Selective Src-Family B Kinase Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction
biorxiv(2021)
摘要
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 Statement
The authors have declared no competing interest.
* ALK1
: Activin receptor-Like Kinase 1
BMPR2
: Bone Morphogenetic Protein Receptor Type 2
BRE
: BMP Response Element
DEG
: Differentially Expressed Gene
EC
: Endothelial Cell
ECD
: Endothelial Cell Dysfunction
FYN
: FYN Proto-Oncogene
HTS
: High Throughput siRNA Screen
ID1,2, and 3
: Inhibitor of Differentiation 1, 2, and 3
LCK
: Lymphocyte specific protein tyrosine kinase
PAEC
: Pulmonary Artery Endothelial Cell
PAH
: Pulmonary Arterial Hypertension
PBMC
: Peripheral Blood Mononuclear Cell
PDGFR
: Platelet derived growth factor receptor
pSMAD1
: Phosphorylated SMAD-1
PBS
: Phosphate Buffered Saline
PTK
: Protein Tyrosine Kinase
ROS
: Reactive Oxygen Species
scRNA-seq
: Single Cell RNA Seq
siRNA
: Small Interfering RNA
si-Fyn
: PAECs subject to Fyn knockout with si-RNA
si-Lck
: PAECs subject to Lck knockout with si-RNA
SRC
: Cellular Sarcoma Related*
TCR
: T-Cell Receptor
TKI
: Tyrosine Kinase Inhibitor
VEGFR
: Vascular Endothelial Growth Factor Receptor
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