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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo. 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 {alpha}-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.

 
Ser119Ala CREB Inhibits cAMP- Dependent Transcription
Cotransfection of the DN CREB construct with the cAMP-dependent {alpha}-846-luc reporter resulted in complete inhibition of transcriptional activity of the promoter of the cAMP-responsive, human glycoprotein {alpha}-subunit gene (25), both in the absence or presence of TSH (Fig. 1BGo). In contrast, TSH stimulation of transcriptional activity of the reporter gene was observed when cells were transfected with the bTgP control vector (Fig. 1BGo). 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. 2Go). 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.

 
DN CREB Mice Are Hypothyroid
Serum hormone measurements of TSH, T4, and T3 are shown in Fig. 3Go. The serum TSH levels were elevated 8-fold in transgenic mice when compared with wild-type controls (Fig. 3AGo). In the transgenic animals, serum T4 levels were 40% of wild-type (Fig. 3BGo), and serum T3 levels were 56% of wild-type (Fig. 3CGo). 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 (~15–20%) 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.

 
Histological Analysis of the Thyroid Gland
The wild-type gland has uniformly distributed follicles with minimal variation in follicular diameter (Fig. 4AGo). A single layer of follicular cells surrounds the lumen of each follicle (Fig. 4AGo). 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. 4CGo). All lumina stained positive upon periodic acid Schiff (PAS) staining (Fig. 4EGo).



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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.

 
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. 4BGo). 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. 4FGo). 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. 4DGo). 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. 4DGo).

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. 5DGo). In contrast, no transgene mRNA was detected in the wild-type gland using this same probe (Fig. 5CGo). As control, the sense probe did not hybridize to mRNA in either wild-type or transgenic thyroids (Fig. 5Go, 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.

 
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. 6Go, 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.

 
In contrast, the transgenic glands showed a marked reduction in cytosol and in the number of cytoplasmic organelles and secretory granules (Fig. 6Go, 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 6EGo 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. 6FGo). 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. 7Go). 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. 8Go). 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 ({alpha}, ß, {Delta}) 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. 2Go). 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. 4Go). 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. 7Go). 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. 8Go). 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{alpha} (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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {Delta}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 {alpha} 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 24–26 °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 University’s Animal Care and Use Committee.

Cell Culture
FRTL-5 (American Type Culture Collection, Manassas, VA) cells were maintained in Ham F12 Coon’s 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 60–75% confluency, 500 ng of the {alpha}-846-luciferase ({alpha}-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 Bouin’s 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 Karnovsky’s 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 manufacturer’s 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 0.7–2 µCi {alpha}-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 15–709 303 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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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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