Volume 131, Issue 2, Pages 538-553 (August 2006) PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway  Frédéric Varnat, Béatrice Bordier–Ten Heggeler, Philippe Grisel, Nathalie Boucard, Irène Corthésy–Theulaz, Walter Wahli, Béatrice Desvergne  Gastroenterology  Volume 131, Issue 2, Pages 538-553 (August 2006) DOI: 10.1053/j.gastro.2006.05.004 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 PPARβ expression in the small intestine and evaluation of Paneth cell deficiency in PPARβ-null mice. (A) Detection of PPARβ mRNA by RPA in intestinal mucosa (n = 3, *P < .05). Du, duodenum; Je, jejunum; il, ileum; Co, colon. L27-radiolabeled antisense riboprobe was used as an internal standard. (B) In situ hybridization revealing PPARβ expression in the adult small intestine using an antisense riboprobe (middle panel), H&E (HE) control of morphology is shown in the left panel, and the control using the sense riboprobe is shown in the right panel. (C) Strong PPARβ mRNA expression at the bottom of the crypts. Cell-by-cell dosimetric quantification (middle panel) of the signal obtained by in situ hybridization (left panel) reveals the gradient of PPARβ mRNA expression from the bottom to the top of the crypts. The dosimetry was performed with National Institutes of Health image software (n = 3; *P < .05). Immunohistochemistry with a specific PPARβ antibody (right panel) shows the nuclear localization and high level of the PPARβ protein in the granule-rich Paneth cells (white arrows indicate cell position 9). Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 PPARβ expression in the small intestine. (A) Immunohistochemistry using an antilysozyme antibody in both PPARβ+/+ and PPARβ−/− mice (n = 5 in each genotype). Paneth cells are stained clearly in brown. (B) Lysozyme protein was quantified by Western blot in the duodenum of PPARβ+/+ (+/+) and PPARβ−/− (−/−) mice, using β-tubulin as an internal control. Cryptdin-1 expression in the duodenum was evaluated by RPA (left panels). Numbers below each lane correspond to the mean relative expression ± SD evaluated in 3 independent animals (*P < .05). (C) Casein zymography assessed the activity of matrix metalloproteinase-7 and trypsin in the duodenal mucosa and the results were normalized to β-tubulin protein. Purified trypsin was used as a standard. Numbers below each lane correspond to the mean relative activity ± SD evaluated in 3 independent animals (*P < .05). (D) The 1-μm sections from duodenum embedded in EPON 812 were stained with methylene blue/azure II/fuchsin (lower panel). The upper panel presents the quantification of the granules according to their diameter. The granules from a total of 20 cells have been counted and measured (bars, 30 μm; *P < .05). (E) Electron microscopy of ultrathin sections from PPARβ+/+ (+/+) and PPARβ−/− (−/−) mice duodenum (upper panels, the apical pole is at the top of pictures; sg, secretory granule; n, nucleus; rer, rough endoplasmic reticulum; bars in the upper panel, 150 nm; bars in the lower panel, 75 nm). Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Roles of PPARβ in promoting Paneth cell differentiation. Adult wild-type and PPARβ-mutant mice were given oral gavage for 12 days with either L-165041, an agonist of PPARβ, or the vehicle alone, as indicated. (A) Detection of L-FABP and cryptdin-1 mRNA in intestinal mucosa by RPA (*P < .05, **P < .001). (B) Average number of lysozyme-positive cells per crypt section in the duodenum of wild-type (+/+) and PPARβ-null mice (−/−) treated or not with L-165041 (n = 5; *P < .05). (C) Bactericidal activity in the intestinal mucosa from PPARβ+/+ (+/+) and PPARβ−/− (−/−) mice treated or not with L-165041. ■, CFUs counted in plates exposed to lysis buffer; □, CFUs counted on plates exposed to duodenal lysates from control animals (treated with vehicle alone); , CFUs counted on plates exposed to duodenal lysates from L-165041–treated animals (n = 5; *P < .05). (D) Quantitative analysis of the small intestinal microbiota in wild-type (□) and PPARβ-null mice (■) (n = 3; *P < .05). Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 PPARβ promotes the postnatal development of Paneth cells. (A) The average number of Paneth cells per duodenal crypt section in PPARβ+/+ (+/+) and PPARβ−/− (−/−) mice at P15 and P21 as assessed by counting lysozyme-positive cells (n = 5; *P < .05). (B) Detection of cryptdin-1 mRNA in intestinal mucosa by RPA at P15 and P21 in wild-type and PPARβ-mutant mice. Number below each lane corresponds to the relative mean expression ± SD evaluated in 3 different animals (*P < .05). (C) Postnatal ontogeny of PPARβ and cryptdin-1 mRNA content in the intestinal mucosa (right panel) as assessed by RPA (n = 3). The left panel shows the parallel evolution of PPARβ (black line) and cryptdin-1 (grey line) mRNA expression, as evaluated by RPA, during the postnatal period. The grey bars represent the average number of Paneth cells per cross-section at P15 and P21 in wild-type mice as shown in A. Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 PPARβ-null mice show a perturbed hedgehog signaling pathway. (A) Evaluation by RPA of Wnt, Notch, and Hedgehog signaling pathway perturbations using total RNA from duodenal mucosa of PPARβ−/− and wild-type adult mice (n = 3; *P < .05). (B) Western blot quantification of Hedgehog (Hh), Ihh, and BMP-4 proteins in total protein extract from duodenal mucosa of PPARβ−/− and wild-type adult mice (n = 3; *P < .05). (C) RPA using total RNA from duodenal mucosa of wild-type mice treated with the PPARβ agonist L-165041 (n = 5, *P < .05, **P < .001). Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 PPARβ controls the differentiation of Paneth cells through the control of the hedgehog signaling pathway. (A) Evaluation of the number of Paneth cells (right panel) and Ptch-1 mRNA accumulation (left panels) in the duodenal mucosa on in vivo treatment with cyclopamine, a potent and specific hedgehog signaling inhibitor or with vehicle, as indicated. The average number of Paneth cells per crypt section were determined by immunohistochemistry using an antilysozyme antibody, and Ptch-1 mRNA level was assessed by RPA (n = 5; *P < .05). (B) Western blot showing the regulation of villin, lysozyme, and Ihh proteins by PPARβ activation in differentiating HT-29 cells. After 16 hours incubation with 1 μm L-165041, HT-29 cells were incubated with 2.5 mmol/L sodium butyrate for 0, 2, and 4 hours. (C) Western blot of control HT-29 cells and HT-29 treated for 24 hours with a combination of 2.5 μg/mL recombinant Shh (rshh) and 1 μm L-165041. (D) Western blot of control HT-29 cells and HT-29 treated for 24 hours with a combination of cyclopamine at 2 μg/mL and 2.5 mmol/L sodium butyrate. Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 PPARβ controls the number of Paneth cells by regulating the differentiation of their precursors. (A) Double immunohistofluorescence showing the colocalization of hedgehog signaling pathway components (Ihh, Ptch-1, and Hip) with lysozyme protein. Red arrows, mature Paneth cells; white arrows, Paneth cell precursors. (B) Average number of Ptch-1–positive cells in crypt epithelium as assessed by immunohistochemistry in the small intestine of wild-type and PPARβ-null mice treated or not with L-165041 ( ) or with cyclopamine (■) (n = 5; *P < .05). □, vehicle. (C) Schematic representation of the hedgehog signaling pathway between mature and precursor Paneth cells. (D) Model for PPARβ action on the level of Ihh, resulting in the alteration of Paneth cell homeostasis. Gastroenterology 2006 131, 538-553DOI: (10.1053/j.gastro.2006.05.004) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions