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.

Previous studies have suggested that the NG2 proteoglycan interacts with type VI collagen. We have further characterized this interaction using a solid phase binding assay in which purified NG2 was shown to bind to pepsin-solubilized type VI collagen. In addition, NG2 bound a recombinant ␣2 (VI) collagen chain but did not appreciably bind to the recombinant ␣1 (VI) chain or the N-terminal domain of ␣3 (VI) (N9 -N2). Binding of NG2 to type VI collagen was shown to be concentration-dependent and saturable and to depend mainly on the NG2 core protein, since chondroitinase-treated NG2 bound the collagen as well as undigested samples. In addition, the binding studies revealed several other possible ligands for NG2, including type II collagen, type V collagen, tenascin, and laminin. Binding of the proteoglycan to these molecules was also shown to be mediated by domains contained within the NG2 core protein. The ability of NG2 to bind to these extracellular matrix molecules was compared with that of the chondroitin sulfate proteoglycan decorin, revealing an almost identical binding pattern of the two proteoglycans to the different collagen types. In addition, decorin was found to effectively inhibit the ability of NG2 to bind to collagen, thus suggesting that the two proteoglycans may bind to some of the same regions on the collagen substrates. In contrast, decorin did not bind tenascin and was ineffective in inhibiting the binding of NG2 to tenascin or laminin, indicating that NG2 may bind these two molecules using a separate domain that is distinct from its collagen binding region.Proteoglycans represent a diverse class of macromolecules the defining feature of which is the addition of one or more glycosaminoglycan sugar chains to a core glycoprotein. Characterization of this group of molecules has expanded in recent years, revealing an enormous diversity in number, structure, and function (for review, see Refs. 1-4). Although early work emphasized the importance of the glycosaminoglycan chains in mediating proteoglycan interactions with other ligands, sequence information obtained from the growing list of cloned proteoglycan core proteins has revealed the presence of binding motifs similar to those found in other proteins (3). This suggests that proteoglycan core proteins may, in fact, be responsible for many of the interactions attributed to proteoglycans. Proteoglycans contained within the extracellular matrix (ECM), 1 which include the large aggregating proteoglycans aggrecan (5) and versican (6), the smaller leucine-rich family of proteoglycans typified by decorin, and the basement membrane proteoglycan perlecan, all have been shown to interact with several other matrix components, including laminin (7), fibronectin (8, 9), tenascin (10), hyaluronic acid (5, 6, 11), and collagen (12-14). These interactions are believed to be important for the proper assembly, maintenance, and function of the ECM. In addition, cell surface proteoglycans such as syndecan and CD44 have been shown to bind matrix molecules, including...

NG2 is a membrane-spanning proteoglycan with a primary structure unique among cell surface or extracellular matrix proteins. To characterize the interaction between NG2 and extracellular matrix proteins, we have used a eukaryotic expression system to produce and purify several recombinant fragments covering not only the entire ectodomain of NG2 but also distinct subdomains of the molecule.