Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation
Daniel O. Kechele, … , P. Kay Lund, Kathleen M. Caron
Daniel O. Kechele, … , P. Kay Lund, Kathleen M. Caron
Published January 17, 2017
Citation Information: J Clin Invest. 2017;127(2):593-607. https://doi.org/10.1172/JCI87588.
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Research Article Gastroenterology

Orphan Gpr182 suppresses ERK-mediated intestinal proliferation during regeneration and adenoma formation

Abstract

Orphan GPCRs provide an opportunity to identify potential pharmacological targets, yet their expression patterns and physiological functions remain challenging to elucidate. Here, we have used a genetically engineered knockin reporter mouse to map the expression pattern of the Gpr182 during development and adulthood. We observed that Gpr182 is expressed at the crypt base throughout the small intestine, where it is enriched in crypt base columnar stem cells, one of the most active stem cell populations in the body. Gpr182 knockdown had no effect on homeostatic intestinal proliferation in vivo, but led to marked increases in proliferation during intestinal regeneration following irradiation-induced injury. In the ApcMin mouse model, which forms spontaneous intestinal adenomas, reductions in Gpr182 led to more adenomas and decreased survival. Loss of Gpr182 enhanced organoid growth efficiency ex vivo in an EGF-dependent manner. Gpr182 reduction led to increased activation of ERK1/2 in basal and challenge models, demonstrating a potential role for this orphan GPCR in regulating the proliferative capacity of the intestine. Importantly, GPR182 expression was profoundly reduced in numerous human carcinomas, including colon adenocarcinoma. Together, these results implicate Gpr182 as a negative regulator of intestinal MAPK signaling–induced proliferation, particularly during regeneration and adenoma formation.

Authors

Daniel O. Kechele, R. Eric Blue, Bailey Zwarycz, Scott T. Espenschied, Amanda T. Mah, Marni B. Siegel, Charles M. Perou, Shengli Ding, Scott T. Magness, P. Kay Lund, Kathleen M. Caron

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Figure 5

Reduced Gpr182 increases lethality and polyp numbers in ApcMin/+ mice.

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Reduced Gpr182 increases lethality and polyp numbers in ApcMin/+ mice. (...
(A) Kaplan-Meier survival curve comparing Gpr182lacZ/lacZ Apc+/+ (black), Gpr182+/+ ApcMin/+ (blue), Gpr182lacZ/+ ApcMin/+ (yellow), and Gpr182lacZ/lacZ ApcMin/+ (red) animals. (B) BW of 5-month-old female and male ApcMin mice from different Gpr182 genotypes. (C) Spleen weight normalized to BW for Gpr182+/+ ApcMin/+, Gpr182lacZ/+ ApcMin/+, and Gpr182lacZ/lacZ ApcMin/+ mice. (D) Small intestine length from Gpr182lacZ/lacZ ApcMin/+ animals compared with controls. (E) Representative images of the number and size of adenomas from Gpr182+/+ ApcMin/+ and Gpr182lacZ/lacZ ApcMin/+ mouse intestines. (F) Quantification of polyp numbers throughout the small intestines of Gpr182+/+ ApcMin/+, Gpr182lacZ/+ ApcMin/+, and Gpr182lacZ/lacZ ApcMin/+ mice. (G) Distribution of the number of polyps throughout the small intestine based on approximate polyp size. (H) Histology for 5-month-old Gpr182+/+ ApcMin/+ and Gpr182lacZ/lacZ ApcMin/+ jejunum polyps labeled with H&E and β-catenin (white). (I and J) Polyp proliferation assessed by Ki67 (blue) staining normalized to DAPI (purple) in Gpr182+/+ ApcMin/+ and Gpr182lacZ/lacZ ApcMin/+ mice. (K) Gpr182 expression in polyps from jejunum of Gpr182+/+ ApcMin/+ and Gpr182lacZ/lacZ ApcMin/+ mice compared with expression levels in Apc+/+ jejunum. Expression was normalized to Gpr182+/+, Gapdh, and 18S. Scale bars: 5 mm (E) and 100 μm (H and I). Biological replicates: n = 25–100 mice per genotype (A); n = 10–20 mice per genotype per sex (B); n = 700–1,700 polyps from 20 to 35 mice per genotype (C, D, F, and G); n = 15–60 polyps from 5 mice (J); and n = 3–5 mice per genotype (K). *P < 0.05, **P < 0.01, and ***P < 0.001, by Mantel-Cox test (A), 1-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparisons test (B–G), Mann-Whitney t test (J), or 1-way ANOVA with Tukey’s multiple comparisons test (K).