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Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Leonard D. Kohn, Cell Regulation Section, Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Building 10, Room 9C101B, National Institutes of Health, Bethesda, Maryland 20892-1800. E-mail: lenk{at}bdg10.niddk.nih.gov
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Abstract |
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Introduction |
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We have recently shown (3) that thyroglobulin (TG) accumulated in the follicular lumen is a potent suppressor of NIS RNA levels in addition to RNA levels of the thyroid-specific or thyroid-restricted genes, i.e. TG, the thyroid peroxidase (TPO), and the TSH receptor (TSHR). This reflects the ability of follicular TG to suppress the expression of thyroid-specific or thyroid-restricted transcription factors that regulate expression of the TG, TPO, TSHR, or NIS genes: thyroid transcription factor-1 (TTF-1), TTF-2, and Pax-8 (3). We showed that the action of TG counterbalanced the action of TSH on several of these genes and speculated that TG might be a negative feedback autoregulator of the function of thyrocytes in vitro and in vivo (3).
The follicle is the functional unit of the thyroid. However, the function of individual follicles with respect to iodide uptake is not synchronized, but heterogeneous (4, 5, 6, 7, 8, 9). Demonstration of TG regulation of iodide uptake in vivo and at the apical membrane requires, therefore, a histochemical approach. Additionally, TSH-regulated iodide uptake by the thyrocyte does not occur as an isolated biological event. The thyroid gland is a hypervascularized tissue, in which a fine capillary network surrounds each thyroid follicle (10). As inorganic iodide is supplied by the blood, the possibility has been considered that there is coordinate TSH regulation of vascular permeability and concentrative iodide transport (11). Exchange of nutrients between the blood stream and the perivascular space surrounding the thyrocyte is regulated by both osmotic pressure (12, 13, 14) and specific factors that alter vascular permeability, i.e. vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) (15, 16, 17). Recent evidence indicates that thyrocytes produce VEGF/VPF and that its synthesis and secretion are regulated by TSH (11, 18, 19). The possibility exists that TSH-enhanced VEGF/VPF alters permeability and enhances the availability of iodide to NIS, thereby facilitating concentrative iodide uptake into the thyrocyte (11). The effect of follicular TG on VEGF/VPF and whether its control is coordinate with the effects of TG on NIS are unknown.
In this report, we focused on demonstrating that the suppressive action of follicular TG on TSH-increased NIS RNA levels was caused by suppression of NIS gene expression at a transcriptional level, as we showed is the case for the TG, TPO, and TSHR genes (3), and, equally importantly, was associated with negative regulation of TSH-increased NIS protein and iodide uptake. These studies were undertaken to test the hypothesis that TG suppression of thyroid-specific or thyroid-restricted genes was associated with a negative autoregulatory effect on function in vitro and in vivo and that TG suppression could, indeed, counterbalance the action of TSH on that function. In the process, we amplify our understanding of the action of TG by showing that TG coordinately suppresses VEGF/VPF and NIS RNA levels in TSH-treated cells in association with decreased iodide uptake. This suggests there may be coordinate control of vascular permeability and NIS to maximize iodide uptake by the thyroid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Iodide uptake measurements
Iodide uptake was measured as previously described (21).
Briefly, cells grown in 24-well plates were washed with HBSS containing
HEPES buffer, pH 7.4, and were incubated with 0.1 µCi carrier-free
125I at 37 C for 40 min. Cells were washed with ice-cold
PBS, pH 7.4, and lysed with ice-cold ethanol by placing them at -20 C
for 20 min, at which point the radioactivity of the iodide taken up by
the cell was measured.
Iodide autoradiography and TG immunohistochemistry
Wistar strain male rats were injected ip with 1 mCi
125I 2 h before animals were killed in accord with an
approved institutional protocol. Thyroids were obtained and fixed in
Bouin’s solution, then embedded in paraffin. Sections were cut, and
autoradiography or immunohistochemical staining was performed as
previously described (5, 22, 23, 24).
RNA isolation and Northern analysis
Total RNA isolation and Northern analysis were performed by a
modification of methods previously described (3, 25, 26). RNA was
prepared using RNeasy Mini Kits (QIAGEN, Valencia, CA) and
minor modifications of the manufacturer’s protocol. Cells were
cultured in 10-cm dishes, washed with PBS, recovered with 600 µl
lysis solution, and passed through a QIAshredder (QIAGEN).
After 600 µl 70% ethanol were added, the mixture was passed through
a spin column and washed with 600 µl RW1 wash solution and twice with
500 µl RPE wash solution. RNA was eluted with 30 µl
diethylpyrocarbonate-treated water.
RNA samples were electrophoresed on denatured agarose gels and capillary blotted on a nylon membrane using a Turboblotter (Schleicher & Schuell, Inc., Keene, NH). After UV cross-linking, hybridization was performed as follows. Probes were labeled with [32P]deoxy-CTP using a Ladderman Labeling Kit (PanVera, Madison, WI) in the presence of random primer, deoxy-NTP, and Bca DNA polymerase. After the Nytran membranes (11 x 14 cm; Schleicher & Schuell, Inc., Keene, NH) were prehybridized with 10 ml QuickHyb Hybridization Solution (Stratagene, La Jolla, CA) for 1 h at 68 C, 1 x 107 cpm radiolabeled probe were added after it had been premixed with 100 µl sonicated salmon sperm DNA (Stratagene, La Jolla, CA), heated at 94 C for 5 min, then chilled on ice. After hybridization for 3 h, membranes were washed in 4 x SSPE with 0.5% SDS for 30 min at 37 C, 2 x SSPE with 0.1% SDS for 20 min at 65 C, and 1 x SSPE with 0.1% SDS for 20 min at 65 C. Reprobing was performed after incubating membranes in 50% formamide, 50 mM Tris-Cl (pH 8.0), and 10% SDS for 1–2 h at 65 C.
The VEGF probe was prepared using RT-PCR and the following primers: 5'-ACAGAAGGGGAGCAGAAA-3' and 5'-GAGGTCTAGTTCCCGAAA-3'. First strand complementary DNA was synthesized using an Advantage RT for PCR Kit (CLONTECH Laboratories, Inc., Palo Alto, CA). PCR reactions were performed as previously described (3, 25) using the Touchdown PCR procedure (27), a GeneAmp 9600 PCR machine (Perkin Elmer Corp., Norwalk, CT), and Pfu DNA polymerase (Stratagene). Probes for TG, NIS, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were described previously (3, 26).
NIS promoter-luciferase chimeric plasmids
Genomic sequence from -420 to -1 bp of the NIS promoter (28)
was amplified by PCR using rat genomic DNA (Promega Corp.,
Madison, WI) and forward and reverse primers containing an adaptor
sequence to facilitate directional cloning. The 5'-primer had a
5'-XhoI restriction site,
5'-ACGCCTCGAGTCCTTATGGAGCCCGGAAG-3'; the 3'-primer had a
HindIII restriction site,
5'-GGCTAAGCTTGGAGACAGGTGACTCGGTG-3'. PCR products were cloned into
plasmid PGL-3Basic (Promega Corp.), and inserts were
sequenced to ensure there was no misincorporation.
Transient expression analysis
Lipofectamine (Life Technologies, Inc.,
Gaithersburg, MD) was used to transfect promoter-reporter gene
constructs into FRTL-5 cells (3, 26). Briefly, cells were grown in
6-well plastic plates to about 50% confluence and washed with 2 ml
warmed (37 C) serum-free culture medium (6H0), and 1 ml of a premade
plasmid/Lipofectamine mixture was added. The plasmid/Lipofectamine
mixture was made by incubating 1 µg plasmid DNA with 10 µl
Lipofectamine and 200 µl 6H0 medium for 30 min at room temperature,
then diluting it with 800 µl 6HO medium. Cells were incubated for
4 h at 37 C in a CO2 incubator before 4 ml complete
medium with serum were added. Fresh medium was added after 24 h,
with or without the added ligands or reagents noted in individual
experiments, and reporter activity was measured 36 h later. To
measure luciferase activity, cells were washed and scraped with 1 ml
Dulbecco’s PBS, then resuspended and lysed with 30 µl 1 x
Reporter Lysis Buffer (Promega Corp.) by repetitive
pipetting, 30 times, using a micropipette with a yellow tip (200-µl
capacity). The lysate was incubated for 30 min at room temperature,
frozen on a dry ice, thawed, then centrifuged 4 C for 10 min. Twenty
microliters of the supernatant were mixed with 100 µl luciferase
assay reagent (Promega Corp.) and analyzed using a
luminometer (Lumat LB9507, Wallac, Inc., Gaithersburg,
MD). Two microliters of the supernatant were taken for protein
measurements using bicinchoninic acid protein assay reagent
(Pierce Chemical Co., Rockford, IL) and the
manufacturer’s protocol.
Western blots
Samples were prepared, and blotting was performed as previously
described (29, 30). Minor modifications were as follows. Sample buffer
containing Tris, SDS, and ß-mercaptoethanol were purchased from
Novex (San Diego, CA), and the blocking buffer was PBS
containing 0.6% Tween-20, 10% milk protein, and 1% crystalline BSA.
Anti-NIS was provided by N. Carrasco (Albert Einstein College of
Medicine, Bronx, NY); donkey antirabbit IgG was obtained from
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) as was the
mol wt marker kit. Detection used ECL reagents (Amersham Pharmacia Biotech, Arlington Heights, IL).
Materials
Highly purified bovine TSH was obtained from the hormone
distribution program of the NIDDK, NIH (NIDDK-bTSH; 30 U/mg). Bovine
follicular 19S TG was prepared by salt extraction and agarose
chromatography as previously described (31, 32, 33); each was a single
330-kDa component by electrophoresis in reducing gels containing SDS
(31, 32, 33). When tested, bovine TG from Sigma Chemical Co.
gave the same results. The source of the other materials was
Sigma Chemical Co. unless otherwise noted.
Statistical significance
All experiments were repeated at least three times with
different batches of cells. Values are the mean ± SD
of these experiments where noted. Significance between experimental
values was determined by two-way ANOVA; P < 0.05 was
significant.
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Results |
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, lane 1), because insulin and
insulin-like growth factor I in the serum are potent positive
regulators of TG, not NIS, gene expression (1, 2, 34). The presence of
TSH increased NIS RNA levels 10- to 20-fold, but increased TG RNA
levels only 2-fold (Fig. 1, A, lane 2, and B), consistent with data in
previous reports (1, 2, 34). When 19S follicular TG was added to the
medium, TSH-increased NIS and TG RNA levels were markedly reduced (Fig. 1, A, lane 4, and C). In the absence of TSH, 19S follicular TG added to
the medium of the cells also decreased basal TG RNA levels (Fig. 1A, lane 3); in the case of NIS, this was small and more difficult to
visualize because of the very low NIS RNA levels in the absence of TSH.
The effect of follicular TG on TG RNA levels in 5H medium, which
contains added insulin and insulin-like growth factor I in the serum,
was associated with its action to decrease TTF-2 levels (3).
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, A–C) and ß-actin (data not shown). The suppressive effect of a
physiological concentration of follicular TG can, therefore, overcame a
maximal effect of TSH, particularly in the case of NIS whose RNA levels
were predominantly increased by TSH.
In our previous report (3), we showed that follicular TG suppression of
the TG, TPO, and TSHR genes reflected an ability of TG to modulate
their expression at a transcriptional level. The same is the case for
the TSH-induced expression of the NIS gene. Thus, TSH increased
expression of a NIS promoter-luciferase chimera transiently transfected
into FRTL-5 cells was decreased by the addition of a physiological
concentration of 19S follicular TG to the medium (Fig. 2). The same concentrations of BSA, IgG,
and mannitol had no effect (data not shown). TSH and TG had no effect
on the activity of the PGL-3Basic vector control. The follicular TG
caused a small decrease in basal NIS promoter activity consistent with
its small effect on basal NIS RNA (Fig. 1, lane 3).
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). Thus, when iodide uptake in
FRTL-5 cells cultured with TSH was evaluated, TG significantly
suppressed uptake as a function of time (Fig. 4A) and TG concentration
(Fig. 4B). The same concentrations of BSA had no such effect (Fig. 4, A
and B), nor did the same concentrations of IgG or mannitol (data not
shown). The effect of 19S follicular TG on iodide transport was similar
to its effect on NIS RNA levels as a function of concentration and
time, as shown below (Figs. 6 and 7). However, the effect was less than
expected based on the effect of the TG on NIS protein. At 24 and
48 h, using 10 mg/ml follicular TG, levels of this NIS protein
were about 10% of control levels and negligible, respectively (Fig. 3). Iodide uptakes at 24 and 48 h, in contrast, were about 30%
and 10% of control values using 10 mg/ml follicular TG (Fig. 4, A and
B). Using 1 mg/ml TG, NIS protein was about 25% of control levels
after 48 h, whereas iodide uptake was significantly higher, about
50% of control values.
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To determine whether follicular TG down-regulated iodide uptake
in vivo, uptake of iodide in individual cells was studied
together with follicular TG binding to the apical membrane. A combined
autoradiographic/histochemistry approach was used. In previous reports,
TG suppression of thyroid-specific genes appeared to be associated with
TG moieties that bound to an apical membrane receptor (3) and was
associated in vivo with TG bound to the rim of the
follicular lumen (39). To determine whether TG that was bound to the
apical membrane might be associated with suppressed iodide uptake in
thyrocytes surrounding the lumen of a follicle, Wistar rats were
injected with 125I, and thyroid tissue sections were
immunostained with anti-TG antibody after autoradiography to visualize
the TG bound to the apical membrane in relation to the radioiodine
incorporated into the thyrocytes. There was a clear inverse correlation
between follicular TG accumulation on the apical surface of the cells
and iodine uptake in cells surrounding the follicular lumen (Fig. 5).
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, TG immunostaining was visible as brown coloration, and
125I was visualized as dark silver grains detected by
autoradiography. Consistent with heterogeneity of follicles (5, 22),
some follicles showed dense radioiodine within cells surrounding the
follicular lumen (Fig. 5A, follicle with single asterisk and
arrows), but others showed almost no uptake (Fig. 5A, follicle with double asterisk). Iodide uptake measured by
the dark silver grains was not associated with TG immunostaining
(brown color) at the apical border of the cells surrounding
the follicular lumen, whereas TG immunostaining was not associated with
iodide uptake in adjacent thyrocytes. In general, a follicle that had
dense iodide uptake in the cells had poor TG immunostaining (Fig. 5A, asterisk), whereas a follicle with no iodide uptake in the
cells had a rim of TG immunostaining along the apical membrane (Fig. 5A, double asterisk). Higher magnification indicates that
the TG exists on the surface of the cells facing the follicular lumen
rather than within the cytoplasm of the follicular epithelium (Fig. 5B), consistent with evidence that there is a rim of poorly iodinated
TG bound to the apical membrane (31, 32, 33, 39). The high power
magnification shows more clearly that even within the same follicle,
localization of radioiodine within a cell and TG accumulation on
the apical membrane exhibit an inverse correlation (Fig. 5, A and B,
lower right follicle). These results are consistent with our
in vitro observations shown in
Figs. 1–4 and are consistent
with our hypothesis (3, 39) that follicular TG regulation of
thyroid-specific genes, by regulating levels of thyroid-specific or
thyroid-restricted transcription factors, will result in altered
thyrocyte function.
Follicular TG coordinately suppresses TSH-increased NIS and
VEGF/VPF RNA levels
One possibility that has been considered, based on previous
VEGF/VPF studies (11), is that TSH-increased VEGF/VPF RNA levels might
reflect the effects of TSH to increase the availability of iodide to
the NIS gene by increasing vascular permeability. If so, there might be
coordinate control of NIS and VEGF/VPF RNA levels.
When 19S follicular TG was added to the medium, it caused a
simultaneous decrease in TSH-increased RNA levels of NIS and VEGF/VPF
as a function of the TG concentration (Fig. 6). TG did not similarly alter the
expression of ubiquitous genes such as GAPDH (Figs. 1, 6, and 7) or ß-actin (data not shown). Adding
the same concentrations of BSA, IgG, or mannitol had no effect on the
expression of VEGF/VPF or NIS RNA levels (Fig. 6).
Suppression of TSH-increased NIS and VEGF/VPF RNA levels was maximal by
24 h, whereas suppression of TG RNA levels was maximal at later
time periods (Fig. 7, A and B). Moreover, withdrawal of TG from the
medium had a similar ability to reverse the suppression of NIS and
VEGF/VPF RNA levels as a function of time (Fig. 7A). Thus, the
suppression of TSH-increased NIS and VEGF/VPF messenger RNA (mRNA)
levels was TG protein specific and not caused just by protein
concentration or osmotic pressure.
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Discussion |
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The present study was aimed at linking these observations to function, i.e. we wanted to show that follicular TG was indeed a negative feedback autoregulator of follicular function and could counterbalance the action of TSH. Additionally, we wanted to amplify our studies of the effect of TG on the NIS gene. Thus, in the previous reports (3, 39), we showed that exogenous 19S follicular TG decreased transcriptional expression of the TG, TPO, and TSHR genes, but we did not demonstrate this for the NIS gene. Additionally, we had not provided evidence for an effect on NIS protein or NIS function, i.e. iodide transport.
In this report, we show that 19S follicular TG, at physiological concentrations, i.e. at concentrations known to exist in the follicular lumen based on needle biopsies (35), decreases TSH-increased NIS promoter activity as well as RNA levels in rat thyroid FRTL-5 cells and that this is concurrent with decreases in TSH-increased NIS protein and TSH-increased iodide transport. We show that this mechanism, which is defined in rat FRTL-5 cells, has an in vivo counterpart. Thus, TG bound to the apical membrane of follicular thyrocytes in vivo is associated with suppressed iodide uptake within adjacent thyrocytes when animals are injected with radioiodine. In sum, we show that follicular TG is a counterregulator of TSH-induced NIS function as well as gene expression in a thyrocyte culture system that has been used to characterize the porter (1, 2) and that this likely to be the case in vivo.
Studies of TSH-increased NIS RNA levels, protein, and iodide transport have noted discrepancies between TSH-induced changes in NIS protein vs. RNA levels and iodide transport (1, 2, 38). They accordingly raised questions about protein half-life and TSH-regulated posttranscriptional regulation of NIS protein (1, 2, 38). The present studies reveal another discrepancy; TG decreases NIS protein levels more than it does iodide transport. This may reflect several possibilities. One is the existence of other iodide-transporting systems or channels in FRTL-5 cells, i.e. an apical membrane porter or other chloride/iodide channels, both of which have been described previously (36, 37). These may not be similarly down-regulated by TG. In a recent report we showed that TG increased the RNA levels of pendrin, the presumptive apical membrane iodide porter (41), i.e. TG may indeed have opposite effects on iodide porters other than NIS in thyrocytes. A second possibility is that there may be active and inactive forms of NIS protein, which are regulated by posttranslational modifications, changes in subcellular localization, or protein half-life (2, 38). Recent studies of the Carrasco group (38) showed that TSH regulated the subcellular localization and phosphorylation of NIS and raised the possibility this could control differences in the effect of TSH on RNA vs. protein levels, protein vs. iodide transport levels, and protein stability. A posttranslational modification induced by TSH, i.e. phosphorylation, could, for example, alter half-life (2, 38). We have recently shown that phosphorylation can also regulate TG suppression (42, 43). In short, the more profound effect of TG on NIS protein, than on iodide uptake, emphasizes 1) the still unresolved complexity of iodide transport in thyrocytes despite the discovery of NIS and 2) the complexity of NIS regulation already evident in other studies (2, 38). The present observations may help in future studies of the regulation of NIS protein vs. function.
NIS is a transmembrane protein located on the basal membrane of the
thyrocyte (1). The TSHR is on the basal membrane, whereas TG appears to
operate from the apical membrane, as evidenced by the in
vivo results in Fig. 5 and suggested in separate reports (31, 32, 33, 39, 41, 42, 43, 44, 45). Thus, in a study of propylthiouracil-treated rats (39),
we showed that a rim of TG bound to the apical membrane of the follicle
in vivo was associated with the suppression of TTF-1 mRNA
and TG bioynthesis. The existence of a protein on the apical membrane
that binds TG that has been vectorially transported to the follicular
lumen has been suggested (31, 32, 33, 39, 44, 45). A suggested function of
the binding protein on the apical membrane is to anchor TG during its
reiterative iodination and sialylation processes by TPO and
sialotransferase, respectively (31, 32, 33, 45). This is consistent with
the fact that TG preparations bound to the apical membrane are poorly
iodinated, poorly sialylated, and have the highest binding activity to
the apical membrane (3, 31, 32, 33, 39, 45). This is also consistent with
the observation that follicular TG preparations from colloid nodules or
iodine deficiency goiters are stronger suppressors than TG from normal
thyroid (3). Thus, these data (Fig. 5) (3, 31, 32, 33, 39, 44, 45) suggest
that TG bound to the apical membrane is an important TG moiety
initiating the suppression. However, which receptor binds the TG and
how binding of TG to this apical membrane receptor initiates
transcriptional suppression of gene expression were unexplained until
recently.
One protein that binds TG and was associated with the apical membrane based 1) on its role to vectorially transport TG to the follicular lumen and 2) its involvement in selective fluid endocytosis (45) had properties of the asialoglycoprotein receptor (ASGPR) on liver membranes (31, 32, 33). In recent reports (42, 43 45A ), we have unequivocally identified the protein that binds TG as the ASGPR, shown that the ASGPR is on the apical membrane, and shown that TG binding to the ASGPR initiates suppression. Localization of the ASGPR on the apical membrane was evidenced in polarized FRT cells with a specific antibody to the recombinant RHL-1 subunit of the rat thyroid ASGPR (45A ). Separate studies (42, 43) showed that the same antibody to the recombinant RHL-1 subunit of the rat thyroid ASGPR prevented the ability of TG to act as a suppressor (42, 43). In summary, the data accumulated in these reports indicates that TG bound to the ASGPR on the apical membrane is the important TG moiety initiating suppression.
In the case of both TSH and follicular TG the effect on NIS protein levels and function is mediated by transcriptional control of NIS gene expression (Refs. 1, 2, 3, 42, 43, 46, 47 and this report). In the case of TSH, transcriptional regulation mediated by the cAMP signal transduction system seems clear (1, 2, 46, 47). In the case of follicular TG, the exact mechanism by which a large extracellularly stored molecule could physiologically exert its regulatory action at a transcriptional level was unclear. The TG interaction with the ASGPR on the cell surface in liver or thyroid cells is not known to activate a signal transducing pathway (31, 32, 33, 43). Nevertheless, TG has been reported to have phosphorylated tyrosine, serine, and threonine residues (48) and to have a kinase A-like activity (49). In recent reports (42, 43), we have shown that okadaic acid, which inhibits serine/threonine dephosphorylation in cells, eliminates the TG-suppressive activity. We also showed (42, 43) that phosphorylating the serine or serine/threonine residues of TG itself, by autophosphorylation or by phosphorylation with protein kinase A (PKA), respectively, also eliminates TG suppression, but, unlike okadaic acid, enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. We concluded (42, 43) that follicular TG suppression of thyroid-restricted genes is mediated by the ASGPR on the thyrocyte apical membrane and is regulated by a signal system in which phosphorylation of serine/threonine residues on the bound ligand, TG, and possibly on the ASGPR, is an important component. These data provide a hitherto completely unsuspected role for the ASGPR in transcriptional signaling as well as endocytosis and establish a functional role for phosphorylated serine/threonine residues on the TG molecule. Whether it is ASGPR-mediated signaling or endocytosis of a TG fragment that mediates transcriptional suppression is unclear. This may be a phosphorylated fragment, one that is not fully glycosylated, and/or one that is poorly iodinated. These possibilities are under investigation.
The thyroid gland, like other endocrine organs, is a hypervascularized tissue in which a fine capillary network surrounds the follicles (10). In the thyroid, increased VEGF/VPF RNA levels are associated with the hypervascularity of Graves’ disease and cystic accumulations of fluid (11, 19). In addition, a recent study has shown that VEGF/VPF can induce fenestrations in the endothelium (17). In the present study, we show that physiological concentrations of follicular TG coordinately regulate VEGF/VPF and NIS RNA levels. This raises the possibility that follicular TG not only suppresses TSH-increased follicular cell function (NIS) directly, but also may alter vascular permeability by secreting VEGF/VPF in paracrine manner. This raises the possibility that TSH and TG coordinately regulate NIS and VEGF/VPF to efficiently improve or inhibit iodide transport into the thyroid follicle and is consistent with the fact that iodide uptake in a whole thyroid correlates with serum iodide levels.
Based on the data herein and those from related reports (3, 39, 42, 43, 44, 50, 51, 52), we propose a model for the autoregulatory function of
accumulated follicular TG on thyroid iodide uptake, thyroid hormone
synthesis, and thyroid hormone secretion in follicular thyrocytes
surrounding each follicle. In a follicle rich with colloidal TG, the
accumulated follicular TG may suppress NIS and VEGF/VPF gene
expression, thereby, minimizing iodide uptake (Fig. 8A). At the same time, the
transcriptionally directed synthesis of TG and TPO are suppressed (Fig. 8A). In that situation, TSH may act predominantly to cause resorption
and degradation of follicular TG and the secretion of thyroid hormones
into the bloodstream (Fig. 8A). Because the rate of resorption and
degradation of TG exceeds new synthesis and replacement of TG in the
colloid (50, 51, 52), the TG concentration in the lumen of such a follicle
will decrease. As the TG content in the follicular lumen decreases
(Fig. 8B), transcriptional suppression would be released, and gene
expression of NIS and VEGF/VPF would be reinitiated, as would TG and
TPO gene expression (Fig. 8B). The synthesized and secreted VEGF/VPF
would induce fenestration of the vascular endothelium; this would
increases vascular permeability and allow NIS, whose expression is also
increased, to take up iodide most efficiently (Fig. 8B). When
accumulation of follicular TG reaches a certain level, TG suppression
of gene expression would again dominate TSH-stimulated gene expression,
and the whole process would be repeated.
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In summary, we demonstrate negative feedback regulation of follicular function by follicular TG, iodide transport in particular. We demonstrate that the TG effect on function is independent of TSH, but is mechanistically coordinated with TSH action. We show that TG counterregulates the effect of TSH on thyroid function and is operative at the level of transcription (3).
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Acknowledgments |
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Received February 1, 1999.
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