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. 2010 Aug;151(8):3728-37.
doi: 10.1210/en.2010-0319. Epub 2010 Jun 9.

Amphiregulin promotes intestinal epithelial regeneration: roles of intestinal subepithelial myofibroblasts

Jinyi Shao  1 , Hongmiao Sheng
Affiliations

Amphiregulin promotes intestinal epithelial regeneration: roles of intestinal subepithelial myofibroblasts

Jinyi Shao et al. Endocrinology. 2010 Aug.

Abstract

Epidermal growth factor family plays critical roles in intestinal epithelial proliferation and differentiation. The precise function of amphiregulin (AREG), a member of the epidermal growth factor family, in intestinal biology is largely unknown. The present study attempted to address the functional roles of AREG in intestinal epithelial regeneration. Total body irradiation was performed, and intestinal regeneration was assessed in AREG knockout mice. Genetically disruption of AREG significantly impaired intestinal regeneration after radiation injury. It is known that prostaglandin E(2) (PGE(2)) exerts radio-protective and growth-stimulatory effects on intestinal epithelium. We found that PGE(2) radio-protective action did not involve AREG. However, PGE(2) growth-stimulatory effects required functional AREG. Localization of AREG expression was determined by immunohistochemistry in regenerative intestine. The immunoreactivity of AREG was predominantly localized in intestinal subepithelial myofibroblasts (ISEMF). Primary ISEMF cultures were established, and growth-stimulatory actions of ISEMF-generated AREG were demonstrated in cell coculture system. In addition, we found that the cAMP/protein kinase A pathway robustly induced AREG in cultured ISEMF. These studies suggest that AREG plays critical roles in intestinal epithelial growth. Modulation of levels of AREG by targeting ISEMF represents a novel strategy for treatment of certain intestinal disorders.

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Figures

Figure 1
Figure 1
AREG in intestinal epithelial regeneration after radiation injury. A, AREG+/+ and AREG−/− mice were exposed to 12-Gy total body irradiation. The ileum was fixed in methacarn fixative 84 h after the irradiation and cross sectioned. H&E staining of AREG+/+ mouse ileum (×200) (a); H&E staining of ARE−/− mouse ileum (×200) (b); BrdU staining of AREG+/+ mouse ileum (×200) (c); BrdU staining of ARE−/− mouse ileum (×200) (d). Arrows, Regenerative crypts. Results shown are representative of three separate experiments. B, AREG+/+ and AREG−/− mice were exposed to 12-Gy total body irradiation. Ileal mucosa was harvested, and RNA was extracted at indicated time points (n = 5). mRNA levels of AREG, EGF, and TGF-α were determined by RT-qPCR and expressed as fold to levels in mice without irradiation (0 h). *, P < 0.05.
Figure 2
Figure 2
PGE2 growth-stimulatory effects in intestinal regeneration. A, AREG+/+ mice were exposed to 12-Gy total body irradiation. Vehicle (V) or dmPGE2 (dmE2; 150 μg/kg of body weight) was administered twice daily via oral gavage. Animals were killed 78 h after the irradiation. The ileum was fixed in methacarn fixative, and cross-sections were prepared. H&E staining (a) and BrdU staining (c) of vehicle-treated AREG+/+ mouse ileum (×200); H&E staining (b) and BrdU staining (d) of dmPGE2-treated AREG+/+ mouse ileum (×200). B, AREG+/+ mice (n = 5) (a) or AREG−/− mice (n = 5) (b) were exposed to 12-Gy total body irradiation. Vehicle or 16,16-dimethyl PGE2 (150 μg/kg) was administered twice daily via oral gavage. AREG+/+ mouse ileum was collected 78 h after irradiation, and AREG−/− mouse ileum was harvested 96 h after irradiation. The percentage of regenerative crypts was determined as described in Materials and Methods. Plotted is the mean ± sd of the results from five animals. *, P < 0.05. AREG+/+ mice (n = 5) (c) or AREG−/− mice (n = 5) (d) were exposed to 12-Gy total body irradiation. Vehicle or 16,16-dimethyl PGE2 (120 μg/kg) was ip injected 1 h before irradiation. AREG+/+ mouse ileum was collected 78 h after irradiation and AREG−/− mouse ileum was harvested 96 h after irradiation. The percentage of regenerative crypts was determined as described in Materials and Methods. Plotted is the mean ± sd of the results from five animals. *, P < 0.05. Results shown are representative of two independent experiments.
Figure 3
Figure 3
PGE2 induction of AREG expression in vivo. dmPGE2 (120 μg/kg) was administered to AREG+/+ mice via ip injection (n = 3). RNA was extracted from intestinal mucosa 2, 6, and 24 h after dmPGE2 injection. AREG mRNA levels were determined by RT-qPCR and expressed as fold to levels in mice without dmPGE2 (0 h). *, P < 0.05.
Figure 4
Figure 4
Localization of AREG in mouse intestine. A, AREG+/+ or AREG−/− mice were exposed to 12-Gy total body irradiation (Rad.). The ileum was fixed in 10% formalin 48 h after the irradiation and then sectioned horizontally. Immunostaining for AREG and α-SMA was performed as described in Materials and Methods. H&E staining of AREG+/+ mouse ileum (×400) (a); AREG staining of AREG−/− mouse ileum (×400) (b); AREG staining of AREG+/+ mouse ileum (×400) (c); AREG staining of AREG+/+ mouse ileum (×1000) (d); α-SMA staining of AREG+/+ mouse ileum (×1000) (e); H&E staining of AREG+/+ mouse ileum (×1000) (f). B, RIE (a) or ISEMF (b) cells received 6-Gy irradiation, and RNA was extracted at the indicated time points. Levels of AREG mRNA were determined by real-time RT-PCR and expressed as fold to levels in cells treated by vehicle for 6 h. *, P < 0.05.
Figure 5
Figure 5
Growth-stimulatory action of ISEMF-generated AREG. A, Establishment of primary AREG+/+ and AREG−/− mouse ISEMF cultures. Primary ISEMF cultures that contain either wild-type AREG gene (AREG+/+) or inactive AREG allele (AREG−/−) were established from AREG+/+ and AREG−/− mouse intestine using the outgrowth method described in Materials and Methods. Morphology of primary ISEMF (a); positive immunoreactivity of α-SMA (b) and negative of desmin in primary ISEMF (c). B, Trophic effects of ISEMF on intestinal epithelial cells. RIE cells were placed in Transwell chambers (0.4 μm), which were then inserted into 24-well plate containing no cells (no cell), AREG+/+ ISEMF (AREG+), or AREG−/− ISEMF (AREG+) in the presence of vehicle (V) or 1 μm PGE2 (E2). RIE/ISEMF cells were cocultured for 48 h. MTT was added into upper chamber and incubated for 2 h. Transwell membrane was removed from the chamber and placed in MTT solvent. After the converted dye was completely dissolved, absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 650 nm using an aliquot of the MTT solvent. Plotted is the mean ± sd of relative values of MTT conversion performed in quadruplicate. *, P < 0.05. All MTT assays were repeated at least three times. C, Trophic effects of ISEMF-generated AREG on intestinal epithelial cells. RIE cells were cocultured with 18Co cells in the presence of vehicle, PGE2, PGE2 plus control IgG (E2-IgG), or PGE2 plus 10 μg/ml AREG neutralizing antibody (E2-α-AREG) as described in B. RIE/18Co cells were cocultured for 48 h. MTT conversion was determined. Plotted is the mean ± sd of relative values of MTT conversion performed in quadruplicate. *, P < 0.05.
Figure 6
Figure 6
Regulation of AREG expression by the cAMP/PKA pathway. A, Forskolin (Fork.) induction of AREG in ISEMF. 18Co cells were grown in serum-free medium for 24 h. Forskolin at indicated concentrations was added. H89 (10 μm) was applied 15 min before 100 μm forskolin (H89). RNA was extracted 2 h after forskolin engagement. Levels of AREG were determined by RT-qPCR and expressed as fold to levels in cells without forskolin (0 μm). *, P < 0.05. B, 18Co cells were grown in serum-free medium for 24 h and then treated with 25 μm forskolin. H89 (10 μm) was applied 15 min before forskolin (H89). RNA was extracted at indicated time points after forskolin engagement. Levels of AREG were determined by RT-qPCR and expressed as fold to levels in control cells (0 h). *, P < 0.05. C, 18Co cells were grown in serum-free medium for 24 h and then treated with 300 μm 8-CPT-cAMP. H89 (10 μm) was applied 15 min before forskolin (H89). RNA was extracted at indicated time points after forskolin engagement. Levels of AREG were determined by RT-qPCR and expressed as fold to levels in control cells (0 h). *, P < 0.05.
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