A Dominant Negative CREB (cAMP Response Element-Binding Protein) Isoform Inhibits Thyrocyte Growth, Thyroid-Specific Gene Expression, Differentiation, and Function
Lynda Q. Nguyen,
Peter Kopp,
Fred Martinson,
Kristina Stanfield,
Sanford I. Roth and
J. Larry Jameson
Division of Endocrinology, Metabolism, and Molecular Medicine
(L.Q.N., P.K., F.M., K.S., J.L.J.) Department of Pathology
(S.I.R.) Northwestern University Medical School Chicago,
Illinois 60611-3008
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ABSTRACT
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cAMP mediates the effects of TSH by regulating
thyroid follicular cell proliferation, differentiation, and function.
To assess the functional importance of the cAMP response element
binding protein (CREB) in thyroid follicular cell regulation in
vivo, we targeted the expression of a dominant negative (DN) CREB
isoform to the thyroid glands of transgenic mice using a
tissue-specific promoter. Transgenic mice exhibited severe growth
retardation and primary hypothyroidism. Serum levels of TSH were
elevated 8-fold above normal levels, and T4 and
T3 levels were low. Histologically, the mutant
thyroid glands were characterized by poorly developed follicles that
were heterogeneous in size with diminished colloid. Ciliated thyroid
epithelial cells were observed in the transgenic thyroid glands,
suggesting a failure of follicular cell differentiation. Consistent
with this hypothesis, the DN CREB transgene inhibited the expression of
an array of genes including thyroglobulin, thyroperoxidase, and the TSH
receptor in semiquantitative RT-PCR experiments. Altered expression of
the thyroid transcription factors Pax-8, TTF-1, and TTF-2 was also
observed. These results demonstrate a critical role for CREB in thyroid
growth, differentiation, and function in vivo.
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INTRODUCTION
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The follicular cells of the thyroid gland produce the two
principal thyroid hormones, T4 and
T3, that are essential for normal growth,
development, and metabolism. In turn, stimulation by the pituitary
glycoprotein hormone TSH via its interaction with the TSH receptor
(TSHR) is required for optimal growth and expression of the
thyroid-specific genes needed for cell survival and hormone production
(1, 2, 3).
It is well established that the effects of TSH are largely mediated by
the cAMP-dependent protein kinase A (PKA) pathway (1, 2). The binding
of TSH to its seven-transmembrane G protein-coupled receptor activates
membrane-bound adenylate cyclase (AC). An ensuing increase in
intracellular levels of cAMP stimulates PKA, leading to the
phosphorylation and activation of specific transcription factors in the
nucleus. Such factors include the cAMP response element binding protein
(CREB) and the cAMP-responsive element modulator (CREM), which belong
to the bZIP class of transcription factors (4, 5). Proteins belonging
to this class contain a leucine zipper (ZIP), an
-helical
coiled-coil structure allowing for homo- or heterodimerization, and an
adjacent basic domain (b), rich in lysine residues that are needed for
direct contact with DNA (4). PKA-mediated phosphorylation of
CRE-binding transcription factors may ultimately lead to the
transcriptional regulation of many cAMP- responsive genes (5, 6).
The importance of the cAMP cascade in thyroid follicular cells
has prompted exploration of the role of CREB as a regulator of gene
expression in this system. TSH/cAMP modulates the expression of most,
if not all, of the thyroid-specific genes, including the TSHR (7, 8),
thyroperoxidase (TPO) (9, 10), thyroglobulin (TG) (10, 11, 12), and
the sodium iodide symporter (NIS) (13, 14, 15). In addition, the expression
and/or activity of the thyroid transcription factors Pax-8, TTF-1, and
TTF-2 have also been shown to be regulated by TSH/cAMP (16, 17, 18, 19, 20).
Transfection of a mutant CREB gene (KCREB), which dimerizes and
inactivates endogenous wild-type CREB, into the rat thyroid follicular
cell line FRTL-5 leads to a reduction in TSH-stimulated cell
proliferation, cAMP-mediated transcription, and reduces iodide uptake,
a hallmark of thyroid follicular cell function (21). These studies
suggest that CREB directly or indirectly mediates some of the actions
of cAMP in the thyroid.
To further explore the role of CREB in thyroid follicular cell
regulation, transgenic mice expressing a mutant CREB isoform in the
thyroid were created. This dominant negative (DN) CREB isoform contains
a serine to alanine substitution at position 119, thus eliminating the
PKA phosphoacceptor site (22). Thyroid-specific expression was achieved
by placing the Ser119Ala CREB mutant under regulation of the bovine TG
promoter. This promoter has been used extensively in other transgenic
models to successfully target specific genes to the follicular cells of
the thyroid gland (23, 24). Transgenic mice expressing the DN CREB
Ser119Ala transgene exhibit dwarfism and are hypothyroid. In addition,
the expression of several thyroid-specific genes and thyroid-specific
transcription factors is decreased. Together, these data provide direct
evidence in vivo for the importance of CREB in thyroid
follicular cell growth and in the expression of thyroid-specific genes
needed for differentiation and function.
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RESULTS
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Targeting Strategy
The transgene construct used for the generation of transgenic mice
expressing the dominant negative CREB mutant in the thyroid is shown in
Fig. 1A
. The Ser119Ala mutant of the
human CREB gene was placed under control of the bovine TG promoter,
allowing for targeted expression of the transgene in the follicular
cells of the thyroid gland. Consistent with previous studies using the
bovine TG promoter (bTgP) to direct transgene expression in the thyroid
(23, 24), RT-PCR analysis confirmed expression of the transgene solely
in the thyroid gland, as transgene mRNA was not detected in other
tissues examined, including the lung, kidney, muscle, and spleen.

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Figure 1. Transgene Construct and Transient Expression in
FRTL-5 Cells
A, Schematic representation of the transgene used to direct DN CREB
expression to the thyroid gland. The S119A mutant of human CREB was
cloned downstream of the -2043 to +9 fragment of the bovine TG (b-Tg)
promoter, allowing for tissue-specific gene expression. The
arrows designate sense and antisense primers used for
PCR-based screening. B. Effect of the DN CREB on cAMP-dependent
transcription. FRTL-5 cells were cotransfected with 500 ng of
-846-luciferase reporter and 500 ng of either the bTgP/control
plasmid or the bTgP/S119A expression vector. After transfection, cells
were treated with or without 1 mIU/ml TSH 48 h before luciferase
assays. Values are the mean relative light units (RLU) ±
SEM of triplicate transfections.
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Ser119Ala CREB Inhibits cAMP- Dependent Transcription
Cotransfection of the DN CREB construct with the cAMP-dependent
-846-luc reporter resulted in complete inhibition of transcriptional
activity of the promoter of the cAMP-responsive, human glycoprotein
-subunit gene (25), both in the absence or presence of TSH (Fig. 1B
). In contrast, TSH stimulation of transcriptional activity of the
reporter gene was observed when cells were transfected with the bTgP
control vector (Fig. 1B
). These findings confirm functional expression
by the bTg promoter and indicate that the DN CREB mutant inhibits the
TSH signaling pathway in FRTL-5 cells.
DN CREB Mice Exhibit Severe Growth Retardation
Although there were no significant differences in the birth
weights between the transgenic and wild-type littermates, all animals
bearing the DN CREB transgene exhibited severe postnatal growth
retardation. Transgenic mice were small, with shortened limb and tail
length (Fig. 2
). Differences in body
weights were apparent as early as 2 weeks after birth, and by 3 weeks
of age, the transgenic mice of both sexes weighed approximately 50% of
the wild-type littermate controls (transgenic, 6.7 g ± 0.4
SEM for n = 37 vs. wild-type, 12.9 g
± 0.6 SEM for n = 27).

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Figure 2. Comparison between Wild-Type and Transgenic
Littermates at 4 Weeks
Three transgenic mice expressing the DN CREB mutant are flanked by two
wild-type littermates.
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DN CREB Mice Are Hypothyroid
Serum hormone measurements of TSH, T4, and
T3 are shown in Fig. 3
. The serum TSH levels were elevated
8-fold in transgenic mice when compared with wild-type controls (Fig. 3A
). In the transgenic animals, serum T4 levels
were 40% of wild-type (Fig. 3B
), and serum T3
levels were 56% of wild-type (Fig. 3C
). Semiquantitative RT-PCR
analysis also revealed that levels of TSH-ß mRNA were elevated 4-fold
in the pituitary glands of transgenic mice compared with wild-type
control animals [transgenic, 3.5 relative density units (RDU) ±
0.3 SEM for n = 5 vs. wild-type, 0.8
RDU ± 0.04 SEM for n = 5],
reflecting decreased feedback inhibition from the reduced serum levels
of thyroid hormone. The mice displayed many of the characteristic
features of rodent hypothyroidism (26), such as reduced stature,
abnormal gait, retarded locomotor activity, rounded forehead, flattened
ears, and coarse hair patterning. A subset of the mice (
1520%)
failed to survive past the weaning age of 21 days.

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Figure 3. Serum T4, T3, and TSH
Values of Transgenic and Wild-Type Mice
The values given for T4, T3 , and TSH represent
the mean ± SEM. Values of n represent the number
of animals used for each group. A, TSH values are expressed in ng/ml.
Wild-type, 213.3 ng/ml ± 25.1 SEM for n =
30 vs. transgenic, 1751.2 ng/ml ± 112.8
SEM for n = 29. B, T4 values are
expressed in micrograms/dl. Wild-type, 3.6 ± 0.1
SEM for n = 22 vs. transgenic,
1.5 ± 0.2 SEM for n = 23. C, T3
values are expressed in nanograms/dl. Wild-type, 65.0 ± 5.2
SEM for n = 20 vs. transgenic,
36.8 ± 4.7 SEM for n = 21.
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Histological Analysis of the Thyroid Gland
The wild-type gland has uniformly distributed follicles with
minimal variation in follicular diameter (Fig. 4A
). A single layer of follicular cells
surrounds the lumen of each follicle (Fig. 4A
). The
calcitonin-producing C cells are located in their normal position
adjacent to the follicular cells in the central portions of the thyroid
gland, as assessed by calcitonin immunostaining (Fig. 4C
). All lumina
stained positive upon periodic acid Schiff (PAS) staining (Fig. 4E
).

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Figure 4. Histological Analysis
Thyroid glands from 3-week-old wild-type and transgenic mice were
fixed, sectioned, and stained with H & E and PAS, or immunostained with
calcitonin. A, H & E stain of wild-type thyroid gland at 10x
magnification. Note the uniformly distributed follicles with minimal
variation in diameter. B. H & E stain of transgenic thyroid gland at
10x magnification. Viewed under the same magnification as the
wild-type gland in panel A, the transgenic thyroid is significantly
smaller in size and is marked by a reduction in follicular cell number
and a reduced number of follicles that are of variable size. A
parathyroid gland is indicated by the white star at the
bottom of the panel. C, Calcitonin immunostaining of wild-type
thyroid gland at 10x magnification. The C cells (stained
brown) are situated beneath the follicular cells and are
dispersed in the central portion of the gland. D, Calcitonin
immunostaining of transgenic thyroid gland at 10x magnification. The C
cells are still situated in the central portion of the gland. C cell
formation and calcitonin production do not seem to be affected in the
transgenic thyroid glands. E, PAS staining of wild-type thyroid gland
at 40x magnification. Intrafollicular staining of colloid demonstrates
normal TG production and storage within the lumina. F, PAS staining of
transgenic thyroid gland at 40x magnification. Colloid staining was
not present in all lumina in the transgenic thyroid (indicated by the
star), suggesting a decrease in TG production and/or
storage. The varying size and shape of the follicles is also a striking
feature in the transgenic thyroid glands expressing the DN CREB mutant.
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The thyroid glands of transgenic mice were very small but located in
the typical position anterio-lateral to the trachea, indicating normal
descent of the gland during development (Fig. 4B
). In contrast to the
wild-type glands, the thyroid glands from transgenic mice displayed the
following atypical histological features: 1) a marked decrease in
follicular cell number; 2) abnormal follicular shape and size; and 3) a
reduction in the number of follicles formed. PAS staining of the
lumina was markedly reduced, and many contained minimal or were
completely devoid of colloid (Fig. 4F
). Intrafollicular TG staining was
positive in the transgenic thyroid glands, albeit reduced when compared
with normal glands (data not shown). The C cells in the transgenic
thyroids were located in their normal position within the centralized
portion of the gland, as observed by calcitonin immunostaining (Fig. 4D
). Gross histological examination suggested a higher proportion of C
cells compared with follicular cells in the transgenic thyroids
compared with wild-type. Quantitative histomorphometry indicated that
the ratio of follicular to C cells is decreased due to a 2-fold
reduction in the average number of follicular cells (transgenic, 113
for n = 4 samples vs. wild-type, 242 for n = 4
samples). However, the number of C cells are not significantly
different, also assessed by histomorphometry (transgenic, 111 for
n = 4 samples vs. wild-type, 119 for n = 4
samples) and calcitonin staining (Fig. 4D
).
Ser119Ala CREB Expression Is Restricted to the Follicular Cells
To demonstrate transgene expression in the thyroid, a 300-bp
antisense probe was designed to recognize RNA corresponding to the
3'-untranslated region of the Ser119Ala CREB transgene. By in
situ hybridization using the antisense probe, expression of the
dominant negative CREB transcript was observed in the follicular cells
of the transgenic thyroid gland, but not in the interfollicular cells
or surrounding muscle tissue (Fig. 5D
).
In contrast, no transgene mRNA was detected in the wild-type gland
using this same probe (Fig. 5C
). As control, the sense probe did
not hybridize to mRNA in either wild-type or transgenic thyroids (Fig. 5
, E and F).

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Figure 5. Transgene Expression Is Restricted to the
Follicular Cells of the Thyroid
The left column represents serial sections from a
4-week-old wild-type control mouse. The right column
represents serial sections from a transgenic littermate. Light
microscopy pictures were taken at 10x magnification. A, H & E staining
of wild-type thyroid gland showing normal morphology. A parathyroid (P)
gland is visible on the top left corner. B, H & E
staining of transgenic thyroid gland showing abnormal morphology. The
follicles are unevenly distributed and of heterogeneous size. The
large dark-staining mass consists of follicular cells
that surround one large follicle. M, Muscle; A, adipose. C, No
transgene mRNA was detected with the antisense probe in the wild-type
thyroid. D, Antisense probe hybridized to transgene mRNA in follicular
cells of the transgenic thyroid. Muscle, adipose, and surrounding
interfollicular cells did not stain positive for transgene mRNA. E, No
transgene mRNA was detected with the sense control probe in the
wild-type thyroid. F, Transgene mRNA was not detected with the sense
control probe in the transgenic thyroid.
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Ultrastructural Analysis of the Thyroid Gland
The wild-type glands of 21-day-old mice displayed the
typical morphological and ultrastructural features that have been
well characterized in the thyroid (27). The follicular cells contained
colloid droplets and the characteristic ultrastructural features of
mitochondria, rough endoplasmic reticulum, Golgi apparatus, secretory
granules, and transport vesicles (Fig. 6
, A and B).

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Figure 6. Electron Microscopy of Thyroids from 3-Week-Old
Wild-Type and Transgenic Mice
A, Normal thyroid gland from wild-type control mouse at 1500x
magnification illustrating normal aspect of thyroid follicular cells
surrounding adjacent lumina. An extensive network of capillaries is
present at the basal surface between the thyroid follicles. B, Normal
thyroid at higher magnification (5000x). Note the abundance of
cytoplasmic organelles, including the rough endoplasmic reticulum,
Golgi apparatus, and mitochondria. C, Thyroid from DN CREB transgenic
mouse. The regular organization of the thyroid epithelium is absent,
and there is a marked reduction in follicle size. D, Thyroid follicular
cells from transgenic thyroid in panel C at higher magnification
(8000x). Noticeable in the mutant thyroid is a marked reduction in
cell volume and cytoplasmic organelles, as well as a loss of
cell-to-cell contact. E, Mutant thyroid at 4000x magnification
illustrating a papilliform projection extending into the lumen in the
top left corner, an alteration frequently seen in the
transgenic thyroid samples examined. Also visible is the loss of a
single layer of follicular cells lining the follicular lumina. The C
cells, recognized by their electron-dense granules, appear normal. F,
Ciliated thyroid follicular cells from a transgenic thyroid gland at
12,000x magnification.
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In contrast, the transgenic glands showed a marked reduction in cytosol
and in the number of cytoplasmic organelles and secretory granules
(Fig. 6
, C and D). In addition, a loss of follicular cell-to-cell
contact, illustrated in panel C, was occasionally encountered in the
transgenic thyroids. Figure 6E
shows a papilliform projection extending
out into the follicular lumen, an alteration frequently seen in the
transgenic thyroids. Also evident in panel E are multicellular layers
of follicular cells that are often found lining the lumina. One
particular feature, which distinguished the transgenic thyroids from
the wild-type, was the increased presence of ciliated follicular cells
that are rare in wild-type follicular cells, especially those derived
from an adult animal (28) (Fig. 6F
). The calcitonin-producing C cells,
recognized by their characteristic electron-dense granules, appeared
normal.
Expression of Thyroid-Specific Genes and Transcription Factors
Semiquantitative RT-PCR was used to assess thyroid-specific gene
expression. Compared with wild-type controls, transgenic thyroid mRNA
levels of TG, TPO, and the TSHR were reduced by 8-fold, 6-fold, and
4-fold, respectively (Fig. 7
). Expression
of the thyroid transcription factors PAX-8, TTF-1, and TTF-2 was
reduced by 4-fold, 3-fold, and 4-fold, respectively, in transgenic
thyroids when compared with wild-type controls (Fig. 8
). Representative autoradiograms of the
RT-PCR products from wild-type and transgenic thyroid RNA are shown in
the upper right corner for each gene.

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Figure 7. Thyroid-Specific Gene Expression
Semiquantitative RT-PCR was performed, and the quantitated values for
TG, TPO, and the TSHR are represented as ratios to GAPDH. Values given
represent the mean RDU ± SEM. Values of n
represent the number of animals used for each group. The
inset on each graph depicts representative PCR products
derived from RNA of wild-type (WT) and transgenic (TG) thyroids. A,
Thyroglobulin gene expression. Wild-type, 2.7 ± 0.40
SEM for n = 5 vs. transgenic,
0.3 ± 0.09 SEM for n = 5. B,
Thyroperoxidase gene expression. Wild-type, 1.5 ± 0.15
SEM for n = 5 vs. transgenic,
0.2 ± 0.07 SEM for n = 5. C, TSHR gene
expression. Wild-type, 0.8 ± 0.2 SEM for n
= 5 vs. transgenic, 0.2 ± 0.05
SEM for n = 5.
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Figure 8. Thyroid Transcription Factor Gene Expression
Semiquantitative RT-PCR was performed, and the quantitated values for
PAX-8, TTF-1, and TTF-2 are represented as ratios to GAPDH. Values
given represent the mean RDU ± SEM. Values of n
represent the number of animals used for each group. The
inset on each graph depicts representative PCR products
derived from RNA of wild-type (WT) and transgenic (TG) thyroids. A,
PAX-8 gene expression. Wild-type, 0.4 ± 0.04 SEM
for n = 5 vs. transgenic, 0.1 ± 0.06
SEM for n = 5. B, TTF-1 gene expression.
Wild-type, 0.5 ± 0.09 SEM for n = 5
vs. transgenic, 0.2 ± 0.04 SEM
for n = 5. C., TTF-2 gene expression. Wild-type, 0.1 ± 0.02
SEM for n = 5 vs. transgenic,
0.04 ± 0.006 SEM for n = 5.
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DISCUSSION
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cAMP modulates a diverse array of cellular responses, including
protein phosphorylation, gene transcription, cell growth, and
differentiation. The effects of cAMP are, however, cell type specific.
It inhibits the proliferation of a number of cell types including
fibroblasts and certain cancer cells (29, 30). However, in endocrine
cells such as somatotrophs (31), corticotrophs (32), and thyroid
follicular cells (2, 33), an elevated level of cAMP is a requisite
signal for both growth and differentiation.
A number of cAMP-responsive proteins, including CREB, orchestrate
responses to a host of extracellular signals to regulate gene
expression in the nucleus (34). In vivo, the function of
CREB has been previously explored in several studies. Targeted
disruption of all functional isoforms of CREB (
, ß,
) generates
mice that die shortly after birth due to impaired T cell development
and respiratory distress (35). Expression of the DN CREB in the
pituitary somatotrophs (31), thymocytes (22), or cardiomyocytes of the
heart (36), inhibits the proliferation and function of these cell
types. Thus for cell types that utilize the cAMP/CREB pathway for
critical cellular functions, the DN CREB mutant is a powerful probe of
these actions in vivo. The role of CRE-binding proteins in
the transcriptional regulation of cAMP-responsive genes in the thyroid
follicular system, however, remains relatively poorly defined despite
the fact that the PKA pathway represents the dominant regulatory
cascade in this cell type.
In this study, animals expressing the dominant negative CREB mutant in
the thyroid gland are hypothyroid and exhibit a dwarf phenotype (Fig. 2
). In many cases, postnatal lethality was observed, presumably as a
consequence of severe congenital hypothyroidism. However,
L-T4 replacement resulted in the
restoration of growth and fertility in the dwarf mice. These features
are consistent with our findings that expression of the dominant
negative CREB mutant is thyroid-specific.
The thyroid glands of the DN CREB transgenic animals are located in the
normal position, but are hypoplastic (Fig. 4
). Immediately apparent is
the reduction in the number of follicular cells (2-fold) in the
transgenic thyroids as assessed by quantitative histomorphometry,
although the number of C cells remains constant when compared with
wild-type controls. This observation indicates a decrease in the
proliferative response of follicular cells to TSH, which is elevated
because of hypothyroidism. There are several potential mechanisms by
which the DN CREB mutant might block cell growth. One possibility is
that the binding of the mutant CREB as a homodimer or as a heterodimer
with endogenous wild-type CREB to CREs located in the promoters of key
target genes prevents gene expression. CREB has been shown to regulate
transcription of several key genes involved in mitogenesis and DNA
replication, including jun, fos, and proliferating
cell nuclear antigen (PCNA). In its hypophosphorylated form, CREB
represses transcription of both the jun and fos
promoters; upon Ser-133 phosphorylation, the repression is relieved and
transcriptional activation is achieved (37, 38). Likewise, the binding
of CREB to the CRE located on the murine PCNA promoter is
necessary for full transcriptional activation after engagement of the
PKA pathway (39). Therefore, it is likely that the
phosphorylation-defective CREB mutant inhibits the transcription of a
variety of genes that are involved in cell proliferation.
Alternatively, it is possible that binding of the DN CREB to the
promoters of cAMP- responsive genes may prevent DNA binding of
other CRE-binding transcription factors such as CREM and activating
transcription factor-1 (ATF-1).
Examination of the thyroid glands from the transgenic mice with
electron microscopy demonstrated the presence of numerous ciliated
follicular cells, a type of cell rarely seen in the adult thyroid gland
(28). Ciliated cells have previously been reported in the thyroid
glands of the mouse (40), guinea pig (41), and dog (28). The mammalian
respiratory tract is lined predominantly by ciliated columnar cells
that are derived from common progenitor cells of the foregut endoderm
(42). It is likely that the ciliated cells observed in the DN CREB
transgenic thyroid glands represent a population of cells that failed
to differentiate into mature and functioning follicular cells.
To explore the possible molecular defects responsible for the thyroid
phenotype, the expression levels of an array of thyroid-specific genes
were examined. Expression of the DN CREB transgene resulted in reduced
expression of the TG, TPO, and TSHR genes (Fig. 7
). In the case of the
TSHR gene, chronically elevated levels of cAMP are known to inhibit
transcription through the inducible cAMP early repressor (ICER) (43).
Elevated levels of circulating TSH in response to lowered thyroid
hormones may partially explain this result in our transgenic mice.
Alternatively, the DN CREB may inhibit the expression of factors that
directly regulate the expression of TG, TPO, and the TSHR genes, such
as the thyroid-specific transcription factors PAX-8, TTF-1, and TTF-2
(20). We found that the expression levels of all three thyroid
transcription factors were significantly lower in the transgenic
thyroids compared with wild-type controls (Fig. 8
). Expression of
PAX-8, TTF-1, and TTF-2 is necessary for the optimal expression
of the TSHR, TPO, and TG genes (8). Decreased expression of these
transcription factors may at least partially result in the decreased
biosynthesis of the TSHR, TPO, and TG proteins. Furthermore, this
result may explain the reduced response to the trophic effects of TSH,
as well as the decrease in intraluminal colloid due to a decrease in TG
synthesis.
The levels of cAMP in the thyroid follicular cell, and the integration
of other signaling pathways, ultimately control the delicate balance
between proliferation and differentiation. The consequences of a
selective blockade of the TSH signaling pathway is illustrated by the
hyt/hyt mouse model, where mice harboring an inactivating
TSHR mutation (Pro556Leu) develop resistance to TSH and consequent
hypothyroidism (26, 44, 45). The opposite extreme is illustrated by two
transgenic mouse models that examine the effects of constitutively
elevated levels of cAMP in the thyroid. In one model, expression of the
activating Gs
(Arg201His) mutation leads to thyroid adenomas,
consistent with the effects of these mutations in patients with
autonomously functioning thyroid nodules (46). In the second model,
overexpression of the A2 adenosine receptor
causes hyperplasia and hyperthyroidism (47). In the current study,
there is presumably an inhibition of the TSH signaling pathway
downstream of both the receptor and PKA via thyroid-specific expression
of the DN CREB. Because CREB can be phosphorylated and activated by
various other signaling cascades reviewed in Ref. 48 , expression of the
phosphorylation-defective CREB mutant would render the cell
unresponsive to most signals leading to CREB phosphorylation and
activation.
These findings confirm a role for CREB in thyroid follicular cell
regulation. Expression of the DN CREB transgene does not affect
migration or formation of the thyroid gland, but does significantly
inhibit follicular cell growth, thyroid-specific gene expression,
terminal differentiation, and hormone synthesis. Selective inhibition
of the TSH signaling pathway at the level of CREB results in severe
thyroid hypoplasia due to a reduced response to the trophic effects of
TSH. Because expression of the TG gene is not initiated until
approximately E14.5 in the mouse (20, 49), these findings only address
the role of CREB during a defined period of thyroid development. Future
studies investigating the developmental expression pattern of CREB and
related bZIP proteins will add further insight into the role of these
transcription factors during thyroid morphogenesis and
differentiation.
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MATERIALS AND METHODS
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Generation of Transgenic Mice
A gene targeting vector was constructed by cloning a 1.659-kb
fragment of the human Ser119Ala CREB mutant (22) into the
EcoRI/BamHI site of the Bluescript II SK +/-
vector (Stratagene, La Jolla, CA), downstream of the
-2043 to +9 region of the bovine TG promoter (23). Ser-119 of the
CREB isoform (CREB 327) corresponds to Ser-133 in the CREB isoform
341. The two isoforms differ due to the presence of a 42-bp fragment,
known as the
domain, located in the 5' end of the coding region of
CREB 341 (50). The function of this domain is not entirely clear.
Transgenic mice were generated at the Northwestern University
Transgenic Facility (Chicago, IL). Purified transgene was microinjected
into the pronucleus of fertilized single-cell eggs from B6/SJL F1 mice
and then surgically transferred into the oviduct of pseudopregnant
females. Animals were bred to the C57BL/6 mouse strain (Harlan Sprague Dawley, Inc., Indianapolis, IN) to obtain
F1 offspring and subsequent generations. Germline
transmission was documented in 9 of 11 founder animals by Southern blot
analysis and PCR-based screening. Three founder lines expressing the
mutant CREB S119A transgene were characterized and used for analysis;
no apparent difference in phenotype severity was seen between these
positive lines.
All mice were housed in microisolator chambers in a barrier facility
(temperature 2426 °C) with a 14-h light, 10-h dark cycle, and
ad libitum access to standard laboratory chow and water. To
ensure transgene transmission to subsequent generations, selected
transgenic mice received sc implants of
L-T4 hormone pellets
(Innovative Research of America, Sarasota, FL) under
Metafane (Schering Plough Animal Health Corp., Union, NJ)
anesthesia. A hormone pellet (5 mg) was inserted through a small
incision on the back just above the scapulae, allowing for slow release
of hormone into the bloodstream for a period of 90 days. All surgical
and experimental procedures were approved and conducted in accordance
with the policies of Northwestern Universitys Animal Care and Use
Committee.
Cell Culture
FRTL-5 (American Type Culture Collection,
Manassas, VA) cells were maintained in Ham F12 Coons modified medium
supplemented with penicillin (100 U/ml; Cellgro Media Tech, Herndon,
VA), streptomycin (100 µg/ml; Cellgro), 5% calf serum, 10 µg/ml
insulin, 5 µg/ml transferrin, and 1 mIU/ml TSH (Sigma,
St. Louis, MO). Cells were kept in a humidified incubator at 37 C/5%
CO2. The medium was changed every 3 to 4
days.
Transfection and Luciferase Assays
FRTL-5 cells were seeded onto 12-well plates in complete medium.
At approximately 6075% confluency, 500 ng of the
-846-luciferase
(
-846-luc) reporter, which contains two cAMP response elements (CRE)
(25), and 500 ng of either the pSK-bovine TG promoter control vector
(pSKbTgP) or the pSK-bovine TG promoter Ser119Ala CREB plasmid
(pSKbTgP-Ser119Ala), as described above, were co-transfected into
appropriate wells using Lipofectamine Plus Reagent (Life Technologies, Inc., Gaithersburg, MD) according to the suggested
protocol. After transfection, the cells were treated with complete
medium without TSH or complete medium containing 1 mIU/ml TSH for 2
days. Luciferase activity was determined approximately 48 h after
hormone treatment using an AutoLumat LB953 luminometer (EG&G, Salem,
MA) as previously described (51).
Serum Hormone Measurement
RIAs for mouse TSH were performed using a
125I RIA kit supplied by the National Hormone and
Pituitary Program. The antigen used for iodination was highly purified
rat TSH (NIDDK-rTSH-I-9) and the reference preparation for mouse TSH
was AFP51718MP. The T3 and
T4 RIAs were measured with DPC Coat-A-Count kits
(Diagnostic Products, Los Angeles, CA).
Histological Analysis and Electron Microscopy
For histological analysis, thyroid glands were fixed in Bouins
fixative for 24 h, processed, and embedded in paraffin.
Four-micron sections were obtained for standard hematoxylin and eosin
(H & E) staining. For electron microscopy, thyroid glands were fixed in
half-strength Karnovskys fixative for 4 h and osmicated in 1%
OsO4. Ninety-nanometer sections were placed on
copper grids and stained with 4% uranyl acetate at 60 C. A JEOL JEM
1220 transmission electron microscope (JEOL USA, Inc.,
Peabody, MA) was used for visualization.
In Situ Hybridization
In situ hybridization was performed using the
nonisotopic mRNA locator-Hyb Kit (Ambion, Inc.,
Austin, TX) according to the manufacturers protocol. For in
vitro transcription to generate RNA probes, a transgene-specific
300-bp fragment of the 3'-untranslated region (3'-UTR) of the Ser119Ala
CREB mutant was cloned into the BamHI/EcoRI sites
of the pPCR-Script Amp SK (+) vector (Stratagene).
In vitro transcription was performed using the Riboprobe
in vitro Transcription System kit (Promega Corp., Madison, WI), and T3 and T7 polymerases were used to
generate sense and antisense probes, respectively. The probes were
labeled with the BrightStar Psoralen-Biotin nonisotopic labeling kit
(Ambion, Inc., Austin, TX).
For localization of transgene message, transgenic and nontransgenic
thyroid tissue was fixed, embedded in paraffin, and sectioned as
described above. After deparaffinization, a 1:10 probe to hybridization
buffer solution was prepared for in situ hybridization. The
mRNA locator-Biotin Detection Kit (Ambion, Inc.) was used to detect transgene mRNA. After sense and
antisense probe hybridization, the thyroid tissue was incubated with a
1:300 streptavidin-alkaline phosphatase conjugate solution, followed by
two washes with 1x Tris buffer. Substrate was added and samples were
incubated at 37 C for 1 h. To terminate the color reaction,
samples were rinsed several times with nuclease-free water and
visualized by light microscopy.
Semiquantitative RT-PCR
Total RNA from transgenic and nontransgenic thyroid and
pituitary tissue was isolated using TRIZOL Reagent (Life Technologies, Inc.). For reverse transcription of transgenic and
nontransgenic thyroid and pituitary RNA, 2 µg of RNA were treated
with DNase I, and first-strand cDNA was synthesized using 250 ng
DNase-treated RNA and 15 U avian myeloblastosis virus reverse
transcriptase (AMV-RT; Promega Corp.).
For PCR amplification, the following genes were amplified using sense
and antisense primers as follows: glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) sense, 5'-CCC TTC ATT GAC CTC AAC TA-3', and
antisense, 5'-CCA AAG TTG TCA TGG ATG AC-3', with the expected product
of 399 bp (52); pituitary TSH-ß subunit (TSH-ß) sense, 5'-GAT ATC
AAT GGC AAA CTG TTT-3', and antisense, 5'-AGA AAA TCC CCC CAG ATA
GAA-3', with the expected product of 249 bp (53); TSHR sense, 5'-AAA
ATG AGG CCA GGG TCC CTG CTG CTG C-3', and antisense, 5'-GGT GGA GCT CCT
TGC AGG TGA C-3', with the expected product of 138 bp (44, 45); TG
sense, 5'-GGG AGG CCA ACA GTT TGT CT-3', and antisense, 5'-GGC CAG GAA
TCG TCT CTG CA-3', with the expected product of 590 bp (54); TPO sense,
5'-TCC TGA CAC CTG CCT GGC AA-3', and antisense, 5'-AAT CAG AGT ACT GGT
CAT CT-3', with the expected product of 240 bp (55); PAX-8 sense,
5'-GGG CCC AGC AGG TGC CTC GG-3', and antisense, 5'-AGA GGT GGG TGG TGC
GCT TG-3', with the expected product of 199 bp (56); TTF-1 sense,
5'-CAG TGT CTG ACA TCT TGA GT-3', and antisense, 5'-AGC GCT GTT CCG CAT
GGT GT-3', with an expected product of 289 bp (57); TTF-2 sense, 5'-TAC
AGC TAC ATC GCA CTC AT-3', and antisense, 5'-GAG CGC TTG AAG CGC TTG
CG-3', with an expected product of 289 bp (49).
The PCR amplification reaction was performed with forward and reverse
primers (37.5 pmol each), GAPDH forward and reverse primers (37.5 pmol
each), 7.5 µl deoxynucleoside triphosphate (10 mM), 10%
1x PCR buffer (67 mM Tris, pH 8.8, 6.7 mM
MgCl2, 16 mM
(NH4)2SO4,
10 mM ß-mercaptoethanol), 10% dimethylsulfoxide, 2.5 U
Taq DNA polymerase, and H2O to 46
µl. A reaction mix (premix) was prepared with all the above reagents
and 0.72 µCi
-32P-dCTP per reaction (250
µCi/µl; Amersham Pharmacia Biotech, Arlington Heights,
IL). Four microliters of cDNA were added to 46 µl of premix, and
reactions were performed at 94 C for 1 min, 53 C for 1 min, 72 C for 3
min for 24 cycles, 72 C for 15 min, and 4 C to terminate. The PCR
products were separated on a 6% polyacrylamide gel and analyzed with a
PhosphorImager (Storm 860, Molecular Dynamics, Inc.,
Sunnyvale, CA). To normalize for differences in the amount of total RNA
added to the reactions, amplification of GAPDH was performed as an
endogenous control. A ratio between the quantified product of interest
and GAPDH product was calculated.
Quantitative Histomorphometry
Tissue sections immunostained for calcitonin were used to
quantify the number of follicular cells and C cells in the thyroid
glands of transgenic and wild-type animals. The cells were counted
using an eyepiece reticule containing a field of one hundred
0.01-mm2 squares. Total numbers of follicular and
C cells were obtained within an area of one hundred squares (1
mm2). Ratios of follicular to C cell numbers were
obtained for each sample.
Statistics
Chi-square test was used for comparisons between two groups. For
comparisons involving multiple groups, nonparametric one-way ANOVA was
used. All P values were two-sided; a P value
< 0.05 was considered to indicate statistical significance.
 |
ACKNOWLEDGMENTS
|
---|
The authors especially thank Dr. J. Leiden for the Ser119Ala
hCREB construct (University of Chicago) and Dr. G. Vassart (Brussels,
Belgium) for the generous gift of the bovine TG promoter construct, M.
Moody for her work on the electron microscopy [Cell Imaging Facility,
Northwestern University Medical School (NUMS)], and NUMS media
services for photographic work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: J. Larry Jameson, M.D., Ph.D., Division of Endocrinology, Metabolism and Molecular Medicine, Northwestern University Medical School, Tarry 15709 303 East Chicago Avenue, Chicago, Illinois 60611.
Received for publication January 6, 2000.
Revision received May 5, 2000.
Accepted for publication May 30, 2000.
 |
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