Volume 124, Issue 7, Pages 1748-1757 (June 2003) Glial-derived neurotrophic factor regulates apoptosis in colonic epithelial cells  Martin Steinkamp, Irmlind Geerling, Thomas Seufferlein, Georg von Boyen, Bernhard Egger, Johannes Grossmann, Leopold Ludwig, Guido Adler, Max Reinshagen  Gastroenterology  Volume 124, Issue 7, Pages 1748-1757 (June 2003) DOI: 10.1016/S0016-5085(03)00404-9

Figure 1 GDNF is up-regulated in the colonic epithelium of rat experimental colitis. (A-D) Immunohistochemistry of GDNF (red) at (A) control, (B) day 1, (C) day 3, and (D) day 21 after induction of colitis. Arrows indicate staining of myenteric plexus. (E) GDNF staining (red) in rat myenteric plexus (arrow). (F) Negative control under omission of first antibody. (G) Blocking control. (H) GFR-α1 (red) in the colonic epithelium of rat. All slides were counterstained with hematoxylin. (Original magnification: A–D, 10×; E, 20×.) Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 2 GDNF is up-regulated in the colonic epithelium in IBD. (A- C) GDNF staining (red) in (A) control, (B) Crohn’s disease, and (C) ulcerative colitis. (D) GFR-α1 staining (red) in human colonic epithelium (arrow). (E) Negative control under omission of GDNF antibody. (F) Blocking control. (G and H) GDNF staining (arrows) of (G) submucosal and (H) myenteric plexus. All slides were counterstained with hematoxylin. (Original magnification: A-C, 20×; D, 40×.) Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 3 GDNF and GFR-α1 expression in the colon of experimental rat colitis and in human IBD. Western blots against GDNF, GFR-α1, and β-actin at various time points in (A) experimental rat colitis and (B) human colon samples from controls, Crohn’s disease (CD), and ulcerative colitis (UC). Densitometric analysis of GDNF expression in (C) rat colitis or (D) human samples normalized against β-actin. Asterisk indicates a significant increase of GDNF in Crohn’s disease samples compared with control (P < 0.05, 2-sided Student t test). Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 4 Immunofluorescent staining of GFR-α1 in (A) HT-29 and (B) SW480 cells. (C) Western blot against GFR-α1 and Ret in protein lysates from HT-29 and SW480 cells. Rat pituitary extract served as a control. (D) Detection of GFR-α1 (upper panel) and Ret (lower panel) messenger RNA expression in HT-29 and SW480 cells by reverse-transcription PCR. Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 5 GDNF activates the MAPK cascade in colonic epithelial cells. (A) HT-29 and (B) SW480 cells were synchronized in serum-starved medium for 18 hours. GDNF was added at various concentrations and incubated for 45 minutes (A and B, lower panel) or the cells were treated with GDNF (100 ng/mL) for various incubation periods (A and B, upper panel). (C and D) GDNF-induced MAPK activation depends on MEK activity. After synchronization, (C) HT-29 and (D) SW480 cells were pretreated with PD98059 (PD, 10 μmol/L) 30 minutes before addition of GDNF (100 ng/mL) where indicated and further cultivated for 45 minutes. Western blots were performed using antibodies against phosphorylated (upper panels) and total (lower panels) p42/44 MAPK. The blots show representative results obtained from 3 independent experiments. Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 6 GDNF activates the PI3K/Akt (PKB) signaling pathway in colonic epithelial cells. (A) HT-29 and (B) SW480 cells were treated with GDNF at various concentrations and incubated for 45 minutes (A and B, lower panel) or treated at 100 ng/mL for various incubation periods (A and B, upper panel). (C and D) GDNF-induced Akt (PKB) activation depends on PI3K activity. (C) HT-29 and (D) SW480 cells were pretreated with LY294002 (LY, 20 μmol/L) 30 minutes before addition of GDNF (100 ng/mL) where indicated and then further cultivated for 45 minutes. Representative Western blots from 3 independent experiments are shown. Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 7 GDNF prevents colonic epithelial cells from TRAIL-induced apoptosis. Confluent SW480 cells were pretreated over 30 minutes with either (A) PD98059 (PD, 10 μmol/L) or (B) LY294002 (LY, 20 μmol/L) where indicated. GDNF (100 ng/mL) was then added, and the cultures were further cultivated for 1 hour. Induction of apoptosis was performed by addition of TRAIL (30 ng/mL), and cells were harvested after an additional 12 hours. The Western blots show the occurrence of the cleaved form of PARP. Representative results obtained from 3 independent experiments are shown. Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)

Figure 8 Caspase 3/7 activity in SW480 cells. SW480 cells were pretreated over 30 minutes with either (A) PD 98059 (PD, 10 μmol/L) or (B) LY294002 (LY, 20 μmol/L). GDNF (100 ng/mL) was then added, and the cultures were further incubated for 1 hour. Thereafter, TRAIL was added at 30 ng/mL. After 4 hours, caspase 3/7 activity assay was performed, and results are expressed as relative fluorescence (∗P < 0.05, 2-sided Student t test) (C and D) 4′,6-Diamidino-2-phenylindole staining of (C) controls and (D) TRAIL-treated SW480 cells 6 hours after induction of apoptosis. Arrows indicate apoptotic cells with chromatin condensation and DNA fragmentation. Gastroenterology 2003 124, 1748-1757DOI: (10.1016/S0016-5085(03)00404-9)