Fig 2: 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)

Fig 3: LRP5 interacts with PHD2. (A) Cardiomyocytes were infected with Ad-LRP5 and exposed to hypoxia as indicated. Whole-cell lysates were subjected to immunoprecipitation using an anti-Myc antibody followed by western blotting using the antibodies indicated to the right of the blot. n = 4/group. (B) Myc-tagged Ad-LRP5 and Ad-PHD2 were co-infected into cardiomyocytes and exposed to hypoxia for 60 min. Whole-cell lysates were subjected to immunoprecipitation using an anti-PHD2 antibody followed by western blotting using the anti-Myc and anti-PHD2 antibodies. n = 4/group. (C) Cardiomyocytes were prepared as in (B), and lysates were subjected to immunoprecipitation using an anti-Myc antibody followed by western blotting using the anti-Myc and anti-PHD2 antibodies. n = 4/group. (D) Proximity ligation assay (PLA) was performed on cardiomyocytes using anti-PHD2 and anti-Myc antibodies. Nuclei were stained with DAPI (blue). Scale bar, 200 µm. (E) Cardiomyocytes were infected with Ad-lacZ or Ad-LRP5, with or without hypoxia exposure. After 90 min, whole-cell lysates were subjected to immunoprecipitation using an anti-PHD2 antibody followed by western blotting using an anti-LRP5 antibody. n = 3/group. (F) Cardiomyocytes were co-transfected with adenoviruses as indicated and exposed to hypoxia. After 90 min, cell lysates were subjected to immunoprecipitation using an anti-PHD2 antibody followed by western blotting using the indicated antibodies. n = 3/group. (G) Cardiomyocytes were infected with adenoviruses as indicated, and whole-cell lysates were subjected to immunoprecipitation using an anti-PHD2 antibody or normal IgG followed by western blotting using the indicated antibodies. n = 3/group. All images shown are representative of those obtained from at least three independent experiments.

Fig 4: Effect of LRP5 on gene expression profile under hypoxia. (A) Extracted total RNA from triplicate biological samples was subjected to mRNA sequencing. GO enrichment analysis in the “Biological Process” category for genes in Ad-lacZ-, Ad-LRP5-, and shLRP5-infected hypoxic cardiomyocytes. DEGs (differentially expressed genes) were included with p < 0.05 and FC = 2.0 vs. normoxic Ad-lacZ. (B) Hierarchial analysis of differentially expressed KEGG pathways (left) and a heatmap of HIF-1a transcriptional target genes (right) in the Ad-lacZ-, Ad-LRP5-, and shLRP5-infected hypoxic cardiomyocytes. Each fold change was calculated relative to the normoxic Ad-lacZ control. p < 0.05, FC = 2.0. (C) The eight most significantly changed genes between Ad-lacZ and Ad-LRP5-infected hypoxic cardiomyocytes. Each fold change was calculated relative to the normoxic Ad-lacZ control. (D) RT-qPCR confirmation of mRNA expression of selected genes under 4 h of hypoxia. Gene expression was normalized to the arithmetic mean of three control genes (GAPDH, ß-actin, and a-tubulin). Fold change was calculated relative to the normoxic Ad-lacZ control or shLamin control, n = 3/group. Means ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 versus each control.

Fig 5: LRP5 aggravates HIF-1a stability in hypoxic cardiomyocytes. (A) Ad-lacZ- and Ad-LRP5-, and (B) shLamin- and shLRP5-infected cardiomyocytes were exposed to either normoxia or hypoxia for 30 and 60 min and harvested for western blotting. Representative western blot analysis showing HIF-1a protein levels in cardiomyocytes incubated for increasing times under hypoxia. n = 4/group. Means ± SD. ### p < 0.001 Ad-LRP5 versus Ad-lacZ; NS, no significance; *** p < 0.001 shLRP5 versus shLamin. (C) HIF-1a transcript levels quantified using RT-qPCR analysis. Gene expression was normalized to the arithmetic mean of three control genes (GAPDH, ß-actin, and a-tubulin). n = 3/group. Means ± SD. NS, no significance. (D) Representative half-life of the HIF-1a protein in cardiomyocytes. Ad-lacZ-, Ad-LRP5-, shLamin-, and shLRP5-infected cardiomyocytes were co-infected with Ad-HIF-1a and treated with CHX (50 µg/mL) for the indicated times and then harvested at various time points for western blotting. Protein levels were normalized to ß-actin levels. n = 4/group. (E) Representative western blots showing HIF-1a expression in cytosolic and nuclear- fractionated lysates after exposure to hypoxic conditions for 60 min. a-tubulin and lamin B were used as cytoplasmic and nuclear protein controls, respectively. n = 4/group. Means ± SD. ** p < 0.01 versus Normoxia; # p < 0.05 Ad-LRP5 versus Ad-lacZ; $$$ p < 0.001 shLRP5 versus Ad-LRP5. (F,G) Transactivation of HRE-luciferase activity. Cardiomyocytes were co-transfected with the HRE luciferase reporter and pRL-TK vector (control for transfection efficiency) along with (F) Ad-lacZ- or Ad-LRP5 (G) shLamin- or shLRP5- infection. Relative luciferase activity was the ratio of luciferase over renilla activity. Means ± SD. *** p < 0.001 versus Normoxia; # p < 0.05 Ad-LRP5 versus Ad-lacZ (F) or *** p < 0.001 versus Normoxia; $ p < 0.05 shLRP5 versus shLamin (G). All images shown are representative of those obtained from at least three independent experiments.