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Am J Physiol Endocrinol Metab 294: E291-E299, 2008. First published November 27, 2007; doi:10.1152/ajpendo.00281.2007
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Connexin-32 acts as a downregulator of growth of thyroid gland

Gaëlle Prost,1,2,3 Françoise Bernier-Valentin,1,2,3 Yvonne Munari-Silem,2 Samia Selmi-Ruby,1,2,3 and Bernard Rousset1,2,3

Institut National de la Santé et de la Recherche Médicale, 1UMR 664 and 2UMR 369, Université Lyon 1; and 3Faculté de Médecine Laennec, Université de Lyon, Lyon, France

Submitted 3 May 2007 ; accepted in final form 26 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Thyroid epithelial cells communicate through gap junctions formed from connexin (Cx)32, Cx43, and Cx26. We previously reported that reexpression of Cx32 in "gap junction-deficient" FRTL-5 and FRT thyroid cell lines induces a reduction of cell proliferation rate and an activation of expression of cell differentiation. The present study aimed at determining whether Cx32 could exert similar regulatory functions in vivo. We investigated morphological and functional characteristics of thyroid gland of Cx32-deficient mice (Cx32-KO), mice overexpressing Cx32 selectively in the thyroid (Cx32-T+), and Cx32-KO mice with a thyroid-selective Cx32 complementation obtained by crossing Cx32-KO and Cx32-T+ mice. In basal conditions, Cx32-KO mice did not present any detectable thyroid alteration, whereas Cx32-T+ mice showed a thyroid hypoplasia (20% reduction) associated with a slight increase in thyroid functional activity. Under thyrotropin stimulation (following sodium perchlorate treatment), Cx32-KO mice developed a larger goiter (≤65% increase) than wild-type littermates, whereas Cx32-T+ mice exhibited the same thyroid hyperplasia as wild-type mice. Restoration of Cx32 expression in the thyroid of Cx32-KO mice abrogated the thyroid growth increase related to Cx32 deficiency. All together, these data show that Cx32 acts as a downregulator of growth of thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to TSH-stimulated thyroid cells.

gap junctions; connexins; connexin gene inactivation; thyroid-targeted transgenesis; thyroid goiter

GAP JUNCTIONS (GJ) consist of intercellular channels made of connexin (Cx) proteins encoded by a family of 21 genes in human and 20 genes in mouse. GJ are ubiquitously distributed, and most tissues coexpress several Cx leading to cell type-specific Cx expression patterns. The functions of GJ are rather well known in organs composed of excitable cells such as the propagation of action potentials in the heart but are less documented in most of other organs. In endocrine glands, GJ play different roles: they are involved in the regulation of insulin secretion by the pancreas (21), in the modulation of adrenal cell responsiveness to corticotropin (25), or in the control of gametogenesis in testis (29) and ovaries (12). One way to investigate the role of GJ in a given tissue or organ is to analyze the phenotype of mice overexpressing a given Cx or mice with a Cx gene null mutation. Such genetic approaches used in a generalized or tissue-specific way have revealed, for example, the importance of Cx43 in heart development (14) and Cx26 in hearing (2).

One of the general and probably fundamental physiological roles of GJ is to contribute to the control of cell growth. Since the pioneering work of Loewenstein (22), this question has been studied in vitro in a variety of cell lines originating from numerous organs. We found that the restoration of cell-to-cell communication by reexpression of Cx32 in "GJ-deficient" thyroid cell lines (FRTL-5 and FRT lines) induces a reduction of the cell proliferation rate and an increase in expression of cell differentiation, i.e., gene expression (32) and three-dimensional cell organization in follicle structures (34). The Cx32-induced reduction of thyroid cell proliferation rate appears to be associated with a delay in the entry in the S-phase of the cell cycle (8). Restoration of intercellular communication by reexpression of Cx43, the other major Cx physiologically expressed in the thyroid gland, does not lead to any detectable change of cell growth or differentiation (8). In thyroid cells as in many other in vitro cell systems, the actions of Cxs appear Cx specific. For a rather long period of time, the regulatory role of Cx on cell proliferation in vitro was thought to be related only to cell-to-cell transfer or maintenance at equilibrium of important signals among GJ-connected cells. There is now evidence for GJ-independent roles of Cx in the control of cell growth and expression of cell differentiation (17). Data from various cells transfected with different Cx genes have often evidenced an absence of correlation between the level of GJ-mediated intercellular communication and the changes in growth properties of Cx-expressing cells (17, 37). It has been postulated that Cx could act on the cell cycle machinery through protein-protein interactions; the list of proteins capable of interacting with Cx is rapidly growing (15). At present, the mechanism(s) through which Cx, either by themselves or by the formation of functional GJ, modulates the rate of cell division remains to be elucidated.

One important question that is not often adressed is whether a given Cx, which exerts a regulatory activity on the growth of given cell lines in vitro, is endowed with a similar regulatory role in the corresponding normal cells in vivo. In the present study, we examined whether the regulatory functions of Cx32 demonstrated on thyroid cells in culture actually occur in the intact organ under physiological conditions. We have investigated the thyroid status of mice with a generalized inactivation of the Cx32 gene (Cx32-KO mice). To demonstrate that the observed changes were specifically related to the absence of Cx32 in the thyroid, we performed a thyroid-selective Cx32 complementation of Cx32-KO mice. For that, we generated transgenic mice overexpressing Cx32 selectively in the thyroid (Cx32-T+ mice) by use of the thyroglobulin gene promoter, and we crossed these mice with Cx32-KO mice. To visualize and quantify exogenous Cx32 expression, the Cx32 gene was fused with the enhanced-green fluorescent protein (EGFP) coding sequence. We report the thyroid status of Cx32-KO, Cx32-T+, and thyroid Cx32-complemented Cx32-KO mice in basal conditions and under thyrotropin stimulation (following sodium perchlorate treatment). We have found that both the lack and the excess of Cx32 affect the growth of the thyroid gland.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Origin and treatments of mice. C57BL/6 mice with a generalized inactivation of the Cx32 gene, i.e., Cx32-KO mice were obtained from Prof. K. Willecke (Institute für Genetik Universität Bonn, Bonn, Germany). The main characteristics of these mice have been described by Nelles et al. in 1996 (26); they are viable and fertile and exhibit a small reduction in body weight compared with wild-type littermates.

Mice with a thyroid-selective overexpression of exogenous Cx32 (Cx32-T+) were generated by conventional transgenesis, i.e., microinjection of a linearized DNA construct in fertilized eggs from OF1 strain mice at the Transgenic Mouse Facilities of Lyon 1 University, Lyon, France. The construct (pTg-Cx32/EGFP) was composed of the rat Cx32 coding sequence in frame in 3' with the EGFP cDNA; the transgene was placed downstream the 2-kb regulatory region (pTg) of the bovine thyroglobulin gene, as previously described (7). Transgenic mouse lines were generated by crossing founder animals with wild-type OF1 mice.

All mice were housed in a conventional animal facility with a 12:12-h light-dark cycle and ad libitum access to standard laboratory chow (Safe Animal Food & Engineering, Augy, France) and water. Groups of mice, at least 8 wk old, received 1% sodium perchlorate (NaClO4; Fluka, St. Louis, MO) in drinking water for up to 20 wk. Mice were anesthetized by intraperitoneal injection of 10 µl/g body wt of a solution composed of 1 volume of 2% xylazine (Rompun; Centravet, Dinan, France), 5 volumes of 5% ketamine (Imalgene; Centravet), and 15 volumes of saline. Animals were bled by cardiac puncture, and the thyroid gland was collected post mortem by careful dissection using a binocular magnifying lens. Thyroid tissue was weighed and subjected to either fixation or freezing (without or with Tissue-TeK immersion) in liquid nitrogen and storage at –80°C until use. All surgical and experimental procedures were conducted in accordance with the french policies in animal use and care.

Genotyping. Wild-type and mutant Cx32 alleles were simultaneously detected by PCR using a set of three primers. A 500-bp fragment was amplified from the wild-type allele with the following primers: 5'-CCATAAGTCAGGTGT AAAGGAGC-3' and 5'-AGATAAGCTGCA GGGACCATAGG-3'; the third primer was complementary to a sequence in the neo resistance cassette of the disrupted allele (5'-ATCATGCGAAACGATCCTCATCC-3'). The PCR reaction generated a 414-bp fragment from the mutant Cx32 allele. PCR was performed in a total volume of 30 µl containing 2 mM MgCl2, 0.25 mM dGTP, dATP, dCTP, and dTTP, 0.3 mM of each primer, 1.5 U Taq DNA polymerase, and 300 ng of genomic DNA. The reaction conditions were 5 min at 95°C followed by 40 cycles (30 s at 95°C, 45 s at 67°C, 90 s at 72°C) and a final extension at 72°C for 10 min.

Integration of the Cx32/EGFP transgene was analyzed by Southern blot using an EGFP cDNA probe (7) and by PCR screening using the primers 5'-CTG GTC GAG CTG GAC GGC GAC G-3' and 5'-GGC GGT CAC GAA CTC CAG CAG GAC C-3' located within the EGFP coding sequence. The PCR conditions included an initial step of denaturation at 94°C for 10 min followed by 35 cycles of amplification (60 s at 94°C, 60 s at 60°C, and 90 s at 72°C) and 10 min at 72°C for final extension. PCR products were fractionated on 2.0% agarose gels and visualized by ethidium bromide staining and UV transillumination.

Histological and immmunofluorescence analyses. For conventional histological examinations by light microscopy, thyroid tissue was fixed in 10% formaldehyde and embedded in paraffin using the standard procedures. Five-micrometer sections were prepared and stained with hematoxylin using AniPath Histology platform facilities (Faculté de Médecine Laennec, Lyon, France). EGFP fluorescence (excitation at 488 nm, emission at 505–530 nm) was detected on frozen thyroid sections by use of a fluorescence-equipped Axiophot microscope (Carl Zeiss, Oberkochen, Germany) or on whole mice (killed by injection of a massive dose of anesthetics) using a FluorImager from Molecular Dynamics (Bondoufle, France) located at the Cecil Imaging platform (IFR Lyon-Est, Faculté de Médecine Laennec, Lyon, France).

For immunofluorescence analyses, mouse thyroid lobes frozen in Tissue Tek (Fisher, Pittsburgh, PA) were processed to prepare 5-µm sections. Thyroid tissue sections were fixed in 4% p-formaldehyde, washed, and incubated with polyclonal anti-Cx32 antibodies produced in rabbit (11) overnight at 4°C; tissue sections were then incubated with a biotinylated anti-rabbit Ig antibody (Rockland, Gilbertsville, PA) for 1 h at 37°C. Immune complexes detected with Alexa 488-labeled streptavidin (Molecular Probes, Eugene, OR) were visualized using a fluorescence-equipped Axiophot microscope.

Real-time PCR. Total RNA was isolated on silica columns (RNeasy minikit; Qiagen, Courtaboeuf, France) according to the manufacturer's protocol. Potentially contaminating genomic DNA was eliminated by DNAse I treatment (RNAse-free DNAse, Qiagen). Total RNA (1 µg) was reverse transcribed in a 20-µl reaction volume containing 200 U MMLV reverse transcriptase (Promega, Madison, WI), 10 nmol of each dNTP, 24 U ribonuclease inhibitor (Promega), and 100 pmol of random hexamers (Amersham Pharmacia, Orsay, France) for 1 h at 37°C. PCR was performed on a LightCycler from Roche (Roche Diagnostics, Meylan, France). After 5 min at 95°C, cDNAs were amplified in duplicate in a final volume of 10 µl using a FastStart DNA Master SYBR Green I kit from Roche. The reaction mixture contained 5 µM of the forward and reverse primers (Table 1) and 5 µl of diluted cDNA template solution corresponding to 2.5 ng of retrotranscribed RNA for Cx32, Cx32/EGFP, Cx43, Cx26, NIS, DIO1, TPO, p21, p27, and to 25 pg for 18S rRNA. PCR conditions included an initial denaturation step of 10 min at 95°C followed by 35–40 cycles (15 s at 95°C, 4 s at 58, 59, 57, 57, 60, 60, 60, 62, 59, or 64°C, and 8 s at 72°C) for the final extension step. Fluorescence intensity measurements were used to determine the crossing point value, i.e., the cycle number at which fluorescence was significantly greater than the background. The specificity of PCR amplification was assessed by determination of the Tm of amplicons, using a fusion program consisting of a temperature increase of 0.1°C/s from 60 to 97°C.


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  		Table 1. Primers used for mRNA assay by quantitative RT-PCR

 
Standard curves were generated using 10 to 106 copies of cloned cDNA (Cx32/EGFP, 18S rRNA, p21, p27) or cloned amplicons (Cx32, Cx43, Cx26) or using dilutions of a calibrator (cDNA generated from a pool of normal mouse thyroid RNA) for DIO1, NIS and TPO mRNA assays. Cloning was carried out in the pGEMT easy vector from Promega Results were expressed in copy number per µg RNA or in fold change compared with the calibrator, after standardization using 18S rRNA. Comparison of the transcript contents of samples was done by Student's t-test.

Hormone measurements. Mouse serum TSH concentration was assayed using a double antibody precipitation radioimmunoassay according to Pohlenz et al. (28) with reagents provided by National Hormone and Pituitary Program (Harbor-UCLA-Medical Center, Torrance, CA). Briefly, the assay mixture contained anti-rat TSH antibodies prepared in guinea pig, 125I-labeled rat TSH, and either mouse serum or a crude mouse TSH/LH reference preparation used as standard. Rat TSH was labeled using 125I-Na and tubes coated with Iodogen (Pierce Chemical, Rockford, IL); its specific radioactivity was ~30 µCi/µg TSH. TSH concentration was measured in 2–50 µl of mouse serum. Goat anti-guinea pig Ig precipitating antibodies (Antibodies, Davis, CA) were used to precipitate immune complexes.

Serum total thyroxine (T4) concentration was measured by radioimmunoassay using anti-T4 antibodies (Valbiotech, Paris, France), 125I-T4 (specific radioactivity: 1,300 mCi/mg) (PerkinElmer, Wellesley, MA), serial dilutions of T4, or 10 µl of mouse serum and sheep anti-rabbit Ig antibodies immobilized on beads (Biogenesis, Brentwood, NH). The sensitivity of the assay was ~60 ng/100 ml.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Thyroid phenotype of Cx32-KO mice. Because the Cx32 gene is located on the X chromosome, the thyroid status of Cx32-depleted male and female mice was analyzed separately. In the basal state, Cx32-KO male mice had a normal thyroid, in terms of both size and structure, compared with wild-type littermates (Figs. 1A and 2). The weight of the gland of 10-wk-old mice was 2.4 ± 0.1 mg (n = 7) in Cx32-KO and 2.4 ± 0.2 mg (n = 8) in wild-type mice. In basal conditions, serum TSH concentration (Fig. 1B) and serum T4 concentration (data not shown) were similar in Cx32-KO and wild-type male mice.


Figure 1
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  		Fig. 1. Alterations of thyroid gland in connexin (Cx)32-depleted male mice. A: comparison of thyroid weight of Cx32-KO male mice (filled columns) and wild-type littermates (open columns) at 10 wk of age in the basal state (no treatment, time 0) and after perchlorate treatment for 2, 8, or 14 wk or after 14 wk of perchlorate treatment and 1 (week 15) or 3 wk (weeks 15–17) without any treatment. Arrow indicates end of perchlorate administration. Each column and vertical bar represents the mean and SE of values obtained from 7–9 mice, except at 8 wk of perchlorate treatment when data deriving from 3 separate experiments and a total of 22 wild-type and 23 Cx32-KO male mice were pooled. *Statistically significant difference compared with wild-type mice, P < 0.01. B: variations in serum TSH concentration of Cx32-KO (closed symbols) and wild-type male mice (open symbols) in response to perchlorate treatment. TSH was measured by RIA developed for mouse TSH as described in MATERIALS AND METHODS. Symbols and vertical bars represent the mean or SE of values from 7–9 mice.

 

Figure 2
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  		Fig. 2. Morphological analyses of thyroid gland of wild-type and Cx32-KO male mice in basal state or under TSH stimulation (after 8 wk of perchlorate treatment). Representative microscope images of p-formaldehyde-fixed, paraffin-embeded thyroid tissue sections visualized after hematoxylin staining. Scale in µm gives magnification of micrographs.

 
Treatment of mice with perchlorate (which inhibits thyroid iodide supply and thus thyroid hormone synthesis) induced a rise in serum TSH concentration and a thyroid hyperplasia (Fig. 1, A and B). Interestingly, the increase in thyroid weight was more rapid and more marked in Cx32-KO than in control male mice. During the first 2 wk of anti-thyroid treatment, the thyroid size of Cx32-KO mice increased ~2.5-fold, from 2.4 ± 0.1 mg (n = 7) (thyroid weight in basal state or time 0 of treatment) to 5.7 ± 0.4 mg (n = 6, P < 0.01), whereas the thyroid weight of wild-type mice increased only by ~40%, from 2.4 ± 0.2 mg (n = 8) to 3.4 ± 0.2 mg (n = 8, P < 0.01). The difference in thyroid size between Cx32-KO and wild-type male mice persisted for up to 14 wk (Fig. 1A); at that time, the thyroid of Cx32-KO mice was still 25% bigger than that of control mice. Noteworthy, serum TSH concentration, which sharply increased during the first weeks of the anti-thyroid treatment, was, at each time point, similar in the two groups of mice. Histological examinations of hyperplastic thyroid tissue did not detect any difference between Cx32-deficient and control male mice (Fig. 2). In the two groups of mice, there was a similar reorganization of the thyroid tissue resulting from the emptying of the lumen of the follicles and from the increase of thyroid epithelial cell volume in response to the strong and prolonged TSH stimulation (33). Upon discontinuation of the perchlorate treatment, the goiter of both Cx32-KO and wild-type male mice rapidly decreased in size. However, goiter involution was more rapid in Cx32-KO than in control mice; after 3 wk, thyroid weight of Cx32-KO mice was no longer different from that of wild-type mice.

We analyzed the thyroid phenotype of three groups of female mice: Cx32–/– (Cx32-KO), Cx32+/– (mice with one normal and one mutated Cx32 gene), and wild-type (Cx32+/+) mice. In basal conditions, there was no difference in thyroid weight (Fig. 3), serum TSH or T4 concentrations (data not shown) between the three groups of mice. Prolonged TSH stimulation (resulting from perchlorate treatment) induced a larger goiter in Cx32-deficient than in wild-type mice: Cx32+/– mice presented an intermediate phenotype. After 20 wk of anti-thyroid treatment, thyroid weight of wild-type, Cx32 +/–, and C32–/– female mice was 7.3 ± 0.5 mg (n = 12), 10.5 ± 1.2 mg (n = 12), and 13.4 ± 1.3 mg (n = 17), respectively. Morphological analyses of thyroid tissue did not show any difference between the three groups of mice (data not shown). Thyroid weight increment between Cx32-KO and wild-type mice was higher in female than in male mice. As in male mice, differences in thyroid growth were not related to differences in TSH stimulation, since serum TSH concentration was similar in the three groups of mice (data not shown).


Figure 3
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  		Fig. 3. Alterations of thyroid gland in Cx32-depleted female mice. A: relationship between changes in thyroid weight and Cx32 genotype of female mice. Cx32–/– (filled columns) Cx32+/– (mice with one normal and one mutated Cx32 gene) (gray columns) and Cx32+/+, or wild-type (open columns) female mice were killed at 10 wk of age in the basal state (no treatment, time 0) or after 15 or 20 additional weeks of perchlorate treatment. Columns and vertical bars represent means and SE of values obtained from groups of 10–11 mice in the basal state or 12–17 mice under perchlorate treatment. *Statistically significant difference compared with wild-type mice, P < 0.01. B: immunofluorescence detection of Cx32 in thyroid of Cx32+/+ (a), Cx32+/– (b), and Cx32–/– (c) female mice after 15 wk of perchlorate administration. Frozen sections of thyroid tissue were fixed with 4% p-formaldehyde and immunostained with anti-Cx32 antibodies. Immune complexes were detected using biotinylated anti-rabbit Ig and Alexa 488-labeled streptavidin. Morphology of thyroid follicles after perchlorate administration for 15 wk, similar in the 3 groups of mice, is shown in d (hematoxylin-stained section); magnification is the same as in a, b and c.

 
The distribution of Cx32 in the thyroid of Cx32+/+, Cx32+/–, and Cx32–/– female mice was analyzed by indirect immunofluorescence (Fig. 3B); the study was performed in mice treated with perchlorate for 15 wk. As expected, thyroid of Cx32–/– mice was completely Cx32-negative (Fig. 3B, c). In the thyroid of Cx32+/+ and Cx32+/– mice, immunolabeled Cx32 appeared as dots, which likely correspond to GJ between thyrocytes organized in follicle structures, as previously reported (13, 24); due to the prolonged TSH stimulation, thyroid follicles had a reduced size (see Fig. 3B, d). The labeling of thyroid sections from Cx32+/– mice was heterogenous; Cx32-positive zones adjoined Cx32-negative zones (Fig. 3B, b). In the Cx32-negative zones, follicle structures are formed from cells no longer expressing Cx32 as a result of the X chromosome inactivation process.

Impact of Cx32 gene inactivation on expression of other Cx in thyroid. Polarized epithelial thyroid cells or thyrocytes, representing up to 70% of cells of the thyroid gland (4), are known to express three connexins, Cx32, Cx43, and Cx26 (13, 23, 24). We examined whether the inactivation of the Cx32 gene in the thyroid was associated with changes in the expression of the other Cx. Analyses were performed at the transcript level on wild-type and Cx32-KO male mice subjected to 8 wk of perchlorate treatment (Fig. 4). Among the three Cx transcripts, Cx32 mRNA was the most abundant: ~2.2 x 107 copies/µg RNA vs. 4.8 x 105 and 2.6 x 104 copies/µg RNA for Cx43 and Cx26, respectively. The Cx43 and Cx26 thyroid transcript contents were similar in wild-type and Cx32-deficient mice. Neither Cx43 nor Cx26 compensates for the absence of Cx32 and the disappearance of Cx32 GJ in the mouse thyroid.


Figure 4
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  		Fig. 4. Does inactivation of the Cx32 gene influence thyroid expression of other Cx or expression of cyclin-dependent kinase inhibitors p21cip1 and p27kip1? Wild-type (open columns) and Cx32-KO male mice (filled columns) at 8 wk of age were subjected to perchlorate treatment for 8 wk. Thyroid RNA was used to generate cDNAs by use of random primers. Cx26, Cx32, and Cx43 transcripts (A) and p21 and p27 transcripts (B) were assayed by quantitative PCR as described in MATERIALS AND METHODS. Results are expressed as means (columns) and SE (bars) of values obtained from 5–7 mice (A) and 13–17 mice (B). Arrow indicates that no Cx32 transcript was detected in the thyroid of Cx32-KO mice. *Statistically significant difference compared with wild-type, P < 0.05.

 
Impact of Cx32 gene inactivation on expression of cyclin-dependent kinase inhibitors p21cip1 and p27kip1. We (8) previously found that Cx32-expressing FRTL-5 or FRT cells characterized by a reduced proliferation rate (compared with wild-type FRTL-5 or FRT cells) exhibited an increased expression of the cyclin-dependent kinase inhibitors p21cip1 and/or p27kip1. We compared the thyroid transcript content corresponding to these two kinase inhibitors in Cx32-KO and wild-type male mice under perchlorate treatment. Data of Fig. 4B show a significant decrease in the p21cip1 transcript content of the thyroid of Cx32-deficient mice and no difference in the level of p27kip1 between Cx32-KO and wild-type mice; this might be explained by the fact that TSH activates the expression of p27kip1 (3).

Specificity of the thyroid phenotype of mice with generalized inactivation of Cx32: complementary approach. To ascertain that the changes in thyroid growth evidenced in Cx32-KO mice were actually the consequence of the thyroid depletion in Cx32, we investigated whether a thyroid-selective Cx32 complementation of Cx32-KO mice could reverse the thyroid growth changes related to Cx32 deficiency. The strategy that we chose required 1) the generation of transgenic mice exhibiting a moderate overexpression of Cx32 selectively in the thyroid (Cx32-T+ mice) and 2) the interbreeding of these mice with Cx32-KO mice.

Generation of mice with thyroid-targeted Cx32 overexpression. The construct used for transgenesis, composed of the Cx32gene in fusion with the EGFP cDNA placed under the control of the thyroglobulin gene promoter, had previously been validated in stably transfected FRTL-5 cells (7). The Cx32/EGFP chimeric protein 1) was capable of forming GJ targeted to the plasma membrane and 2) retained its capacity to slow down cell proliferation as native Cx32. Three mouse lines (lines 2, 4, and 7) were generated; only two of them, line 2 and line 7 expressed Cx32/EGFP. Evidence for integration of Cx32/EGFP transgene in mouse lines 2 and 7 and for its transmission to the progeny is provided in Fig. 5. The level of expression of the transgene in the thyroid of the two mouse lines in basal conditions and under TSH stimulation was analyzed by quantitative RT-PCR and compared with the level of expression of endogenous Cx32 (Fig. 6A). The thyroid Cx32 transcript level was constant whatever the mouse genotype (wild-type or transgenic) or the thyroid activation state (basal or TSH stimulation). The thyroid content in Cx32/EGFP transcripts was very different in the two mouse lines; the ratio between Cx32/EGFP and Cx32 transcript levels was equal to 4 in the mouse line 2 and to only 0.04 in the mouse line 7. As expected, perchlorate treatment led to an increase in the expression of the transgene in both mouse lines. The expression of the Cx32/EGFP transgene was visualized on thyroid cryosections by EGFP fluorescence emission (Fig. 6B). Cx32/EFGP protein appeared as bright spots over the thyroid epithelium. In the mouse line 2, Cx32/EFGP could be used for in situ fluorescence imaging of the thyroid gland in basal conditions (Fig. 6D) or under TSH stimulation (Fig. 6E). Cx32-T+ mice of line 2 maintained in basal conditions had a smaller thyroid (by ~20%) than wild-type littermates (Fig. 7A). This change in thyroid size, which occurred without any alteration of the overall follicle morphology (Fig. 7, B and C) was reproducibly obtained on distinct experimental groups generated over a 2-yr period. The same thyroid hypoplasia was observed on 12-mo-old mice. Under perchlorate treatment (i.e., TSH stimulation), the thyroid weight reduction of Cx32-T+mice (line 2) persisted for 3 wk and then disappeared. Serum TSH concentrations were similar in transgenic and wild-type mice (data not shown). The size of the thyroid of mice from line 7 (with a very low expression of Cx32/EGFP) did not differ from that of wild-type controls whatever the experimental conditions.


Figure 5
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  		Fig. 5. Identification of Cx32/EGFP transgene in mouse genomic DNA by Southern blot. Tail mouse DNA from the founder (F1) and from the progeny (F2) of lines 2 and 7 was analyzed by agarose gel electrophoresis after HindIII digestion. Cx32/EGFP insert (1.9 kb) was detected using a 32P-labeled EGFP cDNA probe.

 

Figure 6
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  		Fig. 6. Characterization of mice with thyroid-targeted Cx32 overexpression. A: comparative analyses of the level of expression of the Cx32/EGFP fusion gene (filled columns) and endogenous Cx32 (open columns) in thyroid of Cx32-T+ mouse lines. Measurements were made at the mRNA level by quantitative RT-PCR. Cx32/EGFP transcripts were assayed using primers located on the EGFP sequence. Wild-type and transgenic mice from two lines (lines 2 and 7) were studied at 8–10 wk of age in the basal state or after 7 additional weeks of perchlorate treatment. Colums and vertical bars represent means and SE of 4–5 determinations. B: visualization of the Cx32/EGFP fusion protein by detection of EGFP fluorescence. Fluorescence microscope observation of a thyroid cryosection from a line 2 mouse in the basal state. FL, follicle lumen. C–E: thyroid imaging based on detection of EGFP fluorescence using a FluorImager from Molecular Dynamics. C, control; nontransgenic mouse; D, mouse (line 2) in the basal state; E, mouse (line 2) treated by perchlorate for 7 wk. Mice were killed by injection of a massive dose of anesthetics, and thyroid fluorescence was visualized after rapid surgical opening of the neck region.

 

Figure 7
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  		Fig. 7. Alterations of the thyroid gland of Cx32-T+ transgenic mice. A: changes in thyroid weight of transgenic mice from line 2 (filled columns) or line 7 (gray columns) compared with wild-type littermates (open columns). Measurements were made on mice at 8–10 wk of age in the basal state (time 0) and after 3 or 7 wk of perchlorate treatment. Each column and vertical bar represents the mean and SE of values from 24–25 wild-type mice, 15–19 mice from line 2, and 12–13 mice from line 7. *Statistically significant difference compared with wild-type mice, P < 0.05. B and C: morphological examinations of thyroid of Cx32-T+ transgenic mice (C) and wild-type controls (B) in the basal state. Representative microscope images of paraffin-embedded tissue sections visualized after hematoxylin staining. Scale in µm gives magnification of micrographs.

 
Despite a reduced thyroid weight, Cx32-T+ mice (line 2) exhibited the same serum thyroid hormone and TSH concentrations as wild-type littermates (Fig. 8). This prompted us to investigate parameters of thyroid activity. The levels of transcripts encoding the Na+/iodide symporter (NIS), type I deiodinase (DIO1) (Fig. 8) or thyroid peroxidase (result not shown) were significantly increased in the thyroid of transgenic mice compared with controls. Noteworthy, these changes indicating an activation of the gland took place at constant serum TSH concentration.


Figure 8
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  		Fig. 8. Investigation of the functional status of thyroid gland of Cx32-T+ transgenic mice. A and B: serum TSH and thyroxine (T4) concentrations of Cx32-T+ mice from line 2 (filled columns) and nontransgenic littermates (open columns) at 8–10 wk of age. Mean and SE of values from 18–19 mice per group. C and D: changes in level of expression of genes involved in thyroid hormone biosynthesis, Na+/iodide symporter (NIS), and type 1 deiodinase (DIO1). Measurements were made at the transcript level by quantitative RT-PCR and expressed as fold change compared with calibrator. Columns and vertical bars represent means and SE of values from 5 mice per group. *Statistically significant difference compared with wild-type mice, P < 0.01.

 
Reintroduction of Cx32 in the thyroid of Cx32-KO mice. Cx32-T+ male mice (from line 2, which exhibited a moderate overexpression of Cx32/EGFP) were crossed with Cx32+/– female mice to generate male mice with the four following genotypes: wild-type, Cx32-KO, Cx32-T+, and Cx32-KO/Cx32-T+ mice, which expressed Cx32 (in the form of Cx32/EGFP) only at the level of thyroid gland. As reported above, Cx32-T+ mice in the basal state had a smaller thyroid glands than wild-type and Cx32-KO mice (Fig. 9). Thyroid weight of Cx32-KO/Cx32-T+ male mice was not different from that of wild-type mice. Under TSH stimulation (after 3 wk of perchlorate treatment), Cx32-KO male mice developed a goiter 25% larger than that of wild-type (as reported before) or Cx32-T+ mice. Interestingly, the thyroid of Cx32-KO/Cx32-T+ mice had the same size as wild-type or Cx32-T+ mice (Fig. 9); this indicates that the reexpression of Cx32 in the thyroid of Cx32-KO mice reversed the thyroid growth alterations related to Cx32 deficiency.


Figure 9
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  		Fig. 9. Reversal of the thyroid phenotype of Cx32-KO mice by reintroduction of Cx32 in the thyroid. Cx32-T+ male mice (from line 2) were crossed with Cx32+/– female mice to generate 4 groups of mice with the genotypes indicated in the figure. Thyroid was analyzed in mice at 10 wk of age maintained in basal conditions and in mice subjected to 3 additional weeks of perchlorate treatment. Columns and vertical bars represent means and SE of values from 12–15 mice per group. *Statistically significant difference between this group of mice and the 3 other groups, P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study discloses a regulatory role of Cx32 and/or Cx32-GJ on growth of the thyroid gland in vivo. We have found that a lack of expression of Cx32 in the thyroid is associated with a marked and persistent increase in thyroid growth in response to TSH stimulation, whereas Cx32 expressed in excess limits thyroid growth under physiological conditions.

As observed in the earliest analyses of the phenotype of Cx32-KO mice by Willecke and coworkers more than 10 years ago (26), we found that the thyroid gland of mice lacking Cx32 was normal in size and activity in physiological (basal) conditions. We evidenced that Cx32 depletion had a marked influence on the way thyroid cells respond to their main mitogenic factor, TSH. Thyroid cells depleted in Cx32 exhibited an increased responsiveness to TSH; indeed, under perchlorate treatment, thyroid hyperplasia or goiter formation (in response to the rise in serum TSH) was markedly accelerated in Cx32-KO mice. The phenomenon was observed in both male and female Cx32-KO mice, but the increment in TSH-induced thyroid hyperplasia appeared higher in female than in male mice. Such sex-related differences, which are often observed in thyroid pathophysiology, have no straightforward explanation. Female mice with one mutated and one intact Cx32 gene exhibited an intermediate phenotype. In these mice, there was a heterogeneous expression of Cx32 among thyroid cells, which probably resulted from the X chromosome inactivation process. A similar observation has previously been made with another X chromosome-linked gene, G6PD (27). The increment in TSH-induced thyroid hyperplasia observed in Cx32-deficient mice was fully reversible. This was demonstrated in Cx32-KO male mice; 3 wk after discontinuation of the anti-thyroid treatment, the size of the gland was similar in Cx32-KO and wild-type male mice, suggesting that apoptosis of epithelial cells, which is involved in the involution of goiter (30), could be more active in Cx32-deficient than in control mice.

A moderate overexpression of Cx32 in the thyroid (4 to 10 times the endogenous Cx32 expression level) led to a new thyroid equilibrium characterized by normal serum TSH and T4 concentrations but a gland smaller than normal and exhibiting signs of activation. The augmentation of thyroid functional activity, evidenced by the increase in thyroid-specific gene expression (e.g., NIS, TPO, DIO1), probably compensates for the reduction in the mass of thyroid parenchyma and contributes to a normal thyroid hormone production and thus to a normal circulating TSH concentration. These in vivo findings are in full agreement with previous in vitro data on FRTL-5 cells showing that the overexpression of Cx32 leads to a reduction of the cell proliferation rate and to an increase in expression of differentiation cell markers (32).

The specificity of our in vivo findings is further documented by the complementation experiment. The reintroduction of Cx32 in the thyroid of Cx32- KO mice led to the abrogation of the thyroid phenotypic changes observed in conditions of Cx32 deficiency. This finding strongly suggests that the other Cx expressed in the thyroid gland, especially Cx43 (13), cannot functionally replaced Cx32. This is in agreement with our previous in vitro data on FRTL-5 and FRT cell lines showing that the Cx32-dependent regulatory actions on cell proliferation or expression of differentiation (8, 34) are not reproduced by Cx43.

The mechanism through which Cx32 exerts regulatory functions on cell growth and differentiation remains largely hypothetical; it could depend either on Cx32-GJ-mediated cell-to-cell communication or on the ability of Cx32 to interact with proteins belonging to signaling cascades.

Cx32 GJ allow the cell-to-cell transfer of cyclic AMP (1), the intracellular messenger of TSH action (5, 18). It is now well demonstrated that thyroid cell proliferation depends on cyclic AMP (31). The enhancement of thyroid growth in response to TSH observed in Cx32-deficient mice could be secondary to an increase in the proportion of cells reaching the cyclic AMP concentration threshold required for the activation of the cell cycle. On the other hand, thyroid hypoplasia observed in mice overexpressing Cx32 in basal conditions could be secondary to an increase in the proportion of cells remaining below a cyclic AMP concentration threshold to undergo division.

As mentioned earlier in this discussion, one has to consider that Cx32 could also regulate cell proliferation through a mechanism independent of GJ-mediated intercellular communication. It has been proposed that Cx32 could exert regulatory actions by interfering with signaling pathways such as the MAPK pathway (19) or the pathway activated through the ERBB/Her-2 tyrosine kinase receptor (10). Importance has been given to direct interactions between Cx and proteins endowed with diverse function(s) (see Ref. 15 for a review). The known interaction partners of Cx32 are calmodulin (35), zonula occludens-1 and -2 (20), E-cadherin, {alpha}-catenin (9), and discs large homolog-1 (Dlgh1) (6). The interaction of Cx32 with the latter protein, very recently described in liver (6), takes on importance since Dlgh1 is involved in cellular growth control via a nuclear translocation mechanism (16). Dlgh1, localized at the plasma membrane, blocks the cell cycle in the G0/G1 phase, and translocation of Dlgh1 to the nucleus has been shown to increase cell proliferation (11). As the loss of Cx32 was found to induce a decrease in the level of Dlgh1 and to cause its nuclear translocation; it was proposed (6) that Cx32 might act in the control of cell proliferation in liver through the maintenance of Dlgh1 at the plasma membrane. If Dlgh1 is expressed in the thyroid gland, the Cx32-dependent control of thyroid growth might be related to the Cx32-Dlgh1 interaction.

In conclusion, we have shown that Cx32 controls growth of normal thyroid gland; an excess of Cx32 limits growth of thyroid cells in the basal state, whereas a lack of Cx32 confers an additional growth potential to stimulated thyroid cells. Our data indicate that Cx32 and Cx32 GJ are probably dispensable in the thyroid gland in steady-state conditions (i.e., basal or physiological conditions) but are essential to limit thyroid hyperplasia caused by high and sustained serum TSH concentration.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from Association pour la Recherche sur le Cancer.


   ACKNOWLEDGMENTS
 
We are grateful to Prof. Klaus Willecke for giving us the opportunity to analyze the thyroid phenotype of Cx32-KO mice. We thank C. Durand and R. Rabilloud for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. ROUSSET, INSERM UMR 664, Faculté de Médecine Laennec, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France (e-mail: rousset{at}sante.univ-lyon1.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
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