Abstract-Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. BMPER (BMP endothelial cell precursor-derived regulator) is a differentially expressed protein in embryonic endothelial precursor cells. In earlier work, we found that BMPER interacts with BMPs and when overexpressed antagonizes their function in embryonic axis formation. In contrast, in a BMPER-deficient zebrafish model, BMPER behaves as a BMP agonist. Furthermore, lack of BMPER induces a vascular phenotype in zebrafish that is driven by disarray of the intersomitic vasculature. Here, we investigate the impact of BMPER on endothelial cell function and signaling and elucidate its role in BMP-4 function in gain-and loss-of-function models. As shown by Western blotting and immunocytochemistry, BMPER is an extracellular matrix protein expressed by endothelial cells in skin, heart, and lung. We show that BMPER is a downstream target of FoxO3a and consistently exerts activating effects on endothelial cell sprouting and migration in vitro and in vivo. Accordingly, when BMPER is depleted from endothelial cells, sprouting is impaired. In terms of BMPER related intracellular signaling, we show that BMPER is permissive and necessary for Smad 1/5 phosphorylation and induces Erk1/2 activation. Most interestingly, BMPER is necessary for BMP-4 to exert its activating role in endothelial function and to induce Smad 1/5 activation. Vice versa, BMP-4 is necessary for BMPER activity. Taken together, BMPER is a dose-dependent endothelial cell activator that plays a unique and pivotal role in fine-tuning BMP activity in angiogenesis. Key Words: BMPER Ⅲ bone morphogenetic proteins Ⅲ vascular biology Ⅲ endothelial cell function Ⅲ signaling A ngiogenesis is a basic biological event that is involved in embryonic development but also in adult physiological and pathological conditions, such as inflammation, tumor growth, atherosclerosis, or response to ischemia. This process depends on the orchestrated function of intra-and extracellular proteins, many of which are conserved from embryonic development through adulthood. 1 Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)- superfamily. Originally, they have been identified by their ability to induce ectopic bone formation and have been extensively studied during embryonic development, in which they control axis formation and organogenesis. Today, more than 20 BMP-related proteins and a number of BMP modulating proteins have been identified. 2 A growing body of evidence suggests that they serve as important regulators in vascular development and disease. 3 BMPs are extracellular proteins that signal through cell surface complexes of type I and type II serine/threonine kinase receptors. On activation, the receptors mediate intracellular signaling mainly through the Smad 1/5 transcription factors. BMP signaling is regulated at several levels: activity of R-Smads (1/5) is modulated by facilitating (eg, Smad 4) or inhibitory (eg,...
BMPER (bone morphogenetic protein [BMP] endothelial precursor cell derived regulator) is an extracellular protein, that interacts with BMPs and thereby modulates BMP dependent vasculogenesis and angiogenesis. Our previous observations suggest a complex regulation of BMPER expression. During embryogenesis BMPER is expressed at the time and at sites of vasculogenesis, whereas in the adult organism it is expressed in heart, lung and skin. Methods and Results: We have cloned the mouse BMPER promoter and appropriate deletion constructs into pGL3 to regulate luciferase expression. As predicted in silicio, we found that Sp1 and Sp1-like transcription factors such as the krueppel-like factors (KLFs) regulate BMPER transcription. KLF-15 resulted in a 4.5 fold upregulation. Accordingly, BMPER expression was inhibited by the Sp-1/SP-1 like inhibitor mitramycin A. Site specific mutation of a proximal KLF-15 binding site reduced the effect of KLF-15 on BMPER expression. Along the same lines, knock down of KLF-15 in HUVEC by siRNA reduced BMPER expression. The transactivating effect of KLF-15 could be competed away by coexpression of Sp-1 suggesting that both factors may compete for the same binding site in the BMPER promoter. In EMSA, an oligo representing a well characterized KLF-15 binding site in the AceCs2 promoter but not an oligo encoding for a NFkappa-B site competed with the oligo coding for the KLF-15 site in the BMPER promoter. In contrast FoxO3A, a member of the FoxO family of transcription factors, serves as an inhibitor of BMPER expression, as shown by gain and lack of FoxO3A experiments. Additionally, we found that BMPER stimulates angiogenesis in a BMP-4 dependent manner in several in vitro and in vivo assays. Vice versa, BMPER is necessary for BMP-4 to exert is angiogenic activity on endothelial cells. Conclusion: BMPER is upregulated by KLF-15 and inhibited by FoxO3a. BMPER has angiogenic activity and is a key modulator of the BMP pathway.
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