Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation

Abstract

Exposure to high levels of ionizing radiation (IR) leads to debilitating and dose-limiting gastrointestinal (GI) toxicity. Using three-dimensional mouse crypt culture, we demonstrated that p53 target PUMA mediates radiation-induced apoptosis via a cell-intrinsic mechanism, and identified the GSK-3 inhibitor CHIR99021 as a potent radioprotector. CHIR99021 treatment improved Lgr5+ cell survival and crypt regeneration after radiation in culture and mice. CHIR99021 treatment specifically blocked apoptosis and PUMA induction and K120 acetylation of p53 mediated by acetyl-transferase Tip60, while it had no effect on p53 stabilization, phosphorylation or p21 induction. CHIR99021 also protected human intestinal cultures from radiation by PUMA but not p21 suppression. These results demonstrate that p53 posttranslational modifications play a key role in the pathological and apoptotic response of the intestinal stem cells to radiation and can be targeted pharmacologically.

Figure 1. PUMA deficiency protects crypt culture and Lgr5+ cells against radiation by blocking apoptosis.

Small intestinal crypts from WT and PUMA KO mice were plated in Matrigel and subjected to 5 Gy, or mock (Un) irradiation, 24 h after plating and harvested at the indicated time points. (A) Representative pictures of enteroids at day 6 and quantitation of enteroids with 5 or more buds. (B) Apoptosis was assessed by TUNEL staining at 24 h in WT and PUMA KO crypts. Left, representative pictures of TUNEL (green) staining. Right, quantitation of TUNEL index. (C) Representative pictures and quantitation of p-H2AX staining in cultured crypts at 4 and 24 h. (D) Representative pictures and quantitation of Ki 67 staining in cultured crypts at 24 h. (E) Expression of PUMA and p21 transcripts was analyzed by real-time RT-PCR. (F) Crypts were isolated from Lgr5-EGFP Cre-ER background. Lgr5+ apoptosis was assessed by TUNEL/GFP staining at 24 h. Left, representative pictures of TUNEL (green), Lgr5 (red) and nuclei (DAPI) staining. Right, the percentage of Lgr5+ crypts containing one or more TUNEL+ cells. (G) Expression of Olfm4, Lgr5, Bmi1 and Hopx transcripts was analyzed by real-time RT-PCR. Values are means ± SD, n = 3 wells from three different mice with each genotype. ***P < 0.001, **P < 0.01. Scale bar, A, 100 μm; B, C, D & F, 20 μm.

Figure 2. CHIR99021 protects crypt culture against radiation and blocks apoptosis.

CHIR99021 (2.5 μM) was added to crypts 24 hr prior to 5 Gy or mock (Un) irradiation, and crypts were harvested at the indicated time points after radiation. (A) Representative pictures of enteroids at day 6 and quantitation of enteroids with 5 or more buds. (B) Apoptosis was assessed by TUNEL staining at 24 h. Left, representative pictures of TUNEL (green) staining. Right, quantitation of TUNEL index. (C) Quantitation of p-H2AX staining in cultured crypts 24 h after radiation. (D) Quantitation of Ki67 staining in cultured crypts 24 h after radiation. (E) Crypts were isolated from Lgr5-EGFP Cre-ER background. Apoptosis was assessed by TUNEL staining at 24 h. Left, representative pictures of TUNEL (green), Lgr5 (red) and nuclei (DAPI) staining. Right, quantitation of Lgr5+ crypts containing one or more TUNEL+ cells. (F) Expression of Olfm4, Lgr5, CD44, Bmi and Hopx transcripts was analyzed by real-time RT-PCR. Values are means ± SD. n = 3 wells from three different mice in each group. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, A, 100 μm; B & E, 20 μm.

Figure 3. CHIR99021 inhibits p53-dependent PUMA induction and p53 K120-acetylation.

Crypt culture were subjected to 5 Gy, or mock (Un) irradiation and harvested at indicated time points. (A) The expression of indicated proteins at 24 h in cultured crypts (3 wells pooled) was determined by western blotting. β-actin (Actin) was used as the control for loading. (B) Expression of PUMA and p21 transcripts was analyzed by real-time RT-PCR. (C) Cultured crypts were infected with an adenovirus encoding PUMA (Ad-PUMA) for 24 h, and PUMA mRNA levels and protein (HA) were analyzed by real-time RT-PCR and western blotting. (D) Cultured crypts were infected by Ad-PUMA at the same time with or without CHIR99021 treatment. Left, quantitation of enteroids with 5 or more buds at day 4. Right, representative pictures of enteroids at day 4. Scale bar, 100 μm. Values are means ± SD, n = 3 wells from three different mice in each group. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4. CHIR99021 protects mice from radiation-induced lethal GI injury.

WT C57BL/6 mice were injected intraperitoneally with CHIR99021 (2 mg/kg) or vehicle (dimethyl sulfoxide) 4 h prior to 14.5 Gy ABI (A) or 15 TBI (B-G). (A) Survival curves of mice after ABI. n = 10 mice in each group. (B) Apoptosis in the intestinal crypts 4 h after TBI was assessed by TUNEL staining (brown). Left, representative pictures of TUNEL staining. Right, quantitation of TUNEL index. (C) Representative pictures and quantitation of p-H2AX staining in the intestinal crypts 4 h and 24 h after TBI. (D) Representative pictures of regenerated crypts identified by BrdU staining 96 h after TBI (brown). (E) Quantitation of regenerated crypts (left) and villus height (right) in D. (F) TUNEL staining in the crypts of Lgr5-EGFP;Cre-ER mice 4 h after TBI. Left, representative pictures of TUNEL (green), Lgr5 (red) and nuclei (DAPI) staining. Right, quantitation of Lgr5+ crypts containing one or more TUNEL+ cells. (G) Expression of Olfm4, Lgr5, CD44, Bmi1 and Hopx transcripts was analyzed by real-time RT-PCR. (B-G), values are means ± SD, n = 3 mice in each group. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar, 25 μm.

Figure 5. CHIR99021 suppresses induction of PUMA and p53 K120 acetylation by radiation in mice.

Intestinal mucosa of WT mice were harvested after 15 Gy TBI with or without CHIR99021 treatment (2 mg/kg) 4 h prior to radiation. (A) The expression of indicated proteins at 4 h was analyzed by western blotting. β-actin was used as the control for loading. (B) The mRNA expression of PUMA and p21 was analyzed by real-time RT-PCR. Values are means ± SD. n = 3 mice in each group. ***P < 0.001, **P < 0.01. (C) The expression of p-H2AX (pooled from 3 mice) was analyzed by western blotting. The lysates were pooled from 3 mice.

Figure 6. CHIR99021 protects human intestinal culture and stem cells against radiation injury.

Two independent human intestinal cultures, EL1 (A-E) and MM1 (F-G), were plated and passaged in Matrigel as described in methods. CHIR99021 (2.5 μM) was added to culture medium 24 h before 5 Gy radiation, and cultures were harvested at indicated times after radiation. (A) Representative pictures of enteroids at day 10 and quantitation of enteroids ≥100 μm. (B) Apoptosis of was assessed by TUNEL staining at 24 h. Olfm4 labels putative stem cells. Left, representative pictures of TUNEL (green), Olfm4 (red) and nuclei (DAPI) staining. Middle, quantitation of TUNEL index. Right, quantitation of Olfm4+ cells containing one or more TUNEL+ cells. (C) Representative pictures (left) and quantitation of p-H2AX staining (right) 24 h after radiation. (D) Expression of PUMA and p21 transcripts was analyzed by real-time RT-PCR. (E) Expression of Olfm4, Lgr5, Ascl2 and CD44 transcripts was analyzed by real-time RT-PCR. (F) Enteroid growth of MM1 culture was analyzed at day 6. (G) The expression of PUMA and p21 transcripts in MM1 culture at 24 h was analyzed by RT-PCR. Values are means ± SD, n = 3 wells. ***P < 0.001, **P < 0.01. Scale bar, A and F, 200 μm; B and C, 30 μm.