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Original Studies |
University Department of Clinical Biochemistry, The Royal Infirmary, Edinburgh, Scotland EH3 9YW; and The Rowett Research Institute (J.R.A., F.N.), Bucksburn, Aberdeen, Scotland AB21 9SB
Address all correspondence and requests for reprints to: Dr. G. J. Beckett, Department of Clinical Biochemistry, The Royal Infirmary, Edinburgh, Scotland EH3 9YW. E-mail: g.j.beckett{at}ed.ac.uk
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Abstract |
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Introduction |
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Three distinct selenium-dependent glutathione peroxidases (GPXs) are expressed by the thyroid, namely cytoplasmic GPX, membrane-bound phospholipid hydroperoxide GPX, and extracellular GPX (10, 11). The rate-limiting step in thyroid hormone synthesis is thought to be the generation of hydrogen peroxide at the apical membrane of the thyrocyte (12, 13, 14, 15). High concentrations of hydrogen peroxide are harmful to the thyrocyte, and it has been suggested that the GPXs serve to prevent the accumulation of toxic concentrations of hydrogen peroxide and lipid hydroperoxides within the thyrocyte during hormone synthesis (16, 17). Thyroidal extracellular GPX may provide an important mechanism for regulating thyroid hormone synthesis by modulating hydrogen peroxide concentrations in the follicular lumen (11). For each of the above-mentioned selenoproteins, Se is incorporated as a specific selenocysteine residue encoded by a TGA triplet.
Experiments with cultured human thyrocytes labeled with
[75Se]selenite have shown that these cells express at
least eight major selenoprotein bands, as determined by SDS-PAGE. The
molecular mass of most of these selenoproteins lies between 14–31 kDa,
but one major selenoprotein has a molecular mass of approximately 57
kDa (11). This 57-kDa selenoprotein has not been characterized, but
possible candidates include SP56 and AP56 (
56 kDa) (18, 19), protein
disulfide isomerase (58 kDa) (20, 21), and thioredoxin reductase
(TR) (22, 23, 24). SP56, AP56, and protein disulfide isomerase (PDI) are
examples of proteins that appear to bind selenium, but cloning and
sequencing have failed to identify TGA-directed selenocysteine
residues. The role of selenium in these binding proteins is
unclear.
TR is a FAD-containing enzyme that, in conjunction with its substrate thioredoxin, forms a redox system that has multiple functions and is found in all organisms (22). Human TR is a dimeric selenoprotein comprising subunits of 55–60 kDa, each having a single selenocysteine residue near the carboxyl-terminus (22, 23, 24). In addition to its ability to reduce oxidized thioredoxin, TR can directly reduce lipid hydroperoxides and hydrogen peroxide in the presence of NADPH (25); thus, if expressed in large amounts by thyrocytes, TR may play an important role in protecting the cell from peroxidative damage.
We now report that TR expression in human thyrocytes is stimulated by the calcium/phosphoinositol signaling pathway, a pathway that also stimulates hydrogen peroxide production during thyroid hormone synthesis.
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Materials and Methods |
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DMEM, Earle’s Balanced Salt Solution (EBS), penicillin, streptomycin, amphotericin B, glutamine, DMEM-Ham’s F-12 nutrient mix plus L-glutamine, 15 mmol/L HEPES, and FCS were obtained from Life Technologies (Paisley, UK). Collagenase was purchased from Worthington Biochemicals Corp. via Lorne Laboratories (Twyford, UK), and dispase was supplied by Boehringer Mannheim U.K. (Lewes, UK). [75Se]Selenite was obtained from the Reactor Center, University of Missouri (Columbia, MO). Antisera to SP56 and protein disulfide isomerase were provided by Prof. D. Medina, Texas Medical Center (Houston, TX). Antiserum to TR was raised in rabbits to TR purified from rat liver as described previously (22). The purified TR showed a single band on SDS-PAGE using silver staining. All other reagents, including control-processed serum replacement 1 (CPSR-1), were supplied by Sigma Chemical Co. (Poole, UK).
Isolation and culture of human thyrocytes and HepG2 cells
Human thyrocytes were isolated from thyroid tissue obtained from patients with Graves’ disease or functional nodules and undergoing thyroid surgery. The tissue was surplus to routine histopathological examination. Cells were isolated using a modified version of a dog thyroid cell culture method (8) and has previously been described in detail (26). Thyrocytes were plated out in DMEM-10% CPSR-1 (FCS treated to remove endotoxins and Igs) into 75-cm2 flasks in 25 mL medium at a density of 107 cells/flask and incubated at 37 C in an atmosphere of 5% CO2. The thyrocytes were then grown for an additional 2 days to achieve confluence.
HepG2 cells, a human fetal liver-derived cell line, were obtained from the European Collection of Cell Cultures (Salisbury, UK) and maintained in DMEM-Ham’s F-12 nutrient mix plus L-glutamine and 15 mmol/L HEPES containing 5% FCS. The cells were cultured in 75-cm3 flasks without antibiotics in a water-saturated atmosphere of 5% CO2 and 95% air at 37 C and regularly tested negative for mycoplasma contamination.
Monitoring selenoprotein expression
Selenoproteins expressed by thyrocytes were monitored by labeling cells with [75Se]selenite as described previously (11). Confluent cultures of thyrocytes were washed with EBS and incubated for an additional 72 h with DMEM-10% CPSR-1 in the presence of 0.02 megabecquerels (MBq)/mL [75Se]selenite. Mimics of the cAMP (8-bromo-cAMP) and calcium-phosphoinositol second messenger cascades (phorbol 12-myristate 13-acetate and the calcium ionophore A23187) and TSH were also added to the culture medium for the 72 h of incubation at the specified concentrations. After labeling, the growth medium was removed, and the cells were washed twice with EBS and harvested into 25 mL EBS by scraping followed by centrifugation at 2000 x g for 10 min. The thyrocytes were resuspended in 1 mL 60 mmol/L Tris buffer, pH 7.8, containing 1 mmol/L dithiothreitol and 1 mmol/L ethylenediamine tetraacetate and lysed by sonication.
After dilution to a common final protein concentration, the sonicated thyrocytes were diluted 2:1 with boiling mix (35 mmol/L SDS, 1.4 mmol/L glycerol, 0.3 mmol/L 2-mercaptoethanol, and 15 mmol/L bromophenol blue) and heat treated at 90 C for 10 min. The [75Se]selenoproteins present in 25 µg protein were separated by SDS-PAGE on a 12% gel, the resulting gel was dried, and the selenoproteins visualized by autoradiography using Kodak X-Omat XAR-5 film (Eastman Kodak, Rochester, NY).
The selenoproteins expressed by HepG2 cells were monitored in an identical fashion.
Western blot analysis
Western blotting using antisera to SP56 and PDI was performed using the method of Towbin et al (27). For TR, proteins resolved by SDS-PAGE were transferred to Immobilon P membranes, which were, in turn, blocked using a 10% solution of horse serum in 25 mmol/L Tris buffer and 500 mmol/L NaCl (pH 7.5) containing 0.05% Tween before being probed with affinity-purified anti-TR antibody at a final dilution of 1:500. Chemiluminescence was used to visualize the immuoreactive proteins (28)
Determination of protein
The protein content of the sonicated thyrocytes was determined using the Bradford dye binding method (29).
Effects of TSH and mimics of second messenger systems on the expression of TR in human thyrocytes and the cell line HepG2
The change in expression of TR in response to the addition of various test compounds was determined from autoradiography of SDS-PAGE gels described above. Various agents were added either singly or in combination, at specific concentrations, for the last 72 h of culture. These agents were 8-bromo-cAMP (10-4 mol/L), TSH (0.3, 1.0, and 10 U/L), phorbol 12-myristate 13-acetate (PMA; 10-5–10-8 mol/L), and the calcium ionophore A23187 (10-5–10-8 mol/L). The effects of PMA (10-6 mol/L) and A23187 (10-6 mol/L) on the expression of TR in HepG2 cells were assessed using the same techniques.
Time course of induction of TR after exposure of human thyrocytes to A23187 and PMA
Human thyrocytes were preincubated with [75Se]selenite (0.02 MBq/mL) for 24 h, after which time PMA (10-6 mol/L) was added together with A23187 (10-6 mol/L), whereas no additions were made to a series of control flasks. Thereafter, expression of TR in the thyrocytes at 2, 4, 6, and 24 h was determined by SDS-PAGE with autoradiography, as described above.
Identification of the 57-kDa selenoprotein as TR
Further identification of the 57-kDa selenoprotein was performed as follows. HepG2 cells were grown to confluence in the presence of 0.02 MBq/mL [75Se]selenite in nine 265-cm2 culture flasks. The cells were then incubated for an additional 72 h with 0.02 MBq/mL [75Se]selenite in the presence of A23187 (10-6 mol/L) and PMA (10-6 mol/L). Cells were then harvested and lysed as described above, and the lysate was centrifuged at 13,000 x g for 10 min to remove cell debris. The 57-kDa selenoprotein was then purified to homogeneity using a purification method based on that used for TR (22). Briefly, ammonium sulfate was added to the cell lysate to 35% saturation and centrifuged at 13,000 x g for 10 min. The resulting supernatant was dialyzed overnight and fractionated on an ion exchange column (diethylaminoethyl cellulose), followed by affinity purification using ADP-Sepharose. Final purification of the 57-kDa [75Se]selenoprotein was achieved by exclusion chromatography using LKB Ultrogel (LKB, Bromma, Sweden). Purification of the 57-kDa [75Se]selenoprotein was monitored by SDS-PAGE, using protein staining and autoradiography. TR enzymic activity was also monitored through the purification described previously (22). The purified protein was subjected to Western blot analysis using antiserum raised to rat liver TR.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human thyrocytes cultured for 72 h in the presence of
[75Se]selenite expressed at least 10 selenoprotein bands,
with most having a molecular mass less than 30 kDa (Fig. 1
). Three selenoproteins with a molecular
mass greater than 50 kDa were expressed by thyrocytes (1 major band and
2 minor bands). Inclusion of TSH (1 U/L) during this 72-h incubation
period produced a marked induction in the expression of a 28-kDa
selenoprotein previously characterized as IDI (26), but TSH had no
effect on the expression of any other selenoprotein (Fig. 1).
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The addition of PMA (10-6 mol/L) alone produced a
small increase in the expression of the selenoprotein band with a
molecular mass of 57 kDa (mean molecular mass determined from three
experiments; Fig. 2). The addition of
A23187 (10-6 mol/L) alone produced a marked increase in
the expression of the same 57-kDa selenoprotein, such that this
selenoprotein comprised approximately 90% of the total selenoproteins,
as determined from 75Se labeling (Fig. 2). The addition of
both PMA (10-6 mol/L) and A23187 (10-6 mol/L)
in combination slightly enhanced the expression of the 57-kDa
selenoprotein above that achieved using A23187 alone (Figs. 2 and 3). Similar results were found in all
thyrocyte preparations regardless of whether the original tissue was
obtained from patients with Graves’ disease or normal thyroid tissue
surrounding a thyroid nodule (data not shown).
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The addition of A23187/PMA had no apparent effect on the expression of
other selenoproteins with the exception of a 14-kDa selenoprotein that
was induced to a small extent (Fig. 3).
Identification of the 57-kDa selenoprotein as TR
The 57-kDa selenoprotein induced by A23187 and PMA reacted with
antiserum to TR (Fig. 4). The sensitivity
of the Western blot procedure was such that no TR band could be
detected in thyrocytes or HepG2 cells grown in the basal state, but TR
was clearly detected in cells treated with PMA/A23187. Antisera to PDI
and SP56 also showed single bands using Western blot analysis, although
each band had a mobility different from that of the 57-kDa
selenoprotein that was induced by PMA/A23187. Furthermore, no induction
of the PDI or SP56 protein bands was observed in cells treated with
PMA/A23187 (data not shown). The 57-kDa selenoprotein obtained from
HepG2 cells grown in the presence of A23187/PMA copurified with TR
activity and the final purified preparation showed a single band on
SDS-PAGE using Coomassie blue and autoradiography. This selenoprotein
also reacted on a Western blot with antiserum raised to rat liver TR
(data not shown). Amino acid sequencing was attempted, but the purified
protein was blocked at the N-terminus.
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The effect of PMA and A23187 on TR expression were dose dependent,
with a maximal induction of TR expression achieved at a PMA
concentration of 10-7 mol/L and a concentration of A23187
10-7 mol/L (Fig. 3).
After the addition of A23187 (10-6 mol/L) with PMA
(10-6 mol/L), a significant induction of TR expression was
observed after 6 h, with maximal induction occurring by 24 h
(Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PMA and A23187 together stimulate activation of the calcium-phosphoinositol cascade (30), and the increased TR expression produced by these agents suggests that this second messenger cascade is an important regulator of TR expression in human thyrocytes. Although A23187 alone had a more marked effect on TR expression than PMA alone, there was a requirement for both of these agents to be present for maximal expression of TR. This is consistent with the two-branch model of cellular responsiveness to second messengers, where both an initial activation of calmodulin and a sustained activation of protein kinase C are necessary to produce the maximum cellular response. Examples of this include vascular smooth muscle contraction and glucose-induced insulin secretion (31).
We have shown previously that IDI expression in human thyrocytes is regulated by the calcium-phosphoinositol and cAMP signaling cascade. Up-regulation of IDI is achieved by TSH or 8-bromo-cAMP, whereas A23187/PMA prevents stimulation of IDI expression by TSH or 8-bromo-cAMP (26). In contrast to IDI, we found that the addition of TSH or 8-bromo-cAMP had no effect on TR expression, nor did these agents modify the ability of A23187/PMA to induce TR expression. These observations indicate that the cAMP signaling pathway is not involved in the regulation of TR.
What is the likely function of TR in thyrocytes? TR has a broad substrate specificity and, with NADPH as a cofactor, reacts with a wide range of substrates, including thioredoxin, peroxides, selenodiglutathione, selenite, and GPX (22). TR thus has the potential to exert a number of important and diverse biological effects on the cell acting either directly or through thioredoxin.
The thioredoxin/TR redox system can act as a cofactor for reactions catalyzed by IDI (32). TR can also detoxify both lipid hydroperoxides and H2O2 and may serve as an important alternative detoxification pathway to the selenium-dependent GPX (25), as recent work using the rat thyrocyte cell line FRTL5 has suggested that GPX may be ineffective at degrading high concentrations of H2O2 (33). This detoxification role for TR may be of particular importance in the thyrocyte because high concentrations of many peroxides are generated during thyroid hormone synthesis.
We thought it possible that induction of TR in response to stimulation of the calcium-phosphoinositol pathway was secondary to increased H2O2 or lipid hydroperoxide generation. However, exposure of thyrocytes to a wide range of H2O2 and tert-butyl-hydroperoxide concentrations that included lethal doses (1–100 mmol/L) had no effect on TR expression (data not shown).
It has been suggested that the thyroid atrophy associated with endemic myxoedematous cretinism is due to concurrent Se and iodine deficiency (16, 17). In this situation, H2O2 production is increased, whereas the Se deficiency gives rise to decreased expression of GPX and, thus, increased peroxidative damage. Se deficiency also gives rise to diminished TR expression (34), and it is possible that loss of thyroidal TR expression in combined iodine and Se deficiency may contribute to the development of thyroid atrophy.
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Acknowledgments |
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Footnotes |
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Received November 18, 1997.
Revised February 19, 1998.
Accepted March 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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