The YAP/TAZ antagonist, PTPN14, stabilizes SMAD4 through direct interactions in endothelial cells: Implications for Hereditary Hemorrhagic Telangiectasia

Hereditary Hemorrhagic Telangiectasia (HHT) is a vascular condition caused by germline heterozygous loss-of-function mutations of ENG, AVCRL1 , and occasionally SMAD4 , encoding components of TGFβ/BMP signaling. Telangiectases occur in most HHT patients, and pulmonary, visceral, or cerebral arteriovenous malformations (AVMs) occur in 20-50%, but our understanding of how HHT mutations disrupt down-stream signaling pathways causing clinical manifestations, and why some patients suffer more serious sequelae, is incomplete. We previously showed that genetic variation within PTPN14 at rs2936018 associates with presentation of PAVM in HHT patients. Here we show rs2936018 is a cis-eQTL for PTPN14 , with lower expression of the HHT at-risk allele, and that in primary human endothelial cells, PTPN14 physically interacts with the transcription factor, SMAD4, protecting it from ubiquitylation to support higher SMAD4 expression levels. In a panel of 69 lung samples, we find Ptpn14 RNA expression correlates with markers of angiogenesis, lymphangiogenesis, cell-cell interaction, BMP signaling and rho kinase signaling, indicating preferential expression in endothelial cells. RNAScope in situ hybridization analysis and use of transgenic Ptpn14 -reporter mice ( Ptpn14 . tm1(KOMP) Vlcg ) show that Ptpn14 is predominantly but not exclusively expressed in lymphatic and vascular ECs of lung, heart and skin. In lung, Ptpn14, Acvrl1, Eng and SMAD4 expression are tightly correlated as well as with Flt1, Ece1, Sash1 , and Mapk3k . Additionally, Ptpn14, Acvrl1, Eng and SMAD4 show strong expression correlation with components of G protein coupled receptor (GPCR) signaling pathways impacting rho kinase, Cdc42, a regulator of cell migration. We report, for the first time, that in primary human endothelial cells PTPN14 binds SMAD4, as demonstrated by co-immunoprecipitation studies and confirmed by proximity ligation assay. In the nucleus, PTPN14 stabilizes SMAD4, protecting it from ubiquitylation and turnover and potentiating basal transcriptional activity from BMP and TGFβ responsive reporters, whereas in the cytoplasm PTPN14 sequesters phospho-TAZ (pTAZ) leading to TAZ turnover. PTPN14 therefore provides a physical link supporting BMP/ALK-1/SMAD4 signaling while inhibiting YAP/TAZ signaling in ECs to regulate a balance between these two pathways to maintain vascular stability. Polymorphisms in PTPN14 may alter tonic BMP/ALK-1/SMAD4 and TGFβ/TGFβRI/SMAD4 signaling to magnify ligand-mediated responses. In this way, genetic variation within PTPN14 may influence clinical outcomes of HHT through its action on SMAD4. ### Competing Interest Statement The authors have declared no competing interest.