Fig. 5.

NGF/TrkA signaling transactivates Wnt signaling via MAPK signaling. a HEK293 cells (upper panel) with ectopic expression of TrkA and Caco2 cells (lower panel) were transfected with the TOPFlash plasmid. Serum-starved cells were treated with either NGF or Wnt3a at indicated concentrations (ng/ml) 24 h prior to the luciferase reporter assay. TCF-binding activities were measured as the readings of the firefly luciferase reporter and were normalized to the reference reporter renilla luciferase. b Serum-starved Caco2 cells transfected with the TOPFlash construct were treated with or without MNAC13 prior to NGF stimulation and then subjected to analyses for luciferase activity (**p < 0.01, n = 3; ANOVA). c Western blotting analyses on the expression of p-β-catenin (Y142/S552/Y654) in serum-starved Caco2 cells after NGF treatment. d Serum-starved Caco2 was treated with NGF for indicated times. Phosphorylation of Lrp5 (T1492) and Lrp6 (S1490) was detected by western blotting. Total Lrp5/6 served as a loading control. Phosphorylation of Akt and Erk1/2 was a positive control for showing the activation of NGF signaling. e Western blotting analyses on the level of phosphorylated forms of Lrp5 (T1492) and Lrp6 (S1490) in the colonic tissues from both control and NMS mice treated with or without intraperitoneal injection of NGF; it is noted that the tissues were isolated for analyses shortly after the completion of NGF/NMS treatment. f The phosphorylation of Lrp6 detected by western blotting was examined for the responses of Caco2 cells to the stimulation of NGF. The cells were pre-incubated with or without U0126 and Wortmannin before NGF treatment. g Luciferase reporter assay for Wnt signaling in Caco2 cells treated with a combination of NGF, Wnt3a, U0126, and Wortmannin (**p < 0.01, ***p < 0.001, n = 3; ANOVA). h Representative images showing the intestinal organoids cultured with/without NGF or R-spondin 1 (scale bars: 100 μm)