BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

Abstract

Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.

Fig. 1

Origin of BMP9 expression. Immunostaining for BMP9 in human (a–f) and mouse (g, inset corresponds to rabbit control immunoglobulins) liver sections. Human and mouse tissue sections were stained with anti-BMP9 antibodies from R&D Systems (a, b, e, f, g) or from Biogenesis (c, d). The specificity of the staining was checked by addition of an excess of recombinant human BMP9 (1 μg/mL) (b, d). Note the strong immunostaining in hepatocytes and biliary ducts. In e, enlargement of a blood sinusoid [arrowhead indicates absence of staining in liver endothelial cells and arrows indicate staining in hepatocytes (H)]. In f, enlargement of a biliary duct [arrowhead indicates intrahepatic biliary epithelial cells and arrows indicate staining in hepatocytes (H)]. Slides were counterstained with haematoxylin. Scale bars 100 μm

Fig. 2

BMP9 circulates in plasma under a high molecular mass form. a BMP9 biosynthesis. BMP9 is synthesized as a precursor protein (Pre-pro-BMP9) composed of 429 amino acids (aa) that include a 22 aa signal peptide, a 297 aa prodomain (33 kDa) and a 110 aa mature protein (12.5 kDa). The pre-pro-BMP9 then homodimerizes (pro-BMP9) and is subsequently cleaved by serine endoproteases. This generates two active forms: the short mature form (25 kDa) and the complexed form (100 kDa) in which the prodomain remains associated with the mature form. b Proteins from human plasma were separated by gel filtration chromatography. In a parallel experiment, recombinant human BMP9 was passed onto the same column. BMP9 activity was then measured in the different fractions using the ALK1-BRE-luciferase assay as described in “Materials and methods”. The data from one representative experiment (out of 4) are represented. c and d Plasma was passed through an anti-human serum albumin column. The plasma (1), the eluate (2) and the flow-through (3) were then analyzed by 10% SDS-PAGE and stained by Coomassie blue (c) and their BMP9 activity using the ALK1-BRE-luciferase assay was also quantified as described in “Materials and methods” (d). Data are expressed as the mean luciferase value ± SD obtained from two independent experiments

Fig. 3

BMP9 circulates in plasma under both active and inactive high molecular mass forms. a Proteins from a pool of five human plasmas were separated through gel filtration chromatography. BMP9 levels were then measured in the different fractions by three different means: the ALK1-BRE-luciferase assay, the BMP9 ELISA and the pro-BMP9 ELISA as described in “Materials and methods”. The ALK1-BRE-luciferase assay data and the data obtained from the BMP9 ELISA are presented as pg/mL of BMP9. The data obtained with the pro-BMP9 ELISA are presented as optical densities (OD). The data obtained in one representative experiment out of 3 are presented. b, c Human fractions (30–42) were treated with or without furin and BMP9 levels were then measured in the different fractions with the ALK1-BRE-luciferase assay (fraction 34, corresponding to the peak of BMP9 activity, was also measured c in the absence (gray square) or presence of anti-BMP9 neutralizing antibodies (black square). The ALK1-BRE-luciferase assay data are presented as pg/mL of BMP9. The data obtained in one representative experiment out of 2 are presented. d Nine human plasma (0.3%) were treated with or without furin and BMP9 activation was then measured using the ALK1-BRE-luciferase assay and the BMP9 ELISA as described in “Materials and methods”. Data are expressed as furin-treated plasma over untreated plasma mean ± SEM from duplicate determinations. e Pie chart representing the percentage of each BMP9 complex circulating in human plasma, as calculated from (d) measured with the ALK1-BRE luciferase assay

Fig. 4

Ontogeny of BMP9 circulating levels in mice. BMP9 levels were measured from pooled diluted plasma (0.5%) taken from mice at the indicated developmental stages using the ALK1-BRE-luciferase assay as described in “Materials and methods”. In order to check that the activity measured by the ALK1-BRE-luciferase assay was attributable to BMP9, the assay was performed (inset) in the absence (gray squares) or the presence of anti-BMP9 neutralizing antibodies (black squares). The results are presented as means ± SD from triplicate determinations (E embryonic day, P post-natal day)

Fig. 5

Rat aortic endothelial cells are physiologically Smad1/5/8 phosphorylated in response to circulating BMP9. a Immunostaining for phosphoSmad1/5/8 of rat aorta cross-sections. The aortas were fixed immediately after surgical removal from the killed animal and processed for phospho-Smad1/5/8 immunostaining. Note the nuclear staining of endothelial cells. b Quantification of the number of phosphoSmad1/5/8-positive nuclei. Rat aortic slices were either fixed immediately after sacrificing the animal or incubated ex vivo for 1 h with PBS (step 1) and then for another hour with rat serum in the absence or presence of either anti-BMP9 neutralizing antibodies or recombinant ALK1ecd (step 2). The rat aorta slices where then fixed and immunostained for phosphoSmad1/5/8. Results are presented as the percentage of phosphoSmad1/5/8 nuclei in endothelial cells per aortic ring. Data are expressed as the mean ± SD of values obtained in 3 independent experiments (***p < 0.001)