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TRH-TSH-THYROID |
Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles (S.C., B.R., J.E.D., S.S.), 6041 Gosselies, Belgium; Departments of Medicine and Pediatrics, University of Chicago (S.R.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Dr. Serge Clément, Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Institut de Biologie et de Médecine Moléculaire, Université Libre de Bruxelles, 6041 Gosselies, Belgium. E-mail: sclement{at}ulb.ac.be
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Introduction |
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Materials and Methods |
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After sequencing, the human IGF-I- and IGF-IR-coding regions were introduced in EcoRI BamHI sites of the pSG5 plasmid containing the ß-globin intron II and the simian virus 40 polyadenylation sequences (polyA). Then, a DNA fragment containing the intron, the cDNA, and the polyA was isolated from PSG5 and inserted in a pBluescript plasmid containing the bovine Tg promoter (15). Transgenes were extracted from the plasmid by XbaI-XhoI restriction.
Production of transgenic mice
Transgenic mice were generated as described by Hogan et
al. (16) by microinjection of the excised construct
into the pronucleus of fertilized eggs from superovulated FVB/N mice.
Transgenic mice were screened by Southern blotting on DNA extracted
from tail tips.
Histological procedures
Tissues samples were fixed for 18 h in 10%
neutral-buffered formalin and embedded in paraffin by standard
procedure. Sections (6 µm) were stained with hematoxylin and eosin.
The area of each follicle was estimated by measuring the smaller inner
diameter (d) and the larger inner diameter (D) of the follicle. The
approximate follicular lumen area was calculated by the formula A
= 
(d/2)(D/2). More than 100 follicles were measured in each thyroid
gland. For each mouse, cell density per mm2 was
estimated by counting number of cells in 20 areas of 0.0373
mm2, and follicle density was estimated by
counting 10 areas of 0.1485 mm2.
RT-PCR procedure
Polyadenylated RNA from thyroid glands of wild-type (WT) and
transgenic mice were isolated using the FastTrack kit
(Invitrogen). RT was performed according to standard
procedure using random hexamer primers on 100 ng mRNA. cDNAs were
amplified by PCR using the following primers, annealing temperatures,
and cycles numbers: IGF-I transgene: forward, 5'-GGC TGC AGG AAT TCG
ATA TC-3'; reverse, 5'-GCT GCA ATA AAC AAG TTC TG-3' (58 C, 35 cycles);
IGF-IR transgene: forward, 5'-GGC TGC AGG AAT TCG ATA TC-3'; reverse,
5'-CAC ATC GGC TTC TCC TCC AT-3' (60 C, 35 cycles); hypoxanthine
guanine phosphoribosyl transferase: forward, 5'-GTG ATT GGC GAT
GAT GAA CCA G-3'; reverse, 5'-GGC TTT TCC ACT TTC GCT AAT G-3' (55 C,
35 cycles); IGF-binding protein-1 (IGFBP-1): forward, 5'-GCC GTT CCT
GAT TCT CCT GT-3'; reverse, 5'-ATA GGT GCT GAT GGC GTT CC-3' (55 C, 35
cycles); IGFBP-2: forward, 5'-GTA CCT GTG AAA AGA GAC GC-3'; reverse,
5'-TGT CCG TTC AGA GAC ATC TTG-3' (58 C, 28 cycles); IGFBP-3: forward,
5'-AGG CAG CCT AAG CAC CTA C-3'; reverse, 5'-TCT GCA CGC TGA GGC AAT
G-3' (58 C, 28 cycles); IGFBP-4: forward, 5'-GAC GAA GCC ATC CAC
TGC CC-3'; reverse, 5'-CTG TTT GGG GTG GAA GTT GC-3' (52 C, 35 cycles);
IGFBP-5: forward, 5'-TTG CCT CAA CGA AAA GAG C-3'; reverse, 5'-TTC ATT
CCG TAC TTG TCC ACA C-3' (55 C, 28 cycles).
Western blotting analysis
Proteins (40 µg/lane) were separated by SDS-PAGE and
transferred to nitrocellulose membranes for 16 h at 30 V and 4 C.
Membranes were incubated in a blocking solution containing 0.5% BSA
and 2% dry milk. Primary antibodies were rabbit polyclonal against
human IGF-I (mouse cross-reactive) and human IGF-IR (mouse
cross-reactive; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA). After washing, peroxidase-labeled donkey antirabbit Igs were
used to detect primary antibodies. Finally, blots were incubated with
enhanced chemiluminescence reagents and autoradiographed.
Tests of thyroid function
Thyroidal radioiodide uptake was measured by counting (Packard
2900TR counter) whole thyroid glands 2 d after ip injection of 50
µCi Na 131I. Blood samples were obtained by
retroorbital puncture under ether anesthesia. Circulating TSH and
T4 hormones were measured in serum samples kept
frozen at -70 C. T4 was measured by double
antibody precipitation RIA (Diagnostic Products, Los
Angeles, CA) modified to measure T4 in mouse
serum with a sensitivity of 0.25 µg/dl. Mouse TSH was measured by a
disequilibrium, heterologous RIA described in detail (17).
The sensitivity of the assay is 5–10 mU/liter.
Methimazole treatment
Mice received a solution containing 0.2% methimazole
(Sigma-Aldrich Corp.) and 10% glucose in drinking
water. Treatment was conducted during 3 months.
Statistical analysis
Statistical significance was analyzed with unpaired t
test excepted for Fig. 5D
where the Mann-Whitney test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, A and D). Both
transgenes were purified and injected separately into the pronucleus of
one-cell FVB/N embryos. Three founders transgenic mice were identified
to carry either the IGF-I or the IGF-IR transgenes by Southern
blotting. Five of these founders transmitted the transgene to their
progeny in a Mendelian fashion: three independent IGF-I (IGF-I
,
IGF-Iß, and IGF-I
) and two independent IGF-IR transgenic lines
(IGF-IR and IGF-IRß) were thus obtained. IGF-I and IGF-Iß
transgenic mice were bred, respectively, with IGF-IR and IGF-IRß
transgenic mice to obtain double transgenic mice overexpressing both
transgenes in the thyroid gland (IGF-I/IGF-IR transgenic mice).
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, B and E). No PCR fragment of
a size corresponding to genomic amplification was ever seen,
confirming the purity of the mRNA preparations.
Overexpression of the transgenes at the protein level was demonstrated
by Western blotting performed on extracts from WT, IGF-I, IGF-IR, and
IGF-I/IGF-IR transgenic thyroid glands using antibodies against human
IGF-I or IGF-IR (Fig. 1, C and F, respectively).
The three types of transgenic animals (IGF-I, IGF-IR, and IGF-I/IGF-IR) showed no obvious abnormalities and a had normal life span. In particular, no visible goiter and no obvious signs of hyper- or hypothyroidism were seen; transgenic animals have normal body weight and normal behavioral activity, two characteristics that are expected to be impaired in severe thyroid disease.
Thyroid gland weight in IGF-I, IGF-IR, and IGF-I/IGF-IR transgenic
mice
Thyroid gland weight was measured at different ages (Fig. 2A). In 7-, 12-, and 16-wk-old IGF-I and
IGF-IR transgenic mice, no significant difference in thyroid gland
weight was observed compared with WT mice. However, when both IGF-I and
IGF-IR transgenes were expressed in the same mouse, the gland weight
was significantly increased. Moreover, the thyroid gland of
IGF-I/IGF-IR transgenic mice continues to enlarge, whereas in 12-wk-old
control, IGF-I, and IGF-IR mice, growth slows down. The glands of 12-
and 16-wk-old IGF-IR transgenic mice showed a propensity to increase in
size without reaching statistical significance compared with WT thyroid
gland. The difference reached statistical significance only in
21-wk-old IGF-IR mice.
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and 3A).
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Electron microscope analysis of the transgenic thyroid glands confirmed
the normal structure of the thyroid cells and the normal organization
of the gland. Aspect and size of the nuclei were not different from
those observed in WT mice, and abundance and structure of the different
organelles were normal (data not shown). In 7-wk-old transgenic mice,
the number of cells and follicles per mm2 were in
the normal range, and follicles sizes were not significantly different
between control and transgenic mice (data not shown). However, in
16-wk-old IGF-I/IGF-IR mice, the average follicular lumen area was
increased (Figs. 3, B and C, and 4A) and the number of cells per
mm2 was slightly decreased when compare to WT
mice (Fig. 4B). The
number of follicles per mm2 in IGF-I/IGF-IR mice
showed also a slight decrease compared with WT mice (Fig. 4C).
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, B and D, and data
not shown). However, the thyroids of IGF-I/IGF-IR transgenic mice kept
large follicular lumina despite goitrogen treatment compared with WT
mice (Figs. 3, D and E, and 4A). Moreover, after methimazole treatment,
the number of cells per mm2 was lower in IGF-IR
and IGF-I/IGF-IR mice compared with WT mice (Fig. 4B). The increased
gland weight observed in these mice therefore does not correspond to a
parallel increase in thyroid follicular cell density.
Thyroidal functions in IGF-I, IGF-IR, and IGF-I/IGF-IR transgenic
mice
As thyroid gland weight was increased in IGF-I/IGF-IR mice, the
presence of abnormal thyroid function was considered. Thyroidal
radioiodide uptake 48 h after administration of the isotope was
not significantly different in IGF-I and IGF-IR transgenic mice
compared with that in WT mice, but was increased by a factor of 2 in
IGF-I/IGF-IR transgenic mice (Fig. 5A).
IGF-I/IGF-IR mice showed thyroid enlargement and increased iodide
uptake, suggesting that the regulation of thyroid hormone synthesis is
impaired. Serum T4 in 7-wk-old IGF-I and IGF-IR
transgenic mice was not significantly different compared with that in
WT mice, but IGF-I/IGF-IR transgenic mice had a mean
T4 level slightly, but significantly, higher than that
in WT mice (Fig. 5B). Methimazole treatment reduced the serum
T4 concentration in all mice, but IGF-I/IGF-IR
transgenic mice conserved a significantly higher
T4 level compared with WT mice receiving the same
antithyroid treatment (Fig. 5B).
The serum TSH level is the most sensitive indicator of thyroid function
state and therefore is required to understand the action of the
IGF-I/IGF-IR system in our transgenic mice. The TSH concentration in
7-wk-old mice was significantly lower in IGF-IR and IGF-I/IGF-IR,
compared with WT, mice (Fig. 5C). IGF-I transgenic mice also showed a
propensity to decreased TSH level, but the mean value did not reach
statistical significance. Serum TSH was also measured after 3 months of
methimazole treatment (Fig. 5D). TSH levels increased in all types of
mice during methimazole treatment. This increase was only moderate in
IGF-IR and IGF-I/IGF-IR transgenic mice (in particular for IGF-I/IGF-IR
mice) compared with WT mice.
IGFBP expression in IGF-I transgenic mice
IGFBP expression was previously shown to be modified in some
transgenic animals in response to IGF-I overexpression (18, 19). As our IGF-I simple transgenic mice show no alteration in
thyroid morphology and function, we investigated whether such a
compensatory mechanism is present in their thyroid. Expression of
IGFBP-1 to -5 in thyroid was investigated by semiquantitative RT-PCR on
thyroid mRNA. No expression of IGFBP-1 and IGFBP-4 was detected in the
mouse thyroid. IGFBP-2 and IGFBP-5 were expressed in the mouse thyroid,
but in a similar way in IGF-I and WT mice (Fig. 6). However, IGFBP-3 expression was
significantly reduced in the thyroids of IGF-I transgenic mice to 50%
of the level observed in WT animals (Fig. 6). When RT-PCR analysis was
performed on mRNA from IGF-I/IGF-IR double transgenic thyroid glands, a
similar down-regulation of IGFBP-3 expression was also observed (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of IGF-I and IGF-IR was successfully targeted to the thyroid glands of transgenic mice using the bovine Tg promoter. Transgene overexpression at the mRNA and protein levels was demonstrated by RT-PCR and by Western blotting, respectively. IGF-I and IGF-IR mice were crossed to obtain transgenic mice overexpressing both transgenes. None of the transgenic mice produced showed external signs of thyroid dysfunction, such as a visible goiter, or signs of hyper/hypothyroidism. The absence of obvious phenotype in these mice may result from the existence of compensatory mechanisms.
A first compensatory mechanism identified is the down-regulation of TSH in IGF-IR, IGF-I/IGF-IR, and most probably IGF-I mice. This indicates that IGF-I and IGF-IR stimulate thyroid function and overproduction of thyroid hormones, which, in turn, decreases pituitary TSH production by the classical negative feedback effect. Consequently, thyroid function is restored to its normal level. This is the classical response to overproduction of thyroid hormones and explains why in such conditions measurement of serum TSH is the most sensitive test of thyroid function. Indeed, in mild cases of hyperthyroidism, only a decrease in serum TSH is observed with little change in thyroid hormone level. Only in more severe disease do thyroid hormone levels increase. So the slightly increased T4 level in IGF-I/IGF-IR (but not in simple transgenic mice) could reflect a more severe phenotype due to a synergic effect of both transgenes. Moreover, the thyroidal enhanced iodine uptake in IGF-I/IGF-IR transgenic mice also indicates that IGF-I/IGF-IR system stimulates thyroid functions. This increased function could be due to a direct effect of IGF-I on thyroid function or to an increased sensitivity of the thyroid to TSH in response to IGF-I.
A second compensatory mechanism may result from the modulation of IGFBP expression in transgenic thyroid cells, as previously seen in other transgenic models (18, 19). IGFBP-3, especially when cell associated, enhances IGF-I action in some cultured cells (20). IGFBP-3 also induces a tissue-specific organomegaly when ubiquitously overexpressed in transgenic mice (21). Thus, IGFBP-3 could enhance IGF-I action, and its reduced expression in IGF-I and IGF-I/IGF-IR transgenic mice may decrease the effects of the transgene on thyroid follicular cells.
These two compensatory mechanisms (TSH and IGFBP-3 down-regulation) may explain the normal thyroid gland structure, the absence of significant enlargement, and the lack of severe perturbations of thyroid function despite expression of the single IGF-I or IGF-IR transgene. The presence of a significantly enlarged thyroid in mice expressing both transgenes probably results from a synergic effect of IGF-I and IGF-IR. However, the consequence of this synergic effect is more an increase in colloid volume than an increase in cell number, and therefore does not reflect marked hyperplasia. Indeed, histological analysis showed an increased follicular lumen area in IGF-I/IGF-IR transgenic mice. Such an increased thyroid follicular area was also observed in mice overexpressing GH (22) and in hereditary dwarf mice treated with GH and/or TSH (23). This effect is probably due to increased Tg synthesis and accumulation in the follicular lumen. A similar IGF-I- or insulin-dependent Tg synthesis has also been demonstrated in vitro in FRTL-5 cells and dog thyroid cells (5, 6). Thus, the increased colloid volume could explain at least in part the increased thyroid gland weight found in IGF-I/IGF-IR transgenic mice.
Methimazole treatment of WT, IGF-I, and IGF-IR mice results in an important increase in TSH level. However, treatment of IGF-I/IGF-IR mice induced only a slight increase in TSH level compared with WT mice. This resistance to antithyroid drug action in double transgenic mice indicates that a much lower increase in TSH is necessary to achieve a similar relative increase in the weight and function of the thyroid.
In our transgenic animals, IGF-I could replace TSH to some extent, but TSH remains necessary for thyroid stimulation. Indeed, the TSH level is reduced, but not absent. This is in agreement with previous findings in patients with hypopituitarism showing deficiency in both TSH and GH and therefore in IGF-I. In these patients GH replacement, which presumably restores the normal IGF-I level, does not increase thyroid volume (24). A similar TSH requirement is observed in IGF-I/IGF-IR transgenic mice.
IGF-I (or insulin at supraphysiological concentrations) is required for the mitogenic action of TSH in dog and human thyroid cells in primary culture, but IGF-I alone has no mitogenic effect in these systems. In rat FRTL-5 and PCCL3 thyroid cells, however, IGF-I alone stimulates the proliferation to a similar extent as TSH (7). Our in vivo results offer new arguments that IGF-I per se has little mitogenic effect on the thyroid cell, but acts with TSH to control its proliferation. Indeed, thyroid weight in IGF-I/IGF-IR transgenic mice is moderately increased compared with that in mice with constitutive activation of the cAMP cascade in thyroid (25, 26). Even in IGF-I/IGF-IR transgenic mice, this increase largely reflects an increased colloid volume, rather than an increase in cell population. Thus, contrary to what could be expected from in vitro work on cell lines, but in agreement with work on primary cultures of dog and human thyroid cells, IGF-I has little mitogenic effect per se in vivo. Recent work on dog thyroid cells defining the different roles of IGF-I and TSH in the generation of the cyclin D3/CDK4 complex that triggers the mitogenic cascade may explain our findings. Indeed, although IGF-I provides the cell with the cyclin D3 necessary to form the cyclin D3/CDK4 complex, TSH activates this complex (27). Moreover, the comitogenic action of IGF-I and TSH is probably regulated by TSH itself, which stimulates the thyroidal autocrine production of IGF-I (8, 9, 10), making TSH the main physiological regulator of thyroid function and growth in vivo.
The finding that in our transgenic mice IGF-I/IGF-IR system enhances thyroid function and growth is reminiscent of previous observations made in acromegalic patients (12, 13, 14). Thyroid enlargement is present in a majority of patients with acromegaly. An inverse relationship between thyroid volume and TSH level has been reported in such patients, suggesting that thyroid enlargement develops independently of TSH or that GH and IGF-I hyperstimulation may result in partial thyroid autonomy. To explain the discrepancy between the frequent impairment of TSH response and the normal concentration of thyroid hormone, Yoshinari et al. (28) suggested that the high IGF-I levels found in acromegaly may have a direct stimulatory effect on the secretion of T4, independently of TSH action. Our double transgenic mice also present a thyroid enlargement and reduced TSH levels. The later abnormality probably results from the stimulation of thyroid hormone synthesis by the IGF-I/IGF-IR system, as suggested by the slight increase in T4 levels in these mice. Our IGF-I/IGF-IR transgenic mice may thus represent a model of thyroid problems in acromegalic patients.
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Acknowledgments |
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Footnotes |
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1 Fellow of the Belgian Fonds National de la Recherche
Scientifique. 
2 Maître de Recherches of the Belgian Fonds National de la
Recherche Scientifique.
Abbreviations: IGF-IR, IGF-I receptor; IGFBP, IGF-binding protein; polyA, polyadenylation sequence; WT, wild-type.
Received May 14, 2001.
Accepted for publication August 28, 2001.
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0.05 compared with WT
mice. B, Serum T4 levels in mice maintained on a normal
diet (
; n = 15–22) or after methimazole treatment (
; n
= 5–9). *, P 
, WT mice (n = 4–7); 
, IGF-IR mice (n = 3–7); 
, IGF-I/IGF-IR mice
(n = 3–7). Values are the mean ± SEM.
Asterisks indicate statistically significant differences
compared with WT mice of the same age. *, P 
