Proc Natl Acad Sci U S A 116(36): 17800-17808

PMC: PMC6731690

PMID: 31431534

Role of soluble endoglin in BMP9 signaling

D Charnock-Jones+3 authors

Supplementary Material

^{Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, United Kingdom;}

^{Department of Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, 2300 RC Leiden, The Netherlands;}

^{Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle upon Tyne NE1 3BZ, United Kingdom;}

^{Reproductive Biology Laboratory, Amsterdam University Medical Centers, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands;}

^{Department of Obstetrics and Gynaecology, University of Cambridge, Cambridge CB2 0SW, United Kingdom;}

^{National Institute for Health Research, Cambridge Biomedical Research Centre, University of Cambridge, Cambridge CB2 0SW, United Kingdom;}

^{Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, United Kingdom}

^{To whom correspondence may be addressed. Email:}ku.ca.mac@522lw.

Edited by Se-Jin Lee, University of Connecticut System, Farmington, CT, and approved July 25, 2019 (received for review September 26, 2018)

Author contributions: A.L., Z.T., M.T., R.E.R., D.S.C.-J., H.M.A., P.t.D., and W.L. designed research; A.L., Z.T., M.T., R.E.R., J.C., M.v.D., D.S.C.-J., and W.L. performed research; G.B.A., D.S.C.-J., H.M.A., P.t.D., and W.L. contributed new reagents/analytic tools; A.L., Z.T., M.T., R.E.R., J.C., M.v.D., N.W.M., G.B.A., D.S.C.-J., H.M.A., P.t.D., and W.L. analyzed data; H.M.A., P.t.D., and W.L. jointly supervised the research; and N.W.M., D.S.C.-J., H.M.A., P.t.D., and W.L. wrote the paper.

^{A.L., Z.T., M.T., and R.E.R. contributed equally to this work.}

Edited by Se-Jin Lee, University of Connecticut System, Farmington, CT, and approved July 25, 2019 (received for review September 26, 2018)

This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY).

Significance

Endoglin (ENG) is a dimeric transmembrane accessory receptor highly expressed on endothelial cells. Its extracellular domain can be cleaved and released into circulation as soluble ENG (sENG). Higher levels of sENG contribute to the pathogenesis of preeclampsia (PE). Bone morphogenetic protein 9 (BMP9), a circulating signaling molecule and vascular quiescence factor, binds sENG with high affinity. Hence sENG has been thought to be a dimeric molecule and an inhibitory ligand trap for BMP9. Here we show that sENG purified from human tissues is monomeric and that the sENG:BMP9 complex is an active signaling molecule, but requires cell-surface ENG for optimal signaling. This study provides insight for understanding the role of sENG in PE and other cardiovascular diseases.

Keywords: BMP9, soluble endoglin, ALK1, placenta, preeclampsia

Significance

Abstract

Endoglin (ENG) is a coreceptor of the transforming growth factor-β (TGFβ) family signaling complex, which is highly expressed on endothelial cells and plays a key role in angiogenesis. Its extracellular domain can be cleaved and released into the circulation as soluble ENG (sENG). High circulating levels of sENG contribute to the pathogenesis of preeclampsia (PE). Circulating bone morphogenetic protein 9 (BMP9), a vascular quiescence and endothelial-protective factor, binds sENG with high affinity, but how sENG participates in BMP9 signaling complexes is not fully resolved. sENG was thought to be a ligand trap for BMP9, preventing type II receptor binding and BMP9 signaling. Here we show that, despite cell-surface ENG being a dimer linked by disulfide bonds, sENG purified from human placenta and plasma from PE patients is primarily in a monomeric form. Incubating monomeric sENG with the circulating form of BMP9 (prodomain-bound form) in solution leads to the release of the prodomain and formation of a sENG:BMP9 complex. Furthermore, we demonstrate that binding of sENG to BMP9 does not inhibit BMP9 signaling. Indeed, the sENG:BMP9 complex signals with comparable potency and specificity to BMP9 on human primary endothelial cells. The full signaling activity of the sENG:BMP9 complex required transmembrane ENG. This study confirms that rather than being an inhibitory ligand trap, increased circulating sENG might preferentially direct BMP9 signaling via cell-surface ENG at the endothelium. This is important for understanding the role of sENG in the pathobiology of PE and other cardiovascular diseases.

Abstract

Endoglin (ENG) is a homodimeric transmembrane glycoprotein that is strongly expressed on endothelial cells (ECs) (1). Loss-of-function mutations in ENG cause hereditary hemorrhagic telangiectasia type I (HHT1) (2), which is characterized by telangiectases affecting the nose, gastrointestinal tract, and skin, as well as larger arteriovenous malformations (AVM) in the brain, lung, and liver. ENG mutations have also been reported in patients with pulmonary arterial hypertension (PAH), a vascular disorder characterized by the remodeling of small pulmonary vessels, resulting in increased right ventricular systolic pressure that ultimately leads to right-sided heart failure (3).

ENG has a large extracellular domain (ECD) and a short cytoplasmic tail, and its ECD can be cleaved from the cell surface under conditions related to endothelial dysfunction and inflammation (4). Cleaved ENG ECD, also known as soluble endoglin (sENG), is markedly elevated in preeclampsia (PE) and contributes to the pathogenesis of PE (5). Circulating sENG is also elevated in PAH and is proposed to be a biomarker for the prognosis of group I PAH patients (6). Intriguingly, administration of sENG reduces cardiac fibrosis in pressure overload-induced heart failure in mice (7).

In preclinical studies, loss of ENG leads to increased EC proliferation, decreased cell migration against flow, and reduced flow-mediated EC elongation (8 –10). How ENG regulates such important cellular functions at the molecular level is not known. ENG was originally discovered as a component of the transforming growth factor-β (TGFβ) family signaling complex (11). TGFβ family ligands, including bone morphogenetic proteins (BMPs), are homodimers, initiating the cellular signaling by forming a signaling complex at the plasma membrane with 2 copies of a type I receptor and 2 copies of a type II receptor. TGFβ type I receptor (TGFβRI), also termed Activin receptor-like kinase (ALK)5, and TGFβ type II receptor (TGFβRII) mediate signaling from TGFβ1, -β2 and -β3, whereas ALK1 has been reported to participate in signaling in response to both TGFβ and BMPs (12, 13). ENG and betaglycan are coreceptors for the TGFβ family signaling, and both are single-pass transmembrane proteins (14). While their transmembrane and cytoplasmic domains show high sequence similarity (71% similarity with 63% identity in human), the extracellular domains of ENG and betaglycan share little sequence homology (11). While the coreceptor function of betaglycan is to capture and display TGFβ2 to its receptors (15), the molecular function of ENG is less well understood with many controversial reports and unanswered questions in the field. For example, using radio-labeled ligands and coimmunoprecipitation, ENG was initially identified as a component of the TGFβ receptor system, binding to TGFβ1 and TGFβ3 but not TGFβ2 (11); hence, sENG was proposed as a ligand trap for TGFβ1 (5). However, subsequent biochemical studies using purified recombinant ENG ECD-Fc fusion protein (ENG-Fc) revealed that ENG ECD binds directly with high affinity only to BMP9 and BMP10, but not to other TGFβ family receptors or ligands; hence, sENG has been proposed to be a ligand trap for BMP9 and BMP10 (16). Moreover, it has been shown that TGFβ1 can signal through ALK1 and ALK5 in endothelial cells, and its signaling through ALK1 requires ENG (12, 17, 18). However, ALK1 was later found to be a specific type I receptor for BMP9 and BMP10 (13, 19). Third, although ENG has been shown to inhibit TGFβ signaling (20), the requirement of ENG for BMP9 signaling has not been unequivocally established (21, 22).

BMP9 is synthesized by the liver and circulates at active concentrations in a prodomain-bound form (pro-BMP9) with its prodomain noncovalently bound to its growth factor domain (GFD) (23). The crystal structure of the sENG N-terminal orphan domain in complex with BMP9-GFD demonstrates that sENG binds to BMP9 at sites overlapping with the prodomain and the type II receptors (24 –26). This implies that ENG ECD will need to displace the prodomain from BMP9 and then dissociate from BMP9 to allow type II receptor binding and the formation of the signaling complex. Using Biacore sandwich complex formation assays and measuring the changes in response units, it has been proposed that BMP9 prodomain can be displaced by the binding of ALK1, type II receptors, and sENG (27), although direct evidence from solution studies is yet to be shown. In order to assimilate the information into a coherent model of sENG function, a number of important questions remain unanswered, such as the physiological form of circulating sENG and whether sENG inhibits BMP9 signaling at physiologically relevant concentrations. The role of cell-surface ENG versus sENG function is also unclear.

To address these questions, we performed a detailed biochemical dissection of the protein–protein interactions between ENG ECD with BMP9 and its binding partners. We provide evidence that circulating sENG is primarily in the monomeric form and that monomeric sENG can readily displace the prodomain from pro-BMP9 to form a stable complex with BMP9. Moreover, sENG is not an inhibitory BMP9 ligand trap; instead, the purified sENG:BMP9 complex can signal in ECs with similar potency and specificity as pro-BMP9, but cell-surface ENG is required for optimal sENG:BMP9 signaling.

Supplementary File

Click here to view.^{(6.2M, pdf)}

Supplementary File

Click here to view.^{(17K, xlsx)}

Supplementary File

Click here to view.^{(15K, xlsx)}

Supplementary File

Click here to view.^{(15K, xlsx)}

Acknowledgments

This work was supported by British Heart Foundation Grants PG/12/54/29734, PG/15/39/31519, and PG/17/1/32532 (to W.L. and N.W.M.); and by Grants RG/12/2/29416 and PG/14/86/31177 (to H.M.A.). P.t.D. acknowledges support from the Cancer Genomics Centre Netherlands and from the Netherlands CardioVascular Research Initiative, the Dutch Heart Foundation, the Dutch Federation of University Medical Centers, The Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON PHEADRA). Cambridge National Institute for Health Research (NIHR) Biomedical Research Centre provided infrastructure support. The views expressed are those of the authors and not necessarily those of the NIHR or the Department of Health and Social Care.

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The microarray data have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. {"type":"entrez-geo","attrs":{"text":"GSE119206","term_id":"119206","extlink":"1"}}GSE119206).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1816661116/-/DCSupplemental.

Footnotes

References

1. Gougos A., Letarte M., Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells. J. Biol. Chem.265, 8361–8364 (1990). [[PubMed]
2. McAllister K. A., et al. , Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat. Genet.8, 345–351 (1994). [[PubMed]
3. Harrison R. E., et al. , Transforming growth factor-beta receptor mutations and pulmonary arterial hypertension in childhood. Circulation111, 435–441 (2005). [[PubMed]
4. Rathouska J., Jezkova K., Nemeckova I., Nachtigal P., Soluble endoglin, hypercholesterolemia and endothelial dysfunction. Atherosclerosis243, 383–388 (2015). [[PubMed]
5. Venkatesha S., et al, Soluble endoglin contributes to the pathogenesis of preeclampsia. Nat. Med.12, 642–649 (2006). [[PubMed][Google Scholar]
6. Malhotra R., et al, Circulating angiogenic modulatory factors predict survival and functional class in pulmonary arterial hypertension. Pulm. Circ.3, 369–380 (2013). [Google Scholar]
7. Kapur N. K., et al. , Reduced endoglin activity limits cardiac fibrosis and improves survival in heart failure. Circulation125, 2728–2738 (2012).
8. Sugden W. W., et al. , Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues. Nat. Cell Biol.19, 653–665 (2017).
9. Mahmoud M., et al, Pathogenesis of arteriovenous malformations in the absence of endoglin. Circ. Res.106, 1425–1433 (2010). [[PubMed][Google Scholar]
10. Jin Y., et al, Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling. Nat. Cell Biol.19, 639–652 (2017). [Google Scholar]
11. Cheifetz S., et al, Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells. J. Biol. Chem.267, 19027–19030 (1992). [[PubMed][Google Scholar]
12. Goumans M. J., et al. , Balancing the activation state of the endothelium via two distinct TGF-beta type I receptors. EMBO J.21, 1743–1753 (2002).
13. David L., Mallet C., Mazerbourg S., Feige JJ., Bailly S., Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood109, 1953–1961 (2007). [[PubMed][Google Scholar]
14. Nickel J., Ten Dijke P., Mueller TD., TGF-β family co-receptor function and signaling. Acta Biochim. Biophys. Sin. (Shanghai)50, 12–36 (2018). [[PubMed][Google Scholar]
15. López-Casillas F., Wrana JL., Massagué J., Betaglycan presents ligand to the TGF beta signaling receptor. Cell73, 1435–1444 (1993). [[PubMed][Google Scholar]
16. Castonguay R., et al, Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain, inhibits blood vessel formation, and suppresses tumor growth. J. Biol. Chem.286, 30034–30046 (2011). [Google Scholar]
17. Goumans M. J., et al. , Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGFbeta/ALK5 signaling. Mol. Cell12, 817–828 (2003). [[PubMed]
18. Lebrin F., et al, Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction. EMBO J.23, 4018–4028 (2004). [Google Scholar]
19. Scharpfenecker M., et al, BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J. Cell Sci.120, 964–972 (2007). [[PubMed][Google Scholar]
20. Lastres P., et al, Endoglin modulates cellular responses to TGF-beta 1. J. Cell Biol.133, 1109–1121 (1996). [Google Scholar]
21. Nolan-Stevaux O., et al, Endoglin requirement for BMP9 signaling in endothelial cells reveals new mechanism of action for selective anti-endoglin antibodies. PLoS One7, e50920 (2012). [Google Scholar]
22. Upton P. D., Davies R. J., Trembath R. C., Morrell N. W., Bone morphogenetic protein (BMP) and activin type II receptors balance BMP9 signals mediated by activin receptor-like kinase-1 in human pulmonary artery endothelial cells. J. Biol. Chem.284, 15794–15804 (2009).
23. Bidart M., et al, BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain. Cell. Mol. Life Sci.69, 313–324 (2012). [[PubMed][Google Scholar]
24. Saito T., et al, Structural basis of the human endoglin-BMP9 interaction: Insights into BMP signaling and HHT1. Cell Rep.19, 1917–1928 (2017). [Google Scholar]
25. Mi L. Z., et al. , Structure of bone morphogenetic protein 9 procomplex. Proc. Natl. Acad. Sci. U.S.A.112, 3710–3715 (2015).
26. Townson S. A., et al. , Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex. J. Biol. Chem.287, 27313–27325 (2012).
27. Kienast Y., et al, Rapid activation of bone morphogenic protein 9 by receptor-mediated displacement of pro-domains. J. Biol. Chem.291, 3395–3410 (2016). [Google Scholar]
28. Zhou A., et al, A redox switch in angiotensinogen modulates angiotensin release. Nature468, 108–111 (2010). [Google Scholar]
29. Yamasaki M., Li W., Johnson D. J., Huntington J. A., Crystal structure of a stable dimer reveals the molecular basis of serpin polymerization. Nature455, 1255–1258 (2008). [[PubMed]
30. Wang X., Fischer G., Hyvönen M., Structure and activation of pro-activin A. Nat. Commun.7, 12052 (2016).
31. Kang J. G., et al. , RsrA, an anti-sigma factor regulated by redox change. EMBO J.18, 4292–4298 (1999).
32. Chen S. H., Hsu J. L., Lin F. S., Fluorescein as a versatile tag for enhanced selectivity in analyzing cysteine-containing proteins/peptides using mass spectrometry. Anal. Chem.80, 5251–5259 (2008). [[PubMed]
33. Liu J., Liu Y., Gao M., Zhang X., An accurate proteomic quantification method: Fluorescence labeling absolute quantification (FLAQ) using multidimensional liquid chromatography and tandem mass spectrometry. Proteomics12, 2258–2270 (2012). [[PubMed]
34. Ellman GL., Tissue sulfhydryl groups. Arch. Biochem. Biophys.82, 70–77 (1959). [[PubMed][Google Scholar]
35. Gregory AL., Xu G., Sotov V., Letarte M., Review: The enigmatic role of endoglin in the placenta. Placenta35 (suppl. A), S93–S99 (2014). [[PubMed][Google Scholar]
36. Tong Z., Morrell N. W., Li W., Pro-BMP9 and sENG monomer:BMP9 complex signalling in human pulmonary arterial endothelial cells after 1.5 hours treatment. Gene Expression Omnibus. . Deposited 29 August 2018. [PubMed]
37. van Baardewijk L. J., et al. , Circulating bone morphogenetic protein levels and delayed fracture healing. Int. Orthop.37, 523–527 (2013).
38. Araki M., et al, Serum/glucocorticoid-regulated kinase 1 as a novel transcriptional target of bone morphogenetic protein-ALK1 receptor signaling in vascular endothelial cells. Angiogenesis21, 415–423 (2018). [[PubMed][Google Scholar]
39. Anderberg C., et al, Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination. J. Exp. Med.210, 563–579 (2013). [Google Scholar]
40. Wang X., et al, LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling. Nature499, 306–311 (2013). [Google Scholar]
41. Gallardo-Vara E., Tual-Chalot S., Botella L. M., Arthur H. M., Bernabeu C., Soluble endoglin regulates expression of angiogenesis-related proteins and induction of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia. Dis. Model. Mech.11, dmm034397 (2018).
42. Valbuena-Diez A. C., et al. , Oxysterol-induced soluble endoglin release and its involvement in hypertension. Circulation126, 2612–2624 (2012). [[PubMed]
43. Vitverova B., et al, Soluble endoglin and hypercholesterolemia aggravate endothelial and vessel wall dysfunction in mouse aorta. Atherosclerosis271, 15–25 (2018). [[PubMed][Google Scholar]
44. Jezkova K., et al, High levels of soluble endoglin induce a proinflammatory and oxidative-stress phenotype associated with preserved NO-dependent vasodilatation in aortas from mice fed a high-fat diet. J. Vasc. Res.53, 149–162 (2016). [[PubMed][Google Scholar]
45. Nemeckova I., et al, High soluble endoglin levels do not induce endothelial dysfunction in mouse aorta. PLoS One10, e0119665 (2015). [Google Scholar]
46. Carty D. M., Delles C., Dominiczak A. F., Novel biomarkers for predicting preeclampsia. Trends Cardiovasc. Med.18, 186–194 (2008).
47. Guerrero-Esteo M., Sanchez-Elsner T., Letamendia A., Bernabeu C., Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II. J. Biol. Chem.277, 29197–29209 (2002). [[PubMed]
48. Long L., et al, Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: Potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation119, 566–576 (2009). [[PubMed][Google Scholar]
49. Yung L. M., et al. , A selective transforming growth factor-β ligand trap attenuates pulmonary hypertension. Am. J. Respir. Crit. Care Med.194, 1140–1151 (2016).
50. Ermini L., et al, A single sphingomyelin species promotes exosomal release of endoglin into the maternal circulation in preeclampsia. Sci. Rep.7, 12172 (2017). [Google Scholar]
51. Wei Z., Salmon R. M., Upton P. D., Morrell N. W., Li W., Regulation of bone morphogenetic protein 9 (BMP9) by redox-dependent proteolysis. J. Biol. Chem.289, 31150–31159 (2014).