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Zinc-induced PTEN Protein Degradation through the Proteasome Pathway in Human Airway Epithelial Cells

2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês

10.1074/jbc.m303318200

ISSN

1083-351X

Autores

Weidong Wu, Xinchao Wang, Wenli Zhang, William Reed, James M. Samet, Young E. Whang, Andrew J. Ghio,

Tópico(s)

Protein Kinase Regulation and GTPase Signaling

Resumo

The tumor suppressor PTEN is a putative negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. Exposure to Zn2+ ions induces Akt activation, suggesting that PTEN may be modulated in this process. Therefore, the effects of Zn2+ on PTEN were studied in human airway epithelial cells and rat lungs. Treatment with Zn2+ resulted in a significant reduction in levels of PTEN protein in a dose- and time-dependent fashion in a human airway epithelial cell line. This effect of Zn2+was also observed in normal human airway epithelial cells in primary culture and in rat airway epithelium in vivo. Concomitantly, levels of PTEN mRNA were also significantly reduced by Zn2+ exposure. PTEN phosphatase activity evaluated by measuring Akt phosphorylation decreased after Zn2+ treatment. Pretreatment of the cells with a proteasome inhibitor significantly blocked zinc-induced reduction of PTEN protein as well as the increase in Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Further study revealed that Zn2+-induced ubiquitination of PTEN protein may mediate this process. A phosphatidylinositol 3-kinase inhibitor blocked PTEN degradation induced by Zn2+, suggesting that phosphatidylinositol 3-kinase may participate in the regulation of PTEN. However, both the proteasome inhibitor and phosphatidylinositol 3-kinase inhibitor failed to prevent significant down-regulation of PTEN mRNA expression in response to Zn2+. In summary, exposure to Zn2+ ions causes PTEN degradation and loss of function, which is mediated by an ubiquitin-associated proteolytic process in the airway epithelium. The tumor suppressor PTEN is a putative negative regulator of the phosphatidylinositol 3-kinase/Akt pathway. Exposure to Zn2+ ions induces Akt activation, suggesting that PTEN may be modulated in this process. Therefore, the effects of Zn2+ on PTEN were studied in human airway epithelial cells and rat lungs. Treatment with Zn2+ resulted in a significant reduction in levels of PTEN protein in a dose- and time-dependent fashion in a human airway epithelial cell line. This effect of Zn2+was also observed in normal human airway epithelial cells in primary culture and in rat airway epithelium in vivo. Concomitantly, levels of PTEN mRNA were also significantly reduced by Zn2+ exposure. PTEN phosphatase activity evaluated by measuring Akt phosphorylation decreased after Zn2+ treatment. Pretreatment of the cells with a proteasome inhibitor significantly blocked zinc-induced reduction of PTEN protein as well as the increase in Akt phosphorylation, implicating the involvement of proteasome-mediated PTEN degradation. Further study revealed that Zn2+-induced ubiquitination of PTEN protein may mediate this process. A phosphatidylinositol 3-kinase inhibitor blocked PTEN degradation induced by Zn2+, suggesting that phosphatidylinositol 3-kinase may participate in the regulation of PTEN. However, both the proteasome inhibitor and phosphatidylinositol 3-kinase inhibitor failed to prevent significant down-regulation of PTEN mRNA expression in response to Zn2+. In summary, exposure to Zn2+ ions causes PTEN degradation and loss of function, which is mediated by an ubiquitin-associated proteolytic process in the airway epithelium. PTEN 1The abbreviations used are: PTEN, phosphatase and tensin homolog deleted on chromosome 10; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol (3,4,5)-triphosphate; EGF, epidermal growth factor; MAPKs, mitogen-activated protein kinases; MEK, MAPK/ERK kinase; RIPA, radioimmune precipitation buffer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (phosphatase and tensin homolog deleted on chromosome 10) was discovered in 1997 as a new major tumor suppressor encoded by a gene localized to human chromosome 10q23 region (1Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4315) Google Scholar, 2Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2526) Google Scholar). Deletions or somatic mutations within the PTEN gene have been observed in many primary human cancers, tumor cell lines, and several familial cancer predisposition disorders (3Eng C. Int. J. Oncol. 1998; 12: 701-710PubMed Google Scholar, 4Waite K.A. Eng C. Am. J. Hum. Genet. 2002; 70: 829-844Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). It is now known that PTEN plays critical roles not only in suppressing cancer but also in embryonic development, cell migration, cell signaling, and apoptosis (5Gu J. Tamura M. Yamada K.M. J. Cell Biol. 1998; 143: 1375-1383Crossref PubMed Scopus (302) Google Scholar, 6Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 7Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1754) Google Scholar, 8Tamura M. Gu J. Takino T. Yamada K.M. Cancer Res. 1999; 59: 442-449PubMed Google Scholar, 9Di Cristofano A. Pandolfi P.P. Cell. 2000; 100: 387-390Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar, 10Vazquez F. Sellers W.R. Biochim Biophys Acta. 2000; 1470: M21-M35PubMed Google Scholar, 11Simpson L. Parsons R. Exp. Cell Res. 2001; 264: 29-41Crossref PubMed Scopus (569) Google Scholar, 12Yamada K.M. Araki M. J. Cell Sci. 2001; 114: 2375-2382Crossref PubMed Google Scholar). PTEN protein consists of 403 amino acids. It displays high homology in its NH2-terminal region to dual specificity phosphatases and also to tensin and auxilin (1Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4315) Google Scholar, 2Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2526) Google Scholar, 10Vazquez F. Sellers W.R. Biochim Biophys Acta. 2000; 1470: M21-M35PubMed Google Scholar, 13Leslie N.R. Downes C.P. Cell. Signal. 2002; 14: 285-295Crossref PubMed Scopus (354) Google Scholar). The COOH terminus is most likely regulatory in nature and is composed of three subdomains: a C2 domain that has been implicated in phospholipid-binding; two tandem PEST domains that regulate PTEN protein stability; and a PDZ-binding domain that associates with PDZ-containing proteins (14Georgescu M.M. Kirsch K.H. Akagi T. Shishido T. Hanafusa H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10182-10187Crossref PubMed Scopus (278) Google Scholar, 15Morimoto A.M. Berson A.E. Fujii G.H. Teng D.H. Tavtigian S.V. Bookstein R. Steck P.A. Bolen J.B. Oncogene. 1999; 18: 1261-1266Crossref PubMed Scopus (31) Google Scholar, 16Wu X. Hepner K. Castelino-Prabhu S. Do D. Kaye M.B. Yuan X.J. Wood J. Ross C. Sawyers C.L. Whang Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4233-4238Crossref PubMed Scopus (338) Google Scholar, 17Adey N.B. Huang L. Ormonde P.A. Baumgard M.L. Pero R. Byreddy D.V. Tavtigian S.V. Bartel P.L. Cancer Res. 2000; 60: 35-37PubMed Google Scholar, 18Tolkacheva T. Boddapati M. Sanfiz A. Tsuchida K. Kimmelman A.C. Chan A.M. Cancer Res. 2001; 61: 4985-4989PubMed Google Scholar). Although PTEN has phosphatase activity in vitro against both lipid and protein substrates (5Gu J. Tamura M. Yamada K.M. J. Cell Biol. 1998; 143: 1375-1383Crossref PubMed Scopus (302) Google Scholar, 12Yamada K.M. Araki M. J. Cell Sci. 2001; 114: 2375-2382Crossref PubMed Google Scholar, 19Myers M.P. Tonks N.K. Am. J. Hum. Genet. 1997; 61: 1234-1238Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20Gu J. Tamura M. Pankov R. Danen E.H. Takino T. Matsumoto K. Yamada K.M. J. Cell Biol. 1999; 146: 389-403Crossref PubMed Scopus (376) Google Scholar), its primary physiological target appears to be a phospholipid rather than a phosphoprotein: specifically, the D3-phosphate group of phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) (13Leslie N.R. Downes C.P. Cell. Signal. 2002; 14: 285-295Crossref PubMed Scopus (354) Google Scholar, 21Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2614) Google Scholar, 22Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13513-13518Crossref PubMed Scopus (1013) Google Scholar, 23Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2120) Google Scholar, 24Downes C.P. Bennett D. McConnachie G. Leslie N.R. Pass I. MacPhee C. Patel L. Gray A. Biochem. Soc. Trans. 2001; 29: 846-851Crossref PubMed Google Scholar). PIP3 is specifically produced from phosphatidylinositol 4, 5-bisphosphate by phosphatidylinositol 3-kinase (PI3K), which is activated by cell receptors including various tyrosine kinase growth factor receptors and integrins (25Stephens L. Eguinoa A. Corey S. Jackson T. Hawkins P.T. EMBO J. 1993; 12: 2265-2273Crossref PubMed Scopus (137) Google Scholar, 26Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 27Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5171) Google Scholar). Accumulation of PIP3 further results in the activation of the serine-threonine protein kinase B/Akt through the recruitment of PI3K-dependent serine/threonine kinases (28Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 29Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1190) Google Scholar, 30Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar), subsequently modulating both proliferative and apoptotic signals (10Vazquez F. Sellers W.R. Biochim Biophys Acta. 2000; 1470: M21-M35PubMed Google Scholar, 31Vazquez F. Ramaswamy S. Nakamura N. Sellers W.R. Mol. Cell. Biol. 2000; 20: 5010-5018Crossref PubMed Scopus (656) Google Scholar). Thus, by dephosphorylating PIP3, PTEN antagonizes growth-promoting and antiapoptotic pathways mediated by PI3K/Akt signaling (32Wu X. Senechal K. Neshat M.S. Whang Y.E. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15587-15591Crossref PubMed Scopus (595) Google Scholar, 33Li J. Simpson L. Takahashi M. Miliaresis C. Myers M.P. Tonks N. Parsons R. Cancer Res. 1998; 58: 5667-5672PubMed Google Scholar, 34Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). Zinc is an essential micronutrient with multiple structural and regulatory cellular functions (35Vallee B.L. Falchuk K.H. Physiol. Rev. 1993; 73: 79-118Crossref PubMed Google Scholar) but also a common airborne metallic contaminant that may contribute to the health effects of ambient and occupational air pollution (36Kuschner W.G. D'Alessandro A. Wong H. Blanc P.D. Environ. Res. 1997; 75: 7-11Crossref PubMed Scopus (85) Google Scholar, 37Martin C.J. Le X.C. Guidotti T.L. Yalcin S. Chum E. Audette R.J. Liang C. Yuan B. Zhang X. Wu J. Am. J. Ind. Med. 1999; 35: 574-580Crossref PubMed Scopus (26) Google Scholar, 38Barceloux D.G. J. Toxicol. Clin. Toxicol. 1999; 37: 279-292Crossref PubMed Scopus (199) Google Scholar, 39Adamson I.Y. Prieditis H. Hedgecock C. Vincent R. Toxicol. Appl. Pharmacol. 2000; 166: 111-119Crossref PubMed Scopus (251) Google Scholar). There has been a recent increase in interest in zinc-induced intracellular signaling (40Maret W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12325-12327Crossref PubMed Scopus (79) Google Scholar). Zn2+ ions have been shown to activate the signaling pathways involving the receptor or non-receptor tyrosine kinases, Ras/mitogen-activated protein kinases (MAPKs) (41Samet J.M. Graves L.M. Quay J. Dailey L.A. Devlin R.B. Ghio A.J. Wu W. Bromberg P.A. Reed W. Am. J. Physiol. 1998; 275: L551-L558Crossref PubMed Google Scholar, 42Wu W. Graves L.M. Gill G.N. Parsons S.J. Samet J.M. J. Biol. Chem. 2002; 277: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 43Wu W. Jaspers I. Zhang W. Graves L.M. Samet J.M. Am. J. Physiol. 2002; 282: L1040-L1048Crossref Scopus (42) Google Scholar), and the PI3K/Akt/p70 S6 kinase pathway (44Kim S. Jung Y. Kim D. Koh H. Chung J. J. Biol. Chem. 2000; 275: 25979-25984Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) and to inhibit the activity of protein tyrosine phosphatases (45Samet J.M. Silbajoris R. Wu W. Graves L.M. Am. J. Respir. Cell Mol. Biol. 1999; 21: 357-364Crossref PubMed Scopus (82) Google Scholar). In contrast to the effects of Zn2+ on cell signaling, overexpression of PTEN has opposite effects, such as blocking downstream signaling of EGF receptor including the Ras/MEK/MAPK cascade and antagonizing PI3K/Akt signaling (5Gu J. Tamura M. Yamada K.M. J. Cell Biol. 1998; 143: 1375-1383Crossref PubMed Scopus (302) Google Scholar, 20Gu J. Tamura M. Pankov R. Danen E.H. Takino T. Matsumoto K. Yamada K.M. J. Cell Biol. 1999; 146: 389-403Crossref PubMed Scopus (376) Google Scholar). This suggested the possibility that Zn2+ exposure decreased PTEN activity. Our study investigated the effects of Zn2+ treatment on PTEN in human airway epithelial cells. We report here that Zn2+ exposure causes a significant decrease in PTEN protein levels and activity through a proteolytic mechanism that depends on PI3K and leads to Akt activation. Cell Culture and in Vitro Exposure—The BEAS-2B (subclone S6) cell line was derived by transforming human bronchial cells with an adenovirus 12-simian virus 40 construct (46Reddel R.R. Ke Y. Gerwin B.I. McMenamin M.G. Lechner J.F. Su R.T. Brash D.E. Park J.B. Rhim J.S. Harris C.C. Cancer Res. 1988; 48: 1904-1909PubMed Google Scholar). BEAS-2B cells (passages 70–80) were grown to confluence on tissue culture-treated Costar 6- or 12-well plates in keratinocyte basal medium supplemented with 30 μg/ml bovine pituitary extract, 5 ng/ml human EGF, 500 ng/ml hydrocortisone, 0.1 mm ethanolamine, 0.1 mm phosphoethanolamine, and 5 ng/ml insulin, as described previously (43Wu W. Jaspers I. Zhang W. Graves L.M. Samet J.M. Am. J. Physiol. 2002; 282: L1040-L1048Crossref Scopus (42) Google Scholar). Cells were placed in keratinocyte basal medium (without supplements) for 20–22 h before treatment with zinc sulfate (Sigma). Normal human bronchial epithelial cells (passages 2–3) were obtained from normal adult volunteers by transbronchoscopic brush biopsy of mainstem bronchi, conducted while following a protocol approved by the Committee on the Protection of the Rights of Human Subjects at the University of North Carolina at Chapel Hill (45Samet J.M. Silbajoris R. Wu W. Graves L.M. Am. J. Respir. Cell Mol. Biol. 1999; 21: 357-364Crossref PubMed Scopus (82) Google Scholar). Normal human bronchial epithelial cells were plated in supplemented bronchial epithelial cell basal medium (0.5 ng/ml human epidermal growth factor, 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 10 μg/ml transferrin, 0.5 μg/ml epinephrine, 6.5 ng/ml triiodothyronine, 50 μg/ml gentamycin, 50 ng/ml amphotericin-B, 52 μg/ml bovine pituitary extract, and 0.1 ng/ml retinoic acid) grown to confluence and then cultured with bronchial epithelial cell basal medium deprived of epidermal growth factor for 12–16 h before challenge with zinc sulfate. Immunoprecipitation—BEAS-2B cells pretreated with MG132 were stimulated with Zn2+ for 1 h. Cells treated with different doses of Zn2+ were lysed with RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS in phosphate-buffered saline, pH 7.4) containing 0.1 mm vanadyl sulfate and protease inhibitors (0.5 mg/ml aprotinin, 0.5 mg/ml trans-epoxy succinyl-l-leucylamido-(4-guanidino)butane (E-64), 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 ng/ml leupeptin). Cell lysates (300 μg) were precleared with protein A-agarose and then incubated with agarose-conjugated ubiquitin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. The precipitates were washed with cold RIPA buffer before immunoblotting using a murine monoclonal anti-human PTEN antibody (Cascade Bioscience, Winchester, MA). Immunoblotting—Cells with or without pretreatment of pharmacological inhibitors were treated with Zn2+ and then lysed in RIPA buffer. Cleared cell lysates or immunoprecipitates were subjected to SDS-PAGE, as described before (42Wu W. Graves L.M. Gill G.N. Parsons S.J. Samet J.M. J. Biol. Chem. 2002; 277: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Proteins were transferred onto nitrocellulose membrane. Membrane was blocked with 5% non-fat milk, washed briefly, incubated with antibodies against human PTEN (Cascade Bioscience), phospho-specific Akt and Akt (Cell Signaling Technology, Beverly, MA), and β-actin (USBiological, Swampscott, MA), respectively, at 4 °C overnight followed by incubating with corresponding horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Immunoblot images were detected using chemiluminescence reagents (Pierce) and the Gene Gynome Imaging System (Syngene, Frederick, MD). Real-time Reverse Transcriptase-PCR—BEAS-2B cells grown to confluence were exposed to 50 μm Zn2+. Cells were lysed with 4 m guanidine thiocyanate (Roche Applied Science), 50 mm sodium citrate, 0.5% sarkoyl, and 0.01 m dithiothreitol. Total RNA (200 ng) was isolated using RNeasy kit (Qiagen Inc., Valencia, CA) and reverse-transcribed into cDNA. Quantitative PCR was performed using TaqMan Universal PCR Master Mix (Roche Applied Science) and an ABI Prism 7700 sequence detector (Applied Biosystems, Foster City, CA) (47Yang F. Wang X. Haile D.J. Piantadosi C.A. Ghio A.J. Am. J. Physiol. 2002; 283: L932-L939PubMed Google Scholar). PTEN mRNA levels were normalized using the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Relative amounts of PTEN and GAPDH mRNA were based on standard curves prepared by serial dilution of cDNA from human BEAS-2B cells. The following oligonucleotide primers and probes were used: PTEN, 5′-TGT TGT TTC ACA AGA TGA TGT TTG A-3′ (sense), 5′-CGT CGT GTG GGT CCT GAA TT-3′ (antisense), 5′-ACT ATT CCA ATG TTC AGT GGC GGA ACT TGC-3′ (probe); GAPDH, 5′-GAA GGT GAA GGT CGG AGT C-3′ (sense), 5′-GAA GAT GGT GAT GGG ATT TC-3′ (antisense), 5′-CAA GCT TCC CGT TCT CAG CC-3′ (probe). In addition, PTEN mRNA expression was also determined in cells pretreated with vehicle or inhibitors (MG132 or LY294002) prior to zinc exposure according to the same procedure. Immunohistochemistry—Male Sprague-Dawley rats (60 days old) were anesthetized with halothane and instilled with either 0.5 ml of saline or 0.5 ml of 50 μm zinc sulfate in saline. 24 h after instillation, the lungs of euthanized rats were fixed with 10% formalin (35 ml/kg of body weight) (Fisher). The immunochemical staining was conducted as described before (48Wang X. Ghio A.J. Yang F. Dolan K.G. Garrick M.D. Piantadosi C.A. Am. J. Physiol. 2002; 282: L987-L995Crossref PubMed Scopus (78) Google Scholar). Tissue sections were mounted on silane-treated slides (Fisher) and air-dried overnight. The slides were heat-fixed at 600 °C in a slide dryer (Shandon Lipshaw, Pittsburgh, PA) and then followed by deparaffinization and hydration to 95% alcohol (xylene for 10 min, absolute alcohol for 5 min, and 95% alcohol for 5 min). Endogenous peroxidase activity was blocked with H2O2 in absolute methanol (30% H2O2 in 30 ml of methanol) for 8 min. Slides were rinsed in 95% alcohol for 2 min, placed in deionized H2O, and washed in phosphate-buffered saline. After treatment with Cyto Q Background Buster (Innovex Biosciences) for 10 min, slides were incubated with mouse anti-human PTEN antibody (Cascade Bioscience, Winchester, MA) diluted 1:100 in 1% bovine serum albumin for 45 min at 37 °C followed by incubation with biotinylated linking secondary antibody from Stat-Q staining system (Innovex Biosciences) for 10 min at room temperature and washed with phosphate-buffered saline, and peroxidase enzyme label from Stat-Q Staining System was applied. Tissue sections were developed with 3,3′-diaminobenzidine tetrahydrochloride and counter-stained with hematoxylin. Coverslips were applied using a permanent mounting medium. Statistics—Data are presented as means ± S.E. Unpaired Student's t tests with conferring correction were used for pairwise comparison. Reduction of PTEN Protein Levels in Human Airway Epithelial Cells Exposed to Zn2 + —Previous studies showed that Zn2+-induced activation of the PI3K/Akt signaling pathway (44Kim S. Jung Y. Kim D. Koh H. Chung J. J. Biol. Chem. 2000; 275: 25979-25984Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) suggested that this metal may affect PTEN protein. To test this assumption, PTEN protein levels were measured in human airway epithelial cells exposed to Zn2+ using Western blotting. As shown in Fig. 1A, exposure to 50 μm Zn2+ for 4 and 8 h significantly decreased PTEN protein levels in BEAS-2B cells. Exposure of BEAS-2B cells to 50 μm Zn2+ for 8 h did not result in significant alterations in cell viability, as assessed by assay of lactate dehydrogenase activity released into the culture medium (data not shown). The magnitude of the Zn2+-induced reduction in PTEN content was proportional to the concentration of Zn2+ administered to the cells (Fig. 1B). PTEN protein level in untreated cells appeared constant over time (Fig. 1A). This finding was reproduced in normal human airway epithelial cells, excluding the possibility that this effect is an artifact of the BEAS-2B cell line (Fig. 1C). In comparison, 50 μm vanadyl sulfate, a potent tyrosine phosphatase inhibitor (45Samet J.M. Silbajoris R. Wu W. Graves L.M. Am. J. Respir. Cell Mol. Biol. 1999; 21: 357-364Crossref PubMed Scopus (82) Google Scholar), produced only a minimal effect on PTEN protein levels in BEAS-2B cells (Fig. 1A). In addition, exposure of cells to 100 ng/ml EGF, a potent EGF receptor ligand (49Carpenter G. Bioessays. 2000; 22: 697-707Crossref PubMed Scopus (305) Google Scholar), for 1, 2, 4, and 8 h did not show a significant effect on PTEN levels (data not shown). These data indicated that Zn2+ treatment specifically reduced PTEN protein content in human airway epithelial cells. To determine whether this effect of Zn2+ also occurs in airway epithelium in vivo, Sprague-Dawley rats were intratracheally instilled with 50 μm Zn2+ or with saline as negative control. Both airway epithelium and alveolar macrophages in normal rat lung tissue were positively stained for PTEN protein that predominantly existed in cytoplasm (Fig. 2A). In contrast, PTEN protein immunostaining decreased markedly in airway epithelium exposed to Zn2+ (Fig. 2B), which was consistent with the in vitro observations. Interestingly, PTEN immunostaining in alveolar macrophages remained unchanged with Zn2+ exposure (Fig. 2B), suggesting that Zn2+-induced PTEN reduction may be cell type-specific. Zn2 + -induced PI3K/Akt Activation in Human Airway Epithelial Cells—Since PTEN antagonizes the PI3K/Akt pathway (13Leslie N.R. Downes C.P. Cell. Signal. 2002; 14: 285-295Crossref PubMed Scopus (354) Google Scholar, 24Downes C.P. Bennett D. McConnachie G. Leslie N.R. Pass I. MacPhee C. Patel L. Gray A. Biochem. Soc. Trans. 2001; 29: 846-851Crossref PubMed Google Scholar, 27Vivanco I. Sawyers C.L. Nat. Rev. Cancer. 2002; 2: 489-501Crossref PubMed Scopus (5171) Google Scholar), reduction of PTEN protein levels induced by Zn2+ should result in Akt activation. To further characterize the inhibitory effect of Zn2+ on PTEN-associated signaling, we next studied the effect of Zn2+ treatment on the activation of Akt in BEAS-2B cells, as measured by the phosphorylation of Akt at serine 473 (50Alessi D.R. James S.R. Downes C.P. Holmes A.B. Gaffney P.R. Reese C.B. Cohen P. Curr. Biol. 1997; 7: 261-269Abstract Full Text Full Text PDF PubMed Google Scholar). Exposure to Zn2+ markedly induced Akt phosphorylation in BEAS-2B cells in a dose- and time-dependent fashion (Fig. 3, A and B). Interestingly, robust Akt phosphorylation was evident 1 h after Zn2+ exposure, when there was minimal effect on the total PTEN protein level. Next, a highly selective inhibitor of PI3K activity, LY294002 (51Ding J. Vlahos C.J. Liu R. Brown R.F. Badwey J.A. J. Biol. Chem. 1995; 270: 11684-11691Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), was further used to ascertain the dependence of Zn2+-induced Akt phosphorylation on PI3K (52Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Akt phosphorylation was clearly inhibited in BEAS-2B cells pretreated with LY294002 (Fig. 3C). As vehicle control, Me2SO appeared to elevate the baseline of Akt phosphorylation in BEAS-2B cells (Fig. 3C). PTEN mRNA Level Was Significantly Reduced by Zn2 + Exposure—Reduction of PTEN protein level and function may be associated with decreased PTEN mRNA expression in several tumor cell lines (53Bruni P. Boccia A. Baldassarre G. Trapasso F. Santoro M. Chiappetta G. Fusco A. Viglietto G. Oncogene. 2000; 19: 3146-3155Crossref PubMed Scopus (129) Google Scholar). To identify the mechanisms responsible for PTEN protein reduction, PTEN mRNA expression in zinc-exposed cells was quantified using real-time reverse transcriptase-PCR. BEAS-2B cells were exposed to different doses of Zn2+ for different periods. As shown in Fig. 4A, reduction of PTEN mRNA expression (about 50%) was observed when cells were exposed to 50 μm Zn2+. Down-regulation of PTEN mRNA expression was only detected upon stimulation of BEAS-2B cells with 50 μm Zn2+ for 8 h (Fig. 4B). Thus, in contrast to PTEN protein levels in BEAS-2B cells, which declined as early as 4 h of exposure, the reduction of PTEN mRNA expression induced by Zn2+ occurred after PTEN protein levels had fallen. PTEN protein is a relatively stable protein with a half-life of 48–72 h (16Wu X. Hepner K. Castelino-Prabhu S. Do D. Kaye M.B. Yuan X.J. Wood J. Ross C. Sawyers C.L. Whang Y.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4233-4238Crossref PubMed Scopus (338) Google Scholar). As shown in Fig. 1A, PTEN protein level in untreated BEAS-2B cells appeared constant within 8 h of exposure. Therefore, these data suggested that the down-regulation of PTEN mRNA levels might not play a major role in Zn2+-induced PTEN reduction. Proteasome-mediated PTEN Degradation in Zn2 + -exposed Cells—The involvement of the proteasome in the degradation and regulation of the function of short-lived proteins, including oncoproteins, tumor suppressors, and cell cycle proteins, has been extensively documented as a mechanism for post-translational regulation of protein levels (54Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1252) Google Scholar). Therefore, we next investigated whether PTEN degradation in response to Zn2+ exposure was proteasome-dependent. BEAS-2B cells were pretreated with a specific proteasome inhibitor, MG132 (55Torres J. Pulido R. J. Biol. Chem. 2001; 276: 993-998Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar), and PTEN protein levels were evaluated by Western blotting. As shown earlier, exposure of cells to 50 μm Zn2+ for 8 h caused significant decreases of PTEN protein levels and Akt activation. In comparison, pretreatment with the MG132 completely prevented both Zn2+-induced PTEN protein loss as well as Akt activation (Fig. 5, A and B). As seen before (Fig. 3C), Me2SO vehicle control appeared to elevate the baseline of Akt phosphorylation (Fig. 5B). To exclude the potential effect of MG132 on PTEN mRNA expression, PTEN mRNA expression induced by Zn2+ in BEAS-2B cells was examined using reverse transcriptase-PCR as described above. Using the same exposure conditions, MG132 had no significant effect on Zn2+-induced PTEN mRNA expression (Fig. 5C). These data strongly implied that the 26 S proteasome played a critical role in Zn2+-induced PTEN degradation. One of the required steps in the proteasome degradation pathway is the formation of an ubiquitin-protein conjugate (56Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). The covalent addition of multiple ubiquitin molecules to the target protein is requisite for efficient recognition and degradation by the 26 S proteasome (18Tolkacheva T. Boddapati M. Sanfiz A. Tsuchida K. Kimmelman A.C. Chan A.M. Cancer Res. 2001; 61: 4985-4989PubMed Google Scholar). Therefore, the state of ubiquitination of PTEN in Zn2+-exposed cells was determined using an immunoprecipitation assay. As shown in Fig. 6, treatment of BEAS-2B cells with Zn2+ induced PTEN ubiquitination in a dose-dependent manner. These data strongly suggested that Zn2+ treatment led to ubiquitination of PTEN protein, targeting PTEN protein to degradation by the proteasome in airway epithelial cells. A PI3K Inhibitor, LY294002, Blocked Zn2 + -induced PTEN Degradation—The role of PTEN in antagonizing the PI3K/Akt pathway has been well recognized (4Waite K.A. Eng C. Am. J. Hum. Genet. 2002; 70: 829-844Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 13Leslie N.R. Downes C.P. Cell. Signal. 2002; 14: 285-295Crossref PubMed Scopus (354) Google Scholar

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