The thyroid hormone receptor-alpha (TRalpha) gene encoding TRalpha1 controls deoxyribonucleic acid damage-induced tissue repair

Abstract

The thyroid hormone (TH) controls, via its nuclear receptor, TH receptor-alpha1 (TRalpha1), 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. TRalpha(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 TRalpha(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 TRalpha mutants. Altogether, in TRalpha(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 TRalpha gene plays a role in crypt cell homeostasis by regulating the rate of cell renewal and apoptosis induced by DNA damage.

Fig. 1.

Analysis of WT and TRα^0/0 Small Intestine Morphology after 8 Gy γ-Ray Irradiation Panels show hematoxylin and eosin staining of paraffin sections of WT (A–E) or TRα^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.

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 TRα^0/0 crypts at different time points after 8 Gy γ-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 TRα^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.

Fig. 3.

Follow-Up of DNA Damage in the Intestine after Irradiation Paraffin sections were from intestine of WT (A–D) or TRα^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.

Fig. 4.

Analysis of p53 Protein Expression after Irradiation A, Number of p53 positive cells per crypt in WT or TRα^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 TRα^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.

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 TRα^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 TRα^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.