SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2

Abstract

<div class="section"> <a class="named-anchor" id="S1"> <!-- named anchor --> </a> <h5 class="section-title" id="d596869e239">Background:</h5> <p id="P1">Hereditary Hemorrhagic Telangiectasia (HHT) is an inherited vascular disorder that causes arterial-venous malformations (AVMs). Mutations in the genes encoding Endoglin ( <i>ENG)</i> and Activin-receptor-like kinase 1 ( <i>AVCRL1</i> encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the <i>SMAD4</i> gene are present in families with Juvenile Polyposis/HHT syndrome that involves AVMs. SMAD4 is a downstream effector of Transforming growth factor-β (TGFβ)/Bone morphogenetic protein (BMP) family ligands that signal via Activin like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific <i>Alk1</i> deletion induce AVMs in mouse models as a result of increased PI3K/AKT signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. </p> </div><div class="section"> <a class="named-anchor" id="S2"> <!-- named anchor --> </a> <h5 class="section-title" id="d596869e256">Methods:</h5> <p id="P2">We generated a tamoxifen-inducible, postnatal endothelial-specific <i>Smad4</i> mutant mice ( <i>Smad4</i> ^i∆EC). </p> </div><div class="section"> <a class="named-anchor" id="S3"> <!-- named anchor --> </a> <h5 class="section-title" id="d596869e270">Results:</h5> <p id="P3">We found that loss of endothelial <i>Smad4</i> resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1 and SMAD4 dependent manner. <i>Smad4</i> ^i∆EC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial <i>Akt1</i> deletion both rescued AVM formation in <i>Smad4</i> ^i∆EC mice. BMP9-induced SMAD4 inhibited Casein Kinase 2 ( <i>CK2</i>) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in <i>Smad4</i> ^i∆EC mice. </p> </div><div class="section"> <a class="named-anchor" id="S4"> <!-- named anchor --> </a> <h5 class="section-title" id="d596869e303">Conclusions:</h5> <p id="P4">Our study reveals SMAD4 as an essential effector of BMP9–10/ALK1 signaling that affects AVM pathogenesis via regulation of <i>CK2</i> expression and PI3K/AKT1 activation. </p> </div>