TGFβ Regulates BMP‐Smad Signaling in Mouse Pup Pulmonary Artery Smooth Muscle Cells via ALK1

Increasingly, TGFβ is becoming recognized as an important regulator of the final stages of pulmonary development. This is because TGFβ‐ and BMP‐signaling element expression is tightly regulated in the developing lung particularly within the maturing lung acini. Moreover, TGFβ activation during newborn lung injury has been found to inhibit cGMP signaling pathways that protect pulmonary development and to disrupt lung microvascular and alveolar assembly. Still, the mechanisms through which TGFβ regulates lung maturation are largely unknown. TGFβ and BMP are classically thought to signal through independent Smad‐regulated mechanisms. In this model, TGFβ engages its type I (R1) and type II (R2) receptors and thereby stimulates Smad2/3 phosphorylation. Subsequently, this activated Smad2/3 joins Smad4, and migrates as a complex into the nucleus where it regulates gene expression. Independently, BMP can interact with its own receptors and stimulate Smad1/5/8 phosphorylation, the nuclear localization of a Smad1/5/8·Smad4 complex, and the transactivation of a different set of genes, including the DNA‐binding protein inhibitor ID1. However, we now report that TGFβ can also cross‐stimulate BMP‐Smad signaling in the newborn lung. Physiologically relevant doses of active TGFβ1 were determined to increase Smad1/5 phosphorylation in primary mouse pup pulmonary artery smooth muscle cells (mPASMC) and lung fibroblasts and in PASMC and fibroblast cell lines. This mechanism likely has in vivo relevance. This is because a proximity ligation analysis method‐‐employing high affinity anti‐Smad isoform antibodies‐‐detected mixed Smad1/5·Smad2/3 complexes, which are indicative of TGFβ‐stimulated BMP‐Smad phosphorylation. These complexes were observed in perivascular smooth muscle and other parenchymal cells of mouse pup lungs. In primary mPASMC we detected also TGFβ‐stimulated Smad1/5 nuclear localization and increased select ID isoform mRNA expression. Studies using small molecule TGFβ and BMP receptor kinase inhibitors suggested that TGFβR1, not BMPR1, but possibly ALK1 mediate this crosstalk mechanism. Although ALK1 was thought not to be expressed outside of endothelial cells in the vasculature, we subsequently detected ALK1 mRNA and protein immunoreactivity in the mPASMC, and ALK1 immunoreactivity in PASMC in the mouse pup lung. Accordingly, we tested the role of this accessory TGFβ/BMP receptor in mediating TGFβ‐stimulated BMP‐Smad signaling. We determined that an antibody generated against the mouse ALK1 extracellular domain inhibited TGFβ1‐stimulated Smad1/5 phosphorylation in mPASMC. These studies demonstrated for the first time a TGFβ‐mediated BMP‐Smad crosstalk mechanism in the developing mouse pup lung and detailed mechanisms that might facilitate TGFβ stimulation of Smad signaling which is usually only ascribed to BMP in PASMC. We speculate that the diversity of canonical Smad signaling induced by TGFβ through this mechanism might have a role in differentially regulating pulmonary vascular development in the newborn lung. Support or Funding Information National Institutes of Health ‐ Heart, Lung, and Blood Institute, R01HL125715