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Molecular Endocrinology, doi:10.1210/me.2007-0278
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Molecular Endocrinology 22 (1): 47-55
Copyright © 2008 by The Endocrine Society

The Thyroid Hormone Receptor-{alpha} (TR{alpha}) Gene Encoding TR{alpha}1 Controls Deoxyribonucleic Acid Damage-Induced Tissue Repair

Elsa Kress, Amelie Rezza, Julien Nadjar, Jacques Samarut and Michelina Plateroti

Université de Lyon, Université Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique, Institut de Génomique Fonctionnelle de Lyon, 69364 Lyon Cedex 07, France

Address all correspondence and requests for reprints to: Michelina Plateroti, Institut de Génomique Fonctionnelle de Lyon, Ecole Normale Supérieure de Lyon, 46 allée d’Italie, 69364 Lyon Cedex 07, France. E-mail: Michela.Plateroti{at}ens-lyon.fr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-{alpha}1 (TR{alpha}1), intestinal crypt cell proliferation in the mouse. In order to understand whether this receptor also plays a role in intestinal regeneration after DNA damage, we applied a protocol of {gamma}-ray irradiation and monitored cell proliferation and apoptosis at several time points. In wild-type mice, the dose of 8 Gy induced cell cycle arrest and apoptosis in intestinal crypts a few hours after irradiation. This phenomenon reverted 48 h after irradiation. TR{alpha}0/0 mutant mice displayed a constant low level of proliferating cells and a high apoptosis rate during the period of study. At the molecular level, in TR{alpha}0/0 animals we observed a delay in the p53 phosphorylation induced by DNA damage. In our search for the expression of the protein kinases responsible for p53 phosphorylation upon irradiation, we have focused on DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The number of cells expressing DNA-PKcs in crypts remained high 48 h after irradiation, specifically in TR{alpha} mutants. Altogether, in TR{alpha}0/0 animals the rate of apoptosis in crypt cells remained high, apparently due to an elevated number of cells still presenting DNA damage. In conclusion, the TR{alpha} gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE THYROID HORMONES (THs), T3 and T4, play multiple roles in vertebrate development and homeostasis (1). T3 acts through nuclear receptors, the TH receptors (TRs), encoded by the TR{alpha} and TRβ loci. The TRs are transcription factors that activate or repress the transcription of target genes (2). The introduction of mutations in the TR genes in mice has allowed further understanding of the mechanisms mediating the multiple actions of TH in target tissues (3). One of the established targets of TH action is the intestine (4). The best-characterized example is its extensive remodeling during the spontaneous or TH-induced amphibian metamorphosis (5).

The intestinal epithelium is a continuously renewing tissue. This depends on the presence of stem cells and committed progenitors located in the crypts of Liberkün. These display a very high proliferation rate (6). Radiation promotes cell death and induces compensatory regenerative proliferation in epithelial cells (7, 8). In light of its characteristic rapid turnover rate, the small intestine epithelium is one of the most radiosensitive tissues (8). The cellular and molecular events responsible for this radiosensitivity are starting to be understood. A few hours after irradiation the crypt cell proliferation is blocked and the cells undergo apoptosis. This process is reverted in a few hours, when apoptosis stops and cell proliferation resumes, refilling again the crypt compartments (8).

The p53 protein plays a major role in this process essentially as a gatekeeper (9). In fact, p53 activation by phosphorylation leads to different and coordinated responses (see scheme in supplemental Fig. 1S, published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org): 1) up-regulation of p21 and DNA mismatch repair gene transcription, favoring cell cycle arrest and DNA repair; 2) up-regulation of an apoptosis-promoting gene (i.e. Bax). The fact that in intestinal crypts p53 activation after {gamma}-ray irradiation preferentially induces apoptosis led to the conclusion that in the small intestine this process is a protective mechanism, which efficiently removes from the tissue stem or progenitor cells with DNA damage (8). This might represent a supplementary mechanism for maintaining very low levels of mutations in crypt stem cells and could help in explaining why the small intestine is less prone to tumorigenesis compared with large intestine (10).

Thyroid hormones control intestinal development at weaning in the mouse (4). Indeed, mice lacking the expression of TR{alpha} gene have reduced cell proliferation in crypts during development and in adulthood (11). The same defect has been demonstrated in congenital or chemically induced hypothyroidism in mice (12, 13). In our search for molecular mechanisms, we have recently shown that the TR{alpha}1 receptor controls, through T3 binding, the expression of key components of the Wnt/β-catenin pathway (11).

This major role of TR{alpha}1 in intestine epithelial proliferation suggested that the TR{alpha} gene might control the regeneration process taking place after {gamma}-ray irradiation. To test this hypothesis, we studied the kinetics of recovery after irradiation of TR{alpha} mutant compared with the WT mice. We show that in TR{alpha} mutants, displaying a lower proliferation rate, the apoptosis in crypts is maintained at high levels for a longer time compared with the WT mice. This correlated with the sustained activation of the p53 protein pathway, which mediates the apoptosis in crypt cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Morphological and Functional Features of the Small Intestine after {gamma}-Ray Irradiation
To assess whether the TR{alpha} gene plays a role in the regeneration of the small intestine, WT and TR{alpha}0/0 mice were exposed to a single dose of 8 Gy of {gamma}-ray irradiation. Histological features have been analyzed at several time points after irradiation and compared with the nonirradiated intestine (Fig. 1Go, A–J). Hematoxylin and eosin staining of WT sections showed a clear-cut disorganization of the crypt epithelium 24 h after irradiation compared with the nonirradiated intestine (Fig. 1Go, B and C vs. A). In fact, those crypts were clearly smaller in size and displayed several apoptotic or ghosts of apoptotic cells (morphological criteria as in Ref.14). However, 48 h after irradiation, these mice displayed emerging but structured crypts (Fig. 1Go, D and E). Hematoxylin and eosin staining of TR{alpha}0/0 intestine sections revealed a similar disorganization of the crypt epithelium 24 h after irradiation, compared with the nonirradiated intestine (Fig. 1Go, G and H vs. F). This can be evaluated by the presence of several apoptotic cells in each crypt, as also described in the WT crypts. However, 48 h after irradiation TR{alpha}0/0 still exhibited small and disorganized crypts compared with the WT at the same time point (Fig. 1Go, I and J vs. D and E).


Figure 1
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  		Fig. 1. Analysis of WT and TR{alpha}0/0 Small Intestine Morphology after 8 Gy {gamma}-Ray Irradiation

Panels show hematoxylin and eosin staining of paraffin sections of WT (A–E) or TR{alpha}0/0 (F–J) small intestine. The intestinal morphology has been studied in nonirradiated mice (A and F) or in mice 24 (B, C, G, and H) or 48 h after irradiation (D, E, I, and J). ve, Villus epithelium; ce, crypt epithelium; ct, connective tissue; ml, muscle layers. The red dotted bars define the limit between the crypts and the villi compartments. Some crypts (yellow dots) are highlighted in panel A. The arrows in B, G, and I point to the ghost of apoptotic cells. Scale bar, 15 µm in panels A, B, D, F, G, and I; scale bar, 7 µm in panels C, E, H, and J.

 
Analysis of Cell Proliferation and Apoptosis
The process of the small intestine regeneration implies both a massive apoptosis of the progenitors with DNA damage followed by an increased proliferation of the surviving ones (8). To determine whether the TR{alpha} gene is implicated in these two processes, we analyzed the proliferative and the apoptotic indexes of the crypt compartment of WT or TR{alpha}0/0 mice at different time points after irradiation. To this end, paraffin-embedded sections of small intestine were immunostained for bromodeoxyuridine (BrdU) incorporation (supplemental Fig. S2) and cleaved-caspase 3 expression (supplemental Fig. S3), and the number of positive cells per crypt was determined by counting the cells under the microscope (Fig. 2Go). In WT mice, the number of proliferating cells significantly decreased by 50% 4 h after irradiation and remained unchanged for the following 24 h (Fig. 2AGo). A clear and significant increase of cell proliferation was evident 48 h after irradiation. As expected, the basal proliferative rate of crypt cells in TR{alpha}0/0 mice was significantly lower than in the WT (Fig. 2AGo), confirming our previous data (11). This low proliferation rate remained unaltered over the time of observation. Finally, no clear-cut increase in the number of proliferating progenitors has been observed 48 h after irradiation.


Figure 2
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  		Fig. 2. Follow-Up of Cell Proliferation and Apoptosis in Intestinal Crypt Cells after Irradiation

Number of proliferating (panel A) and number of apoptotic cells (panel B) in WT and TR{alpha}0/0 crypts at different time points after 8 Gy {gamma}-irradiation. Proliferation and apoptosis have been studied by BrdU and cleaved-caspase 3 immunolabeling, respectively. The number of positive cells per crypt for each marker has been counted under a Zeiss Axioplan microscope on well-oriented sections from three animals per experimental group. Thirty crypts per experimental condition have been evaluated under the microscope. Histograms illustrate means ± SDs. C, WB analysis of the indicated proteins in the whole intestine lysate of WT or TR{alpha}0/0 animals, at different time points after irradiation. The blot is representative of three independent experiments. Each lane represents pooled extracts from two animals (50 µg/lane). Actin has been used as loading control. Histograms in the lower panels (means ± SDs) summarize densitometry analyses (by ImageQuant software) from three independent experiments. Data are normalized by the amount of actin in each sample. *, P < 0.05; and **, P < 0.01 by Student’s t test. When not indicated, the comparisons have been realized with the preceding time point. #, P < 0.05; and ##, P < 0.01 by Student’s t test, compared with the WT at the same time point.

 
Next we analyzed the number of apoptotic cells (Fig. 2BGo). In the WT as well as in TR{alpha}0/0 animals, there were no cleaved-caspase 3-positive cells in crypts in untreated condition. This was in agreement with published data (15). However, after the irradiation the kinetics of apoptosis in the crypts of animals of the two genotypes was strongly different. WT crypt cells displayed a peak of apoptosis 8 h after irradiation, and then the number of apoptotic cells significantly decreased over time. Surprisingly, TR{alpha}0/0 mice showed a strong increase in the number of cleaved-caspase 3-positive cells 4 h after irradiation. However, in contrast to the WT, this level remained very high over the 48 h of observation (Fig. 2BGo).

To correlate the data obtained at cellular level, we analyzed the expression of markers of cell cycle arrest such as p21, and markers of cell cycle progression such as cyclin D1. The levels of these two proteins were studied by Western blot (WB) and quantified by densitometry (Fig. 2CGo). p21 amount was undetectable in control condition of both WT and TR{alpha} mutants. In WT the level significantly increased 4 h after irradiation, remained high at 8 h, and then declined. In TR{alpha}0/0 mice the induction of p21 at 4 h was lower than in the WT. Moreover, the level of the protein continued to increase at 8 and 48 h after irradiation. Cyclin D1 expression levels inversely correlated with those of p21, but were generally lower in the TR{alpha} mutant at almost all time points analyzed. Despite these differences at the protein level, we couldn’t observe any difference in the kinetics of p21 (supplemental Fig. S4) and Cyclin D1 (data not shown) mRNA expression in TR{alpha}0/0 compared with the WT mice.

To check whether there was a difference in the kinetics of removal of cells displaying DNA damage in TR{alpha}0/0 mice compared with the WT, we analyzed the expression of the phospho-H2A.X protein, a well-characterized sensor of DNA damage (16). We have analyzed the number of positive cells by an immunohistochemistry (IHC) approach on tissue slides (Fig. 3Go, A–H), and counted them under the microscope (quantification illustrated in Fig. 3IGo). A few scattered positive nuclei were present in crypts and in lamina propria cells in both WT and TR{alpha}0/0 animals in nonirradiated controls (Fig. 3Go, A and E). Four hours after irradiation this number greatly increased in both genotypes (Fig. 3Go, B and F). However, at 48 h after irradiation it was clear that a higher number of p-H2A.X-positive cells was present in TR{alpha}0/0 crypts compared with the WT (Fig. 3Go, G and H vs. C and D). Figure 3IGo summarizes the counting of the p-H2A.X-positive cells. This clearly shows that in TR{alpha}0/0 crypt there is almost twice the number of positive cells compared with the WT mice 48 h after irradiation. This difference is statistically significant. This was not due to a defect in the expression of the genes involved in DNA repair such as Mlh1, Msh1, and Msh2 (data not shown).


Figure 3
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  		Fig. 3. Follow-Up of DNA Damage in the Intestine after Irradiation

Paraffin sections were from intestine of WT (A–D) or TR{alpha}0/0 (E–H) in control (A and E), 4 h (B and F) and 48 h after irradiation (C, D, G, and H). DNA damage has been revealed by the pH2A.X immunolabeling. I, Quantification of the pH2A.X-positive cells in intestinal crypts. The number of positive cells per crypt has been counted under a Zeiss Axioplan microscope on well-oriented sections from three animals per experimental group. Thirty crypts per experimental condition have been evaluated under the microscope. Histograms illustrate means ± SDs. ve, Villus epithelium; ce, crypt epithelium; ml, muscle layer; c, crypt. The black dotted bars define the limit between the crypt and the villi compartments. The arrows in A and E point to few scattered positive cells. Scale bars, 15 µm in panels A, B, D, E, F, H; scale bar, 40 µm in panels C and G. **, P < 0.01 by Student’s t test. ##, P < 0.01 by Student’s t test, compared with the WT at the same time point.

 
Altogether, the morphological feature of the TR{alpha}0/0 small intestine crypts, 48 h after irradiation, can be explained both by a high level of apoptosis and a low level of proliferation, resulting in a delay of the regeneration of the intestinal mucosa.

p53 Activation Cascade
p53 is the key effector of the DNA damage-sensing pathway (scheme in supplemental Fig. S1). Then we monitored its expression as well as that of proteins that directly or indirectly control its activation. We first monitored the mRNA level of p53 by RT-quantitative PCR (QPCR). Its expression was not significantly modified by {gamma}-ray irradiation, either in WT or in TR{alpha}0/0 mice during the whole analysis (supplemental Fig. S4). These data are in agreement with the fact that the activation of p53 goes through the phosphorylation of the preexisting protein (9, 17).

We then analyzed the expression of p53 protein in crypt cells by an IHC approach. The p53-positive cells in crypts were counted under the microscope and the results were reported in Fig. 4AGo. The kinetics of p53-positive cells during regeneration overlaps with that of apoptotic cells in both WT and TR{alpha} mutants, clearly showing a persistence of p53-positive cells in TR{alpha}0/0 irradiated intestine.


Figure 4
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  		Fig. 4. Analysis of p53 Protein Expression after Irradiation

A, Number of p53 positive cells per crypt in WT or TR{alpha}0/0 mice at different time points after irradiation. The positive immunolabeled cells have been counted under a Zeiss Axioplan microscope on well-oriented sections from three different animals per experimental condition. Thirty crypts per experimental condition have been evaluated under the microscope. Histograms illustrate means ± SDs. B, WB analysis of the indicated proteins in whole-intestine lysates of WT and TR{alpha}0/0 animals, at different time points after irradiation. The blot is representative of three independent experiments. Each lane represents pooled extracts from two animals (50 µg/lane). For loading control, membranes have been stained with Ponceau Red before incubation with the antibodies. The figure illustrates some representative protein bands. Histograms in the lower panels (means ± SDs) summarize densitometry analyses (by ImageQuant software) from three independent experiments. Data are normalized by the amount of actin in each sample. *, P < 0.05; **, P < 0.01 by Student’s t test. When not indicated, the comparisons have been realized with the preceding time point. #, P < 0.05; ##, P < 0.01 by Student’s t test, compared with the WT at the same time point.

 
These changes correlate with the overall level of expression of p53 in the whole intestinal mucosa as assessed by WB analysis and quantified by densitometry (Fig. 4BGo). In the WT small intestine, the level of p53 was almost undetectable at the baseline. Then it started increasing after 4 h, reached a peak at 8 h after irradiation, and then declined greatly toward the basal level. Interestingly, in TR{alpha}0/0 intestine the basal level of p53 was significantly higher than in the WT, remained stable until 8 h, reached a peak at 24 h, and started decreasing at 48 h but it was still significantly higher compared with the WT at the same time point. Some discrepancies with the results obtained by microscopy can be due to the tissue heterogeneity in the lysates from the whole intestine.

We have considered the possibility that the different amounts of total p53 in the two genotypes could depend on different levels of the phosphorylated Mdm2 (serine 166). It is worth pointing out that phospho-Mdm2 protein is the active form that binds to p53 and targets its degradation through the proteasome (18). However, by using WB and IHC approaches we failed to see any significant difference in the phospho-Mdm2 protein expression in the two genotypes during the whole experiment (data not shown).

We also analyzed the expression of phospho-p53 (serine 15), which represents the active form of p53 (9, 17). The results are illustrated in Fig. 4BGo. Phospho-p53 is detectable in irradiated WT mice after 4 h. Its level increased at 8 h and then dropped. In TR{alpha}0/0 animals the phospho-p53 became detectable 8 h after irradiation and then its level slightly decreased. However, the protein was still present at 48 h in these animals in contrast to the WT (Fig. 4BGo, compare lanes 48 h).

The sustained expression of the phosphorylated active form of p53 in the intestine of the TR{alpha}0/0 animals is then thought to be the cause of the higher apoptosis rate in these mice 48 h after irradiation.

Finally we analyzed the expression of the protein kinases involved in the Ser15-phosphorylation of p53 at mRNA and protein levels. However, we couldn’t observe any major difference in the levels of checkpoint kinase (Chk)-1 and -2, ataxia telangiectasia mutated kinase (ATM), and ataxia telangiectasia RAD3-related kinase (ATR) in the TR{alpha}0/0 compared with the WT mice at all time points analyzed (data not shown). It has been reported that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is one of the key proteins quickly recruited on DNA after damage. It phosphorylates p53 on serine-15 (19). To evaluate whether the expression of DNA-PKcs was altered in TR{alpha}0/0 compared with the WT mice, we performed RT-QPCR experiments (Fig. 5AGo). In WT animals there was a clear-cut and significant increase of the DNA-PKcs mRNA 4 h after irradiation. Then the amount decreased at the basal levels. In TR{alpha}0/0 animals, we could observe a similar and significant increase of the DNA-PKcs mRNA 4 h after irradiation. However, the levels remained high and almost unchanged until 24 h after irradiation. Interestingly, when we looked at the expression of DNA-PKcs at protein level in crypt cells by using an IHC approach, we could also detect qualitative and quantitative differences in the two genotypes (Fig. 5Go, B–K). In both WT and TR{alpha}0/0 animals in control condition, we observed a few DNA-PKcs-positive cells, sometimes clearly in nonepithelial cells (Fig. 5Go, B and G). There was a clear-cut increase 8 h after irradiation in DNA-PKcs-positive cells mostly in crypts in the two genotypes (Fig. 5Go, C, D, H, and I). The scoring of the number of DNA-PKcs-positive cells per crypt under the microscope indicated a similar result in the animals of the two genotypes (WT: 3.2 ± 0.79, n = 30; TR{alpha}0/0: 3.6 ± 0.84, n = 30). However, it was clear that in TR{alpha}0/0 mutants the number of DNA-PKcs-positive cells remained high at 48 h compared with the WT animals (Fig. 5Go, J and K vs. E and F). Moreover, the counting under the microscope indicated that this difference was statistically significant (WT: 0.7 ± 0.48; TR{alpha}0/0: 2.3 ± 0.67; P < 0.01 by Student’s t test, n = 30).


Figure 5
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  		Fig. 5. Analysis of DNA-PKcs Expression after Irradiation

A, Quantitative real-time RT-PCR analysis of DNA-PKcs mRNA in intestine of WT and TR{alpha}0/0 irradiated mice. The figure is representative of two independent experiments, using three animals per experimental condition. Histograms illustrate means ± SDs. **, P < 0.01 by Student’s t test. #, P < 0.05 by Student’s t test, compared with the WT at the same time point. B–K, Immunolabeling of DNA-PKcs on paraffin sections from WT (B–F) or TR{alpha}0/0 (G–K) mice. Intestinal sections have been studied in nonirradiated mice (B and G), 8 h after irradiation (C, D, H, and I) or 48 h after irradiation (E, F, J, and K). Pictures show the merging of DNA-PKcs immunolabeled (red) and nuclear staining by Hoechst (blue). ve, Villus epithelium; ce, crypt epithelium; ml, muscle layers. The white dotted bars define the limit between the crypt and the villi compartments. The arrows in B and G indicate the DNA-PKcs-positive cells. Scale bar, 15 µm in panels B, C, E, G, H, and J; scale bar, 7 µm in panels D, F, I, and K.

 
Altogether, these data strongly suggested a shift in p53 protein phosphorylation after irradiation of TR{alpha}0/0 mice, which correlates with the persistence of apoptotic cells in the crypts and the delayed regeneration of the tissue.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The TRs play multiple roles during postembryonic development and physiological homeostasis. They are encoded by TR{alpha} and TRβ genes and are widely expressed throughout the tissues (20). The control by TRs of postnatal organ/tissue maturation has been compared with the multiple remodeling programs taking place during amphibian metamorphosis (5). Therefore it might be expected that TRs could play some role in tissue repair and regeneration in adults after insults such as irradiation.

The THs are involved in several cellular responses such as cell proliferation, cell differentiation, and apoptosis (1). In the small intestine of mice with altered TH/TR pathway, we have extensively documented their major role in epithelial cell proliferation (4). In these same models, we had also analyzed the levels of apoptosis. However, we failed to distinguish any difference between hypothyroid and/or TR{alpha}0/0 animals compared with the euthyroid WT (our unpublished observations). In the present work we show that intestinal injury induced by {gamma}-ray leads to a sustained apoptotic response in crypt cells of TR{alpha} mutant mice. These data show that TR{alpha} controls the apoptosis only upon stress-induced tissue injury. Along the same lines, a role of TH in rat liver regeneration after partial hepatectomy has also been illustrated. This involves a negative control of apoptosis and the activation of cell proliferation to accelerate liver cellularity (21).

It has been widely documented that the regeneration of the intestinal epithelium after irradiation implies both a rapid cell death to eliminate cells with high DNA damage, and a proliferation burst to refill the crypt compartment (7, 8). When we looked at the kinetics of cell apoptosis and cell proliferation, it is clear that the regeneration properties of the TR{alpha}0/0 animals are much different from that of the WT. First of all, TR{alpha} mutants do not respond to high DNA damage with a clear-cut reduction of cell proliferation. One explanation could be that the cell cycle in those animals, even under control conditions, is already very slow. Second, the apoptotic step is longer, probably due a defect in the DNA-damage sensing, as suggested by our results. Altogether, the slow proliferation rate and the sustained apoptosis 24–48 h after irradiation in TR{alpha}0/0 animals would explain their impaired epithelial regeneration as compared with the WT.

Tissue repair after stress-induced injury is largely controlled by the p53 pathway (9, 17). The cellular levels of the p53 are maintained constant by a negative regulatory loop. The Mdm2 protein binds to p53 and targets its degradation via the proteasome (22). In turn, the Mdm2 gene is directly regulated by p53 (23). It is worth pointing out that the Mdm2 gene expression has also been shown to be controlled by TH. In fact, a TH response element is located in the first intron of the gene, near the p53 response element (24). However, when we looked at the expression of the Mdm2 at mRNA level, we could not observe any difference in the TR{alpha} mutants compared with the WT animals (data not shown). A possible explanation is that TR{alpha} plays a secondary role in controlling the expression of this gene via the enhancer in the intron 1, in response to DNA damage.

One important observation in our study is that p53 protein levels, but not mRNA levels, were higher in the intestine of the TR{alpha}0/0 animals compared with the WT in control conditions. Because this was not due to differences in the expression of its main controller Mdm2, other factors must be involved.

It has been shown that p53 and the TRβ1 physically interact in vitro. This leads to the inhibition of the transactivation activities of both TRβ1 and p53 on their respective target genes (25, 26). No data are available concerning a cross talk between TR{alpha}1 and p53 protein. However, several observations rule out such a possibility in intestinal epithelial progenitors. Because the mRNA levels of p53 direct targets (i.e. p21 and Bax in supplemental Fig. S4) were not increased in TR{alpha}0/0 mice, it is unlikely that TR{alpha}1 and p53 inhibit each other through functional interaction. We can exclude any involvement of TRβ, because crypt cells express only the products of the TR{alpha} gene (our unpublished data).

p53 is activated by several cellular stresses, such as irradiation-induced DNA damage (9, 17). This results in the activation of several kinases comprising DNA-PKcs, ATM, Chk1, and ChK2, which in turn phosphorylate the preexisting p53 (9). DNA-PKcs forms a platform on the broken DNA to ensure the assembly of a multiprotein complex allowing DNA repair and phosphorylates p53 on serine 15, thereby activating it (19). DNA damage-dependent recruitment of DNA-PKcs is supposed to be a process involving posttranslational modifications such as phosphorylation (19).

An interesting, even surprising, observation was that a few hours after irradiation there was an increase in the levels of DNA-PKcs mRNA. This suggests that DNA damage signals also via the activation of DNA-PKcs transcription. However, because both WT and TR{alpha} mutant mice respond similarly, we can exclude the possibility that the up-regulation of the mRNA depends on the TR{alpha} gene. On the other hand, the fact that the levels of the mRNA and the protein remained high in the TR{alpha}0/0 animals suggests that the TR{alpha} gene can play a role in its repression. However, this control is indirect because we couldn’t observe any regulation of the DNA-PKcs mRNA in WT mice with altered TH status, or in intestine epithelium primary cultures treated with T3 (data not shown).

In conclusion, the TR{alpha} gene is involved in the regeneration of the intestinal epithelium, both by allowing fast elimination of damaged cells through apoptosis and by enhancing the proliferation of surviving progenitors. This control might reside at very early steps of DNA damage-dependent signaling, through as yet undefined mechanisms. Such a regeneration model would be valuable in identifying novel functions of the TR{alpha} gene. Finally, these data might be of therapeutic interest for patients who undergo abdominal radiotherapy. Tissue complications caused by radiation-induced intestine stem cell depletion may result in structural and functional alterations of the gastrointestinal tract. It would therefore help to have a better understanding of the regeneration process on the intestine as mediated by TH after irradiation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animal Treatment and Tissue Preparation
We used 16-wk-old female TR{alpha}0/0 mice, lacking the expression of the TR{alpha} gene (27), as well as WT female animals having the same genetic background. The mice were housed and maintained with approval from the animal experimental committee of the Ecole Normale Supérieure de Lyon (Lyon, France), and in accordance with European legislation on animal care and experimentation. The animals received 8 Gy of whole-body irradiation, using a 137Cesium {gamma}-source (dose rate, 0.67 Gy/min). For the analysis of cell proliferation rate, 30 min before euthanasia the animals were injected with 100 µl of 4 mg/ml of BrdU solution in PBS. Animals were killed at the indicated time points after irradiation, and the intestine was quickly removed. The proximal and the distal parts of the small intestine were fixed in 4% paraformaldehyde for the histology and the immunohistochemistry, or frozen in liquid nitrogen and used for RNA and/or protein extraction. The protocol for mice irradiation was approved by the Regional Committee for the animal experimentation (CREEA; agreement no. 087). Under the recommendations of this committee, the irradiated mice were killed no later than 48 h after irradiation to avoid any long-term suffering.

Immunostaining
Paraffin sections (5 µm thickness) were used for indirect immunostaining. Briefly, sections were deparaffined in methylcyclohexane for 10 min, hydrated in ethanol, and washed with PBS. Slides were then subjected to antigen retrieval, using microwave heating in 0.01 M citrate buffer (pH 6), and incubated for 1 h at room temperature with blocking buffer (10% normal goat serum, 1% BSA, and 0.02 Triton X-100 in PBS). The slides were incubated with primary antibodies overnight at 4 C followed by incubation with fluorescent antimouse or antirabbit antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). All nuclei were stained with Hoechst (Sigma Chemical Co., St. Louis, MO). For the immunohistoenzymatic approach, we used the Histomouse kit (Zymed Laboratories, Inc., South San Francisco, CA). Fluorescence and bright-field microscopy was performed on a Zeiss Axioplan microscope (Carl Zeiss, Thornwood, NY). To quantify the number of immunolabeled cells, they have been counted under the microscope on well-oriented sections from at least three different animals per experimental condition.

Western Blotting
Whole-protein extracts were obtained by homogenizing mouse small intestine in RIPA buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1 mM phenylmethylsulfonylfluoride; 1 mM EDTA; 5 µg/ml aprotinin; 5 µg/ml leupeptin; 1% Triton X-100; 1% sodium deoxylate; 0.1% sodium dodecyl sulfate; 40 µl/ml protease inhibitors). Proteins (50 µg per lane) were separated on 8 or 10% acrylamide-bis acrylamide (29:1) gel and transferred to a nitrocellulose membrane (Hybond ECL, Amersham) before saturation and incubation with the first antibody. This was followed by incubation with secondary antirabbit or antimouse IgG-horseradish peroxidase (Promega Corp., Madison, WI). The signal was analyzed using the enzymatic chemiluminescence detection kit (LumiLight; Roche Clinical Laboratories, Indianapolis, IN).

Antibodies
For the immunolabeling on sections or WBs we used the following antibodies: anti-BrdU (Amersham Pharmacia Biotech, Piscataway, NJ; reconstituted with the nuclease buffer and used undiluted); anticleaved caspase-3 (diluted 1:100), antiphospho-p53 (Ser15) (diluted 1:500), antiphospho-Mdm2 (Ser166) (diluted 1:50 for immunofluorescence (IF) and 1:100 for WB), antiphospho-Chk2 (Thr387) (diluted 1:500), antiphospho-ATM (Ser1981) (diluted 1:500) (Cell Signaling Technology, Beverly, MA); anticyclin D1 (Lab Vision Corp., Fremont, CA; diluted 1:500); anti-p53 (diluted 1:50 for IF and 1:200 for WB), anti-p21 (diluted 1:500), anti-DNA PKcs (diluted 1:50 for IF and 1:200 for WB) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-phospho-histone H2A.X (Upstate Biotechnology, Inc., Lake Placid, NY; diluted 1:50).

RNA Extraction and Analysis
RNA was extracted from tissues using the QIAGEN RNeasy kit (QIAGEN, Chatsworth, CA). To avoid the presence of contaminating DNA, DNase digestion was performed in all preparations. Reverse transcription was performed using MuMLV reverse transcriptase (Promega) on 1 µg of total RNA according to the manufacturer’s instructions using random hexanucleotide priming (Invitrogen, Carlsbad, CA). QPCR analyses were performed with SYBR green PCR master mix (QIAGEN) in a MXP3000 apparatus (Stratagene, La Jolla, CA). The data from the PCR were normalized to that of 36B4 levels in each sample. The primers used are listed in supplemental Table 1.

Statistics
Numerical results (graphs) are presented as means ± SD. Groups were compared by using Student’s two-tailed t test, with P < 0.05 considered significant.


   ACKNOWLEDGMENTS
 
We thank Nadine Aguilera for animal handling. The histological analyses were performed by the Anipath platform of Rhône-Alpes Genopole.


   FOOTNOTES
 
This work was supported by the Agence Nationale pour la Recherche (Grant ANR-06-BLAN-0232-01) and the program Equipe Labellisée of the Ligue Nationale contre le Cancer. E.K. and A.R. studentships are supported by the Ligue Nationale contre le Cancer.

Disclosure Statement: The authors have nothing to disclose.

First Published Online September 13, 2007

Abbreviations: BrdU, Bromodeoxyuridine; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; IF, Immunofluorescence; IHC, immunohistochemistry; QPCR, quantitative PCR; TH, thyroid hormone; TR, TH receptor; WB, Western blot; WT, wild type.

Received for publication June 1, 2007. Accepted for publication September 5, 2007.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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