Figure 5.

Bmp4 synergizes with Wnt/β-catenin to promote expression of NOTUM at the promoter level. A and B: promoter regions for human NOTUM (A) and AXIN2 (B) reveal putative binding sites for TCF and SMAD1/5 transcription factors. FIMO; P value < 0.0001 for TCF motifs and P value < 0.001 for SMAD motifs. C: addition of Wnt3a conditioned medium alone induced the relative luciferase activity of human NOTUM and AXIN2 promoters. Cotreatment of Wnt3a and recombinant (r)BMP4 induced a higher transcriptional activation of NOTUM promoter compared with individual treatments. D: as opposed to NOTUM promoter, no significant induction was observed in the luciferase activity of AXIN2 promoter when cells were treated with rBMP4 compared with control. Cotreatment of Wnt3a and rBMP4 increased luciferase activity compared with treatment with Wnt3a. One-way ANOVA, n = 3; *P < 0.05, ***P < 0.001, ****P < 0.0001. E: analysis of publicly available ChIP-seq studies performed on human pulmonary artery endothelial cells (HPAECs) identified SMAD1/5 binding sites in the first exon and intron of NOTUM, with 1 binding site overlapping partially with the reporter sequence used in this study.