Role of soluble endoglin in BMP9 signaling.

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.British Heart Foundation, Cancer Genomics Centre Netherlands and from the Netherlands CardioVascular Research Initiative, the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development, and the Royal Netherlands Academy of Sciences (CVON PHEADRA)

CD44 acts as a co-receptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, Heligmosomoides polygyrus, down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3+ regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown. We noted that truncated TGM1, lacking D4/5, showed reduced potency. Combination of D1/2/3 and D4/5 as separate proteins did not alter potency, suggesting that a physical linkage is required and that these domains do not deliver an independent signal. Coprecipitation from cells treated with biotinylated D4/5, followed by mass spectrometry, identified the cell surface protein CD44 as a coreceptor for TGM1. Both full-length and D4/5 bound strongly to a range of primary cells and cell lines, to a greater degree than D1/2/3 alone, although some cell lines did not respond to TGM1. Ectopic expression of CD44 in nonresponding cells conferred responsiveness, while genetic depletion of CD44 abolished enhancement by D4/5 and ablated the ability of full-length TGM1 to bind to cell surfaces. Moreover, CD44-deficient T cells showed attenuated induction of Foxp3 by full-length TGM1, to levels similar to those induced by D1/2/3. Hence, a parasite protein known to bind two host cytokine receptor subunits has evolved a third receptor specificity, which serves to raise the avidity and cell type–specific potency of TGF-β signaling in mammalian cells

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-β (TGF-β) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis

Autocrine Bone Morphogenetic Protein-9 Signals through Activin Receptor-like Kinase-2/Smad1/Smad4 to Promote Ovarian Cancer Cell Proliferation

Bone morphogenetic proteins (BMPs) act as central regulators of ovarian physiology and may be involved in ovarian cancer development. In an effort to understand these processes we characterized TGFβ/BMP receptor and Smad expression in immortalised ovarian surface epithelial cells (IOSE) and a panel of ovarian cancer cell lines. These studies prompted us to evaluate the potential role of BMP9 signalling in ovarian cancer. Using siRNA, ligand trap, inhibitor and ligand stimulation approaches we demonstrate that BMP9 acts as a proliferative factor for IOSE and ovarian cancer cell lines, signalling predominantly via an ALK2/Smad1/Smad4 pathway rather than via ALK1, the major BMP9 receptor in endothelial cells. Importantly, we find that some ovarian cancer cell lines have gained autocrine BMP9 signalling which is required for proliferation. Furthermore, immunohistochemistry analysis of an ovarian cancer tissue microarray reveals that approximately 25% of epithelial ovarian cancers express BMP9 whereas normal human OSE specimens do not. Our data indicate that BMP9 signalling via ALK2 may be a novel therapeutic target in ovarian cancer

Mutational analysis of sclerostin shows importance of the flexible loop and the cystine-knot for Wnt-signaling inhibition.

The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured β-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different

Anti-Sclerostin Antibody Inhibits Internalization of Sclerostin and Sclerostin-Mediated Antagonism of Wnt/LRP6 Signaling

Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with $[^{125} I]$ Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses

Inhibiting Endothelial Cell Function in Normal and Tumor Angiogenesis Using BMP Type I Receptor Macrocyclic Kinase Inhibitors

Angiogenesis, i.e., the formation of new blood vessels from pre-existing endothelial cell (EC)-lined vessels, is critical for tissue development and also contributes to neovascularization-related diseases, such as cancer. Vascular endothelial growth factor (VEGF) and bone morphogenetic proteins (BMPs) are among many secreted cytokines that regulate EC function. While several pharmacological anti-angiogenic agents have reached the clinic, further improvement is needed to increase clinical efficacy and to overcome acquired therapy resistance. More insights into the functional consequences of targeting specific pathways that modulate blood vessel formation may lead to new therapeutic approaches. Here, we synthesized and identified two macrocyclic small molecular compounds termed OD16 and OD29 that inhibit BMP type I receptor (BMPRI)-induced SMAD1/5 phosphorylation and downstream gene expression in ECs. Of note, OD16 and OD29 demonstrated higher specificity against BMPRI activin receptor-like kinase 1/2 (ALK1/2) than the commonly used small molecule BMPRI kinase inhibitor LDN-193189. OD29, but not OD16, also potently inhibited VEGF-induced extracellular regulated kinase MAP kinase phosphorylation in ECs. In vitro, OD16 and OD29 exerted strong inhibition of BMP9 and VEGF-induced ECs migration, invasion and cord formation. Using Tg (fli:EGFP) zebrafish embryos, we found that OD16 and OD29 potently antagonized dorsal longitudinal anastomotic vessel (DLAV), intra segmental vessel (ISV), and subintestinal vessel (SIV) formation during embryonic development. Moreover, the MDA-MB-231 breast cancer cell-induced tumor angiogenesis in zebrafish embryos was significantly decreased by OD16 and OD29. Both macrocyclic compounds might provide a steppingstone for the development of novel anti-angiogenesis therapeutic agents

Modeling of a Sclerostin-LRP6E1E2 complex indicates additional interactions apart from the NXI motif.

<p>(A) Interaction scheme of a 7 mer peptide representing the loop tip amino acid sequence of Sclerostin (residues Leu90 to Arg96) bound into the cleft of the β-propeller domain 1 of LRP6 (PDB entry 3SOV, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081710#pone.0081710-Bourhis1" target="_blank">[27]</a>). Two main binding determinants were identified in the Sclerostin-derived peptide, Asn92 and Ile94, which are engaged in several polar bonds (Asn92) as well as hydrophobic interactions (Ile94) to facilitate recognition and binding. Residues of LRP6 involved in the interaction with Asn92 and Ile94 are shown in the magnification on the left. (B) Theoretical model of the structured core domain of Sclerostin docked onto the propeller 1-2 fragment of LRP6. The two cavities formed by propeller domain 1 and 2 are separated by 50 Å. The extended architecture of Sclerostin (coincidentally measuring also 50 Å in length) might therefore contact propeller domain 1 and 2 (the latter with parts of Sclerostin finger 1 or 2) possibly explaining the higher binding affinity of Sclerostin to LRP6 fragments containing both propeller domains compared to LRP6 propeller 1 alone. (C) Surface representation of murine Sclerostin (left and right presentation are rotated by 180° around the y-axis) color-coded on the basis of an amino acid sequence alignment (D). The colors mark the level of amino acid identity/homology between Sclerostin and Wise proteins of different organisms with red highlighting invariant residues, orange for exchanges by homologous amino acids, and light and dark blue marking variable residue positions. (D) Sequence alignment of the core domain of Sclerostin and the related Wnt inhibitor Wise from different species. Secondary structure, architectural elements and disulfide bond pattern (1,2,3 mark the cystine-knot forming disulfides) are indicated.</p