Rudhira-mediated microtubule stability controls TGFβ signaling during mouse vascular development

The Transforming Growth Factor β (TGFβ) signaling pathway is critical for survival, proliferation, and cell migration, and is tightly regulated during cardiovascular development. Smads, key effectors of TGFβ signaling, are sequestered by microtubules (MTs) and need to be released for pathway function. Independently, TGFβ signaling also stabilizes MTs. Molecular details and the in vivo relevance of this cross-regulation remain unclear, understanding which is important in complex biological processes such as cardiovascular development. Here, we use rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) , a MT-associated, endothelium-restricted and developmentally essential proto-oncogene, as a pivot to decipher cellular mechanisms in bridging TGFβ signaling and MT stability. We show that Rudhira regulates TGFβ signaling in vivo, during mouse cardiovascular development, and in endothelial cells in culture. Rudhira-MT association is essential for the activation and release of Smad2/3 from MTs. Consequently, Rudhira depletion attenuates Smad2/3-dependent TGFβ signaling thereby impairing cell migration. Interestingly, Rudhira is also a transcriptional target of Smad2/3-dependent TGFβ signaling essential for TGFβ-induced MT stability. Our study identifies an immediate early physical role and a slower, transcription-dependent role for Rudhira in cytoskeleton-TGFβ signaling crosstalk. These two phases of control could facilitate temporally– and spatially restricted targeting of the cytoskeleton and/or TGFβ signaling in vascular development and disease. Significance statement The developmental TGFβ pathway is essential for cell migration, cell-cell communication, adhesion, apoptosis, and matrix remodeling. Dysregulation of TGFβ signaling leads to aberrant vascular patterning and angiogenesis during mouse embryogenesis. Pathway activation involves phosphorylation and nuclear transport of Smads. Microtubules sequester Smads in the cytoplasm, thereby inhibiting TGFβ signaling. Conversely, TGFβ signaling stabilizes microtubules. However, the molecular components involved, and biological relevance of this cross-regulation remain unclear. We show that the oncoprotein Rudhira/BCAS3 facilitates Smad-MT dissociation upon ligand-mediated TGFβ receptor activation. Interestingly, Smad-dependent TGFβ signaling activation enhances rudhira transcription, essential for microtubule stabilization during cardiovascular development. This dual regulation of TGFβ signaling and microtubule stability by Rudhira allows sustained temporal control essential for development, and its dysregulation has pathological outcomes. ### Competing Interest Statement The authors have declared no competing interest.