Transduction:

doi: 10.1074/jbc.M410690200 originally published online February 8, 20052005, 280:16916-16924.J. Biol. Chem. 

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Mitochondrial H2O2 Regulates the Angiogenic Phenotype viaPTEN Oxidation*

Received for publication, September 16, 2004, and in revised form, December 23, 2004Published, JBC Papers in Press, February 8, 2005, DOI 10.1074/jbc.M410690200

Kip M. Connor‡, Sita Subbaram‡, Kevin J. Regan‡, Kristin K. Nelson‡,Joseph E. Mazurkiewicz§, Peter J. Bartholomew¶, Andrew E. Aplin¶, Yu-Tzu Tai�,Julio Aguirre-Ghiso**, Sonia C. Flores‡‡, and J. Andres Melendez‡§§

From the ‡Centers for Immunology and Microbial Disease, §Neuropharmacology and Neuroscience, and ¶Cell Biology andCancer Research, Albany Medical College, Albany, New York 12208, �Harvard Medical School, Dana-Farber CancerInstitute, Boston, Massachusetts 02115, **Structural and Cell Biology, Department of Biomedical Sciences, School ofPublic Health, State University of New York, Albany, Rensselaer, New York 12144, and ‡‡Webb-Waring Institute forCancer, Aging and Antioxidant Research, University of Colorado Health Sciences Center, Denver, Colorado 80262

Recent studies have demonstrated that the tumor sup-pressor PTEN (phosphatase and tensin homolog deletedfrom chromosome 10), the antagonist of the phosphos-phoinositol-3-kinase (PI3K) signaling cascade, is suscep-tible to H2O2-dependent oxidative inactivation. Thisstudy describes the use of redox-engineered cell lines toidentify PTEN as sensitive to oxidative inactivation bymitochondrial H2O2. Increases in the steady state pro-duction of mitochondrial derived H2O2, as a result ofmanganese superoxide dismutase (Sod2) overexpres-sion, led to PTEN oxidation that was reversed by thecoexpression of the H2O2-detoxifying enzyme catalase.The accumulation of an oxidized inactive fraction ofPTEN favored the formation of phosphatidylinositol3,4,5-triphosphate at the plasma membrane, resulting inincreased activation of Akt and modulation of its down-stream targets. PTEN oxidation in response to mito-chondrial H2O2 enhanced PI3K signaling, leading to in-creased expression of the key regulator of angiogenesis,vascular endothelial growth factor. Overexpression ofPTEN prevented the H2O2-dependent increase in vascu-lar endothelial growth factor promoter activity and im-munoreactive protein, whereas a mutant PTEN (G129R),lacking phosphatase activity, did not. Furthermore, mi-tochondrial generation of H2O2 by Sod2 promoted endo-thelial cell sprouting in a three-dimensional in vitro an-giogenesis assay that was attenuated by catalasecoexpression or the PI3K inhibitor LY2949002. More-over, Sod2 overexpression resulted in increased in vivoblood vessel formation that was H2O2-dependent as as-sessed by the chicken chorioallantoic membrane assay.Our findings provide the first evidence for the involve-ment of mitochondrial H2O2 in regulating PTEN func-tion and the angiogenic switch, indicating that Sod2 canserve as an alternative physiological source of the po-tent signaling molecule, H2O2.

Reactive oxygen species (ROS)1 have long been established toplay an important role in many disease pathologies and havealso emerged as efficient signaling molecules. The principalmediator of ROS-dependent signaling is the two electron re-duction product of oxygen (O2), hydrogen peroxide (H2O2).H2O2 is generated in response to receptor stimulation and is anefficient signal transducing molecule by its ability to reversiblyoxidize active site cysteines (1, 2). Many protein tyrosine phos-phatases are particularly susceptible to H2O2-dependent inac-tivation because of the lowered pKa of the active site cysteine(3–5). The essential cysteine residue in the signature active sitemotif Cys-(X)5-Arg exists as a thiolate anion (Cys-S�), which atneutral pH is susceptible to nucleophilic attack by H2O2. Oxi-dation of the active site cysteine generates a sulfenic derivative(Cys-SOH), leading to enzyme inactivation that can be reversedby cellular thiols (6).

The tumor suppressor PTEN (phosphatase and tensin ho-molog deleted from chromosome 10), also known as TEP-1(TGF-�-regulated and epithelial cell-enriched phosphatase) orMMAC1 (mutated in multiple advanced cancers), is reversiblyinhibited by H2O2, resulting in the formation of a disulfidebetween the active site cysteine (Cys124) and a vicinal cysteine(Cys71) (4, 7). PTEN functions by removing the 3�-phosphate ofphosphatidylinositol 3, 4,5-triphosphate (PtIns(3,4,5)P3) gener-ating PtIns(4,5)P2, thereby arresting phosphoinositide 3-ki-nase (PI3K) signaling (8–10). Until the discovery of PTENoxidation, the only known regulation of PTEN was throughphosphorylation by casein kinase-2 in the PDZ domain locatedin its C-terminal tail (11–13). Phosphorylation prevents PTENfrom docking with the scaffolding protein membrane-associ-ated guanylate kinase invertase-2 (14) and targeting to theplasma membrane. Sequestration of PTEN in the cytosol al-lows for PtIns(3,4,5)P3-dependent accumulation and activationof the PI3KAkt pathway (11, 12, 15).

Oxidative inactivation of PTEN likely occurs close to thesite of H2O2 production in response to receptor activation.

* This work was supported by United States Public Health ServiceGrants CA77068 and CA095011 (to J. A. M.) and National Institutes ofHealth Predoctoral Fellowship AI49822 (to K. M. C.). The costs of pub-lication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§§ To whom correspondence should be addressed: Center for Immu-nology and Microbial Disease, Albany Medical College, MC 151, 47 NewScotland Ave., Albany, NY 12208. Tel.: 518-262-8791; Fax: 518-262-6161; E-mail: melenda@mail.amc.edu.

1 The abbreviations used are: ROS, reactive oxygen species; PI3K,phosphoinositide-3 kinase; Akt, protein kinase B; BLMV, bovine lungmicrovessels; CMV, cytomegalovirus; GSK3�, glycogen synthase ki-nase-3�; H2O2, hydrogen peroxide; HIF-1�, hypoxia-inducible factor �;PtIns(4,5)P2, phosphatidylinositol-4,5-diphosphate; PtIns(3,4,5)P3,phosphatidylinositol-3,4,5-triphosphate; O2

., superoxide dismutase;Sod2, manganese superoxide dismutase; TEMED, N,N,N�,N�-tetra-methylethylenediamine; GFP, green fluorescent protein; eGFP, en-hanced GFP; YFP, yellow fluorescent protein; CFP, cyan fluorescentprotein; 5-IAF, 5�-fluoresceinated iodoacetamide; PBS, phosphate-buffered saline; Ab, antibody; CAM, chicken chorioallantoic membrane;PH, pleckstrin homology.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 17, Issue of April 29, pp. 16916–16924, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org16916

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However, the exact source of H2O2 in response to receptorengagement is an area of controversy and may involve acti-vation of the noninflammatory NADPH oxidase family mem-bers and subsequent spontaneous or enzymatic dismutationof superoxide (O2

.) to H2O2 (16). The mitochondria are also asource for the generation of intracellular H2O2 under physi-ologic conditions (17) and in response to receptor stimulation(18, 19). However, whether mitochondrial derived H2O2 con-tributes to the oxidative inactivation of PTEN or other phos-phatases has not been established.

In this study, we report that alterations in the steady stateproduction of mitochondrial H2O2 by antioxidant enzyme over-expression modulate the redox state of PTEN. This mechanismis responsible for modulating PI3K/Akt signaling, VEGF pro-duction, and angiogenesis. These findings define a novel rolefor mitochondrial H2O2 and Sod2 in regulating the angiogenicswitch via PTEN phosphatase inactivation.

EXPERIMENTAL PROCEDURES

Cell Lines and Reagents—Human HT-1080 fibrosarcoma cells werecultured in minimum Eagle’s medium supplemented with 10% fetal calfserum, 1000 units/ml penicillin, 500 �g/ml streptomycin, and 1 mg/mlneomycin, in a 37 °C humidified incubator containing 5% CO2. HT-1080fibrosarcoma cell lines transfected with CMV (empty vector), Sod2,and/or catalase were described in detail previously (20).

Measurement of Oxidation Rate in Intact Cells—Measurement ofROS levels in redox-engineered cell lines was performed using theredox-sensitive dye Redox Sensor RedTM CC-1 (Molecular Probes). Cellswere harvested with phosphate-buffered saline (PBS) solution contain-ing 1 mM EDTA. Cells were washed with PBS and resuspended inHanks’ balanced salt solution containing 6.7 g/liter glucose to a finaldensity of 5 � 105 cells/ml. Redox Red CC-1 was added to a finalconcentration of 10 �M at room temperature. Samples were analyzed ina flow cytometer (BD Biosciences) at a wavelength of �540/600 nm.

Western Blotting—Cell lysates were collected in 0.05 mM KPi buffercontaining an array of phosphatase and protease inhibitors, loaded onan SDS-polyacrylamide gel, and electroblotted onto polyvinylidene flu-oride. The primary Abs used included rabbit anti-human PTEN (1:1000), phospho-PTEN (1:1000), Akt (1:1000), phospho-Akt (1:1000),GSK3-� (1:1000), phospho-GSK3-� (1:1000) (Cell Signaling), VEGF (1:1000), and HIF-1� (1:1000) (Santa Cruz Biotechnology). This was fol-lowed by an incubation with horseradish peroxidase-conjugated goatanti-rabbit IgG (1:10,000) (Amersham Biosciences) as the secondary Ab.Antibodies were used according to manufacturer’s recommendations. 30�g of protein was loaded per well with the exception of Akt (50 �g). Theprimary was applied overnight in 5% bovine serum albumin at 4 °C.

Identification of Reduced and Oxidized PTEN by Immunoblot Anal-ysis—Cells were harvested in PBS/EDTA, washed once with PBS, andresuspended in 0.2 ml of 100 mM Tris-HCl (pH 6.8) containing 2% SDSand 40 mM N-ethylmaleimide; 30 �g of protein per cell line was sub-jected to SDS-PAGE under nonreducing conditions as described previ-ously (7). The separated proteins were then transferred to polyvinyli-dene fluoride membrane and immunoblotted with a rabbit anti-PTEN1oAb (1:1000). Immune complexes were detected by a horseradish per-oxidase-conjugated 2oAb (1:10,000) (Amersham Biosciences) and en-hanced chemiluminescence reagents (Pierce).

Sod Zymography—Lysates from cell lines were harvested in 0.005 M

potassium phosphate buffer at pH 7.8 containing 0.1 mM EDTA. Lysatesupernatants were analyzed by electrophoresis in a discontinuous poly-acrylamide gel, consisting of a 5% stacking gel (pH 6.8) and 10%running gel. The gel was stained with a solution containing 2.5 mM

nitro blue tetrazolium, 0.008 mM riboflavin, 30 mM TEMED, and 0.005M potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA, wasincubated for 15 min in the dark at room temperature, and was washedtwice in deionized water. The gels were then exposed to fluorescentlight until clear zones of SOD activity were evident.

5�-Fluoresceinated Iodoacetamide (5-IAF) Labeling—Cells were lysedin 0.05 mM KPi buffer at pH 7.0 containing 10 mM iodoacetamide(Sigma). Samples were rocked at room temperature in the dark for 10min, and free cysteines were blocked at this step. Lysates were thenimmunoprecipitated for PTEN (Sigma) by using protein G-Sepharosebeads (Pierce). This step also removed any excess iodoacetamide. Afterimmunoprecipitation, the bead-antibody-protein complex was resus-pended in 200 �l of 0.05 mM KPi buffer at pH 7.0 containing 10 mM

dithiothreitol (Roche Applied Science). This denatured the proteins,

breaking all disulfides, thereby freeing previously oxidized cysteines.The proteins were then acetone-precipitated in order to remove thedithiothreitol. Samples were then resuspended in buffer containing 5mM 5-IAF and were then vortexed and kept at 4 °C for 30 min. The5-IAF (Molecular Probes) will now bind previously oxidized cysteines.Lysates were then run on an SDS-polyacrylamide gel and subjected toWestern blot using anti-fluorescein isothiocyanate antibody (MolecularProbes).

Transient Transfections of hVEGF Promoter Construct and Domi-nant Negative Constructs—Cell lines were transfected with the varioushVEGF (obtained from Dr Kenneth Anderson at Harvard MedicalSchool and described by Tai et al. (21)). The VEGF construct andpCMV.SPORT�-gal were cotransfected with Lipofectamine Plus rea-gent according to the manufacturer’s instructions (Invitrogen). Thecells were lysed 18 h post-transfection, and the luciferase reporteractivity was determined using the Promega assay system. All of theresults were reported after normalization for transfection efficiency bymeasuring �-galactosidase activity.

In Vitro Angiogenesis Assay—The in vitro angiogenesis assay wasdescribed previously by Nakatsu et al. (22). In brief, bovine lung mi-crovessels (BLMV) were complexed with Cytodex 3 microcarrier (Am-ersham Biosciences) in 1 ml of EGM-2 (Clonetics) at a concentration of400 BLMVs per bead. The bead and cell solutions were shaken every 20min for 4 h at 37 °C and 5% CO2 to allow the cells to bind to the beads.Following the incubation, the cell-bead complex was grown overnight ina T-25-cm2 tissue culture flask (Corning Glass) in 5 ml of EGM-2 at37 °C and 5% CO2. After incubating, the cells were washed three timesin EGM-2 and resuspended at a concentration of 200 cell-coatedbeads/ml in EGM-2 containing 2.5 mg/ml of fibrinogen (Sigma) with0.15 units of aprotinin (ICN). 500 �l of the above solution was added to0.625 units of thrombin (Sigma) in a 24-well tissue culture plate. Thesolution was allowed to clot for 5 min at room temperature and then at37 °C and 5% CO2 for 20 min. One milliliter of EGM-2 containing 0.15units aprotinin was added per well and allowed to equilibrate with theclot for 30 min at 37 °C and 5% CO2. EGM-2 was removed and replacedwith 1 ml EGM-2 containing 0.15 units of aprotinin and 20,000 cells ofthe indicated redox-engineered cell lines (CMV, Sod2, Sod2mCAT,eGFP, and Sod2GFP). For a positive control, 2.5 ng/ml of VEGF wasadded to the CMV control cell line, and for a negative control, 30 �M ofthe PI3K inhibitor LY294002 was added (Calbiochem). To test if theeffects were VEGF-specific, 0.16 �g/ml of a monoclonal mouse anti-human IgG2B VEGF neutralizing antibody (R & D Systems) or IgG2B

isotype control antibody (Pharmingen) was added to the Sod2-overex-pressing cell lines, and the effect on sprout formation was analyzed.Clots were grown for 24 h, and the number of sprouts/bead were ana-lyzed. Images of beads were captured on a Nikon Diaphot microscopewith a 4� objective. The number of sprouts per bead were counted, andall experiments were repeated 10 times with similar results.

Chicken Embryo Chorioallantoic Assay—The CAM assay was per-formed as described previously by Brooks et al. (23) with minor varia-tions. Fertilized chicken eggs were incubated with constant rocking at37 °C under 60% humidity. On the 9th day, the CAM was pulled awayfrom the shell by first drilling a small shallow hole at the end of the eggthat contains the natural air sac; an additional hole was made on thebroad side of the egg directly over the embryonic blood vessels, asdetermined by candling. Gentle suction was applied to the 1st hole,thereby displacing the air sac to the side of the egg shell where the 2ndhole was made. This allowed the CAM to be pulled away from the shellon the side of the egg. A window was opened above the air pocket in theshell and sealed with a piece of sterile tape. A sterile rubber O-ring wasplaced on the CAM, and 2 � 105 cells in a total volume of 50 �l of PBSwere placed in the center of the ring. No cells were added to the control.24 h later the allantoic vessels around the CAM were photographedusing a digital camera, five fields per egg. Vessel density was thenquantified by placing a grid over the photo in Adobe Photoshop 7.0 andcounting the number of vessels per field.

Plasmid Constructs—The pEGFP-N1 vector was obtained from Clon-tech. Human Sod2 was PCR-amplified with 5�-AvaI and 3�-KpnI ends.AvaI was added 4 bp upstream of the Sod2 initiation codon and KpnIwas added prior to the Sod2 termination codon and directionally in-serted in-frame into the pEGFP-N1. The constitutively active myr-Aktand kinase dead (K170M) Akt DNA was kindly provided by Dr. AndrewAplin, Albany Medical College. The wild type PTEN and phosphatasemutant (G129R) PTEN DNA were obtained from Dr. William Sellers,Harvard Medical School, and were described previously (24). The Akt-PH-YFP and PLC�-PH-CFP constructs were obtained from TobiasMeyer, Stanford University. The mito-GFP construct was obtained fromYisang Yoon, University of Rochester.

Mitochondrial Redox Control of Angiogenesis 16917

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Confocal Microscopy—Cell cultures were imaged on an inverted LSM510 Meta laser-scanning confocal microscope (Zeiss, Thornwood, NY) byusing a Plan-Apo 63 � 1.4 NA oil immersion objective. Pinhole diameterwas �1 airy unit. (a) For cells labeled with the single fluorescencereporter YFP, the 514 laser line from an argon laser was used to exciteYFP, and images were collected using an HFT 458/514 dichroic mirrorand a bandpass filter of 535–590 nm at an 8-bit intensity resolutionover 1024 � 1024 pixels at a pixel dwell time of 6.4 �s. (b) For cellslabeled with GFP and mitoTracker Red®, the multitrack mode wasused in which the 488 nm laser line from an argon laser was used toexcite GFP; the 543 laser line from a HeNe laser was used to excite themitoTracker Red®, and images were collected by using an HFT 488/543/633 dichroic mirror and a 500–530 nm bandpass filter for GFP andLP 560 nm filter for the mitoTracker Red® at a 12-bit intensity reso-lution over 1024 � 1024 pixels at a pixel dwell time of 3.2 �s. (c) Forcells labeled with the fluorescence reporter combinations of CFP/GFP orGFP/YFP, images were collected at a 12-bit intensity resolution over1024 � 1024 pixels at a pixel dwell time of 3.2 �s by using spectralimaging followed by linear unmixing to separate the fluorescence con-tribution of each fluorescent protein on a pixel by pixel basis (25, 26). Aseries of � stack X-Y images was collected using the Zeiss METAdetector over a spectrum of wavelengths between 462 and 633 nm withbandwidths of 10.7 nm during excitation with the 458 nm laser line anda HFT458 dichroic mirror for the CFP/GFP pair. For the GFP/YFP pair,a spectrum of wavelengths between 494 and 633 nm with bandwidths of10.7 nm was collected during excitation with the 488 nm laser line andan HFT 488 dichroic mirror. Reference � stack images were collectedand stored for each of the fluorescence reporters alone to provide spec-tral signatures for GFP, YFP, and CFP that were used in the linearunmixing procedure. For time lapse studies, images were captured at512 � 512 pixels, one image every 15 s for a total of 5 min. The ZeissLSM Physiology software package and Microsoft Excel were used toanalyze the time-lapse data.

Statistics—Analysis of variance with � � 0.05 was used for process-ing the data. A two-sample t test was used as post-test unless otherwiseindicated.

RESULTS

Analysis of Redox Engineered Cells—To define moleculartargets that are sensitive to alterations in the mitochondrialproduction of H2O2, we have developed a line of redox-engi-neered HT-1080 fibrosarcoma cell lines using antioxidant en-zyme-based expression systems. We have focused our analyseson six redox-engineered cell lines that have been thoroughlycharacterized in terms of their antioxidant and oxidant profile(20, 27). Enforced expression of mitochondrial manganese-con-taining superoxide dismutase (Sod2) led to a significant in-crease in intracellular H2O2, which was reversed by cytosolic ormitochondrial expression of catalase (Fig. 1A). Sod2 overex-pression led to a 2-fold increase in the rate of oxidation of theH2O2-responsive fluorophore Redox-RedTM that was attenu-ated upon catalase coexpression (Fig. 1A). Sole catalase over-expression also decreased the basal rate of Redox-RedTM oxi-dation. These findings demonstrated that the enhanced rate ofoxidation was attributed to the Sod2-dependent increases inthe steady state production of H2O2 that was subsequentlyreversed upon expression of the H2O2-detoxifying enzymecatalase.

Redox-dependent Oxidation of PTEN—PTEN is a dual spec-ificity phosphatase that is reversibly sensitive to inactivationby H2O2 (7) (Fig. 1B). The oxidation status of PTEN resulted inthe conversion of the sulfhydryl groups to a disulfide, resultingin a more compact protein structure that can be visualizedunder nonreducing conditions (Fig. 1B) (7). The Sod2-depend-ent generation of H2O2 showed accumulation of the oxidizedform of PTEN (Fig. 1B, lane 4) that was prevented by coexpres-sion of catalase (Fig. 1B, lanes 5 and 6). Addition of exogenousH2O2 (0.5 mM) caused a prevalent accumulation of the oxidizedform of PTEN that was blocked by mitochondrial targetedcatalase (Fig. 1B, lane 6). PTEN inactivation resulting frommutational loss or oxidation increased the phosphorylation ofthe serine-threonine kinase Akt (4, 24, 28). Coordinate to the

increase in PTEN oxidation upon Sod2 overexpression was apronounced elevation in phosphorylation at serine 473 of Akt(Fig. 1B, lane 4), which was attenuated by the overexpressionof catalase in either the cytosolic or mitochondrial compart-ment (Fig. 1B, lanes 5 and 6, respectively).

The substrates for Akt are numerous and are involved inregulating many diverse cellular functions, including prolifer-ation, glucose utilization, angiogenesis, and cell survival (29).Glycogen synthase kinase 3� (GSK3�), a negative regulator ofcyclin D1, was inhibited by Akt-dependent phosphorylation.GSK3� phosphorylation was also reversibly sensitive to alter-ations in the steady state production of H2O2 by antioxidantenzyme overexpression (Fig. 1B). The dominant negative iso-form of Akt (K179M) can prevent phosphorylation of native Aktat serine 473 (30), and expression of this mutant in our Sod2overexpressing cell line blocked the increase in Akt and GSK3�

FIG. 1. Antioxidant enzyme overexpression modulates thesteady state production of H2O2 and Akt signaling. A, oxidation ofthe redox-sensitive dye, RedoxSensorTM Red CC-1, by the Sod2- andcatalase-overexpressing cell lines. The redox-engineered cell lines thatwere used include CMV (empty vector), Sod2, CMVCAT (cytosolic cat-alase), CMVmCAT (mitochondrial catalase), Sod2CAT (Sod2 and cyto-solic catalase), and Sod2mCAT (Sod2 and mitochondrial catalase), In-set, �rmf/s, � relative mean fluorescence s�1 of indicated cell lines. B,H2O2-dependent regulation of PTEN oxidation and downstream tar-gets. Cell lines that were used are as follows: CMV (lane 1), CMVCAT(lane 2), CMVmCAT (lane 3), Sod2 (lane 4), Sod2CAT (lane 5), andSod2mCAT (lane 6). Immunoblot analysis was used to evaluate PTEN,Akt, and GSK3� as indicated. PTEN oxidation was confirmed by treat-ment with 0.5 mM H2O2 for 20 min. Lysates were analyzed by immu-noblot for PTEN under either reducing or nonreducing conditions orp-Akt, p-GSK-3�, p-PTEN, Akt, and GSK3�. As controls immunoblotanalysis of total PTEN, Akt, and GSK-3� are shown. The kinase deadmutant Akt (K179M) (lane 7) or constitutively active myr-Akt (lane 8)were transfected into Sod2 or Sod2mCAT cell lines, respectively. Sam-ples were subjected to immunoblot analysis for p-Akt and its down-stream target p-GSK-3�.

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phosphorylation (Fig. 1B, lane 7). Enforced expression of a con-stitutively active myristoylated-Akt rescued the catalase-depend-ent inhibition of both Akt and GSK3� phosphorylation in theSod2-overexpressing cell lines (Fig. 1B, lane 8). The PI3K com-plex was found in the plasma membrane, whereas PTEN islargely a cytoplasmic enzyme. It has been established previouslythat PTEN can be negatively regulated by phosphorylation ofresidues (Ser380, Thr382, and Thr383) in its C-terminal tail (11–13). Given this, we sought to examine if PTEN phosphorylationwas regulated in a redox-dependent manner. PTEN phosphoryl-ation status did not change in any of our redox-engineered celllines, indicating that its primary mode of redox regulation isthrough oxidation. These results suggested that reversible alter-ations in the steady state mitochondrial production of H2O2 mod-ulated the redox state of PTEN and the phosphorylation state ofboth Akt and the downstream target GSK3�.

Sod2-dependent Oxidation of PTEN and PI3K Signaling—Clonal isolation of transfected cells, as in the case of the redox-engineered cell lines, may give rise to variants whose alteredsignaling characteristics may be independent of the gene beingstudied. To further confirm the role of Sod2 in modulatingPI3K/Akt via PTEN oxidation, we utilized a Sod2/GFP fusionconstruct to isolate populations of GFP-fluorescing cell lineswith differing levels of Sod2 expression. Sod2 was targeted tothe mitochondria via a mitochondrial sequence peptide locatedin its N terminus. Prior to fluorescent-activated cell sorting,mitochondrial localization of the Sod2GFP fusion construct inthe HT-1080 cells was confirmed by fluorescent microscopy.Analysis of Sod2GFP-transfected HT-1080 cells showed dis-tinct perinuclear GFP staining that colocalized with the mito-chondrial specific dye, Mitotracker RedTM (Fig. 2B). By usingfluorescent-activated cell sorting, distinct populations of GFP-positive cells were isolated into low, medium, and high fluo-rescing cell lines (Sod2lo, Sod2m1, Sod2m2, and Sod2hi). A purepopulation of GFP-expressing cells was also isolated whoseGFP fluorescence was similar to that of the Sodhi cell lines. Sodzymography of the distinct cell populations showed that Sod2

activity increased relative to the level of GFP intensity in thesorted cell populations and that Sod1 levels were unaffected bySod2GFP transfection (Fig. 2C). Furthermore, GFP expressionalone had no effect on Sod2 activity. The numerous bands inthe region of Sod2 activity represented the various combina-tions of the native Sod2 monomer and the Sod2GFP fusionmonomer that comprises the active tetrameric enzyme.

We next analyzed the redox state of PTEN in the sorted cellpopulations. Analysis of PTEN under nonreducing conditionsshowed Sod2-dependent increase in the levels of its oxidizedform (Fig. 2C). Akt and GSK-3� phosphorylation were alsodose-dependently sensitive to increases in Sod2 activity, whichcorrelated with PTEN oxidation, whereas total Akt andGSK-3� levels remain unchanged (Fig. 2C). Cyclin D1 expres-sion was quite sensitive to alterations in the mitochondrialredox environment as even a low level of Sod2 expressionincreased the levels of its immunoreactive protein.

To characterize further the PTEN oxidation state in thesecell lines, we utilized a method for detecting free cysteines byusing 5�-fluoresceinated iodoacetamide (5-IAF) labeling (5, 31)(Fig. 2A). Cysteine oxidation of PTEN increased in response toSod2 expression, indicating that the Sod2-dependent produc-tion of H2O2 had a direct effect on PTEN oxidation (Fig. 2A).These findings are the first to demonstrate that changes in thesteady state mitochondrial production of H2O2 in tumor cellscan modulate the redox state of PTEN and signaling pathwaysunder its control.

Sod2-derived H2O2 Regulates Phosphoinositide Distribu-tion—PTEN plays an important role in restricting the genera-tion and distribution of 3�-phosphoinositides following receptortyrosine kinase engagement (32). Accumulation of an oxidizedinactive fraction of PTEN would likely favor the distribution ofPtIns(3,4,5)P3 at the plasma membrane compartment (Fig.3A). To test this hypothesis, the various control and redox-engineered cell lines were transfected with chimeras of greenfluorescent protein and the pleckstrin homology domain ofeither phospholipase C-� or Akt, to monitor distribution of

FIG. 2. Dose-dependent oxidation of PTEN, Akt phosphorylation, and activation of downstream targets by Sod2. A, PTEN wasimmunoprecipitated (IP) from the control (eGFP), Sod2GFPLo, and Sod2GFPHi cells, and oxidized cysteines were analyzed by 5-IAF labeling andimmunoblotting against fluorescein isothiocyanate (FITC) (see “Experimental Procedures”). B, HT-1080 fibrosarcoma cells were transfected withan Sod2GFP fusion construct, and mitochondrial specific staining was monitored using Mitotracker RedTM. The merging of Sod2GFP andMitotracker Red fluorescence (lower) demonstrates localization of Sod2GFP in the mitochondrial compartment. C, upper panel, Sod2GFP-transfected cells were purified using fluorescence-activated cell sorting into distinct (low (lo), medium 1 (m1), medium 2 (m2), and high (hi))populations based on the GFP fluorescence. Purified populations were then evaluated for Sod2 activity using a SOD native PAGE (see“Experimental Procedures”). Upper bands represent areas of endogenous and GFP-fused Sod2, and lower bands show Sod1 activity. Lower panels,PTEN oxidation was determined using nonreducing PAGE and immunoblot analysis. In parallel, immunoblot analysis of p-Akt, p-GSK3-�, andcyclin D1 was performed. As controls, immunoblot analysis for total Akt and GSK3-� was performed. Red, reduced; Ox, oxidized.

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PtIns(4,5)P2 or PtIns(3,4,5)P3, respectively (Fig. 3A) (33). Forsimplicity, the localization of CFP-PLC�-PH and Akt-PH-YFPhereafter will represent accumulation of PtIns(4,5)P2 andPtIns(3,4,5)P3, respectively. H2O2 has been shown to activatePI3K leading to the production of PtIns(3,4,5)P3 (34, 35) andserves as an effective control to monitor PtIns(3,4,5)P3 accumu-lation at the plasma membrane. Treatment of HT-1080 cells with0.5 mM H2O2 showed a rapid (30 s) linear increase in the local-ization of PtIns(3,4,5)P3 to the plasmalamellar surface (Fig. 3B).In untreated cells, PtIns(3,4,5)P3 binding construct remainedcytosolic (Fig. 3, B and C, 1), and no time-dependent change in itslocalization was observed (Fig. 3B). The pattern of PtIns(3,4,5)P3

distribution in the Sod2-overexpressing cell lines was distinctfrom that of the controls and showed prominent redistribution tothe plasma membrane (Fig. 3C, 2), which was prevented bycoexpression of mitochondrial targeted catalase (Fig. 3C, 3).

Analysis of phosphoinositide distribution in the Sod2GFPand GFP-expressing cell lines was performed with cyan andyellow fluorescent chimeric proteins of either the PLC�-PH orthe Akt-PH domains, respectively. In the GFP-expressing cells,PtIns(4,5)P2 localized to the plasma membrane (Fig. 3C, 5), andits distribution was distinct from that of the GFP itself, whichis largely cytoplasmic (Fig. 3C, 4), whereas the YFP-Akt-PHprotein remained largely cytosolic, due to the lack of accumu-lation of PtIns(3,4,5)P3, and its localization was coincident withGFP (Fig. 3C, 8). These findings agree with previous reportsindicating that in the absence of active receptor signaling bothPtIns(4,5)P2 and PtIns(3,4,5)P3 are either membrane-bound orabsent, respectively (36). The distribution of PtIns(4,5)P2 andPtIns(3,4,5)P3 in the Sod2GFPhi cells was also evaluated (Fig.3D). Sod2GFP overexpression led to an abundant localizationof PtIns(4,5)P2 and PtIns(3,4,5)P3 to a perinuclear organelle

FIG. 3. Analysis of phosphoinositide distribution in the redox-engineered cells. A, diagram of PtIns(4,5)P2 and PtIns(3,4,5)P3 bindingconstructs. The PH domain of PLC� and Akt bind with high affinity and specificity to PtIns(4,5)P2 or PtIns(3,4,5)P3, respectively. B, quantitationof cellular membrane distribution of PtIns(3,4,5)P3 using time lapse video microscopy and the PtIns(3,4,5)P3 binding construct (YFP-Akt-PH) inresponse to 0.5 mM H2O2. Images were captured every 15 s for a total of 5 min. *, p � 0.05 at 90 s and reaches p � 0.001 at 270 s. Analysis ofPtIns(4,5)P2 and PtIns(3,4,5)P3 binding construct distribution in the redox-engineered cell lines (C). PtIns(3,4,5)P3 distribution was detected by theYFP-Akt-PH construct in control CMV, Sod2-overexpressing, and Sod2/catalase-coexpressing cell lines (1–3). For simplicity, the localization ofCFP-PLC�-PH and Akt-PH-YFP reflects the accumulation of PtIns(4,5)P2 and PtIns(3,4,5)P3, respectively, and is labeled accordingly. The eGFPcell lines were transfected with either CFP-PLC�-PH or YFP-Akt-PH (5 and 8). D, Sod2GPF-overexpressing cell lines containing CFP-PLC�-PHor YFP-Akt-PH (2 and 5). Control CMV cell lines were transfected with a mitochondrial localized GFP (mito-GFP) expression construct (7 and 10)and either CFP-PLC�-PH or YFP-Akt-PH (8 and 11). These findings indicate that Sod2-derived mitochondrial H2O2 can modulate the mitochon-drial localization of phospholipids signaling molecules (overlay 3 and 6).

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compartment (Fig. 3D, 2 and 5). The phosphoinositides alsocolocalized with the Sod2GFP protein indicating their mito-chondrial distribution (Fig. 3D, 3 and 6).

The distinct distribution of the phosphoinositide-bindingconstructs in the Sod2GFP cells relative to the Sod2-overex-pressing cells was likely due to the higher enzymatic activity ofthe Sod2GFP sorted population (45- versus 15-fold). Alterna-tively, the GFP protein of the Sod2GFP chimera when presentin the mitochondrial compartment may alter the distribution ofthe phosphoinositide-binding constructs. To test this latter pos-sibility, HT-1080 cell lines were transfected with a mitochon-drial targeted GFP (mito-GFP), and phosphoinositide distribu-tion was evaluated. Expression of the compartmentalized GFPin the mitochondria did not mediate mitochondrial accumula-tion of either phosphoinositide (Fig. 3D, 9 and 12). Further-more, PtIns(4,5)P2 and PtIns(3,4,5)P3 distribution was similarto that observed in the control GFP-expressing cell linesresiding in either the membrane or cytosolic compartments,respectively (Fig. 3D, 8 and 11). These findings imply thatmitochondrial derived H2O2 generated as a result of Sod2overexpression and independent of receptor engagementcontributed to a shift in the production and subsequentmobilization of both PtIns(4,5)P2 and PtIns(3,4,5)P3.

Sod2-dependent Regulation of VEGF Expression—BecausePTEN and reactive oxygen species have been postulated to playan important role in the angiogenic switch (37, 38), we nextexamined the effects of alteration in the mitochondrial produc-tion of H2O2 on expression of angiogenic markers and activity.First, the promoter activity and endogenous protein expressionof a key stimulator of endothelial cell growth, vascular endo-thelial derived growth factor, were assessed. Overexpression ofSod2 led to a dramatic increase in the levels of endogenousVEGF immunoreactive protein as well as the activity of thetransfected VEGF luciferase promoter construct (Fig. 4, A andB). However, coexpression of catalase in either the cytosolic or

mitochondrial compartment reversed the Sod2-dependent en-hancement in VEGF promoter activity as well as its immuno-reactivity (Fig. 4, A and B). Both the transcription factors Ets-1and HIF-1� contribute to the activation of the angiogenic phe-notype (39, 40) and respond to changes in the mitochondrialredox environment (41, 42). We have reported previously thatSod2 enhanced Ets-1-regulated gene expression in an H2O2-de-pendent fashion (43). Fig. 4B demonstrates that HIF-1� pro-tein expression is also sensitive to alterations in the Sod2-de-pendent mitochondrial production of H2O2 that is reversed bycoexpression of both cytosolic and mitochondrial catalase. Theincreased HIF-1� expression was also associated with aug-mented VEGF promoter-driven luciferase activity that is re-versed by mitochondrial catalase overexpression (Fig. 4A).These studies support a role for mitochondrial H2O2 in theregulation of factors critical to the angiogenic switch.

PTEN plays an important role in the regulation of VEGFexpression by blocking Akt-mediated VEGF gene transcription(37, 44). To define whether the Sod2-dependent increase inVEGF expression may be attributed to the loss of PTEN activ-ity and altered Akt signaling, Sod2-overexpressing cell lineswere transfected with a wild type PTEN-GFP fusion or a dom-inant negative, kinase dead Akt (K179M) construct. Enforcedexpression of either PTEN or the Akt (K179M) prevented theSod2-dependent increase in VEGF expression (Fig. 4B).Sod2GFP overexpression also led to an increase in VEGF ex-pression that was not observed in the eGFP-overexpressing celllines. This increase was also attenuated by the enforced expres-sion of PTEN or the Akt (K179M). A myristoylated, constitu-tively active Akt (Myr-Akt) was used to confirm that VEGFexpression in the eGFP HT-1080 cell line was sensitive toalterations in Akt signaling (Fig. 4B). In Fig. 4A, both basaland Sod2-dependent promoter activity were abolished by en-forced expression of PTEN, whereas the PTEN (G129R) mutantlacking both lipid and protein phosphatase activity did not

FIG. 4. Sod2-dependent generation of H2O2 regulates VEGF expression. A, indicated cell lines were transiently transfected with a VEGFluciferase reporter construct alone or in combination with wild type PTEN or a mutant PTEN (G129R) lacking phosphatase activity. The cells werelysed 18 h post-transfection. All transfections were normalized to �-galactosidase and values expressed as raw counts/min. † represents p � 0.01with respect to the control CMV cell line. B, immunoreactive VEGF protein in the indicated redox-engineered cell lines. The eGFP cell lines weretransfected with a constitutively active myr-Akt, whereas the Sod2 and Sod2GFPHi cell lines were transfected with wild type PTEN or a kinasedead Akt (K179M), and VEGF levels were analyzed. Immunoblot analysis of HIF-1� in the indicated cell lines.

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significantly alter VEGF promoter activity in any of the celllines tested. These findings established that alterations in thesteady state mitochondrial production of H2O2 by antioxidantenzymes can modulate Akt-dependent signaling cascades in-volved in regulating VEGF production.

Mitochondrial H2O2 Regulates Angiogenic Activity—We nextinvestigated whether mitochondrial redox-dependent alter-ations in VEGF production impact angiogenic activity. By us-ing a recently developed three-dimensional in vitro angiogene-sis assay, we were able to evaluate whether factors secretedfrom the fibrosarcoma cells were able to promote endothelialcell sprouting (22). BLMV were grown on Cytodex beads andembedded in fibrin gels followed by incubation with the variousredox-engineered cell lines. In Fig. 5, A and B, only rudimen-tary sprout formation was observed in the vector-transfectedcontrol cell lines (CMV and eGFP) and Sod2mCAT coexpres-sors, whereas Sod2 expression led to a significant increase inthe length and number of sprouts (Fig. 5, A and B). Treatmentof the control cell lines with VEGF (2.5 ng/ml) led to an increasein sprout formation to levels observed in the Sod2 cell lines.Addition of SodGFPhi cells to the three-dimensional matrix alsostimulated sprout formation that was not observed with theaddition of eGFP-overexpressing cell lines. Treatment of either

the Sod2 or Sod2GFP cell lines with the PI3K inhibitor,LY2949002, completely blocked sprout formation in both celllines, indicating that PI3K signaling contributed to redox-de-pendent angiogenic activity. The above assays were also per-formed in the absence of a primary fibroblast cell line that wasshown previously to be required for sustained sprout formationin the presence of VEGF due to their ability to secrete addi-tional factors critical for sprout development (22). In the com-plete absence of a fibroblast population, no endothelial sprout-ing was observed (Fig. 5, A and B). To test if this response wasspecific to VEGF, 0.16 �g/ml of a VEGF-neutralizing antibodywas added to wells containing either Sod2 or Sod2GFP-ex-pressing cell lines. Coincubation of the VEGF-neutralizing an-tibody with the Sod2-overexpressing cells resulted in an inhi-bition of angiogenic sprout formation. Furthermore, when theSod2-overexpressing cells were incubated with an isotype con-trol antibody, no hindrance in sprout formation was observed.The present study indicates that increases in the cellular pro-duction of mitochondrial H2O2 leads to the secretion of neces-sary factors, including VEGF that can promote endothelialsprout formation, and that these factors are, at least in part,dependent on PI3K signaling.

In Vivo Determination of Angiogenic Activity—To determinethe redox-dependent regulation on in vivo angiogenesis, welooked at capillary development on the CAM, a widely adoptedin vivo method for studying angiogenesis. Sod2 expression re-sulted in an increase in the development of new embryonicblood vessels, many of which displayed a winding morphologyas compared with that of the control CMV cell lines, a charac-teristic commonly associated with potent angiogenic activity(Fig. 6, A and B). CAMs incubated with the CMV control celllines did not show a significant increase in blood vessel densitycompared with CAMs incubated without cells. Coexpression ofcatalase resulted in blood vessel density similar to that of thecontrol. These studies indicate that Sod2 expression increasesthe angiogenic phenotype in vivo and that coexpression of theH2O2-detoxifying enzyme, catalase, reverses this phenotype.

FIG. 5. Mitochondrial H2O2 enhances in vitro angiogenesis.Bovine lung microvessel cells were bound to Cytodex 3 microcarriersand embedded in a fibrogen/thrombin clot. The indicated cell lines werelayered on top of the clot to analyze their ability to promote angiogenicsprout formation. A, quantitation of angiogenic sprout formation. Thecontrol CMV and Sod2 or Sod2GFP cell lines were treated with 2.5 ngof VEGF or 30 �M of the PI3K inhibitor LY294002 (LY). The Sod2 orSod2GFP expressing cell lines were coincubated overnight with 0.16�g/ml of a VEGF neutralizing antibody; as a control the Sod2 express-ing cells were coincubated with a nonspecific isotype control antibody.Values are mean � S.E. of two separate experiments, n � 4 per exper-iment. Analysis of variance with � � 0.05 was used for processing thedata. Paired t tests were used as post-test. *, p � 0.0001 for theindicated cell lines and treatments, and n. d. indicates not determined.The Sod2GFP values were used to compare eGFP with Sod2. B, repre-sentative data.

FIG. 6. CAM assay showing the redox-dependent regulation ofthe angiogenic phenotype. 9-Day-old chick embryos were incubatedwith the indicated cell lines for 24 h. Following incubation, that area ofthe CAM was analyzed for blood vessel density. Five fields per egg werephotographed and analyzed for blood vessel density. Values are mean �S.E. of 4–5 separate eggs. †, p � 0.0001. No cells were added to the CAMcontrol. Arrow indicates vessel with winding morphology.

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DISCUSSION

In recent years it has become more evident that ROS are notmerely toxic by products of metabolism but serve an importantregulatory role in numerous signaling pathways (45). Our datasuggest a mechanism by which alterations of mitochondrialantioxidant enzyme expression can modulate the levels of in-tracellular H2O2 and subsequently alter the activity of thePI3K signaling cascade through oxidation of PTEN. Further-more, we have defined an alternative redox-sensitive pathwayutilizing H2O2 of mitochondrial origin to control angiogenesis.

The reversible oxidative inactivation of the tumor suppressorPTEN by H2O2 has emerged as a second mode of inhibitoryregulation independent of PDZ phosphorylation (7). The mem-brane-bound NADPH oxidase family members are the primarycandidates for the production of oxidants in the plasma mem-brane microenvironment, and their inhibition can attenuateAkt phosphorylation in activated macrophages (4). Our find-ings indicate that the H2O2-dependent enhancement of thePI3K signaling axis can be attenuated by enforced expressionof PTEN or dominant negative isoforms of Akt, emphasizingthe vital role of PTEN in the redox regulation of this signalingcascade. This is also the first evidence for the oxidative loss ofPTEN function in the absence of mutation in a human tumorcell line by mitochondrial derived H2O2.

Several reports indicate that PtIns(3,4,5)P3 accumulationoccurs at the plasma membrane in response to ROS (4, 46). Thecurrent findings using redox-engineered cell lines are consist-ent with the hypothesis that mitochondrial oxidants can impactthe distribution of these same bioactive phosphoinositides (Fig.3, B and C). Furthermore, mitochondrial localization of thelipid-binding chimeras was also observed in response to Sod2overexpression (Fig. 3D). Akt has been shown to distribute tothe mitochondria and phosphorylate the mitochondrial GSK-3�isoform (47). It is likely that phosphoinositides themselvesrecruit Akt as significant accumulation of inositol phosphatesto the mitochondria has been observed (48). Thus, the enforcedexpression of Sod2 and the production of H2O2 may recruitimportant signaling factors to the mitochondria that mediatePI3K signaling and augment vascular recruitment.

H2O2 itself has been shown to regulate proangiogenic re-sponses in a variety of systems, including human retinal pig-ment epithelial cells (49), cultured keratinocytes (50), and bo-vine pulmonary artery endothelial cells (41). Monte et al. (51)has demonstrated that H2O2 contributes to promoting the an-giogenic activity of tumor-bearing lymphocytes and that thisactivity can be inhibited by coadministration of the H2O2-detoxifying enzyme catalase but not Sod. In vitro angiogenesisof bovine thoracic aorta can be induced with relatively lowconcentrations (1 �M) of H2O2 and can be blocked by catalase(41). The Nox1-dependent generation of H2O2 is a potent trig-ger of angiogenesis, increasing the vascularity of tumors andinducing molecular markers of angiogenesis, including vascu-lar endothelial growth factor (VEGF), VEGF receptors, andmatrix metalloproteinase activity in cultured cells and in tu-mors. Coexpression of catalase blocks the increased activity ofthe angiogenic markers, indicating that hydrogen peroxide sig-nals part of the switch to the angiogenic phenotype (38). Fur-thermore, metalloproteinases are important contributors to theangiogenic switch, and their expression is redox-responsive (27,43). Finally, Ets-1 is also an important regulator of angiogen-esis (52) and is sensitive to H2O2 production (41). Thus, theintracellular production of H2O2 activates the angiogenicswitch enhancing the activity of a variety of signaling path-ways that lead to production of proangiogenic factors.

Several reports have demonstrated that VEGF promoter ex-pression is responsive to H2O2. Cho et al. (53) has determined

that a region from �449 to �126 was required for VEGFpromoter activity in macrophages in response to 1 mM H2O2.More detailed analysis of the VEGF promoter has identified aGC-rich region between �95 and �51 (54), containing an SP1-binding site that is responsive to relatively low (50 �M) concen-trations of H2O2 in human keratinocytes. Hocker and co-work-ers (55) have also shown that enhanced binding at the two SP1and SP3 sites at �73/�66 and �58/�52 represent the coremechanism of oxidative stress-triggered VEGF transactiva-tion. We have demonstrated previously (43) that the bindingactivity of SP1 is regulated in our redox-engineered cell lines.Thus, the SP1 sites at position �73 and �58 may also contrib-ute to the regulation of VEGF expression in response to mito-chondrial derived H2O2.

Active Akt leads to an increase in HIF-1� stabilization andsubsequent transcriptional activation of VEGF and the angio-genic switch (56–58). Loss of PTEN function leads to the in-creased production of VEGF (28). The present study providesevidence for the redox-sensitive link between these two parallelfindings and strongly suggests that mitochondrial H2O2 canalso modulate the angiogenic switch through oxidative inacti-vation of the tumor suppressor PTEN. In the present study wehave established that mitochondrial H2O2 oxidizes PTEN lead-ing to a concomitant increase in Akt signaling, VEGF expres-sion, and angiogenic activity. The oxidant-dependent PTENinactivation also enhances cyclin D1 expression in an Akt-de-pendent fashion and may regulate cell cycle progression. Sod2overexpression protects from programmed cell death in re-sponse to various apoptotic stimuli (59–61), and PTEN oxida-tion may contribute to this resistance. Future studies will bedirected at defining whether a relationship between Sod2,PTEN, and apoptosis exists.

In recent years H2O2 has come to the forefront as a keydeterminant of many redox-sensitive signaling pathways (2).The mitochondrion is the primary source for the intracellularproduction of H2O2, yet little emphasis has been placed on therole of this organelle in signaling processes. Receptor engage-ment leading to oxidant-dependent signaling has traditionallybeen attributed to the Rac-dependent activation of the phago-cytic oxidase family members (62). Further support for thishypothesis was put forth with the discovery of the noninflam-matory oxidases (63). However, a number of reports haveemerged indicating a role for mitochondrial derived oxidants inreceptor-dependent signaling process. Engagement of the �5�1integrin leads to alterations in cell shape via a process thatleads to increased mitochondrial ROS production and subse-quent expression of MMP-1 (18). Finkel and co-workers (64)have identified a feedback regulatory pathway whereby in-creased mitochondrial metabolic flow leads to enhanced oxi-dant production followed by JNK activation. JNK inhibitsGSK3� and shifts glucose utilization toward increased glyco-gen synthesis by restricting the generation of diffusible H2O2.Thus, mitochondrial oxidants deliver a cytosolic signal thatprevents their own production.

In the present study, JNK activation is also observed inresponse to Sod2 overexpression (data not shown) with a cor-responding increase in GSK3� phosphorylation. However,GSK3� inhibition is attributed to Akt activation (Fig. 1B, lane7). In addition, mitochondrial H2O2 production is not feedback-inhibited because its production is driven by the enforced CMV-dependent expression of Sod2.

Sod2 is positioned to efficiently dismutate O2. to H2O2 at near

diffusion limiting rates and is the only antioxidant enzyme thatis induced in response to growth factors, cytokines, ionizingradiation, and redox cycling drugs (65). We propose that themitochondria may play an important role in regulating the

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angiogenic switch by serving as a potent source of the smalldiffusible signaling molecule H2O2. Furthermore, the enhancedexpression of Sod2 under varying pathophysiologic conditionsmay not solely be to prevent the deleterious actions of oxidantsbut also serves to modulate key regulatory pathways that con-trol cellular function by its ability to generate H2O2.

Acknowledgments—We thank Steve Lotz for technical work andDrs. William Sellers, Tobias Meyer, and Yisang Yoon for the gifts of themutant PTEN, YFP-AKt-PH and CFP-PLC�-PH constructs, and themitochondrial eGFP constructs, respectively. Support for the Zeiss LSM510-NLO laser-scanning microscope was provided by NCRR Grant NN-BGEN-1S10RR017926-01 (to J. E. M.).

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