Growth factors protect intestinal stem cells from radiation-induced apoptosis by suppressing PUMA through the PI3K/AKT/p53 axis

Abstract

Gastrointestinal toxicity is the primary limiting factor in abdominal and pelvic radiotherapy, but has no effective treatment currently. We recently showed a critical role of the BH3-only protein p53 upregulated modulator of apoptosis (PUMA) in acute radiation-induced GI damage and GI syndrome in mice. Growth factors such as insulin-like growth factor 1 (IGF-1) and basic fibroblast growth factor (bFGF) have been shown to protect against radiation-induced intestinal injury, although the underlying mechanisms remain to be identified. We report here the suppression of PUMA through the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/p53 axis in the intestinal stem cells as a novel molecular mechanism of growth factor-mediated intestinal radioprotection. IGF-1 or bFGF impaired radiation-induced apoptosis and the expression of PUMA and p53 in the crypt cells and intestinal stem cells. Using colonic epithelial cells that undergo PUMA-dependent and radiation-induced apoptosis, we found that a PI3K inhibitor, dominant-negative PI3K or Mdm2 antagonist restored the induction of PUMA, p53 and apoptosis in the presence of growth factors. In contrast, overexpression of AKT suppressed the induction of PUMA and p53 by radiation. Furthermore, inhibiting PI3K or activating p53 abrogated growth factor-mediated suppression of apoptosis and PUMA expression in the intestinal crypts and stem cells after radiation.

Figure 1. Growth factors protected against apoptosis induced by radiation in the intestinal crypts and stem cells in mice

(A) Apoptosis in the intestinal crypts of WT C57/BL6 mice subjected to various treatments 4 hr after 15 Gy WBR was assessed by TUNEL staining (brown), magnification × 400. a: untreated mice (UN). b: mice irradiated by 15 Gy (IR). c: mice injected i.v. with 3.5 μg human recombinant IGF-1 followed by 15 Gy radiation (IR+IGF-1). d: mice injected i.v. with 3.5 μg human recombinant bFGF followed by 15 Gy radiation (IR+bFGF). (B) Apoptotic index measured by TUNEL staining as in (A) was quantitated. Values are means ± SD; n=3 in each group. *, P<0.05. (C) Left, The fractions of crypts containing at least one TUNEL-positive CBC were calculated by counting 100 crypts with well preserved Paneth cell areas. Values are means ± SD; n = 3 in each group. Right, an example of MMP-7/TUNEL double staining in the crypts 4 hr after 15 Gy with a CBC, magnification × 600. The nuclei were counterstained with DAPI. The CBCs are in between the MMP-7⁺ Paneth cells at the bottom of the crypt. (D) Regenerated crypts at 96 hours after 15 Gy WBR were analyzed by the micro colony assay and quantitated. Values are means ±SD; n=3 in each group.

Figure 2. Growth factors blocked radiation-induced PUMA expression in the intestinal mucosa

(A) PUMA mRNA expression in the jejunal mucosa of mice following 15 Gy WBR with or without treatment of growth factors was evaluated by quantitative real-time RT-PCR. Values are means ± SD; n = 3 in each group. (B) The expression of PUMA, p53, and p21 in the jejunal mucosa of WT mice with the indicated treatments was determined by Western blotting. β-actin (Actin) was used as the control for loading.

Figure 3. Growth factors protected against radiation-induced apoptosis through the PI3K/AKT pathway in vitro

(A) HCT116 p21 KO cells with or without growth factor treatment were irradiated by 15 Gy. Apoptosis was determined by nuclear staining with DNA binding dye Hoechst 33258 48 hr after irradiation. Those with fragmented and condensed nuclei were counted as apoptotic cells. (B) PUMA mRNA expression in HCT116 p21 KO cells following 15 Gy radiation with or without the treatment of growth factors was evaluated by quantitative real-time RT-PCR. Values are means ± SD. Each experiment was repeated for 3 times. (C) The expression of PUMA, p53, Akt, and p-Akt in HCT116 p21 KO cells with the indicated treatments was determined by western blotting. (D) The effect of the PI3K inhibitor LY294002 on the expression of PUMA and p53 in HCT116 p21 KO cells with the indicated treatments. The levels of the proteins were determined by western blotting. LY: LY294002, PI3K inhibitor. (E) The expression of PUMA and p53 in the indicated cell lines 24 hr after radiation was determined by western blotting. α-tubulin was used as the control for loading in (C-E). The gene or protein expression in (B)-(E) was determined 24 hr after irradiation.

Figure 4. Growth factors inhibited PUMA induction by radiation through a p53-dependent mechanism in vitro

(A) The effect of dominant negative PI3K (DN PI3K) on the expression of PUMA, p53, AKT, p-AKT, Foxo3a, and p-Foxo3a in HCT116 p21 KO cells with the indicated treatments. The protein levels were determined by western blotting. (B) The effects of AKT on the indicated proteins in HCT116/p21 KO cells were determined by western blotting. The cells were transfected with WT or constitutively active AKT for 24 h followed by irradiation, and collected for analysis 24 hr later. (C) The effects of a MDM2 antagonist nutlin-3a on the expression of PUMA, p53, MDM2, and p-MDM2 in HCT116 p21 KO cells with the indicated treatments. The protein levels were determined by western blotting. α-tubulin was used as the control for loading. (D) The effects of LY294002 and nutlin-3a on radiation-induced apoptosis in HCT116 p21 KO cells. (E) HCT116 p21 KO cells were transfected with control or Foxo3a siRNA 24 hr before irradiation. Foxo3a and PUMA expression was analyzed by western blotting. The gene or protein expression in (A-C) and (E) was determined 24 hr after radiation, while the apoptosis in (D) was determined 48 hr after radiation.

Figure 5. Growth factors protected the intestinal stem cells from radiation-induced apoptosis through the PI3K-AKT-p53-PUMA axis

(A) p-AKT expression in the intestinal crypts of mice with various treatments was evaluated by immunohistochemistry. a: UN; b: IR; c: IR+IGF-1; d: IR+bFGF. The CBC areas were circled in the groups with growth factor treatment. (B) An example of MMP-7/p-AKT double staining used to identify CBCs in the crypts of mice 4 hr after irradiation. Magnification × 600. Arrowheads indicate p-AKT expressed in CBCs. (C) The effect of the PI3K inhibitor LY294002 (LY) on the apoptosis and expression of indicated proteins in mice subjected to the indicated treatments. Top, apoptotic index in the CBCs measured by TUNEL staining and quantitated. Values are means ± SD; n=3 in each group. Middle, apoptotic index in the crypts measured by TUNEL staining and quantitated. Values are means ± SD; n=3 in each group. Bottom, the expression of PUMA, p53, and p-Akt in the intestinal mucosa was determined by western blotting. β-actin was used as the control for loading. (D) The effects of the MDM2 antagonist nutlin-3a on apoptosis of the CBCs and crypts, and the expression of the indicated proteins were analyzed as in (C).

Figure 6. PUMA deficiency protected the CBCs against radiation-induced apoptosis better than p53 deficiency

Apoptosis in the intestinal crypts of mice with indicated genotype 4 hr or 24 hr after 15 Gy WBR was assessed by TUNEL staining as in Fig. 5C. The fraction of crypts with at least one TUNEL-positive CBC was calculated by counting 100 crypts with well preserved Paneth cell areas. Values are means ± SD; n = 3 in each group. *p < 0.05.