A heterodimer formed by bone morphogenetic protein 9 (BMP9) and BMP10 provides most BMP biological activity in plasma

Abstract

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

Keywords: angiogenesis; blood; bone morphogenetic protein (BMP); heterodimer; liver; vascular biology.

Figure 1.

A, schematic representation of recombinant BMP9 and BMP10 (pro-BMPs), containing respectively a His₆ epitope and a myc epitope downstream of the furin-cleavage site. B, Western blot (WB) analysis of CM from HEK-293-transfected cells. HEK-293 cells were stably transfected with plasmids encoding His-BMP9, myc-BMP10, or both plasmids (indicated BMP9–BMP10). Proteins were detected with antibodies directed against the mature domain of BMP9 or BMP10 as indicated. All samples were run under non-reduced conditions. NT, nontransfected cells. C–E, HisTrap^TM chromatography of CM from cells cotransfected with His-BMP9/myc-BMP10. C, CM applied under native conditions. D, CM denaturated by 8 mol/liter of urea and heated at 65°C for 20 min before being loaded onto the column. E, CM denatured as above, then reduced by DTT and alkylated before loading. Flow-through (FT) was collected. Columns were washed with 20 mmol/liter of imidazole (wash) and eluted by 200 mmol/liter of imidazole (elution). Fractions were analyzed by Western blot with the indicated antibodies under non-reduced (C and D) or reduced conditions (E). Note that the anti-myc antibody gives a strong non-specific band around 100 kDa (*). In C and E, elution tracks are surrounded by a black line to highlight spliced 10 mm imidazole washing fractions. F and G, heterodimerization of a mutant BMP and a wildtype (WT) counterpart. Fully-cleaved mature WT BMP9 and BMP10 have been produced without any tag sequence. BMP9 and BMP10 furin-cleavage sites have been mutated to create uncleavable sequences, respectively, RQAA and RIAA. Wildtype and mutant BMPs have been solely transfected (0.5 ng of plasmid in each condition), or co-transfected (0.5 ng of plasmid coding for WT BMP and 1.5 ng of mutant BMP) into HEK-293 cells. CM were analyzed by Western blotting with the indicated antibodies directed against mature BMPs (F) or for their BRE activity on ALK1-transfected 3T3 cells (G). Arrows in F show the bands corresponding to mature BMPs. Arrowheads point to a 65-kDa band resulting from the dimerization of a wildtype BMP and a mutant pro-BMP. In G, co-transfection with a mutant BMP leads to a dominant-negative effect on ALK1–BRE stimulation. Values represent relative firefly luciferase normalized to Renilla luciferase activity.

Figure 2.

Recombinant BMP9–10 heterodimer is active on endothelial cells through the ALK1 receptor. A, Western blotting (WB) detection of purified BMP9–10 heterodimer under reduced (BMP9) or unreduced (BMP10) conditions. BMP9–10 was purified through a HisTrap^TM column followed by a anti-myc-agarose column and bound heterodimer was eluted by glycine, pH 2 (see Fig. S3). BMP9–10 was quantified using Image Lab (Bio-Rad) after Western blotting. Band intensity was measured on the mature form (23 and 14 kDa for nonreduced or reduced, respectively) and compared with known amount of commercial (comm) BMP9 or BMP10. The figure shows lanes with 5 ng of each BMP. B, HUVECs were transfected with pGL3(BRE)2-luc and pRL-TK-luc and stimulated with BMP ligands in a range from 25 to 1000 pg/ml. Results show the fold-induction over control unstimulated cells. Data are the mean ± S.E. of 4 independent experiments conducted in duplicate. For each dose, statistical analysis was performed and indicated no significant difference between the 3 ligands (two-way analysis of variance multiple comparison test followed by a Tukey post test). C, HUVECs were transfected with duplex siRNA (scramble versus two different ALK1 siRNA) and further transfected 48 h later with pGL3(BRE)2-luc and pRL-TK-luc. They were then stimulated with 250 pg/ml of ligands and results are expressed as the fold-induction over nonstimulated cells (NS). The figure shows one representative experiment out of two conducted in duplicates.

Figure 3.

BMP9–10 heterodimer is present in mouse plasma and is the only active ALK1-stimulating form. A–C, ELISA measurements of circulating BMP9 (A), BMP10 (B), or BMP9–10 heterodimer (C) in plasma from WT, Bmp9-KO mice, and Bmp10-cKO mice. BMP9–10 ELISA was realized using a capture antibody against mature BMP9 and a detection antibody against BMP10. Recombinant BMP9 and BMP10 or purified BMP9–10 heterodimer were used as standards. n = 7 to 15 mice. Results are presented as the median ± interquartile range (Imm) of BMP immune reactive BMP. D, ALK1–BRE activity of plasma from WT, Bmp9-KO, and Bmp10-cKO mice using a BRE assay on 3T3-transfected cells. Values are presented as mean ± S.E. from 8 to 15 mice. E and F, immunoblotting of primary human endothelial cell lysates (E, HMVEC-ds; F, HUVECs) using phospho-Smad1–5 antibody. Cells were starved for 90 min and stimulated for 20 min with 3% plasma from WT, Bmp9-KO, or Bmp10-cKO mice. Blots show 2 different mice per condition. The position of the 50-kDa marker is indicated by the line on the right of each blot. Stimulation values were normalized to actin expression and represented as the % of stimulation with WT plasma taken as 100% (mean ± S.E.). Quantification has been made on 4 different mice for each condition. Stimulation with 100 pg/ml of commercial BMP9 under the same conditions is shown as a control in E. Statistical comparison of KO-mice versus WT mice was performed using a Mann-Whitney test: *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Figure 4.

BMP9–10 heterodimer is present in human plasma and is the main ALK1-stimulating form. A, quantification of BMP9–10 heterodimer in 19 healthy human plasma. BMP9–10 ELISA was realized using a capture antibody against mature BMP10 and a detection antibody against BMP9. Results are presented as the median ± interquartile range (Imm). B–E, purification and activity of BMP9–10 heterodimer from human plasma. BMP9–10 heterodimer was purified by affinity chromatography: 10 ml of a pool of human plasma (selected for its high BMP9–10 immune reactivity, 8–10 ng/ml) was applied to an anti-BMP10 column, and eluted by glycine, pH 2.7. B–D, ELISAs: plasma, FT, and elution fractions were quantified by ELISA to detect BMP9 (B), BMP10 (C), or BMP9–10 heterodimer under the same conditions as in A (D). y axes represent the total amount of immune reactive BMP9, BMP10, or BMP9–10. E, ALK1–BRE activity of the different fractions was measured on 3T3 cells. Recombinant BMP9 was used as a standard to quantify the total amount of active BMP in each fraction. Values are the mean ± S.D. of duplicates measured by ELISA or BRE-luc assay from one representative experiment out of 3.

Figure 5.

Hepatic stellate cells synthesize both BMP9 and BMP10. A, Bmp10 mRNA expression in human tissues (Clontech, 9 tissues out of 20 analyzed) determined by quantitative RT-PCR. Results are shown as the mean ± S.D. of Bmp10 mRNA level normalized to RPL13 mRNA levels. B and C, localization of Gdf2 (Bmp9) or Bmp10 mRNA by RNAscope on mouse adult heart sections (B) or liver sections (C). Paraffin-embedded sections were labeled with Gdf2 probe for BMP9 (left panels) or Bmp10 probe (right panel) revealed by fast-red pink staining and counterstained with hematoxylin. In B, right atria (RA), ventricle (V). Insets show higher magnification of the right atria (scale bar, 25 μm). In C, upper panels show low magnification with several positive pink cells (arrowheads). Lower panels are high magnifications showing hepatocytes (H), a Kupffer cell (K), lining the wall of a sinusoid and stellate cells (S) between hepatocytes and sinusoids. Note the pink labeling of stellate cells.