RT Journal Article
SR Electronic
T1 Selective Src-Family B Kinases Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.09.27.462034
DO 10.1101/2021.09.27.462034
A1 Adam M. Andruska
A1 Md Khadem Ali
A1 Xuefei Tian
A1 Edda Spiekerkoetter
YR 2021
UL http://biorxiv.org/content/early/2021/09/27/2021.09.27.462034.abstract
AB Protein tyrosine kinases (PTKs) are essential for eukaryotic signaling. By targeting select PTKs, the group of drugs known as Tyrosine kinase inhibitors (TKIs) have proven to be effective for treating multiple diseases ranging from cancer to pulmonary fibrosis. However, some TKIs also paradoxically lead to the development of adverse conditions such as pulmonary arterial hypertension (PAH) by promoting endothelial cell dysfunction (ECD). We hypothesize that (1) subsets of PTKs may disproportionately modulate signaling pathways critical for endothelial homeostasis, such as BMPR2 signaling, and (2) inhibiting those pro-endothelial PTKs can promote the development of ECD. Herein we use an agnostic high-throughput siRNA screen to investigate how PTKs affect the canonical BMPR2 signaling pathway. Our major finding is that within the Src-family of non-receptor PTKs, the Src-B family promotes canonical BMPR2 signaling while the Src-A family suppresses it. We focus on two representative members of each family, Lck (for Src-B) and Fyn (for Src-A) that are the strongest activators or inhibitors of BMPR2 signaling in the screen. We confirm that Lck is expressed in the endothelium of pulmonary arteries and show that Lck knockout (termed si-Lck) in pulmonary artery endothelial cells (PAECs) suppresses canonical BMPR2 signaling while Fyn knockout (termed si-Fyn) promotes canonical BMPR2 signaling. Furthermore, Lck and Fyn are responsible for opposing functional behaviors in PAECs: si-Lck promotes apoptosis and interferes with tube formation while si-Fyn suppresses apoptosis and promotes tube formation. After analyzing the whole-transcriptome signature of si-Lck and si-Fyn PAECs we find that in addition to BMPR2 signaling suppression, si-Lck (and not si-Fyn) increases a broad number of ECD markers and increases canonical NF-κβ signaling. In summary, for the first time we show that Src-A and B Family of PTKs exert differential control over key endothelial cell signaling pathways resulting in direct phenotypic consequences. This knowledge may help to guide the design of more precise TKIs which avoid adverse drug reactions brought about through endothelial cell dysfunction.Competing Interest StatementThe authors have declared no competing interest.ALK1Activin receptor-Like Kinase 1BMPR2Bone Morphogenetic Protein Receptor Type 2BREBMP Response ElementDEGDifferentially Expressed GeneECEndothelial CellECDEndothelial Cell DysfunctionFYNFYN Proto-OncogeneHTSHigh Throughput siRNA ScreenID1,2, and 3Inhibitor of Differentiation 1, 2, and 3LCKLymphocyte specific protein tyrosine kinasePAECPulmonary Artery Endothelial CellPAHPulmonary Arterial HypertensionPBMCPeripheral Blood Mononuclear CellPDGFRPlatelet derived growth factor receptorpSMAD1Phosphorylated SMAD-1PBSPhosphate Buffered SalinePTKProtein Tyrosine KinaseROSReactive Oxygen SpeciesscRNA-seqSingle Cell RNA SeqsiRNASmall Interfering RNAsi-FynPAECs subject to Fyn knockout with si-RNAsi-LckPAECs subject to Lck knockout with si-RNASRCCellular Sarcoma Related*TCRT-Cell ReceptorTKITyrosine Kinase InhibitorVEGFRVascular Endothelial Growth Factor Receptor