The Human Type 2 Iodothyronine Deiodinase Is a Selenoprotein Highly Expressed in a Mesothelioma Cell Line
2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês
10.1074/jbc.c100325200
ISSN1083-351X
AutoresCyntia Curcio, Munira Muhammad Abdel Baqui, Domenico Salvatore, Bertrand H. Rihn, Steve Mohr, John W. Harney, P. Reed Larsen, Antônio C. Bianco,
Tópico(s)Redox biology and oxidative stress
ResumoTypes 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with75Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have ∼40-fold higher levels ofhDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T4) to 3,5,3′-triiodothyronine with typical characteristics of D2 such as low K m (T4), 1.3 nm, resistance to propylthiouracil, and a short half-life (∼30 min). D2 activity is ∼30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from theDio2 mRNA sequence precipitates a 75Se protein of the predicted 31-kDa size from 75Se-labeled mesothelioma cells. Bromoadenosine 3′5′ cyclic monophosphate increases D2 activity and 75Se-p31 ∼2.5-fold whereas substrate (T4) reduces both D2 activity and 75Se-p31 ∼2–3-fold. MG132 or lactacystin (10 µm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and 75Se-p31 3–4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T4) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T4 activation. Types 1 and 3 iodothyronine deiodinases are known to be selenocysteine-containing enzymes. Although a putative human type 2 iodothyronine deiodinase (D2) gene (hDio2) encoding a similar selenoprotein has been identified, basal D2 activity is not selenium (Se)-dependent nor has D2 been labeled with75Se. A human mesothelioma cell line (MSTO-211H) has recently been shown to have ∼40-fold higher levels ofhDio2 mRNA than mesothelial cells. Mesothelioma cell lysates activate thyroxine (T4) to 3,5,3′-triiodothyronine with typical characteristics of D2 such as low K m (T4), 1.3 nm, resistance to propylthiouracil, and a short half-life (∼30 min). D2 activity is ∼30-fold higher in Se-supplemented than in Se-depleted medium. An antiserum prepared against a peptide deduced from theDio2 mRNA sequence precipitates a 75Se protein of the predicted 31-kDa size from 75Se-labeled mesothelioma cells. Bromoadenosine 3′5′ cyclic monophosphate increases D2 activity and 75Se-p31 ∼2.5-fold whereas substrate (T4) reduces both D2 activity and 75Se-p31 ∼2–3-fold. MG132 or lactacystin (10 µm), inhibitors of the proteasome pathway by which D2 is degraded, increase both D2 activity and 75Se-p31 3–4-fold and prevent the loss of D2 activity during cycloheximide or substrate (T4) exposure. Immunocytochemical studies with affinity-purified anti-hD2 antibody show a Se-dependent increase in immunofluorescence. Thus, human D2 is encoded by hDio2 and is a member of the selenodeiodinase family accounting for its highly catalytic efficiency in T4 activation. thyroxine 3,5,3′-triiodothyronine type 1 iodothyronine deiodinase selenocysteine type 2 iodothyronine deiodinase propylthiouracil human type 3 iodothyronine deiodinase immunoprecipitation cycloheximide 8-bromoadenosine 3′5′ cyclic monophosphate fetal bovine serum human embryonic kidney polyacrylamide gel electrophoresis phosphate-buffered saline Sec insertion sequence A group of three specific deiodinases monodeiodinate thyroxine (T4)1 to 3,5,3′-triiodothyronine (T3), the active thyroid hormone, or 3,3′,5′-triiodothyronine, an inactive metabolite. The first of these enzymes to be cloned was the type 1 iodothyronine deiodinase (D1), which revealed a rare structural characteristic,i.e. the presence of the selenocysteine (Sec) codon, UGA, in the active center of the enzyme conferring an ∼200-fold higher catalytic efficiency than sulfur in the deiodination reaction (1Berry M.J. Banu L. Larsen P.R. Nature. 1991; 349: 438-440Crossref PubMed Scopus (744) Google Scholar). It also revealed a requirement for a stem loop structure in the 3′-untranslated region, the Sec insertion sequence (SECIS) element (2Berry M.J. Banu L. Chen Y.Y. Mandel S.J. Kieffer J.D. Harney J.W. Larsen P.R. Nature. 1991; 353: 273-276Crossref PubMed Scopus (515) Google Scholar), which is found in all eukaryotic selenoprotein mRNAs described to date (3Low S.C. Berry M.J. Trends Biochem. Sci. 1996; 21: 203-208Abstract Full Text PDF PubMed Scopus (389) Google Scholar). The most recently cloned deiodinase is the type 2 (D2), a propylthiouracil (PTU)-resistant, low K m(T4) obligate outer ring deiodinase that catalyzes T4 to T3 conversion (4Croteau W. Davey J.C. Galton V.A. St Germain D.L. J. Clin. Invest. 1996; 98: 405-417Crossref PubMed Scopus (331) Google Scholar, 5Salvatore D. Bartha T. Harney J.W. Larsen P.R. Endocrinology. 1996; 137: 3308-3315Crossref PubMed Scopus (227) Google Scholar). The major open reading frame of the hD2 cDNA encodes a putative ∼31-kDa protein with high similarity of the Sec-containing active center of the putative D2 enzyme with those of D1 and the other member of this group, type 3 iodothyronine deiodinase (D3) (6St. Germain D.L. Schwartzman R.A. Croteau W. Kanamori A. Wang Z. Brown D.D. Galton V.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7767-7771Crossref PubMed Scopus (119) Google Scholar). Furthermore, a SECIS element has been identified in the extreme 3′-untranslated region of both the human and highly homologous chicken Dio2 genes (7Buettner C. Harney J.W. Larsen P.R. J. Biol. Chem. 1998; 273: 33374-33378Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar,8Gereben B. Bartha T. Tu H.M. Harney J.W. Rudas P. Larsen P.R. J. Biol. Chem. 1999; 274: 13768-13776Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Human, mouse, rat, chicken, and frog D2 all contain a putative in-frame UGA codon in the deduced amino acid sequence of the active center (position 133 in hD2) and therefore all are considered to be selenoenzymes (8Gereben B. Bartha T. Tu H.M. Harney J.W. Rudas P. Larsen P.R. J. Biol. Chem. 1999; 274: 13768-13776Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Despite this evidence, there is vigorous disagreement that D2 is a selenoprotein, with some investigators claiming that Dio2encodes a "virtual" or artificial selenoprotein that is not expressed in humans or rats (9Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1991; 266: 13477-13480Abstract Full Text PDF PubMed Google Scholar, 10Safran M. Farwell A.P. Rokos H. Leonard J.L. J. Biol. Chem. 1993; 268: 14224-14229Abstract Full Text PDF PubMed Google Scholar, 11Farwell A.P. Safran M. Dubord S. Leonard J.L. J. Biol. Chem. 1996; 271: 16369-16374Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 12Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 271: 16363-16368Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 13Leonard J.L. Leonard D.M. Safran M. Wu R. Zapp M.L. Farwell A.P. Endocrinology. 1999; 140: 2206-2215Crossref PubMed Scopus (27) Google Scholar, 14Stachelek S.J. Kowalik T.F. Farwell A.P. Leonard J.L. J. Biol. Chem. 2000; 275: 31701-31707Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Three major arguments support this position, namely 1) the failure of Se deficiency to reduce D2 catalytic activity either in vivo or in vitro (9Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1991; 266: 13477-13480Abstract Full Text PDF PubMed Google Scholar, 15Chanoine J.P. Safran M. Farwell A.P. Tranter P. Ekenbarger D.M. Dubord S. Alex S. Arthur J.R. Beckett G.J. Braverman L.E. Leonard J.L. Endocrinology. 1992; 130: 479-484Crossref Scopus (63) Google Scholar, 16Chanoine J.P. Safran M. Farwell A.P. Dubord S. Alex S. Stone S. Arthur J.R. Braverman L.E. Leonard J.L. Endocrinology. 1992; 131: 1787-1792Crossref PubMed Scopus (49) Google Scholar), 2) the inability to identify a 75Se-labeled protein of the expected size in cells expressing D2 (13Leonard J.L. Leonard D.M. Safran M. Wu R. Zapp M.L. Farwell A.P. Endocrinology. 1999; 140: 2206-2215Crossref PubMed Scopus (27) Google Scholar), and 3) the inability to identify immunoreactive protein by Western analysis, immunoprecipitation (IP), or immunocytochemistry using antibodies prepared against peptides deduced from the sequence of the putative D2 mRNA (4Croteau W. Davey J.C. Galton V.A. St Germain D.L. J. Clin. Invest. 1996; 98: 405-417Crossref PubMed Scopus (331) Google Scholar). An alternative scenario put forward is that D2 is a large protein complex (200 kDa) containing one or more 29-kDa substrate-binding subunits (p29), an ∼60-kDa cAMP-induced activation protein, and one or more catalytic subunits (12Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1996; 271: 16363-16368Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Rat p29 was identified in astrocytes byN-bromoacetyl-T4 labeling (17Farwell A.P. Leonard J.L. J. Biol. Chem. 1989; 264: 20561-20567Abstract Full Text PDF PubMed Google Scholar, 18Safran M. Leonard J.L. J. Biol. Chem. 1991; 266: 3233-3238Abstract Full Text PDF PubMed Google Scholar, 19Leonard D.M. Stachelek S.J. Safran M. Farwell A.P. Kowalik T.F. Leonard J.L. J. Biol. Chem. 2000; 275: 25194-25201Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), has no enzymatic activity, and is highly similar to Dickkopf-3 (20Glinka A. Wu W. Delius H. Monaghan A.P. Blumenstock C. Niehrs C. Nature. 1998; 391: 357-362Crossref PubMed Scopus (1331) Google Scholar). Although many of these apparent discrepancies could be explained by low expression of a highly efficient enzyme, reservations as to the identity of D2 are expressed even in recent reviews of the subject (21Kohrle J. Cell. Mol. Life Sci. 2000; 57: 1853-1863Crossref PubMed Scopus (169) Google Scholar,22Kohrle J. Acta Med. Austriaca. 2000; 27: 1-7Crossref PubMed Scopus (35) Google Scholar). A mesothelioma cell line, MSTO-211H, was recently shown to express large amounts of Dio2 mRNA by microarray analysis (accession number U53506), offering a potential system to resolve the issue of D2 identity (23Rihn B.H. Mohr S. McDowell S.A. Binet S. Loubinoux J. Galateau F. Keith G. Leikauf G.D. FEBS Lett. 2000; 480: 95-100Crossref PubMed Scopus (70) Google Scholar). MG132 and lactacystin were obtained from Calbiochem (La Jolla, CA) and dissolved in Me2SO. MG132 is a reversible proteasome inhibitor. Lactacystin blocks proteasome activity by targeting the catalytic β-subunit and covalently inhibiting the chymotrypsin- and trypsin-like activities. T4, cycloheximide (CX), and 8-bromoadenosine 3′5′ cyclic monophosphate (8-Br-cAMP) were from Sigma and were dissolved in 40 mm NaOH (T4) or Me2SO. Protein G plus/protein A-agarose solution was obtained from Oncogene Research Products (Boston, MA). Outer ring-labeled [125I]T4 (specific activity, 4400 Ci/mmol) was from PerkinElmer Life Sciences. Na2[75Se]O3 was kindly provided by the University of Missouri Research Reactor, courtesy of Drs. Marla Berry and Dolph L. Hatfield. All other reagents were of analytical grade. Mesothelioma (MSTO-211H) and mesothelial (MeT-5A) cell lines were obtained from American Type Culture Collection (ATCC; Manassas, VA) and made available through Dr. Rihn. Cells were plated in 60-mm dishes and grown until confluence in RPMI or M-199, respectively, and supplemented with 0–10% fetal bovine serum (FBS). Human embryonic kidney epithelial cells (HEK-293) cells were incubated in Dulbecco's modified Eagle's medium with 0 or 10% fetal bovine serum. Each experiment was performed with duplicate dishes for each condition, and control plates contained the respective vehicles, 0.2% Me2SO and/or 0.6 mm NaOH. At the appropriate times, cells were harvested and processed for D2 activity in the presence of 1 mm PTU as described previously (24Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (86) Google Scholar). The results are reported as fmol T4deiodinated/mg protein·min ± S.D. In some experiments D2 was transiently expressed in HEK-293. Cells were transfected by the CaPO4 method as described previously (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar) with plasmids containing wild type D2 (KD2-SelP), a D2 mutant in which the Sec-133 (but not Sec-266) was replaced by Cys (CysD2), or a CysD2 in which the SECIS element was deleted (CysD2ΔXba). This was performed as described previously (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar) after cells were labeled with 4–6 µCi of Na2[75Se]O3/dish. The lysis buffer contained 25 mm Tris-HCl (pH 7.4), 300 mm NaCl, 1 mm CaCl2, 0.5% Triton X-100, 10 µg/ml leupeptin, 0.2 units/ml aprotinin, 1 mmphenylmethylsulfonyl fluoride. The 2,000-rpm supernatant of each cell lysate was incubated for 16 h at 4 °C with D2 rabbit antisera 85254 at final dilution of 1:100 (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar). 30 µl of protein G plus/protein A-agarose solution were added per tube and incubated under slow agitation for 2 h at 4 °C. The IP pellets were washed once with lysis buffer and once with 25 mm Tris-HCl (pH 7.4), 140 mm NaCl, 1 mm CaCl2. Pellets were re-suspended in sample loading buffer and analyzed in 12% SDS-PAGE. In some experiments IP was processed in unlabeled cells to allow measurement of D2 activity. For the affinity purification of the antiserum 45618 (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar) the keyhole limpet hemocyanin conjugate of the hD2 peptide (EVRSWLEKNFSKR; residues 253 to 265) used to immunize the rabbits was coupled to agarose resin using Amino-Link agarose gel (Pierce) following the manufacture's instructions. The column was regenerated by washing with 3 m sodium acetate (pH 4.5) and equilibrated with phosphate-buffered saline (PBS) prior to each cycle. The D2 antiserum (45618) was passed through the column five times and then washed 10 times with PBS. Bound antibodies were eluted with 3 msodium acetate (pH 4.5), and eluates were dialyzed against PBS at 4 °C for 2 days. This affinity-purified antiserum (AP-45618; 1:400) was used in the immunocytochemical and Western analysis of MSTO-211H or MeT-5A cells as described (26Gereben B. Goncalves C. Harney J.W. Larsen P.R. Bianco A.C. Mol. Endocrinol. 2000; 14: 1697-1708Crossref PubMed Scopus (125) Google Scholar). This was performed as described previously (27Baqui M.M. Gereben B. Harney J.W. Larsen P.R. Bianco A.C. Endocrinology. 2000; 141: 4309-4312Crossref PubMed Scopus (104) Google Scholar). MSTO-211H and MeT-5A cells were grown directly in glass slides for 24 h, fixed with 3.7% paraformaldehyde in PBS (pH 7.4), permeabilized with 0.5% Triton X-100 in PBS for 10 min, and then blocked for 30 min with 1.0% anti-goat serum, 1.0% bovine serum albumin in PBS. Affinity-purified rabbit D2 antibody (AP-45618) was used at 1:500 followed by incubation with 1.25 µg/ml goat anti-rabbit fluorescein isothiocyanate (Molecular Probes, Eugene OR). In the co-localization experiments, the affinity-purified anti-goat GRP78/BiP (dilution 1:20) antibody (Research Diagnostics, Inc., Flanders, NJ) was used simultaneously with anti-D2 antibody. This was followed by incubation with donkey anti-goat rhodamine F(ab′)2 fragment (Jackson ImmunoResearch Laboratories, West Grove, PA) and with goat anti-rabbit fluorescein isothiocyanate. Mesothelioma (MSTO-211H) cell sonicate converts T4to T3 with kinetic properties typical of native D2, namely a K m for T4 of ∼1.0 nm and insensitivity to inhibition by 1 mm PTU as shown in the Lineweaver-Burk plot (Fig.1 A). Neither D1 nor D3 activities were detected nor did the transformed normal mesothelial cells (MeT-5A) express D2 activity (not shown). D2 activity in MSTO-211H cells was highly Se-dependent. Basal activity was decreased 4-fold by reducing the concentration of FBS containing endogenous selenoproteins (Fig. 1 B). That these changes are specifically caused by Se deficiency and not by other components in FBS is evident from the fact that Se supplementation progressively increased D2 activity at each concentration of FBS, from 2–3-fold at 10% FBS up to ∼30-fold when no FBS was present (Fig. 1 B). The Lineweaver-Burk plots from cells incubated in the absence of FBS with or without 100 nm Na2SeO3 for 24 h shows an ∼16-fold increase in V maxwith no change in the K m (Fig. 1 A). A dose-response curve of D2 activity versusNa2SeO3 concentration indicated that the maximum stimulatory effect is obtained at 100 nm. A time-response curve showed that an effect of Se is present after 2 h, but the full effect requires 8 h (not shown). Similar effects of Se to increase D2 activity were observed in HEK-293 cells transiently expressing Sec-encoding D2 mRNA. Exposure to 100 nm Na2SeO3 for 24 h increased D2 activity by ∼10-fold suggesting this effect is primarily post-transcriptional (Fig. 1 B). To confirm that the MSTO-211H cells express a selenoprotein containing a deduced D2 epitope, cells were labeled with Na2[75Se]O3 for 24 h and processed for D2 IP using an anti-hD2 peptide antiserum (85254) (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar). Mesothelial (MeT-5A) cells were used as negative controls. Four75Se-labeled bands of ∼70, 58, 25, and 18 kDa (but not 31 kDa) are present in the SDS-PAGE of total cell lysates of MSTO-211H or MeT-5A cells (Fig. 1 C, b). These75Se-labeled proteins are increased by incubation with 100 nm Na2SeO3 in MSTO-211H cells but are decreased in MeT-5A (Fig. 1 C, b). SDS-PAGE analysis of the anti-D2 IP of the MSTO-211H cell lysate revealed an ∼31-kDa 75Se-labeled doublet band (p31) of the predicted size of hD2, the intensity of which is increased ∼2-fold in the presence of 100 nm Na2SeO3 (Fig.1 C, c and d). A doublet is seen when hD2 cDNA is transiently expressed because of the presence of a second UGA at the 3′-end of the open reading frame that can be translated either as a Sec or as a stop codon (28Salvatore D. Harney J.W. Larsen P.R. Biochimie (Paris). 1999; 81: 1-4Crossref Scopus (32) Google Scholar). The content of all other 75Se-labeled proteins is reduced in the anti-D2 IP pellets (Fig. 1 C, c). IP of non-labeled cell lysates reduced D2 activity ∼20%, and ∼13of this activity can be recovered from the washed IP pellet (Fig.1 D). Addition of the antiserum to the cell lysate did not inhibit D2 activity nor did exposure to preimmune serum or protein A-Sepharose (not shown). Despite these IP results, we were unable to consistently visualize D2 by Western blot analysis of MSTO-211H cell lysates using affinity-purified anti-D2 antibody (AP-45618). The hD2 gene contains a canonical cAMP-response element-binding protein-binding site about 90 base pairs 5′ to the most 5′-transcription start site (29Bartha T. Kim S.W. Salvatore D. Gereben B. Tu H.M. Harney J.W. Rudas P. Larsen P.R. Endocrinology. 2000; 141: 229-237Crossref PubMed Scopus (89) Google Scholar). D2 activity increases in cells treated for 6 h with 8-Br-cAMP in a dose-dependent fashion (Fig. 2 A). To test whether there is a correlation between 8-Br-cAMP-induced D2 activity and p31, cells treated with 1 mm 8-Br-cAMP were labeled with 75Se and processed for D2 IP. The intensity of the75Se-p31 band increased ∼2.5-fold in 8-Br-cAMP-treated cells (Fig. 2 F). No effects of insulin (0.1–5 µm) or tumor necrosis factor-α (0.01–10 ng/ml) on D2 activity were detected (data not shown). Both endogenous and transiently expressed D2 have a short-life (<1 h) that is reduced further by exposure to substrates such as 3,3′,5′-triiodothyronine or T4 (24Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (86) Google Scholar, 25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar, 30Leonard J.L. Kaplan M.M. Visser T.J. Silva J.E. Larsen P.R. Science. 1981; 214: 571-573Crossref PubMed Scopus (173) Google Scholar, 31St. Germain D.L. Endocrinology. 1986; 119: 840-846Crossref PubMed Scopus (39) Google Scholar, 32Leonard J.L. Biochem. Biophys. Res. Commun. 1988; 151: 1164-1172Crossref PubMed Scopus (83) Google Scholar). This is explained by ubiquitin conjugation and subsequent proteasomal degradation of the ubiquitinated D2 (25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar, 26Gereben B. Goncalves C. Harney J.W. Larsen P.R. Bianco A.C. Mol. Endocrinol. 2000; 14: 1697-1708Crossref PubMed Scopus (125) Google Scholar). Treatment of cells with 100 µm CX for 1 h resulted in a rapid loss of D2 activity, compatible with a D2 half-life of <30 min (Fig.2 B). The decrease in D2 activity was prevented by the proteasome inhibitor, MG132 (10 µm) (Fig. 2 B). Furthermore, exposure to MG132 increased D2 activity up to ∼4-fold, with similar results obtained with 10 µm lactacystin (Fig. 2 C). Remarkably, anti-D2 precipitable75Se-p31, but not other labeled proteins, was also specifically increased by treatment with MG132 (Fig. 2 F). Exposure to substrate (0.01–10 µm T4 in 10% FBS) for 1 h caused a concentration-dependent reduction in D2 activity of up to 10-fold (Fig. 2 D), which again was blocked by MG132 (Fig. 2 E). This was accompanied by a reduction in the anti-D2 precipitable 75Se-p31 (Fig.2 F). MSTO-211H and MeT-5A cells were also analyzed by immunocytochemistry using an affinity-purified anti-D2 antibody (AP-45618). In MSTO-211H cells incubated in serum-free medium for 24 h, the cytoplasm showed mild staining for D2 (Fig.3 A). Treatment with 100 nm Na2SeO3 for 24 h substantially increased the intensity of the staining without changing the distribution pattern (Fig. 3 B). No fluorescence was detected in MSTO-211H cells incubated with secondary antibody alone (Fig. 3 C) or in Na2SeO3–treated MeT-5A cells incubated with affinity-purified anti-D2 antibody (Fig.3 D). To study the subcellular distribution of endogenously expressed D2, MSTO-211H cells were stained with D2 antibody (Fig.3 F) and co-stained with antibody raised against the endoplasmic reticulum resident protein GRP78/BiP (Fig. 3 G), shown previously to colocalize with transiently expressed D2 in HEK-293 and neuroblastoma cells (27Baqui M.M. Gereben B. Harney J.W. Larsen P.R. Bianco A.C. Endocrinology. 2000; 141: 4309-4312Crossref PubMed Scopus (104) Google Scholar). Co-localization was confirmed by superimposition of confocal images (Fig. 3 H). The Se-supplemented MSTO-211H cells have the highest D2 activity reported to date in a human tissue (∼140 fmol/min/mg protein), ∼40% higher than in Graves' thyroid tissue (33Salvatore D. Tu H. Harney J.W. Larsen P.R. J. Clin. Invest. 1996; 98: 962-968Crossref PubMed Scopus (169) Google Scholar). This high D2 expression permits the unequivocal demonstration that endogenous hD2 is a selenoprotein encoded by the Dio2 gene (5Salvatore D. Bartha T. Harney J.W. Larsen P.R. Endocrinology. 1996; 137: 3308-3315Crossref PubMed Scopus (227) Google Scholar). The present results satisfy a number of important criteria establishing the identity of hD2 as the product of the Dio2 gene. Furthermore, they allow confirmation that the endogenous human enzyme behaves as predicted from a number of studies using transiently expressed protein. The first important criterion to establish D2 as a selenoprotein is to demonstrate its Se dependence. Past studies showed that the elevated D2 in cerebral cortex of thyroidectomized rats is not reduced by Se deficiency (15Chanoine J.P. Safran M. Farwell A.P. Tranter P. Ekenbarger D.M. Dubord S. Alex S. Arthur J.R. Beckett G.J. Braverman L.E. Leonard J.L. Endocrinology. 1992; 130: 479-484Crossref Scopus (63) Google Scholar) and that astroglial cells depleted of Se for 3–5 days did not have lower basal D2 activity (9Safran M. Farwell A.P. Leonard J.L. J. Biol. Chem. 1991; 266: 13477-13480Abstract Full Text PDF PubMed Google Scholar). Although there was about 50% reduction of the D2 activity in cAMP-treated primary astrocyte cultures after 7 days of Se deprivation, basal D2 activity was still not affected (34Pallud S. Lennon A.M. Ramauge M. Gavaret J.M. Croteau W. Pierre M. Courtin F. St. Germain D.L. J. Biol. Chem. 1997; 272: 18104-18110Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). However, the central nervous system is well known to retain Se with high efficiency, which could explain why D2 synthesis is not impaired by Se deficiency (35Behne D. Hilmert H. Scheid S. Gessner H. Elger W. Biochim. Biophys. Acta. 1988; 966: 12-21Crossref PubMed Scopus (390) Google Scholar). In MSTO-211H cells, on the other hand, Se depletion for 24 h reduced basal D2 activity by ∼4-fold. This decrease in D2 activity was specifically reversed by Se supplementation (Fig. 1, Aand B). In fact, addition of Se increased D2 activity by ∼30-fold in Se-depleted cells, in a dose- and time-dependent fashion, a phenomenon that also occurs in HEK-293 cells transiently expressing D2 (Fig. 1, A andB). Selenium depletion of MSTO-211H cells also markedly reduces the levels of another selenoprotein, glutathione peroxidase (36Sandstrom B.E. Marklund S.L. Biochem. J. 1990; 271: 17-23Crossref PubMed Scopus (80) Google Scholar). A second criterion met by the present studies is the 75Se labeling and immunological recognition of D2. A recent study could not identify the 75Se-D2 protein by IP using antibodies that were raised against the COOH terminus of full-length rat D2 or against the catalytic core of the enzyme (13Leonard J.L. Leonard D.M. Safran M. Wu R. Zapp M.L. Farwell A.P. Endocrinology. 1999; 140: 2206-2215Crossref PubMed Scopus (27) Google Scholar). This is not the case in the present studies (see Fig. 1 C and Fig. 2 F). Aside from potential differences in the antisera per se, which might be an issue, the D2 activity in rat tissue sonicates or stimulated primary astrocyte cultures are only ∼10% of that in MSTO-211 cells. Because of the higher D2 expression, we can demonstrate IP of D2 activity, as well as of a 75Se-labeled protein of the predicted size (31 kDa) from MSTO-211H cells (Fig. 1 C). This is the first demonstration of endogenous 75Se-D2. The75Se-labeled 31-kDa protein is increased by Na2SeO3, mirroring changes in D2 activity (Fig.1 C). A correlation between changes in D2 activity and the amount of 75Se-p31 is also evident during treatment with proteasome inhibitors (see below), 8-Br-cAMP, or D2 substrate (Fig.2). Additional immunological identity criteria were obtained by immunocytochemical studies using the affinity-purified anti-hD2 antibody. Staining was only present in MSTO-211H cells and was markedly increased by Se supplementation (Fig. 3, A–D). Furthermore, D2 co-localized with the endoplasmic reticulum-specific marker BiP as found previously in HEK-293 and neuroblastoma cells transiently expressing hD2 (Fig. 3, E–H) (27Baqui M.M. Gereben B. Harney J.W. Larsen P.R. Bianco A.C. Endocrinology. 2000; 141: 4309-4312Crossref PubMed Scopus (104) Google Scholar). Despite successful IP of the D2 protein and activity, we were unable to visualize D2 by Western blot analysis presumably because of low antibody affinity for denatured protein, low D2 expression, or both. A short half-life and the acceleration of endogenous D2 degradation by exposure of cells to substrates observed in pituitary tumor cells and in cells transiently expressing D2 are reproduced in these experiments. A short half-life (<1 h) is characteristic of D2 in all cells studied (11Farwell A.P. Safran M. Dubord S. Leonard J.L. J. Biol. Chem. 1996; 271: 16369-16374Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 24Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (86) Google Scholar, 31St. Germain D.L. Endocrinology. 1986; 119: 840-846Crossref PubMed Scopus (39) Google Scholar) and is explained by the fact that D2 is the target of selective ubiquitination and proteolysis by the proteasomal system (24Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (86) Google Scholar, 25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar, 26Gereben B. Goncalves C. Harney J.W. Larsen P.R. Bianco A.C. Mol. Endocrinol. 2000; 14: 1697-1708Crossref PubMed Scopus (125) Google Scholar). This is also the case in MSTO-211H cells, in which D2 activity and 75Se-D2 protein levels were extremely sensitive to inhibitors of the proteasomal pathway (Fig. 2,B–E). It is remarkable that in GH4C1 rat pituitary tumor and transfected HEK-293 cells, treatment with proteasomal inhibitors for 1 h increases D2 activity by only ∼20% (24Steinsapir J. Harney J. Larsen P.R. J. Clin. Invest. 1998; 102: 1895-1899Crossref PubMed Scopus (86) Google Scholar, 25Steinsapir J. Bianco A.C. Buettner C. Harney J. Larsen P.R. Endocrinology. 2000; 141: 1127-1135Crossref PubMed Scopus (89) Google Scholar) whereas in the present study a similar treatment resulted in a 3–4-fold increase in activity (Fig. 2 C). This can be explained by a more rapid turnover of the enzyme in mesothelioma cells. Furthermore, as in pituitary tumor cells and transfected HEK-293 cells, exposure to T4, the preferred D2 substrate, accelerates the turnover, increasing ubiquitination and subsequent proteolysis, a phenomenon that can be specifically inhibited by treatment with proteasomal inhibitors (Fig. 2, D–E). These results demonstrate that the targeting of D2 to the ubiquitin-proteasomal system and the acceleration of its degradation are characteristic of this protein, regardless of the cell type in which it is present or whether it is transiently expressed or endogenous. In conclusion, the evidence developed in the present study unequivocally establishes that endogenous hD2 is a short-lived selenoprotein, the product of the human Dio2 gene, as well as the 90–95% homologous frog, chicken, mouse, and rat proteins. These findings are also consistent with preliminary data reported recently that targeted disruption of the mouse Dio2 gene eliminates D2 activity in brain, pituitary gland, and brown adipose tissue (37Schneider M. Fiering S. St. Germain D.L. Galton V.A. 12th International Thyroid Congress, Kyoto, Japan, October 22–27, 2000. Asia and Oceania Thyroid Association, Kyoto, Japan2000Google Scholar). From a broader perspective it is interesting to note that this is the second example of high levels of a selenodeiodinase in human tumor cells derived from a tissue that normally does not express deiodinase, the other being the high levels of D3 in hemangiomas (38Huang S.A. Tu H.M. Harney J.W. Venihaki M. Butte A.J. Kozakewich H.P. Fishman S.J. Larsen P.R. N. Engl. J. Med. 2000; 343: 185-189Crossref PubMed Scopus (396) Google Scholar). It will be of interest to determine whether primary mesotheliomas also express high levels of D2 and what, if any, effects of increased rate of T4 to T3 conversion might have in these cells. We thank Dr. Stephen Huang for assaying MSTO-211H cell lysates for D3 activity.
Referência(s)