Transforming Growth Factor-beta Stimulates Smad1/5 Signaling In Pulmonary Artery Smooth Muscle Cells Of The Mouse Newborn Lung

BackgroundTGF? has a role in regulating early pulmonary development.The intracellular signaling mechanisms through which TGF? regulates pulmonary development are incompletely understood.Canonical TGF? signaling involves Smad2/3 phosphorylation,Smad2/3·Smad4 complex formation and nuclear localization and gene regulation.Although TGF? stimulates Smadl/5/8 phosphorylation in adult human aortic and PASMC and in adult mouse PASMC,TGF?-mediated BMP R-Smad phosphorylation appears to be variable.It is unknown whether TGF? regulates Smadl/5 phosphorylation and signaling in PASMC in the newborn lung.ObjectiveIn the current studies,we tested whether TGFp stimulates BMP R-Smad phosphorylation in interstitial cells from the developing lung.Methods1.Here we demonstrate that physiologic levels of TGF? stimulate Smadl/5 phosphorylation in primary mouse pup PASMC and lung fibroblasts and in PASMC and lung fibroblast cell lines.Moreover,we provide support for the potential in vivo relevance of this cross-talk pathway by demonstrating in the mouse pup lung the presence of mixed TGF? and BMP R-Smad complexes.2.Then we test whether TGF? stimulate nuclear translocation of pSmadl/5 and induced the mRNA expression of prototypical BMP-regulated genes though IF and RT-PCR.3.To better define the mechanisms through which TGF? regulates Smadl/5 phosphorylation in mPASMC,we used small molecule inhibitors of TGF?RI and BMPRI.Results1.We show that physiologically relevant TGF?1 levels stimulate Smad 1/5 phosphorylation,typically a mediator of bone morphogenetic protein(BMP)signaling,in mouse pup pulmonary artery smooth muscle cell(mPASMC)and lung fibroblasts and other interstitial lung cell lines.This crosstalk mechanism likely has in vivo relevance because mixed Smadl/5/8·Smad2/3 complexes,which are indicative of TGF?-stimulated Smadl/5 activation,were detected in the developing mouse lung using a proximity ligation analysis.2.TGF? stimulates nuclear localization of phosphorylated Smadl/5 and induces the expression of prototypical BMP-regulated genes in the mPASMC.3.Small molecule kinase inhibitor studies suggested that TGF?-regulated Smadl/5 phosphorylation in these cells is mediated by TGF?-type ? receptors,not BMP-type ? receptors,but possibly the accessory ALK1 receptor.ConclusionsTGF?-mediated BMP R-Smad signaling might also have a role in regulating lung and pulmonary vascular development.