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Department of Endocrinology and Metabolism (A.I., F.K., H.S.), Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan; and Department of Internal Medicine II (A.I., K.O., T.H.), Nagoya University School of Medicine, Nagoya 466-8550, Japan
Address all correspondence and requests for reprints to: Fukushi Kambe, Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute of Environmental Medicine, Furo-cho, Chikusa-ku, Nagoya University, Nagoya 464-8601, Japan. E-mail: kambe{at}riem.nagoya-u.ac.jp
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thyroid hormone synthesis is tightly regulated by TSH, the prime regulator for thyroid function: the transcription of the TG, TPO, TSH receptor and sodium/iodide symporter genes is under control of TSH (2, 3, 4, 5, 6). In addition, production of H2O2, which is necessary for iodide oxidation at the step of iodide incorporation into TG, is also regulated by TSH (7, 8). Furthermore, TSH regulates proliferation of thyroid follicular cells (9, 10).
In various types of cells, it has been demonstrated that the cellular reduction-oxidation (redox) status modulates various aspects of cellular function (11). Oxidative stress can induce cell proliferation as well as growth inhibition or cell death. It has been also shown that change in intracellular redox status regulates the function of transcription factors, leading to alteration in gene expression (12, 13). Recently, we and others have demonstrated that the DNA-binding activities of Pax-8, TTF-1, and TTF-2 are regulated posttranslationally by redox state of the molecule (14, 15, 16, 17). It has been also demonstrated in some cells that reactive oxygen intermediates (ROIs) such as H2O2 and O2- act as an intracellular signal-transducing molecule to regulate cellular function and proliferation (18, 19, 20).
Based on these findings, we postulated that H2O2 or other ROIs, generated in response to TSH, might play a role in regulating the function of thyroid follicular cells. In the present study, we therefore investigated the effects of antioxidants, which scavenge ROIs, on expression of Pax-8, TTF-1, TG, and TPO, and on proliferation of rat thyroid FRTL-5 cells. We used two kinds of antioxidants, pyrrolidine dithiocarbamate (PDTC) and N-acetyl-L-cysteine (NAC). NAC enters cells readily and scavenges ROIs directly or indirectly by its conversion to L-cysteine and by increasing intracellular glutathione (21). PDTC contains two thiol moieties and is thus generally recognized as an antioxidant (22). However, it has been recently shown that PDTC can chelate and transport redox-active metal ions into cells, and exert prooxidant effect on rat thymocytes (23). The present study will show that PDTC and NAC exert their effects on the function and proliferation of thyroid cells with a conspicuous difference that could be attributable to the prooxidant effect of PDTC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
FRTL-5 cells (ATCC CRL8305), which were less than 10 passages
after the purchase from ATCC were cultured in Coon’s modified Ham’s
F-12 medium (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 5% heat-inactivated calf serum (Life Technologies, Inc.) and with six hormones (6H) including
hydrocortisone (10 nM), transferrin (5 µg/ml),
somatostatin (10 ng/ml),
glycyl-L-histidyl-L-lysine acetate (10 ng/ml),
bovine TSH (1 mU/ml) and insulin (10 µg/ml). The F-12 medium contains
2.5 ng/ml of CuSO4
5H2O and 830 ng/ml of
FeSO4 7H2O. The cells were
treated with 10 µM to 80 µM PDTC or 5
mM to 20 mM NAC for various lengths of time. In
some experiments, the cells maintained in the TSH-deprived medium (5H)
for 5 days were used.
Northern blot analysis
Total RNA was extracted from FRTL-5 cells by the method of
Chomczynski and Sacchi (24). Northern blot and
hybridization were carried out as described previously
(25). In brief, total RNA (15 µg) was fractionated in
0.8% agarose gels, and then was transferred onto a nylon membrane
(Gene Screen Plus; NEN Life Science Products, Boston, MA).
Heat-denatured complementary DNA (cDNA) probes were labeled with
[32P]dCTP using the random primed DNA labeling
kit (Roche Molecular Biochemicals). Preparation of cDNAs
for rat Pax-8, TTF-1, TPO, TG, and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) were described previously (26, 27).
After hybridization and wash, the membrane was subjected to imaging by
Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) to quantify radioactivities of bands. The
membrane was then autoradiographed.
Preparation of antirat Pax-8 antiserum
The polypeptide named PACP (CSAPPTSATAFDHL) spanning
amino acid residues 445–457, C-terminal region of rat Pax-8, was
synthesized and purified to more than 95% purity by HPLC. Additional
cysteine (in italic) was introduced at N-terminal
of PACP to facilitate conjugation with a carrier protein. PACP was
mixed with methylated bovine albumin (28): 180 µl of
PACP (10 mg/ml in 0.15 M NaCl), 720 µl of
methylated bovine albumin (2.5 mg/ml in 0.15 M
NaCl) and 300 µl of 0.15 M NaCl were mixed and
incubated at 25 C for 30 min. The mixture was stored at -30 C as 100
µl aliquots.
Three female rabbits (Nihon Hakushoku-shu) weighing around 2.5 kg were purchased from Nippon Bio-Supply Center (Tokyo, Japan). We used 150 µg PACP for each immunization. One hundred µl of PACP mixture, 400 µl of 0.15 M NaCl, and 500 µl of complete Freund adjuvant (Difco Laboratories, Detroit, MI) were completely mixed to emulsion using glass syringes. After preimmune blood was drawn from an ear vein, the emulsion (1 ml) was injected intradermally into 10 to 15 separate positions on the back. Freshly prepared emulsion was used for booster immunizations at 2, 4, and 8 weeks after the first injection. Blood samples were collected 4, 6, 8, and 9 weeks after the first immunization. Antibody titer was assessed by dot blot ELISA (enzyme-linked immunosorbent assay) as described (29). All the sera obtained after the last bleeding exhibited specific binding to PACP and were successfully used for immunoprecipitation and supershift analysis in electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA)
Preparation of whole cell extracts and nuclear extracts, and
procedures of EMSA were described previously (27). Protein
concentration of the extracts was determined by a microassay kit
(Bio-Rad Laboratories, Inc., Richmond, CA) using BSA as a
standard. The nucleotide sequence of EMSA probes are as follows: oligo
P which contains recognition sites for Pax-8/TTF-1 in the rat TPO
promoter (1), 5'-TGTCTAAGCTTGAGTGGGCATCAGA-3'; oligo EN,
which contains a recognition site for Pax-8 in the E2 region of the
human TPO enhancer (14, 30),
5'-CTAGAACCAGGGATTCTTCACACTTCATAGAGCACCTCTAG-3'; oligo Z, which
contains a recognition site for TTF-2 in the rat TPO promoter
(1), 5'-AGAAATACTAAACAAACAGAATCG-3'; oligo K, which
contains a recognition site for TTF-2 in the rat TG promoter
(31), 5'-TGACTAGCAGAGAAAACAAAGTGAGC-3'; GADD45, which
contains a p53-binding site in the promoter of growth-arrest DNA-damage
inducible gene 45 (32),
5'-AGCAGAACATGTCTAAGCATGCTGGGCTCG-3'. The probes were prepared by
annealing sense- and antisense-oligonucleotides and by extending the
nucleotides with Klenow enzyme in the presence of
[32P]dCTP. Preparation of oligo Ct, which was
used for the competition experiment was described previously
(14). Whole cell extract (20 µg) or nuclear extract (10
µg) was preincubated in a final volume of 20 µl containing 40
mM HEPES-KOH (pH 7.9), 75 mM KCl, 0.2
mM EDTA-NaOH (pH 8.0), 10% glycerol, 0.5 mM
DTT, and 2 µg poly (dI-dC) (Amersham Pharmacia Biotech, Uppsala, Sweden). In some experiments, DTT was omitted.
After 15-min incubation on ice, 0.03 pmol of a
32P-labeled probe and 3 pmol of competitor
oligonucleotide for competition experiment were added, and incubated
for 30 min at 25 C. In experiments using anti-Pax-8 antiserum or
anti-p53 antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), it was added after this incubation, and further incubated
for 1 h at 4 C. The reaction mixture without dye was loaded
directly onto a 4% polyacrylamide gel (30: 1, acrylamide:
bisacrylamide) in 45 mM Tris-HCl (pH 8.0), 45
mM boric acid and 1 mM EDTA-NaOH (pH 8.0) and
electrophoresed at 160 V at 4 C. The gel was dried on filter paper and
then autoradiographed.
Metabolic labeling and immunoprecipitation
Metabolic labeling was carried out 12 h after treatment
with PDTC (40 µM) or NAC (20 mM). Detailed
procedure was reported previously (33). In brief, the
cells were preincubated in methionine-free Eagle’s MEM (Nissui
Pharmaceutical Co., Tokyo, Japan) for 2 h and then incubated in
methionine-free MEM containing [35S]methionine
(Amersham Pharmacia Biotech, Arlington Heights, IL) and
incubated for an additional 4 h. Immunoprecipitation was performed
using antirat Pax-8 antiserum and Staphylococcus aureus
protein A (Pansorbin; Calbiochem, San Diego, CA). The
precipitates were subjected to 12% SDS-PAGE. After fixation, gels were
incubated in an enhancer (Amplify, Amersham Pharmacia Biotech) for fluorography, dried, and exposed to BAS 2000
system. The gel was then subjected to fluorography.
Transfection
The 395-bp rat TPO promoter sequence (the position 1 to 395 in
EMBL/DDBJ accession number x61170) was cloned by PCR, and ligated to
the upstream of luciferase gene in pGL2-basic plasmid (Promega Corp., Madison, WI). The plasmid was named pTPOpro. The
construction of rat Pax-8-expressing plasmid (pPAX8) was described in
our previous report (14). In brief, the full-length cDNA
for rat Pax-8 was ligated into mammalian expression vector pRC/RSV5.2
(Invitrogen Corp., San Diego, CA). FRTL-5 cells were grown
in 6H media in 12-well plates. Transient transfections were carried out
using calcium-phosphate precipitation method with 1 µg of reporter
plasmid pTPOpro or promoter-less plasmid pGL2-basic together with or
without 1 µg of pPAX8. In all the experiments, 0.1 µg of
pSVß-galactosidase plasmid (Promega Corp.) was
cotransfected, and the total amount of plasmid DNA was kept constant by
addition of pRC/RSV5.2 carrying no cDNA insert. After overnight
exposure to the DNA-CaPO4 precipitate, the medium
was replaced with the fresh medium containing 40 µM PDTC.
After a 30-h incubation, the cells were lysed by addition of 100 µl
glycylglycine buffer (25 mM glycylglycine pH 7.8, 15
mM MgSO4, 4 mM EGTA, 1
mM DTT) containing 0.2% Triton x100. Aliquots (20 µl)
were mixed with 300 µl of glycylglycine buffer containing 2
mM ATP and 15 mM potassium phosphate. One
hundred microliters of glycylglycine buffer containing 0.2
mM D-luciferin was injected to the mixture
using automatic device in LUMAT LB9501 (Berthold, Germany) and
luciferase activity was measured for 10 sec. The ß-galactosidase
activity was measured using Luminescent ß-gal detection kit
(CLONTECH Laboratories, Inc., Palo Alto, CA) and LUMAT
LB9501 according to the instruction from the supplier. The luciferase
activities were normalized by ßgalactosidase activities.
Assays for cell proliferation
Cell proliferation was assessed by WST-1 assay and
3H-thymidine incorporation method. WST-1 assay
was performed using Cell Counting Kit (Dojindo, Kumamoto, Japan).
FRTL-5 cells (3 x 103/well) were plated in
96-well microplates. After preincubation for 48 h, they were
exposed to various concentrations of PDTC or NAC for 24 h. After
the exposure, 10 µl of a solution of the Cell Counting Kit was added
to the wells, followed by the incubation for 2 h. This solution
contains water-soluble tetrazolium salt,
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium,
monosodium salt (WST-1). In viable cells, WST-1 is enzymatically
converted into a water soluble and noncytotoxic formazan dye. Optical
densities at 450 nm of the media were measured using a microplate
reader (Bio-Rad Laboratories, Inc. Richmond, CA)
(34, 35).
3H-thymidine incorporation was performed as follows. Cells were plated in 12-well culture plates at a density of 5 x 104 cells per well, and then treated with various concentration of PDTC or NAC for 12 h. During the last 2 h, 10 µCi/ml of 3H-thymidine (37MBq/ml; NEN Life Science Products) was added to the medium. The incorporation of 3H-thymidine was determined after precipitation of acid-insoluble material with ice-cold 10% trichloroacetic acid. The acid-insoluble material was dissolved in 2% SDS and counted in a liquid scintillation counter.
H2O2 determination
Concentration of
H2O2 in cell culture media
was determined by homovanillic acid fluorescence assay (7)
(8) (36). FRTL-5 cells were maintained in 5H
media in 12-well culture plates. The medium was then replaced with 0.85
ml/well HEPES-buffered medium (pH 7.4) containing 1 mU TSH alone or in
combination with 300 U/ml catalase (purified from mouse liver,
Sigma), 40 µM PDTC, and 20 mM
NAC. After a 2-h incubation, 440 µM homovanillic acid and
0.5 U/ml horseradish peroxidase were added to the medium. The
HEPES-buffered medium consisted of 10 mM HEPES, 134
mM NaCl, 4.7 mM KCl, 1.2 mM
KH2PO4, 1.2 mM
MgSO4, 2.0 mM
CaCl2, 2.5 mM
NaHCO3, 5 mM glucose, and 0.1% BSA.
After 45-min incubation at 37 C, the dishes were chilled on ice to
terminate the reaction. A portion of the medium was collected, and
fluorescence of the medium was measured using a Hitachi
650–10S fluorescence spectrophotometer (Hitachi Co. Ltd.,
Tokyo, Japan). The excitation wavelength was set at 315 nm, and the
emission wavelength at 425 nm. A standard curve was prepared by
measuring the samples containing a different amount of
H2O2. The activity of
horseradish peroxidase per se was not affected by the
presence of 40 µM PDTC or 20
mM NAC (data not shown).
Determination of intracellular concentration of copper and
iron
FRTL-5 cells cultured in 6H medium were treated with PDTC (40
µM) alone or together with BCS (0.4 mM) for
12 h. After washing the cells with PBS several times, the whole
cell extracts were prepared as described previously (14).
Copper and iron contents were determined by using a
Hitachi Z6100 atomic absorption spectrophotometer
(Hitachi), and corrected by protein contents.
Statistical analysis
Statistical analysis was carried out by using one-way ANOVA
followed by Fisher’s protected least significant difference analysis.
The P value less than 0.05 is considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, single bands of TPO mRNA (3.7 kb in
size), TG mRNA (8.5 kb), Pax-8 mRNA (3.0 kb), and TTF-1 mRNA (2.5 kb)
were detected in the control, nontreated cells. The sizes of these
mRNAs were comparable to the previous reports (26, 37).
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Surprisingly, treatment of the cells with 20 mM NAC had only a marginal effect on TPO mRNA and no effect on Pax-8 mRNA. Contrary to the effect of PDTC, NAC decreased GAPDH mRNA.
As shown in Fig. 2, the decreased level
of TPO mRNA was apparent at 12 h after treatment with 40
µM PDTC, whereas it increased GAPDH mRNA as early as at
3 h, followed by a gradual increase. TG mRNA levels were not
altered at any time point studied. These results demonstrate that PDTC
specifically reduces Pax-8 and TPO mRNA, and increases GAPDH mRNA level
in FRTL-5 cells.
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, a protein with a molecular weight of
approximately 50 kDa was detected in nontreated control cells, being
compatible with the estimated molecular weight of murine Pax-8
(38). However, this protein was not precipitated with
preimmune serum. Treatment with PDTC markedly decreased Pax-8
synthesis, which is compatible with the decrease in the mRNA level.
Note that NAC had almost no effect on the synthesis.
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We at first characterized the binding of the transcription factors to
oligo P, which corresponds to the P region of the rat TPO promoter. As
shown in Fig. 4A, three protein/DNA
complexes with different mobility were detected by EMSA using this
oligonucleotide as a probe (lane 1). These three complexes were
displaced by excess amount of cold oligo P (lane 2). When oligo Ct,
which has a complete TTF-1 binding site in rat TG promoter but lacks
Pax-8 binding site, was used as a competitor, the slowest-migrating
complex was completely displaced (lane 3), indicating that this complex
represents TTF-1/oligo P complex. Unrelated competitor oligo K, which
is recognized by TTF-2, did not displace any of the complexes (lane 4).
When antirat Pax-8 antiserum was added to the EMSA reaction buffer, the
complex in the middle disappeared, and a supershifted complex as
indicated by an asterisk appeared (lane 5), indicating that
the middle complex represents Pax-8/oligo P complex. The
fastest-migrating complex was not characterized.
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). At 6 h after PDTC (40 µM), a
substantial decrease in the Pax-8 binding was detected, and the
reduction continued until 12 h, whereas TTF-1 binding was only
marginally affected. The reduction of Pax-8 binding by PDTC was
reproduced using another probe, oligo EN (Fig. 4C), which had been
shown to bind with Pax-8 predominantly (14). The Pax-8
binding was decreased at 6 and 12 h after PDTC. In contrast, 20
mM NAC had no effect on the Pax-8 binding (data not shown).
It should be noted that reduction of Pax-8 binding precedes the
decrease in TPO mRNA level. On the other hand, TTF-2 binding to oligo Z
and K (1) was not affected by PDTC (Fig. 4, D and E).
These results strongly suggest that attenuation of DNA-binding of Pax-8
to the promoter is involved in the decreased expression of TPO mRNA by
PDTC. We previously demonstrated that the reduction of Pax-8 is required for its full binding activity (14). To examine whether PDTC affects the redox status of Pax-8, the cell extracts were prepared without DTT from the cells treated with or without PDTC, and subjected to EMSA in the absence of DTT. Pax-8 in the extracts prepared with or without DTT bound to oligo EN to a similar extent (data not shown), indicating that it is fully reduced in the presence of TSH and that PDTC does not affect the redox status of Pax-8.
Overexpression of Pax-8 restores TPO promoter activities
To confirm the involvement of Pax-8 in the decreased expression of
TPO gene by PDTC, we examined whether introduction of Pax-8-expressing
plasmid could restore the TPO promoter activity by transient
transfection study (Fig. 5). When TPO
promoter-luciferase plasmid (pTPOpro) was transfected into FRTL-5
cells, the luciferase activity was markedly increased above that of
promoterless plasmid (pGL2-basic). Treatment of the transfected cells
with PDTC (40 µM) significantly decreased the luciferase
activity. Cotransfection of Pax-8-expressing plasmid (pPAX8) resulted
in the restoration of the TPO promoter activities, indicating that the
decreased TPO gene expression by PDTC is caused by the reduced
expression of Pax-8.
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, treatment with 40 µM
and 80 µM PDTC for 24 h significantly increased the
cell number when compared with that of nontreated cells.
3H-thymidine incorporation was also increased by
PDTC (Fig. 6B). In contrast, NAC at the concentration of 10
mM or 20 mM had no effects on the cell
growth.
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, when
the nuclear extracts prepared from FRTL-5 cells were subjected to EMSA
using GADD45 as a probe, a single band of protein/DNA complex was
detected. This band was completely displaced by the excess amount of
cold GADD45 oligonucleotide, and the density of the band was decreased
by addition of anti-p53 antibody, indicating that the single band
represents p53/GADD45 complex. Because p53 has been shown to bind DNA
as a homotetramer, the antibody might prevent the tetramer formation or
its DNA binding itself.
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, DNA-binding activity of p53 was decreased as early as 3 h after
PDTC. In contrast, 20 mM NAC had no effect on p53 binding
(data not shown).
Does H2O2 play a role in modulation of
thyroid function?
PDTC and NAC are generally recognized as antioxidants. FRTL-5
cells are demonstrated to produce
H2O2 under control of TSH
(7) (8). We therefore examined the capacity
of these antioxidants to scavenge
H2O2. Homovanillic
acidfluorescence assay was employed to determine
H2O2 content in medium. As
shown in Fig. 8A, TSH increased the
generation of H2O2 by more
than 2-fold when compared with the level of the cells cultured in the
absence of TSH (5H). Addition of purified catalase, which metabolizes
H2O2 into
H2O, into the medium markedly reduced the
fluorescence, indicating that more than 90% of the fluorescence is
derived from the H2O2
generated by the cells. Treatment of the cells with TSH along with 40
µM PDTC or 20 mM NAC almost completely
scavenged the generated
H2O2. Considering the
different effects of PDTC and NAC on TPO and Pax-8 expression and on
the cellular proliferation, it is suggested that the effects of PDTC
cannot be attributed to its
H2O2-scavenging action.
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).
Even in the absence of TSH, PDTC significantly decreased the levels of
TPO and Pax-8 mRNAs and increased GAPDH mRNA. In contrast, NAC had no
effects. These results again suggest that
H2O2-scavenging action of
PDTC does not account for its effect on TPO and Pax-8 expression and on
the cell proliferation.
Noncell-permeable chelator of copper (BCS) inhibits PDTC
effects
It has been shown that PDTC has not only antioxidant property but
also prooxidant property. As a prooxidant, PDTC binds and transports
extracellular redox active copper into the cells (23). We
thus determined intracellular levels of copper in FRTL-5 cells. As
shown in Table 1, treatment of the
cells with PDTC (40 µM) for 12 h resulted in a
significant increase in the intracellular copper. Treatment of the
cells with 0.4 mM BCS which is a noncell-permeable chelator
of Cu1+ (41) and thereby inhibits
PDTC-dependent transport of Cu1+, prevented the
increase in the intracellular copper by PDTC. In contrast,
intracellular iron levels were not altered by PDTC and BCS.
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, the treatment
with BCS abrogated the PDTC-induced decrease in TPO and Pax-8 mRNA
levels in a dose-dependent manner. Note that BCS alone had no effect on
the mRNA levels. This result indicates that elevation of intracellular
Cu1+ concentration causes the decreased
expression of TPO and Pax-8 genes.
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, the
PDTC-induced decrease in p53 binding was reversed by the treatment with
BCS, indicating that the reduced binding of p53 is also due to
elevation of intracellular Cu1+. To test whether
the increase in copper ion affects p53 binding, nuclear extracts from
FRTL-5 cells were incubated with various concentrations of
CuSO4 in vitro. As shown in Fig. 9C, p53
binding was almost completely inhibited by addition of
CuSO4 at a concentration of 100 µM,
suggesting that the direct interaction of copper with p53 reduces its
DNA binding. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Interestingly, despite similar organizations of cis-regulatory elements for Pax-8, TTF-1 and TTF-2 in the TPO and TG promoters (1), the expression of TG mRNA was not affected by PDTC. This might indicate that the TPO gene expression is more tightly regulated by Pax-8 than TG gene. A previous report that showed that transfection of a plasmid expressing Pax-8 activates TPO promoter better than TG promoter (1), supports this notion. It was also shown by the in situ hybridization method using human specimens of the follicular adenomas that Pax-8 mRNA level correlated well with that of TPO mRNA (42).
On the other hand, PDTC treatment of FRTL-5 cells increased GAPDH mRNA levels and stimulated cell proliferation rates. It was also demonstrated that PDTC induced a decrease in the DNA-binding activity of p53. Because it has been shown that the role of p53 in G1 arrest is largely dependent on transcriptional activation of its target gene p21 (43), and that p53-deficient cells exhibit accelerated proliferation rates (44), the reduction of p53 binding might be responsible for the PDTC-dependent promotion of growth of FRTL-5 cells.
Difference in PDTC and NAC effects on thyroid cells
It was reported that
H2O2 acts as an
intracellular signal-transducing molecule for platelet-derived growth
factordependent cell proliferation in vascular smooth muscle cells
(18). Therefore, we postulated a similar role of
H2O2 in thyroid follicular
cells and expected that PDTC and NAC would have similar effects on
thyroid cell functions by scavenging
H2O2. To our surprise, the
effect of PDTC was different from that of NAC: PDTC promoted cell
growth and inhibited the TPO and Pax-8 expression, whereas NAC had
almost no effects on these parameters. Because PDTC and NAC scavenged
the cellular H2O2 to a
similar extent, TSH-induced production of
H2O2 seems not to play a
role in thyroid cell growth and in Pax-8 and TPO expression.
Increase in intracellular copper by PDTC
PDTC also acts as a chelator of ions and exerts a prooxidant
effect by transporting the external redox-active ions into cells. We
thus examined the alteration in intracellular contents of copper and
iron by PDTC. It was clearly shown that PDTC increased the
intracellular concentration of copper, whereas iron contents were not
altered. The copper increased by PDTC must be a form of
Cu1+, because BCS, a noncell-permeable chelator
of Cu1+, inhibited the PDTC-dependent transport
of copper. Similar observations were reported in thymocytes and breast
cancer cell line MCF-7. In thymocytes, it was shown that PDTC increases
intracellular copper and induces oxidative stress, resulting in toxic
effects on the cells including apoptotic cell shrinkage and chromatin
fragmentation (23). In MCF-7 cells, PDTC increases the
intracellular copper, and down-regulates p53 DNA-binding activity
(40).
Consistent with the latter report, the present study demonstrated that PDTC induced a decrease in p53 binding activity and that BCS abrogated the effects of PDTC, indicating the causal effect of the increase in intracellular Cu1+ in FRTL-5 cells. Our study further showed that the PDTCdependent reduction of TPO and Pax-8 expression was also reversed by BCS treatment. However, it remains to be studied how Cu1+ decreases the expression of Pax-8. Copper is an essential transition metal that plays an important role in function of some transcription factors and enzymes. Intracellular copper exists in several oxidation status, which can change from one redox status to another under physiological conditions. This redox cycling is responsible for physiological and toxic potential of this metal. Recently, it was shown that Pax-8 mRNA levels are under control of TSH (45, 46). It is thus speculated that Cu1+ might affect redox-sensitive molecules in the intracellular signaling cascades of TSH.
Modification of p53 by PDTC
Recent study demonstrated that p53 directly binds with
Cu1+, and loses its DNA-binding activity in
vitro (47). Thus, the observed PDTC effect on p53 in
the present study might be explained by a direct interaction between
p53 and Cu1+. Accordingly, our in
vitro study demonstrated that CuSO4 at as
low as 100 µM inhibits the p53 binding.
Another possible mechanism for PDTC effect on p53 is that PDTC might disrupt the structure of DNA-binding domain of p53 by chelating zinc ion because it was demonstrated that zinc ion is required for the formation of normal structure of the domain (48). It was also reported that PDTC promotes the oxidation of cysteine residue in p53 and inhibits p53-mediated transactivation (49). Thus, redox modification of cysteine residue in p53 could result in loss of its binding ability.
Physiological role of intracellular copper
It was reported that the average copper level in normal human
hepatic tissues is 11.43 ± 4.74 µg/g of tissue (180 ± 74
µM), and it increases in the cirrhotic tissues
(15.53 ± 5.90 µg/g) (50). The present study
demonstrated that an increase in the intracellular copper levels from
10.6 ± 1.4 µg/g to 16.0 ± 1.2 µg/g by PDTC affected the
thyroid-specific gene expressions and proliferation. Furthermore, the
treatment of the cell extracts with 100 µM
CuSO4 markedly reduced the p53 binding
activities. These results indicate that the change in intracellular
copper levels in a physiological range influences the function of
thyroid follicular cells.
In conclusion, the present study demonstrated that intracellular copper levels and its redox state are crucial for the TPO and Pax-8 expressions, and p53 function. Recently, it has been shown that the loss-of-function mutation in the p53 gene, usually found in the region for the DNA-binding domain, affects thyroid differentiation phenotypes: introduction of mutated p53 into differentiated cell line abolished TPO and TG expression as well as Pax-8 and TTF-2 expression, and induced TSH-independent cell growth (51). Conversely, introduction of wild-type p53 into undifferentiated thyroid carcinoma cell line induced reexpression of TPO gene (52). Collectively, it is suggested that the redox regulation including intracellular copper levels and its redox cycle plays a role in control and maintenance of the differentiated phenotype and growth of thyroid follicular cells by modulating the p53 function.
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Acknowledgments |
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Footnotes |
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Received July 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B
by tumor necrosis factor-
and its inhibition by antioxidants in rat
thyroid FRTL-5 cells. Endocrinology 139:1715–1722This article has been cited by other articles:
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  X. Cao, F. Kambe, X. Lu, N. Kobayashi, S. Ohmori, and H. Seo Glutathionylation of Two Cysteine Residues in Paired Domain Regulates DNA Binding Activity of Pax-8 J. Biol. Chem., July 8, 2005; 280(27): 25901 - 25906. [Abstract] [Full Text] [PDF]
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 R. Murakami, F. Kambe, H. Mitsuyama, K. Okumura, T. Murohara, S. Niwata, R. Yamamoto, and H. Seo Cyclosporin A Enhances Interleukin-8 Expression by Inducing Activator Protein-1 in Human Aortic Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., November 1, 2003; 23(11): 2034 - 2040. [Abstract] [Full Text] [PDF]
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