Mitochondrial H2O2 Regulates the Angiogenic Phenotype via PTEN Oxidation

Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2. Recent studies have demonstrated that the tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), the antagonist of the phosphosphoinositol-3-kinase (PI3K) signaling cascade, is susceptible to H2O2-dependent oxidative inactivation. This study describes the use of redox-engineered cell lines to identify PTEN as sensitive to oxidative inactivation by mitochondrial H2O2. Increases in the steady state production of mitochondrial derived H2O2, as a result of manganese superoxide dismutase (Sod2) overexpression, led to PTEN oxidation that was reversed by the coexpression of the H2O2-detoxifying enzyme catalase. The accumulation of an oxidized inactive fraction of PTEN favored the formation of phosphatidylinositol 3,4,5-triphosphate at the plasma membrane, resulting in increased activation of Akt and modulation of its downstream targets. PTEN oxidation in response to mitochondrial H2O2 enhanced PI3K signaling, leading to increased expression of the key regulator of angiogenesis, vascular endothelial growth factor. Overexpression of PTEN prevented the H2O2-dependent increase in vascular endothelial growth factor promoter activity and immunoreactive protein, whereas a mutant PTEN (G129R), lacking phosphatase activity, did not. Furthermore, mitochondrial generation of H2O2 by Sod2 promoted endothelial cell sprouting in a three-dimensional in vitro angiogenesis assay that was attenuated by catalase coexpression or the PI3K inhibitor LY2949002. Moreover, Sod2 overexpression resulted in increased in vivo blood vessel formation that was H2O2-dependent as assessed by the chicken chorioallantoic membrane assay. Our findings provide the first evidence for the involvement of mitochondrial H2O2 in regulating PTEN function and the angiogenic switch, indicating that Sod2 can serve as an alternative physiological source of the potent signaling molecule, H2O2. Reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; PI3K, phosphoinositide-3 kinase; Akt, protein kinase B; BLMV, bovine lung microvessels; CMV, cytomegalovirus; GSK3β, glycogen synthase kinase-3β; H2O2, hydrogen peroxide; HIF-1α, hypoxia-inducible factor α; 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′-tetramethylethylenediamine; GFP, green fluorescent protein; eGFP, enhanced GFP; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; 5-IAF, 5′-fluoresceinated iodoacetamide; PBS, phosphate-buffered saline; Ab, antibody; CAM, chicken chorioallantoic membrane; PH, pleckstrin homology. 1The abbreviations used are: ROS, reactive oxygen species; PI3K, phosphoinositide-3 kinase; Akt, protein kinase B; BLMV, bovine lung microvessels; CMV, cytomegalovirus; GSK3β, glycogen synthase kinase-3β; H2O2, hydrogen peroxide; HIF-1α, hypoxia-inducible factor α; 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′-tetramethylethylenediamine; GFP, green fluorescent protein; eGFP, enhanced GFP; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; 5-IAF, 5′-fluoresceinated iodoacetamide; PBS, phosphate-buffered saline; Ab, antibody; CAM, chicken chorioallantoic membrane; PH, pleckstrin homology. have long been established to play an important role in many disease pathologies and have also emerged as efficient signaling molecules. The principal mediator of ROS-dependent signaling is the two electron reduction product of oxygen (O2), hydrogen peroxide (H2O2). H2O2 is generated in response to receptor stimulation and is an efficient signal transducing molecule by its ability to reversibly oxidize active site cysteines (1Rhee S.G. Chang T.S. Bae Y.S. Lee S.R. Kang S.W. J. Am. Soc. Nephrol. 2003; 14: S211-S215Crossref PubMed Google Scholar, 2Finkel T. IUBMB Life. 2001; 52: 3-6Crossref PubMed Scopus (246) Google Scholar). Many protein tyrosine phosphatases are particularly susceptible to H2O2-dependent inactivation because of the lowered pKa of the active site cysteine (3Meng T.C. Fukada T. Tonks N.K. Mol. Cell. 2002; 9: 387-399Abstract Full Text Full Text PDF PubMed Scopus (877) Google Scholar, 4Leslie N.R. Bennett D. Lindsay Y.E. Stewart H. Gray A. Downes C.P. EMBO J. 2003; 22: 5501-5510Crossref PubMed Scopus (487) Google Scholar, 5Chiarugi P. Fiaschi T. Taddei M.L. Talini D. Giannoni E. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 33478-33487Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The essential cysteine residue in the signature active site motif Cys-(X)5-Arg exists as a thiolate anion (Cys-S–), which at neutral pH is susceptible to nucleophilic attack by H2O2. Oxidation of the active site cysteine generates a sulfenic derivative (Cys-SOH), leading to enzyme inactivation that can be reversed by cellular thiols (6Chiarugi P. Cirri P. Trends Biochem. Sci. 2003; 28: 509-514Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10), also known as TEP-1 (TGF-β-regulated and epithelial cell-enriched phosphatase) or MMAC1 (mutated in multiple advanced cancers), is reversibly inhibited by H2O2, resulting in the formation of a disulfide between the active site cysteine (Cys124) and a vicinal cysteine (Cys71) (4Leslie N.R. Bennett D. Lindsay Y.E. Stewart H. Gray A. 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Full Text PDF PubMed Google Scholar). mitochondrial derived H2O2 to the oxidative inactivation of PTEN or phosphatases been that in the steady state production of mitochondrial H2O2 by enzyme overexpression the state of This is for signaling, and findings a role for mitochondrial H2O2 and Sod2 in regulating the angiogenic PTEN phosphatase inactivation. and in and in a cell lines Sod2, catalase in Biol. PubMed Scopus Google Scholar). of Oxidation in of in redox-engineered cell lines was the phosphate-buffered and in to a of was to a of at in a at a of in an of phosphatase and an and The used PTEN Akt and This was by an as the used to of protein was the of Akt The was in bovine at of and PTEN by in PBS, and in of and of protein cell was to as S.R. Yang K.S. Kwon J. Lee C. Jeong W. Rhee S.G. J. Biol. Chem. 2002; 277: 20336-20342Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). The to membrane and a by a and enhanced from cell lines in at pH by in a of a and The was a TEMED, and was for in the at and was in The to fluorescent of activity in at pH at in the for and cysteines at for PTEN by protein This also the was in of at pH This the thereby oxidized The in to the in and and at for The oxidized an and to of and lines the from at and by D. Lin B. G. M. 2002; PubMed Scopus Google The and to the The and the activity was the assay of the for by in vitro angiogenesis assay was by M. 2003; PubMed Scopus Google Scholar). bovine lung in of at a of The and cell for at and to the to to the the the was in a in of at and the in and at a of in of of of the was to of in a The was to for at and at and for of was and to the for at and was and of and of the redox-engineered cell lines Sod2, eGFP, and a of was to the cell and for a of the PI3K inhibitor was the of a or was to the cell and the formation was for and the of of a a The of and assay was as by M. T. G. Cell. Full Text PDF PubMed Scopus Google chicken at the the was from the by first a at the of the that the an was the of the the blood as by was to the thereby the to the of the the was This the to be from the the of the was the in the and a of was the CAM, and in a of of in the of the to the the the a was by a the in and the of was from Sod2 was and was of the Sod2 and was to the Sod2 and the The active and kinase Akt was by The PTEN and phosphatase mutant PTEN from and Sci. A. PubMed Scopus Google Scholar). The and from The was from of an by a was the YFP, the from an was used to YFP, and an and a of at an at a of and the was used in which the from an was used to GFP; the from a was used to the and by an and a for and for the at a at a of the of or at a at a of by by to the of fluorescent protein a by G. R. 2001; PubMed Scopus Google Scholar, R. G. 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Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). expression of mitochondrial superoxide dismutase (Sod2) led to a increase in H2O2, which was reversed by or mitochondrial expression of catalase Sod2 overexpression led to a increase in the of oxidation of the that was attenuated catalase coexpression catalase overexpression also the of findings demonstrated that the enhanced of oxidation was to the in the steady state production of H2O2 that was reversed expression of the H2O2-detoxifying enzyme catalase. Oxidation of is a phosphatase that is reversibly sensitive to inactivation by H2O2 S.R. Yang K.S. Kwon J. Lee C. Jeong W. Rhee S.G. J. Biol. Chem. 2002; 277: 20336-20342Abstract Full Text Full Text PDF PubMed Scopus (821) Google The oxidation of PTEN resulted in the of the to a resulting in a protein that can be S.R. Yang K.S. Kwon J. Lee C. Jeong W. Rhee S.G. J. Biol. Chem. 2002; 277: 20336-20342Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). The generation of H2O2 accumulation of the oxidized of PTEN that was prevented by coexpression of catalase and of H2O2 a accumulation of the oxidized of PTEN that was by mitochondrial catalase PTEN inactivation resulting from or oxidation increased the of the kinase Akt (4Leslie N.R. Bennett D. Lindsay Y.E. Stewart H. Gray A. Downes C.P. EMBO J. 2003; 22: 5501-5510Crossref PubMed Scopus (487) Google Scholar, Sci. A. PubMed Scopus Google Scholar, C. J. H. Lee M. 2003; PubMed Scopus Google Scholar). to the increase in PTEN oxidation Sod2 overexpression was a in at of Akt which was attenuated by the overexpression of catalase in the or mitochondrial and The for Akt are and are in regulating many cellular angiogenesis, and cell M.A. J. 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A. 2001; Google Scholar). to PTEN was in a PTEN did in of redox-engineered cell indicating that its of is that in the steady state mitochondrial production of H2O2 the state of PTEN and the state of Akt and the downstream Oxidation of PTEN and PI3K of as in the of the redox-engineered cell to signaling be of the the role of Sod2 in PTEN a to of cell lines of Sod2 Sod2 was to the a mitochondrial in its to cell mitochondrial of the in the was by fluorescent of that the mitochondrial cell of and cell lines and of was also was to that of the cell of the cell that Sod2 activity increased to the of in the cell and that by Furthermore, expression Sod2 The in the of Sod2 activity the of the Sod2 and the that the active the state of PTEN in the cell of PTEN increase in the of its oxidized Akt and also sensitive to in Sod2 activity, which PTEN whereas Akt and expression was sensitive to in the mitochondrial as a of Sod2 expression increased the of its immunoreactive the PTEN oxidation state in cell a for cysteines by 5′-fluoresceinated P. Fiaschi T. Taddei M.L. Talini D. Giannoni E. Raugei G. Ramponi G. J. Biol. Chem. 2001; 276: 33478-33487Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, Y. Kwon K.S. Rhee S.G. PubMed Scopus Google oxidation of PTEN increased in response to Sod2 indicating that the production of H2O2 a PTEN oxidation findings are the first to that in the steady state mitochondrial production of H2O2 in tumor can the state of PTEN and signaling its H2O2 an important role in the generation and of receptor tyrosine kinase M. J. Sci. 2001; PubMed Google Scholar). of an oxidized inactive fraction of PTEN the of PtIns(3,4,5)P3 at the plasma membrane the and redox-engineered cell lines of green fluorescent protein and the pleckstrin of or Akt, to of or T. Biol. 2001; PubMed Scopus Google Scholar). the of and accumulation of and H2O2 been to PI3K leading to the production of PtIns(3,4,5)P3 Sci. A. 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Bae Y.S. Lee S.R. Kwon J. Sci. Scholar). Our a by which of mitochondrial enzyme expression can the of H2O2 and the activity of the PI3K signaling oxidation of Furthermore, have an alternative H2O2 of mitochondrial to The oxidative inactivation of the tumor suppressor PTEN by H2O2 emerged as a of of S.R. Yang K.S. Kwon J. Lee C. Jeong W. Rhee S.G. J. Biol. Chem. 2002; 277: 20336-20342Abstract Full Text Full Text PDF PubMed Scopus (821) Google Scholar). The are the for the production of in the plasma membrane and can Akt in (4Leslie N.R. Bennett D. Lindsay Y.E. Stewart H. Gray A. Downes C.P. EMBO J. 2003; 22: 5501-5510Crossref PubMed Scopus (487) Google Scholar). 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