BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells

Bo Tao,Jan R Kraehling,Siavash Ghaffari,Cristina M Ramirez,Sungwoon Lee,Joseph W Fowler,Warren L Lee,Carlos Fernandez-Hernando,Anne Eichmann,William C Sessa

doi:10.1074/jbc.ra120.015680

Bo Tao, Jan R Kraehling + Show 8 more

Open Access

https://doi.org/10.1074/jbc.ra120.015680

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Abstract

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

Highlights

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence
human umbilical vein endothelial cell (HUVEC) were surface labeled with a cell-impermeable biotin analog and treated with BMP-9 followed by Western blot analysis of biotin-labeled plasma membrane (PM) proteins
The central goal of this study was to explore the potential crosstalk between BMP-9 and low-density lipoprotein (LDL) as proteins that bind to ALK-1

Summary

Introduction

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Work from our group has shown that a member of the TGFb type 1 receptor family, activin-like kinase 1 (ALK-1), can serve as a binding protein and potential receptor for the uptake and transcytosis of LDL and very low density lipoprotein, but not oxidized LDL or high density lipoprotein across the vascular endothelium [3]. The goal of the present study was to examine the relationship between BMP-9 and LDL as ligands for ALK-1, to discern the pathways mediating their internalization and to interrogate the actions of BMP-9 on LDL transcytosis in endothelial cells

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