Asymmetric crosstalk between the BMP and TGFb pathways resolves signaling ambiguity

The BMP and TGFβ signaling pathways control cellular fate decisions in diverse biological contexts, often playing opposing roles. Despite extensive knowledge of these pathways, understanding how cells respond to environments containing these opposing cues remains a challenge. Here, we systematically analyze the activation of BMP and TGFβ pathways under combinatorial signaling environments. We find that TGFβ ligands inhibit BMP signaling, while BMP ligands enhance TGFβ transcriptional response, across concentrations, ligand variants, and cell types. This asymmetric crosstalk results in the activation of a single specific transcriptional response, even under mixed signaling conditions, effectively reducing signal ambiguity. Furthermore, we show that this crosstalk originates downstream of the SMAD proteins phosphorylation. Using mathematical models we predict, and experimentally verify, that promiscuous interactions between SMAD proteins provide the mechanism for the observed crosstalk. Our findings challenge the canonical models of signaling pathways, suggesting an active role for mediator proteins in determining biological responses. To examine the activation of the BMP and TGFb pathways under complex signaling environment we established an NMuMG dual reporter cell line. Cells contained transcriptional reporters for BMP (Smad1/5/8 response element) controlling the expression of mCherry and TGFb (Smad2/3 response element) controlling the expression of mCitrine. We then examined the gene expression profile of the dual reporter cells using bulk RNA-sequencing in cells cultured under four ligand conditions.