Abstract Body (Do not enter title and authors here): Background: Pulmonary vascular remodeling, a hallmark of pulmonary arterial hypertension (PAH), is driven, at least in part, by hyperproliferation and apoptosis resistance of resident pulmonary vascular cells. We previously reported that HIPPO components mammalian Ste20-like protein kinases 1 and 2 (MST1/2) play non-canonical pro-proliferative pro-survival role in pulmonary artery (PA) smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) acting through BUB3 and FOXO3, respectively, but the mechanisms are not completely understood.
Goal: To identify downstream effectors of HIPPO-MST1/2 shared between human PAH PASMCs and PAAFs.
Methods: Data mining, siRNA and mammalian vector transfection, immunoblot, cell counts, bromodeoxyuridine (BrdU) incorporation and apoptosis assays.
Results: Protein interaction analysis using STRING database predicted cell division cycle protein 20 (CDC20) as a potential functional connectivity link among MST1/2, BUB3, and FOXO3. Analysis of publicly available datasets (GSE117261, GSE144274, GSE144932) revealed significantly increased CDC20 expression in lung tissues and isolated PASMCs and PAAFs from PAH patients compared to non-diseased subjects. CDC20 protein levels were increased in human PAH PASMCs and PAAFs compared to controls. siRNA mediated knockdown of MST1 or MST2 as well as selective MST1/2 inhibitor XMU-MP1 significantly decreased CDC20 protein levels in human PAH PASMCs and PAAFs. XMU-MP1 significantly reduced proliferation (evaluated by BrdU incorporation and cell count assays) and induced apoptosis (assessed by In Situ Cell Death Detection Kit and cell viability assay) in human PAH PASMCs and PAAFs. These effects were reversed by transfection with mammalian vector expressing human CDC20 but not control plasmid.
Conclusion: These data identify CDC20 as a novel downstream effector of non-canonical HIPPO-MST1/2 signaling driving hyperproliferation and apoptosis resistance of PASMCs and PAAFs from human PAH lungs and suggest a new link between HIPPO signaling and cell cycle regulation.