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First published online 20 February 2007
doi: 10.1242/jcs.002949

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BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis

¹ Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands
² Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands
³ Human Genome Sciences, Inc., Rockville, MD 20850, USA
⁴ CoGenesys, Rockville, MD 20850, USA

View larger version (42K): [in this window] [in a new window]   Fig. 1. BMP-9 binds to ALK1 and endoglin in the absence of type-II receptors. COS-7 cells were transiently transfected with cDNAs for ALK1, ALK2, BMPR-II, ActRII-B or endoglin and affinity-labelled with [125I]BMP-9. Crosslinked complexes were immunoprecipitated with specific antisera (marked with a circle) and subjected to SDS-PAGE and autoradiography. (A) [125I]BMP-9 binds with high affinity to ALK1, but not to ALK2 in the absence of type-II receptors. BMP-9 binding to ALK2 is strongly enhanced when BMPR-II or ActR-IIB are co-expressed. (B) [125I]BMP-9 binds to endoglin in the absence of type-I or type-II receptors.

View larger version (37K): [in this window] [in a new window]   Fig. 2. BMP-9 binds to endogenous receptors in endothelial cells. (A,B) BAECs (A) or HUVECs (B) were affinity-labelled with [125I]BMP-9 and crosslinked ligand-receptor complexes were immunoprecipitated with specific antisera as indicated. BMP-9 predominantly binds to ALK1 and BMPR-II, but also to ALK2, ActR-IIA, ActR-IIB and endoglin. (C) Competition of [125I]BMP-9 binding to ALK1 in HUVECs with either excess unlabelled (cold) BMP-9, activin, BMP-7 or TGF-. Only cold BMP-9 can compete with [125I]BMP-9 binding to ALK1.

View larger version (31K): [in this window] [in a new window]   Fig. 3. BMP-9 binds to endogenous receptors in non-endothelial cells. (A-C) Autoradiography of crosslinked complexes of [125I]BMP-9 with cell surface receptors in C2C12 myoblasts (A), XTH-1 breast cancer cells (B), and T98G glioblastoma cells (C). Ligand-receptor complexes were immunoprecipitated with specific antisera as indicated in the figure and subjected to SDS-PAGE. BMP-9 binds to ALK2 in all cell lines, to ALK1 in glioblastoma cells and to endoglin in breast cancer and glioblastoma cells.

View larger version (45K): [in this window] [in a new window]   Fig. 4. BMP-9 activates Smad1 and Smad5, and upregulates ID1 and endoglin in endothelial cells. (A-C) BAECs (A), HUVECs (B) or HDMVECs (C) were incubated with increasing doses of BMP-9 and BMP-6 as indicated in the figure. After 45 minutes, cells were lysed and samples subjected to SDS-PAGE and subsequent western blotting. Membranes were either probed with an antibody that specifically recognises phosphorylated Smad1 and Smad5 or with an antibody directed against ID1. An anti--actin antibody was used to confirm equal loading. (D) Effect of BMP-9 and TGF- on endoglin mRNA expression in BAECs as measured by quantitative real-time PCR.

View larger version (31K): [in this window] [in a new window]   Fig. 5. BMP-9 activates the Smad1- and Smad5-responsive BRE luciferase reporter through ALK1. (A-B) BAECs were transiently transfected with an ID1-promoter-derived luciferase reporter construct (BRE) and a construct for -galactosidase as internal expression control. Before stimulation, cells were serum-starved for 8 hours and then incubated with the respective ligands overnight. (A) BRE activity is induced by BMP-9 (100 ng/ml) and BMP-6 (100 ng/ml) but not by TGF- (5 ng/ml). (B) BMP-9 (5 ng/ml) stimulated BRE-induction can be inhibited by a 15-fold molar excess of ALK1-Fc. (C,D) shRNA constructs against ALK1 or ALK2 were cloned into the pSuper vector and co-transfected into BAECs. ALK1 shRNA blocks BMP-9-stimulated (5 ng/ml) (C), but not BMP-6-induced (25 ng/ml) BRE activation (D). (E) ALK1 shRNA-mediated reduction of BRE activity is rescued by overexpression of human ALK1, but not by human ALK2. *P<0.01 compared with BMP-9-stimulated control; #P<0.01 compared with ALK1 RNAi after BMP-9 stimulation.

View larger version (39K): [in this window] [in a new window]   Fig. 6. BMP-9 attenuates migration and inhibits proliferation of ECs. (A) BAECs were allowed to grow to confluence and serum-starved overnight. Monolayers were wounded and stimulated with either 30 ng/ml bFGF or 10 ng/ml BMP-9. Wound closure was measured after 24 hours using the Olympus Analysis software. (B) Scratched BAEC monolayers were incubated with 10 ng/ml BMP-9 and different concentrations of bFGF as indicated in the figure and cell migration was measured after 24 hours. (C,D) 3000 BAECs were seeded in 96-well plates and stimulated with different concentrations of BMP-9, 30 ng/ml bFGF or combinations of bFGF and BMP-9. Cell proliferation was determined after 2 and 3 days by adding the MTS reagent and measuring the absorbance at 490 nm. B9, BMP-9. ##P<0.001 compared with bFGF treatment; #P<0.01 compared with control; **P< 0.005 and *P<0.05 compared with the respective bFGF treatment without BMP-9.

View larger version (119K): [in this window] [in a new window]   Fig. 7. BMP-9 blocks endothelial network formation. Metatarsals of 17-day-old mouse foetuses were prepared, transferred to cell culture plates and allowed to adhere for 4 days. Medium was refreshed and bones were stimulated for 7 days with BMP-9 (100 ng/ml), VEGF (50 ng/ml) or both. Cultures were fixed and vessel-like structures were visualised by anti-CD31 staining. BMP-9 inhibits baseline formation of the endothelial network. Incubation with VEGF strongly stimulated the formation of vessel-like structures, which was completely abrogated by co-stimulation with BMP-9. Six bones were stimulated per experimental group and one representative picture of each group is shown.