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• W2024396222 abstract "Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H2O2)-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H2O2 produced in response to thyrotropin (TSH). Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H2O2. In addition, methimazole induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhances the elimination of H2O2 produced by TSH in FRTL-5 cells. Treatment with 500 μm H2O2 causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e. terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling, BAX expression, and poly(ADP-ribose) polymerase cleavage. Overexpression of Prx I and Prx II reduces the amount of H2O2-induced apoptosis measured by these assays. These results suggest that Prx I and Prx II are involved in the removal of H2O2 in thyroid cells and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H2O2 in thyroid cells. Peroxiredoxins (Prxs) play an important role in regulating cellular differentiation and proliferation in several types of mammalian cells. One mechanism for this action involves modulation of hydrogen peroxide (H2O2)-mediated cellular responses. This report examines the expression of Prx I and Prx II in thyroid cells and their roles in eliminating H2O2 produced in response to thyrotropin (TSH). Prx I and Prx II are constitutively expressed in FRTL-5 thyroid cells. Prx I expression, but not Prx II expression, is stimulated by exposure to TSH and H2O2. In addition, methimazole induces a high level of Prx I mRNA and protein in these cells. Overexpression of Prx I and Prx II enhances the elimination of H2O2 produced by TSH in FRTL-5 cells. Treatment with 500 μm H2O2 causes apoptosis in FRTL-5 cells as evidenced by standard assays of apoptosis (i.e. terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling, BAX expression, and poly(ADP-ribose) polymerase cleavage. Overexpression of Prx I and Prx II reduces the amount of H2O2-induced apoptosis measured by these assays. These results suggest that Prx I and Prx II are involved in the removal of H2O2 in thyroid cells and can protect these cells from undergoing apoptosis. These proteins are likely to be involved in the normal physiological response to TSH-induced production of H2O2 in thyroid cells. thyrotropin peroxiredoxin methimazole terminal deoxynucleotidyl transferase deoxyuridine triphosphate-biotin nick end labeling poly(ADP-ribose) polymerase 2′,7′-dichlorofluorescein 2′,7′-dichlorfluorescein diacetate Thyroid epithelial cells are constantly exposed to reactive oxygen species because they produce a large amount of hydrogen peroxide (H2O2) in response to thyrotropin (TSH)1 (1.Kimura T. Okajima F. Sho K. Kobayashi I. Kondo Y. Endocrinology. 1995; 136: 116-123Crossref PubMed Scopus (60) Google Scholar, 2.Bjorkman U. Ekholm R. Endocrinology. 1992; 130: 393-399Crossref PubMed Scopus (67) Google Scholar, 3.Leseney A.M. Deme D. Legue O. Ohayon R. Chanson P. Sales J.P. Pires de Carvalho D. Dupuy C. Virion A. Biochimie. 1999; 81: 373-380Crossref PubMed Scopus (56) Google Scholar, 4.Kimura T. Okajima F. Kikuchi T. Kuwabara A. Tomura H. Sho K. Kobayashi I. Kondo Y. Am. J. Physiol. 1997; 273: E639-E643PubMed Google Scholar, 5.Raspe E. Dumont J.E. Endocrinology. 1995; 136: 965-973Crossref PubMed Scopus (56) Google Scholar). A high level of H2O2 can induce an oxidative stress response in thyrocytes, which signals the cell nucleus to arrest growth and undergo apoptosis (6.Riou C. Tonoli H. Bernier-Valentin F. Rabilloud R. Fonlupt P. Rousset B. Endocrinology. 1999; 140: 1990-1997Crossref PubMed Scopus (36) Google Scholar, 7.Riou C. Remy C. Rabilloud R. Rousset B. Fonlupt P. J. Endocrinol. 1998; 156: 315-322Crossref PubMed Scopus (29) Google Scholar, 9.Bretz J.D. Arscott P.L. Myc A. Baker Jr., J.R. J. Biol. Chem. 1999; 274: 25433-25438Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 10.Bretz J.D. Rymaszewski M. Arscott P.L. Myc A. Ain K.B. Thompson N.W. Baker Jr., J.R. J. Biol. Chem. 1999; 274: 23627-23632Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 11.Giordano C. Stassi G. De Maria R. Todaro M. Richiusa P. Papoff G. Ruberti G. Bagnasco M. Testi R. Galluzzo A. Science. 1997; 275: 960-963Crossref PubMed Scopus (540) Google Scholar). Because H2O2can directly damage DNA (12.Nishida S. Nakano T. Kimoto S. Kusunoki T. Suzuki K. Taniguchi N. Murata K. Tomura T.T. FEBS Lett. 1997; 416: 69-71Crossref PubMed Scopus (10) Google Scholar) and other biological macromolecules (13.Fayadat L. Niccoli-Sire P. Lanet J. Franc J.L. J. Biol. Chem. 1999; 274: 10533-10538Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), it has been suggested that thyrocytes should have mechanisms to control the intracellular level of H2O2. Although thyroid cells utilize several cellular defense systems against oxidative damage, including antioxidant proteins, superoxide dismutase (14.Cadet J. Delatour T. Douki T. Gasparutto D. Pouget J.P. Ravanat J.L. Sauvaigo S. Mutat. Res. 1999; 424: 9-21Crossref PubMed Scopus (533) Google Scholar), catalase (15.Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59Crossref PubMed Scopus (575) Google Scholar, 16.Nakamura Y. Makino R. Tanaka T. Ishimura Y. Ohtaki S. Biochemistry. 1991; 30: 4880-4886Crossref PubMed Scopus (33) Google Scholar), and glutathione (17.Villette S. Bermano G. Arthur J.R. Hesketh J.E. FEBS Lett. 1998; 438: 81-84Crossref PubMed Scopus (22) Google Scholar), the exact mechanisms involved in regulating intracellular H2O2 are not known. The antithyroid drug methimazole (MMI) is an immunomodulatory agent (18.Cho B.Y. Shong M. Yi K.H. Lee H.K. Koh C.S. Min H.K. Clin. Endocrinol. 1992; 36: 585-590Crossref PubMed Scopus (46) Google Scholar, 19.Mozes E. Zinger H. Kohn L.D. Singer D.S. J. Clin. Immunol. 1998; 18: 106-113Crossref PubMed Scopus (22) Google Scholar) that has been used to restore euthyroidism and stop progression of autoimmune disease. Several immunological actions of MMI have been described including alteration of lymphocyte function (20.Balazs C. Kiss E. Leovey A. Farid N.R. Clin. Endocrinol. 1986; 25: 7-16Crossref PubMed Scopus (53) Google Scholar) and modulation of MHC class I and class II expression (21.Montani V. Shong M. Taniguchi S.I. Suzuki K. Giuliani C. Napolitano G. Saito J. Saji M. Fiorentino B. Reimold A.M. Singer D.S. Kohn L.D. Endocrinology. 1998; 139: 290-302Crossref PubMed Scopus (39) Google Scholar, 22.Saji M. Shong M. Napolitano G. Palmer L.A. Taniguchi S.I. Ohmori M. Ohta M. Suzuki K. Kirshner S.L. Giuliani C. Singer D.S. Kohn L.D. J. Biol. Chem. 1997; 272: 20096-20107Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). It has also been suggested that MMI can scavenge free radicals (20.Balazs C. Kiss E. Leovey A. Farid N.R. Clin. Endocrinol. 1986; 25: 7-16Crossref PubMed Scopus (53) Google Scholar, 23.Heufelder A.E. Wenzel B.E. Bahn R.S. J. Clin. Endocrinol. Metab. 1992; 74: 737-742Crossref PubMed Scopus (40) Google Scholar), but the molecular basis of this action is not known. Recently, the ability of peroxiredoxins (Prxs) to eliminate H2O2 was described in a variety of cells in response to external stimuli (24.Lee T.H., Yu, S.L. Kim S.U. Kim Y.M. Choi I. Kang S.W. Rhee S.G. Yu D.Y. Gene. 1999; 234: 337-344Crossref PubMed Scopus (32) Google Scholar, 25.Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (202) Google Scholar). Prx exists as multiple isoforms in mammalian cells (26.Lyu M.S. Rhee S.G. Chae H.Z. Lee T.H. Adamson M.C. Kang S.W. Jin D.Y. Jeang K.T. Kozak C.A. Mamm. Genome. 1999; 10: 1017-1019Crossref PubMed Scopus (28) Google Scholar), namely Prx I (also known as NKEF A, MSP 23, and PAG), Prx II (NKEF B), Prx III (MER 5 and Aop1), and Prx IV (AOE 372). The amino acid sequence of Prx is similar to thioredoxin peroxidase, a 25-kDa peroxidase initially identified in yeast (27.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 28.Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar) that reduces H2O2 using thioredoxin (29.Netto L.E.S. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Most Prx family members include two conserved cysteine residues (2-Cys Prx) (30.Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar). Recombinant Prx reduces H2O2 using electrons from the nonphysiological electron donor dithiothreitol (15.Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59Crossref PubMed Scopus (575) Google Scholar,30.Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar). Although the physiological donor remains unknown, Prx is activein vivo as a peroxidase when overexpressed in NIH 3T3 cells (27.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 31.Jin D.Y. Chae H.Z. Rhee S.G. Jeang K.T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Most isoforms of Prx are abundant in the cytosol of almost every tissue (29.Netto L.E.S. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 30.Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar, 31.Jin D.Y. Chae H.Z. Rhee S.G. Jeang K.T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). However, the mechanisms that control the level of intracellular peroxiredoxins are still obscure (32.Matsumoto A. Okado A. Fujii T. Fujii J. Egashira M. Niikawa N. Taniguchi N. FEBS Lett. 1999; 443: 246-250Crossref PubMed Scopus (130) Google Scholar, 33.Lim M.J. Chae H.Z. Rhee S.G., Yu, D.Y. Lee K.K. Yeom Y.I. Gene. 1998; 216: 197-205Crossref PubMed Scopus (43) Google Scholar). This study describes the expression and functions of Prx I and Prx II in FRTL-5 thyroid cells. These two Prx isoforms are constitutively expressed, but the expression of Prx I is modulated by TSH and MMI. Prx I and Prx II play a role in the elimination of H2O2 produced in response to TSH. In addition, Prx I and Prx II modulate the apoptotic response to H2O2 in thyroid cells. These results are the first evidence that peroxiredoxins are involved in regulating the level of intracellular H2O2 induced by TSH and H2O2-induced apoptosis in the thyroid gland. Highly purified bovine TSH was from Sigma. The antibodies to peroxiredoxin isoforms were provided by Dr. Rhee (NHLBI, National Institutes of Health, Bethesda, MD). These antibodies were generated by injecting rabbits with a keyhole limpet hemocyanin conjugated peptide that corresponds to the sequences in the COOH-terminal region of Prx isoforms (27.Kang S.W. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6303-6311Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). [α-32P]dCTP (3000 Ci/mmol) was from NEN Life Science Products. The source of all other materials was Sigma, unless otherwise noted. FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD) were a fresh subclone (F1) that had all properties previously detailed (34.Kohn, L. D., Valente, W. A., Grollman, E. F., Aloj, S. M., and Vitti, P. (September 2, 1986) U. S. Patent 4,609,622Google Scholar). Their doubling time with TSH was 36 ± 6 h; without TSH, they did not proliferate. After cells were maintained for 6 days in medium lacking TSH, addition of 1 milliunit/ml TSH stimulated thymidine incorporation into DNA by at least 10-fold. Cells were diploid and between their 5th and 20th passages. Cells were grown in 6H medium consisting of Coon's modified F12 supplemented with 5% calf serum, 1 mm nonessential amino acids and a mixture of six hormones: bovine TSH (1 milliunit/ml), insulin (10 μg/ml), cortisol (0.4 ng/ml), transferrin (5 μg/ml), glycyl-l-histidyl-l-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml). Fresh medium was added to all cells every 2 or 3 days, and cells were passaged every 7–10 days. In individual experiments, cells were shifted to 5H medium lacking TSH and 5% calf serum; after incubation in this medium, TSH, MMI, or other agents were added as noted. For Northern analysis, total RNA was prepared from the tissues of a Harlan Sprague-Dawley rat according to the standard method (35.Kingston R.E. Chomczynski P. Sacchi N. Ausubel F.M. Current protocol in Molecular Biology. 1. John Wiley & Sons, Inc., New York1993: 4.2.1-4.2.8Google Scholar) In FRTL-5 thyroid cells, total cellular RNA was isolated by standard procedures and Northern analysis were performed as described (36.Suzuki K. Mori A. Ishii K.J. Saito J. Singer D.S. Klinman D.M. Krause P.R. Kohn L.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2285-2290Crossref PubMed Scopus (172) Google Scholar, 37.Taniguchi S.I. Shong M. Giuliani C. Napolitano G. Saji M. Montani V. Suzuki K. Singer D.S. Kohn L.D. Mol. Endocrinol. 1998; 12: 19-33Crossref PubMed Scopus (32) Google Scholar). Final washes are carried out at 65 °C in 1× saline/sodium phosphate/EDTA (150 mm NaCl, 10 mm NaH2PO4, 1 mmEDTA, pH 7.4). The Prx I and Prx II cDNA probes were described previously (38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Rat β-actin probe was kindly provided by B. Paterson (NCI, National Institutes of Health). All probes were radiolabeled by random priming (Amersham Pharmacia Biotech). The eukaryotic overexpression vectors for Prx I and Prx II were prepared as previously described (38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 39.Ohmori M. Ohta M. Shimura H. Shimurat Y. Suzuki K. Kohn L.D. Mol Endocrinol. 1996; 10: 1407-1424PubMed Google Scholar). Prx I and Prx II coding sequences were prepared by polymerase chain reaction from pETprxI and pETprxII, respectively (38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). The polymerase chain reaction products were subcloned into pCR3.1TM basic vector (Invitrogen, Carlsbad, CA) to yield pCRprx I and pCRprx II. Clones containing the coding sequences in the correct orientation were selected and used for transfection. All plasmid preparations were purified twice by CsCl gradient centrifugation (40.Montani V. Taniguchi S.I. Shong M. Suzuki K. Ohmori M. Giuliani C. Napolitano G. Saji M. Fiorentino B. Reimold A.M. Ting J.P. Kohn L.D. Singer D.S. Endocrinology. 1998; 139: 280-289Crossref PubMed Scopus (27) Google Scholar). Transient transfections were carried out with FRTL-5 cells at 80% confluency (41.Park E.S. You S.H. Kim H. Kwon O.Y. Ro H.K. Cho B.Y. Taniguchi S.I. Kohn L.D. Shong M. Thyroid. 1999; 9: 601-612Crossref PubMed Scopus (15) Google Scholar) and 20 μg of pCRprx I and pCRprx II or equivalent molar amounts of the pCR3.1TM basic vector. Transient transfection used an electroporation technique (Gene Pulser II, Bio-Rad). Cells were harvested, washed, and suspended at 1.5 × 107 cells/ml in 0.8 ml of electroporation buffer (272 mm sucrose, 7 mm sodium phosphate at pH 7.4, and 1 mm MgCl2). Cells were pulsed (330 V; capacitance, 900 microfarads), plated (approximately 6 × 106 cells/dish), and cultured for 48 h. Cell viability was approximately 80%. Immunoblot analyses were performed using anti-Prx I or anti-Prx II antibody (38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Adherent FRTL-5 cells were stimulated in the presence or absence of MMI (1 mm), TSH (1 milliunit/ml) or H2O2 (100 μm) for 4 h at 37 °C. The treated cells were scraped, lysed by addition of SDS sample buffer (62.5 mmTris-HCl (pH 6.8), 6% (w/v) SDS, 30% glycerol, 125 mmdithiothreitol, 0.03% (w/v) bromphenol blue) and separated by 10% SDS-polyacrylamide gel electrophoresis along with biotinylated molecular weight standards. The proteins were transferred to a nitrocellulose membrane by electrotransfer for 2 h. After soaking the membrane in blocking buffer (1× Tris-buffered saline, 0.1% Tween-20 with blocking reagent 5% milk) the membrane was incubated with the primary antibodies (anti-Prx I and anti-Prx II antibodies) overnight at 4 °C. Blots were developed using horseradish peroxidase-linked anti-rabbit secondary antibody and chemiluminescent detection system (Phototope®-horseradish peroxidase Western blot detection kit, New England Biolabs). Intracellular H2O2 was assayed in FRTL-5 cells with a fluorescent dye, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), as described (42.Ohba M. Shibanuma M. Kuroki T. Nose K. J. Cell Biol. 1994; 126: 1079-1088Crossref PubMed Scopus (436) Google Scholar). Briefly, phosphate-buffered saline-washed FRTL-5 cells were stimulated with TSH (1 milliunit/ml), rapidly washed once with Krebs-Ringer solution, and then incubated in Krebs-Ringer solution containing DCFH-DA (5 μg/ml). DCFH-DA is nonpolar and readily diffuses into cells, where it is hydrolyzed to the nonfluorescent polar derivative DCFH and thereby trapped within the cells. In the presence of H2O2, DCFH is oxidized to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). DCF fluorescence was measured with a Zeiss Axiovert 135 inverted microscope equipped with a X20 Neoflur objective and Zeiss LSM410 confocal attachment. To avoid photooxidation of DCFH, fluorescent images were collected with a single rapid scan (four-line average; total scan time, 4.33 s) and identical parameters such as contrast and brightness for all samples. The cells were then examined by differential interference contrast microscopy. Five groups of 10–20 subconfluent cells or 20–30 confluent cells were randomly selected from the image for each sample. The average fluorescence intensity for each group was calculated from the fluorescence intensity per cell. Averages were from five group values. Apoptosis of FRTL-5 cells were evaluated by using an detection kit (Promega, Inc., Madison, WI). FRTL-5 cells that were transfected with pCRprx I and pCRprx II were cultured on glass coverslips for 48 h after reaching confluency. The cell-coated coverslips were rinsed three times with phosphate-buffered saline and fixed with 4% paraformaldehyde at 4 °C for 20 min. Coverslips were rinsed with phosphate-buffered saline, and the cells were made permeable by incubating in 0.2% Triton® X-100 in phosphate-buffered saline at 4 °C for 15 min. Cells with fragmented nuclear DNA were detected using terminal deoxynucleotidyl transferase (0.5 units/μl) and fluorescein isothiocyanate-labeled dUTP (0.5 nmol/μl) from Promega (Madison, WI); incubations were performed according to the manufacturer's instructions. Fluorescein isothiocyanate-dUTP fluorescence was detected using the following filter combinations: BP 450–490/LP520 installed on an Episcopic fluorescence microscope from Nikon, Inc. (Melville, NY). The proportion of apoptotic cells was determined by dividing the number of cells with a TUNEL-positive nucleus, measured on 10–20 randomly taken fields by the total number of cells in the corresponding fields. Protein concentration was determined by the Bradford method (Bio-Rad) and used recrystallized bovine serum albumin as the standard. All experiments were repeated at least three times with different batches of cells. Values are the mean ± S.E. Significance between experimental values was determined by two-way analysis of variance. The expression of Prx I and Prx II was examined by Northern hybridization analysis using RNA from rat tissues (Fig.1 A). Hybridization conditions were stringent in order to avoid cross-hybridization of the Prx I and Prx II cDNA probes. Single Prx I and Prx II transcripts were detected at variable expression levels in the tissues examined. Prx I mRNA was expressed at a lower level in the brain than in testis, kidney, muscle, liver, lung, spleen, thyroid, and heart (Fig. 1 A, top panel). Prx II mRNA was expressed at much higher level in heart than in other tissues (Fig. 1 A). Prx II mRNA was expressed at a comparable level in thyroid, testis, kidney, and liver. The regulation of Prx gene expression was studied in FRTL-5 thyroid cells treated with TSH, H2O2, forskolin, or MMI (Fig. 1 B). Cells were cultured in 6H5% medium until reaching confluence, maintained in 5% 5H medium lacking TSH for 7 days, and treated with TSH, H2O2, forskolin, or MMI for 2 h. The expression of Prx I and Prx II mRNA was up-regulated by the addition of TSH, H2O2, or forskolin (Fig. 1 B). The expression of Prx I increased in the presence of MMI, but the expression of Prx II was not changed by this reagent (Fig. 1 B). The TSH-induced expression of Prx I and Prx II peaked within 2 h after the addition of TSH to FRTL-5 cells (data not shown). Prx I and Prx II protein expression was readily detected by Western blot of extracts from FRTL-5 cells (Fig.2 A, lane 1), and the level of Prx I protein increased and was maintained at a high level in the presence of TSH, H2O2 or MMI (Fig. 2 A, top panel, lanes 3–5). However, the level of Prx II protein did not increase in the presence of TSH, H2O2 or MMI (Fig. 2 A, middle panel). Iodide did not change the level of Prx I or Prx II protein expression (Fig. 1 A, lane 2). TSH stimulated the expression of Prx I maximally after approximately 4 h (Fig. 2 B, top panel, lane 1 versus lanes 5 and6), but it did not stimulate the expression of Prx II until 6 h of exposure (Fig. 2 B). These results indicate that Prx I and Prx II are expressed in thyroid cells and that Prx I is up-regulated by TSH, H2O2, and MMI. H2O2 is produced by TSH in thyroid cells. The effect of Prx I and Prx II on this process was examined by transiently overexpressing mouse Prx I and II in FRTL-5 cells. Cells were co-transfected with a β-galactosidase reporter construct to normalize values and correct for differences in transfection efficiency. The intracellular concentration of H2O2 was monitored with the oxidation-sensitive fluorescent probe DCFH-DA and confocal microscopy (Fig.3 A). The addition of exogenous H2O2 increases the DCF fluorescence in these cells (data not shown). Similarly, DCF fluorescence rapidly increases after the addition of TSH to TSH-starved cells (Fig. 3). DCF fluorescence reached its maximal level within 10 min after TSH treatment (Fig. 3 B). This system monitors the intracellular concentration of H2O2 in cells under different conditions or in different cell lines. The following experiment was carried out with FRTL-5 cells transiently transfected with vector (pCR3.1) or expression plasmids for Prx I (pCRprx I) or Prx II (pCRprx II). As expected, TSH stimulated DCF fluorescence in cells with vector alone (Fig. 4). Interestingly, overexpression of Prx I or Prx II inhibited the TSH-induced increase in DCF fluorescence (Fig. 4, B and C). Enhanced expression of Prx I and Prx II after transfection were confirmed by immunoblot analysis (Fig. 4 A). These findings suggest that Prx I and Prx II are involved in the elimination of H2O2 produced by TSH in FRTL-5 cells.Figure 4Effect of Prx overexpression on TSH-induced H2O2 in FRTL-5 cells. FRTL-5 cells were cultured as described in the legend to Fig. 3 and transiently transfected with the indicated expression plasmids. The expression of Prx I and Prx II was measured by immunoblot analysis (A). After 3 days, DCF fluorescence was measured with a confocal microscope after incubation of the cells in the presence of TSH (1 milliunit/ml) for 5 min (B). Relative fluorescence intensity per cell was calculated as described under “Experimental Procedures.” Data shown are means ± S.E. of the values from five groups of 20–30 cells (C). pCR 3.1 is the vector without an insert.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the above findings indicate that Prx I and Prx II are involved in eliminating H2O2 in thyroid cells, it seemed possible that Prx I and Prx II could inhibit H2O2-mediated apoptosis in thyroid cells. H2O2 (0.1–1 mm) induces apoptotic events in FRTL-5 cells without cycloheximide or actinomycin D (data not shown). FRTL-5 cells were transiently transfected with Prx I and Prx II expression vectors or the vector control plasmid and cultured for 24 h in 6H medium with 5% calf serum. Cells were transferred to 5H medium lacking serum and TSH for 36 h and treated with 500 μmH2O2 for 36 h. As shown in Fig.5 A, this treatment induces apoptosis in FRTL-5 cells transfected with the control plasmid pCR3.1 (Fig. 5 A, panel 2); about 60% of these cells were positive in the TUNEL assay (Fig. 5 B). In contrast, FRTL-5 cells overexpressing Prx I or Prx II were less apoptotic, and fewer cells were positive in the TUNEL assay (Fig. 5 B). Prx I and Prx II were approximately equally effective in inhibition of H2O2-mediated apoptosis (Fig.5 B). In a parallel experiment, expression of the proapoptotic proteins BAX and poly(ADP-ribose) polymerase (PARP) was monitored by Western blot. Prx I- and Prx II-transfected cells were treated with 500 μm H2O2 for 36 h (Fig.6). FRTL-5 cells transfected with the control plasmid pCR3.1 expressed a very low level of BAX expression (Fig. 6, lane 1), which increased dramatically in cells treated with H2O2 (Fig. 6, lane 2). Expression of Prx I or Prx II significantly inhibited the expression of BAX after treatment with H2O2. These results suggest an antiapoptotic role for Prx I and Prx II in cells treated with H2O2. Further support for this idea was obtained by monitoring PARP cleavage after treating cells with H2O2. A small amount of the 85-kDa cleaved PARP fragment was present in FRTL-5 cells, which may be related to the serum starvation of these cells prior to treatment with H2O2. In cells transfected with the control vector, the level of uncleaved PARP was significantly reduced by treatment with H2O2. In contrast, expression of Prx I and Prx II inhibited PARP cleavage, so that a normal level of uncleaved PARP protein was maintained after treatment with H2O2. These findings support the suggestion that Prx I and Prx II protect thyroid cells against H2O2-induced apoptosis. This study provides evidence for two significant conclusions: 1) Prx I and Prx II are involved in eliminating H2O2 produced by thyroid cells in physiological response to TSH, and 2) Prx I and Prx II protect thyroid cells from H2O2-induced apoptosis. At present, at least six forms of Prx are known in mammalian cells (25.Chae H.Z. Kang S.W. Rhee S.G. Methods Enzymol. 1999; 300: 219-226Crossref PubMed Scopus (202) Google Scholar,43.Knoops B. Clippe A. Bogard C. Arsalane K. Wattiez R. Hermans C. Duconseille E. Falmagne P. Bernard A. J. Biol. Chem. 1999; 274: 30451-30458Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Prx I and Prx II are cytosolic proteins with peroxidase activity (28.Chae H.Z. Robison K. Poole L.B. Church G. Storz G. Rhee S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7017-7021Crossref PubMed Scopus (706) Google Scholar, 29.Netto L.E.S. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The recombinant forms of Prx I and Prx II reduce hydrogen peroxide using thioredoxin as electron donor (29.Netto L.E.S. Chae H.Z. Kang S.W. Rhee S.G. Stadtman E.R. J. Biol. Chem. 1996; 271: 15315-15321Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 30.Chae H.Z. Rhee S.G. Biofactors. 1994; 4: 177-180PubMed Google Scholar). In addition, Prx I and Prx II reduce H2O2 in cells stimulated with growth factors (31.Jin D.Y. Chae H.Z. Rhee S.G. Jeang K.T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar) and inhibit activation of NF-κB by H2O2 or tumor necrosis factor-α (31.Jin D.Y. Chae H.Z. Rhee S.G. Jeang K.T. J. Biol. Chem. 1997; 272: 30952-30961Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). These results indicate that Prx I and Prx II function as peroxidasesin vivo and may be components of signaling cascades for which H2O2 is an intracellular messenger (38.Kang S.W. Chae H.Z. Seo M.S. Kim K. Baines I.C. Rhee S.G. J. Biol. Chem. 1998; 273: 6297-6302Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Thyroid cells generate large amounts of H2O2 in response to TSH for synthesis of thyroid hormone (44.Carvalho D.P. Dupuy C. Gorin Y. Legue O. Pommier J. Haye B. Virion A. Endocrinology. 1996; 137: 1007-1012Crossref PubMed Scopus (37) Google Scholar, 45.Corvilain B. van Sande J. Laurent E. Dumont J.E. Endocrinology. 1991; 128: 779-785Crossref PubMed Scopus (154) Google Scholar); however, the mechanisms regulating the intracellular concentration of H2O2 are not well understood (46.Ekholm R. Bjorkman U. Endocrinology. 1997; 138: 2871-2878Crossref PubMed Scopus (59) Google Scholar). This report shows that Prx I and Prx II are constitutively expressed in many rat tissues, including the thyroid gland and in rat thyroid FRTL-5 cells (Figs. 1 and 2). In addition, Prx I is up-regulated by TSH in these cells. These results suggest that Prx proteins are involved in regulating intracellular H2O2 levels; for example, TSH-induced Prx I expression may be a mechanism for controlling the H2O2 generated in response to TSH. The mechanism by which TSH up-regulates Prx I expression is not yet known. It is possible that TSH-induced Prx I expression is a response to the increase in H2O2 level resulting from TSH-stimulation. Alternatively, transcription of Prx I and Prx II genes may be induced by activated redox-sensitive transcription factors (47.Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (327) Google Scholar,48.Adler V. Yin Z. Tew K.D. Ronai Z. Oncogene. 1999; 18: 6104-6111Crossref PubMed Scopus (597) Google Scholar). Prx II is expressed constitutively at a higher level than Prx I (Fig. 2), but Prx II protein is not induced by TSH (although its mRNA level is up-regulated by TSH). This result suggests that Prx II may eliminate cytosolic H2O2 constitutively in resting and TSH-stimulated thyroid cells. The mechanism by which H2O2 triggers apoptosis is not well understood (49.Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1479) Google Scholar, 50.von Harsdorf R. Li P.F. Dietz R. Circulation. 1999; 99: 2934-2941Crossref PubMed Scopus (515) Google Scholar). However, H2O2and other reactive oxygen species induce apoptosis in many cell types (51.Vollgraf U. Wegner M. Richter-Landsberg C. J. Neurochem. 1999; 73: 2501-2509Crossref PubMed Scopus (160) Google Scholar, 52.Madeo F. Frohlich E. Ligr M. Grey M. Sigrist S.J. Wolf D.H. Frohlich K.U. J. Cell Biol. 1999; 145: 757-767Crossref PubMed Scopus (874) Google Scholar, 53.Clement M.V. Pervaiz S. Free Radical Res. 1999; 30: 247-252Crossref PubMed Scopus (140) Google Scholar), including thyrocytes (7.Riou C. Remy C. Rabilloud R. Rousset B. Fonlupt P. J. Endocrinol. 1998; 156: 315-322Crossref PubMed Scopus (29) Google Scholar, 9.Bretz J.D. Arscott P.L. Myc A. Baker Jr., J.R. J. Biol. Chem. 1999; 274: 25433-25438Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). However, the physiological mechanisms that protect against apoptosis induced by reactive oxygen species are not clearly elucidated (54.Kamata H. Hirata H. Cell Signal. 1999; 11: 1-14Crossref PubMed Scopus (1013) Google Scholar). Interestingly, FRTL-5 cells that overexpress Prx I or Prx II are resistant to H2O2-mediated apoptosis. The number of cells that scored positive in the TUNEL assay for apoptosis decreased in Prx I- and Prx II-transfected FRTL-5 cells compared with control cells treated with 500 μm H2O2. In addition, the proapoptotic protein Bax was expressed in control cells treated with H2O2, but it was expressed at a lower level in cells expressing Prx I or Prx II (Fig. 6). At present, the mechanism by which Prx I and Prx II protect against H2O2-mediated apoptosis is unclear. However, it is likely that this effect is related to the ability of Prx proteins to eliminate intracellular H2O2 and other reactive oxygen species (55.Zhang P. Liu B. Kang S.W. Seo M.S. Rhee S.G. Obeid L.M. J. Biol. Chem. 1997; 272: 30615-30618Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). TSH is involved in antiapoptotic processes in human (56.Feldkamp J. Pascher E. Perniok A. Scherbaum W.A. Horm. Metab. Res. 1999; 31: 355-358Crossref PubMed Scopus (27) Google Scholar) and rat (57.Li X. Lu S. Miyagi E. Katoh R. Kawaoi A. Endocrinology. 1999; 140: 5962-5970Crossref PubMed Scopus (23) Google Scholar) thyroid cells. This property of TSH may be mediated by decreasing P27 protein, increasing cyclin D expression, and promoting transition from the G1 to the S phase of the cell cycle (57.Li X. Lu S. Miyagi E. Katoh R. Kawaoi A. Endocrinology. 1999; 140: 5962-5970Crossref PubMed Scopus (23) Google Scholar). The results presented here suggest another possible mechanism for this action of TSH; the antiapoptotic effects of TSH may involve its regulation of Prx I protein expression. Similarly, the antithyroid drugs MMI and propylthiouracil have been suggested as oxygen free radical scavengers (8.Imamura M. Aoki N. Saito T. Ohno Y. Maruyama Y. Yamaguchi J. Yamamoto T. Acta Endocrinol. 1986; 112: 210-216Crossref PubMed Scopus (41) Google Scholar, 20.Balazs C. Kiss E. Leovey A. Farid N.R. Clin. Endocrinol. 1986; 25: 7-16Crossref PubMed Scopus (53) Google Scholar, 23.Heufelder A.E. Wenzel B.E. Bahn R.S. J. Clin. Endocrinol. Metab. 1992; 74: 737-742Crossref PubMed Scopus (40) Google Scholar). This study demonstrates that exposure of FRTL-5 cells to MMI enhances expression of Prx I (Fig. 1 B), suggesting that MMI-mediated radical scavenging may depend on its ability to stimulate expression of Prx I. In conclusion, this study demonstrates that two Prx isoforms, Prx I and Prx II, are involved in eliminating H2O2 in thyroid cells and in protecting these cells from H2O2-induced apoptosis. These observations are likely to be physiologically important because large amounts of H2O2 are produced in thyroid cells in response to TSH. Therefore, this study contributes to understanding of the mechanism for regulating intracellular H2O2 in the cells of the thyroid gland. We are grateful to Dr. Zee-Won Lee, Biomolecule Research Team, Korea Basic Science Institute (Taejon, South Korea) for the technical assistance in measuring intracellular H2O2 in thyroid cells." @default.
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• W2024396222 title "Role of Peroxiredoxins in Regulating Intracellular Hydrogen Peroxide and Hydrogen Peroxide-induced Apoptosis in Thyroid Cells" @default.
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