Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells

Abstract

ALK1 is an endothelial-specific type I receptor of the TGF␤ receptor family whose heterozygous mutations cause hereditary hemorrhagic telangiectasia type 2. Although TGF␤1 and TGF␤3 have been shown to bind ALK1 under specific experimental conditions, they may not represent the physiological ligands for this receptor. In the present study, we demonstrate that BMP9 induces the phosphorylation of Smad1/5/8 in microvascular endothelial cells, and this phosphorylation lasts over a period of 24 hours. BMP9 also activates the ID1 promoter-derived BMP response element (BRE) in a dosedependent manner (EC 50 ‫؍‬ 45 ؎ 27 pg/ mL), and this activation is abolished by silencing ALK1 expression or addition of ALK1 extracellular domain. Overexpression of endoglin increases the BMP9 response, whereas silencing of both BMP-RII and ActRIIA expressions completely abolishes it. BMP10, which is structurally close to BMP9, is also a potent ALK1 ligand. Finally, we demonstrate that BMP9 and BMP10 potently inhibit endothelial cell migration and growth, and stimulate endothelial expression of a panel of genes that was previously reported to be activated by the constitutively active form of ALK1. Taken IntroductionActivin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGF␤ receptor family that is implicated in the pathogenesis of the Rendu-Osler disease also known as hereditary hemorrhagic telangiectasia (HHT). 1 The disease is an autosomal dominant vascular dysplasia affecting 1 in 10 000 people. The clinical abnormalities in HHT are caused by direct arteriovenous connections without an intervening capillary bed. The resulting telangiectases occur in the oral cavity (lips and tongue), in the nose, and on the fingertips. Larger arteriovenous malformations (AVMs) can be encountered in the lung, brain, and liver. 2 There is wide variation in the penetrance and severity of these symptoms in patients even within the same family, suggesting that environmental or other genetic factors influence the phenotype. The majority of cases are caused by mutations in either Endoglin (ENG) or ALK1 (ACVRL1) genes, thus defining HHT1 and HHT2, respectively. Recently, mutations in SMAD4 have also been described in a few cases with combined juvenile polyposis and HHT syndromes. 3 Each of the 3 genes implicated in HHT (ENG, ACVRL1, and SMAD4) encode receptors or signaling molecules from the TGF␤ family. Most TGF␤ family ligands bind to heteromeric complexes of type I and type II serine/threonine kinase receptors (for review, see Shi and Massague 4 ). In addition, the type III receptors (betaglycan and endoglin) act as coreceptors that can potentiate the signaling cascade. Upon ligand binding, the type II receptor phosphorylates and activates the type I receptor, also known as activin receptor-like kinase (ALK), which in turn phosphorylates a receptor-regulated Smad protein (Smad1, Smad2, Smad3, Smad5, or Smad8). This phosphorylated Smad dimerizes with a common partner, Smad4, and this complex translocates to the nucleus where ...

BMP-9-induced muscle heterotopic ossification requires changes to the skeletal muscle microenvironment

“…Figure 5 A shows staining for AlkP activity in mrSCs treated with BMP-9 in the presence of 5Â and 20Â molar equivalents of soluble ALK1-Fc receptor, which can bind and specifically inhibit BMP-9. (15) Our results indicated that the level of osteogenesis of mrSCs induced by BMP-9 was inversely proportional to the level of soluble ALK1-Fc receptor added.…”

“…(14) BMP-9 is the physiologic ligand of the endothelial type I ALK1 receptor in association with the type II BMP receptor (BMPRII). (15) ALK1 is one of the seven type I receptors for TGF-b family members. (16) The activation of ALK1 induces the phosphorylation of receptor-regulated Smad-1 and Smad-5 and their accumulation in the nucleus.…”

Osteoinductive signals and heterotopic ossification

“…While BMP-9-induced heterotopic ossification was shown to be mediated through the ALK1 type 1 receptor, the study does not address whether ALK1 acts independently of other BMP type 1 receptors or other receptors contribute to heterotopic ossification, perhaps through type 1 BMP receptor heterodimer formation with ALK1. Although experimental data support that ALK1 binds BMP-9 and activates the BMP-Smad1/5/ 8 pathway in response to BMP-9, (11) there is additional evidence that BMP-9 binds to and signals through receptors other than ALK1, including ALK2, (12,13) the BMP type 1 receptor that is mutated in patients with FOP. (4) It is interesting to speculate that in these experiments, BMP-9 in the context of injury is inducing heterotopic ossification by activating ALK1-ALK2 heterodimers or that a sequential series of BMP signaling events mediated independently by ALK1 and ALK2 is required for induction of heterotopic ossification.…”

“…Interestingly, ALK1 was identified initially as an endothelial-specific type 1 receptor. (11) It also cannot be overlooked that heterotopic bone is a tissue that frequently forms through endochondral ossification and is comprised of multiple cell types. While the brunt of investigations of heterotopic bone formation has focused on osteoblast formation and differentiation, recruitment and differentiation of additional cell types are critical to the process as well.…”

Genetic considerations relevant to intracranial hemorrhage and brain arteriovenous malformations

“…Two recent reports show that ALK-1 may instead signal through BMP-9 and that endoglin can potentiate the signal, suggesting that BMP-9 may represent a physiologically relevant endothelial signaling pathway for HHT pathogenesis [17,52]. Endothelial cell-specific ablation of the murine Alk-1 gene causes AVM formation during development, whereas mice harboring an EC-specific knockout of Alk-5 (the type I TGF-β receptor) or TbRII (the type II TGF-β receptor) show neither AVM formation nor any other perturbation in vascular morphogenesis [42].…”