Fig 2: (A) QRT-PCR receptor levels of Alk1 in the cell lines indicated (left panel). Human microvascular endothelial cells served as the positive control for Alk1 expression [55]. Side-by-side comparison of Alk1, 2, 3, and 6 in PA1 and P76 (right panel). (B-C) Western blotting of either normal breast cell line MCF10A and FTEC (P211) or (C) transformed breast and ovarian cancer cells in the presence of increasing doses of GDF2 for 30 minutes as indicated. Total SMAD1/5 served as loading control. (D) Western blotting for pSMAD2/3 in P76 cells treated with GDF2 (10 ng/ml) for the indicated times. Lysates for TGF-β–treated cells were used as a positive control. (E) GDF2 specifically induces BRE-luciferase promoter activity. Luciferase activity normalized to untreated HEK293 cells transfected with either BRE-Luc, PE2.1 Luc, or P3tP Luc reporter plasmids treated with BMP2 (10 nM), GDF2 (10 ng/ml), or TGFβ (100 pM) for 24 hours is presented.

Fig 3: Models of unprocessed BMP‐10 dimer and processed BMP‐10 CPLX. (A) Model of the unprocessed BMP‐10 dimer showing the α1‐helix (marked in blue) masking ALK‐1‐binding residues Y358, P359, and I371 on the GF (marked in red). (B) Model of the processed BMP‐10 CPLX where the same GF residues (marked in cyan) are accessible for ALK‐1 receptor engagement. Processing reduces the distance between PD arms by about 20 Å as indicated. PPC cleavage sites are marked in light blue.

Fig 4: Working model describing targeting, sequestration, and activation of BMP‐10 depending on its processing status. BMP‐10 is produced systemically by hepatic stellate cells and locally by cardiovascular resident cells. Its activation depends on the localization of PPCs. When PPCs are present intracellularly, BMP‐10 is first processed and secreted in a bioactive form, which is then sequestered by fibrillin microfibrils in latent pools. From these pools, BMP‐10 can be reactivated locally through PD‐proteolytic cleavage, releasing the bioactive GF. Conversely, when PPCs are present extracellularly, BMP‐10 is secreted in an unprocessed, latent form. Activation occurs via PPC‐mediated cleavage of the covalent bond between the PD and the GF, which increases flexibility in the α1 helix, enabling specific GF residues to interact with ALK‐1 and initiate long‐range BMP signaling. These distinct activation pathways mediate either short‐range or long‐range signaling, activating unique signaling cascades during development and homeostasis.

Fig 5: Processed BMP‐10 CPLX interacts with fibrillin‐1 and is rendered into a closed ring‐shape conformation. (A) TEM analysis after incubation of the N‐terminal fibrillin‐1N‐terminal region with a 1:1 mixture of processed and unprocessed BMP‐10. (top, left) Representative negative staining TEM images showing the globular shape of the N‐terminal region of fibrillin‐1 (start_EGF4). (top, right; bottom left) TEM analysis of unbound BMP‐10 molecules. Scale bars: 30 nm (overview) or 20 nm (magnified micrographs). (bottom, right) Quantification of unbound BMP‐10 particles showing a significant increase of molecules with wide angle (80%) versus tight angle (20%). (B) TEM analysis reveals the presence of ring‐shaped BMP‐10 molecules upon the addition of fibrillin‐1 (start_EGF4). The apparent position of globular N‐terminal fibrilin‐1 molecules is indicated by white arrow heads in one representative example. Scale bar: 20 nm. (C) Model of the closed‐ring BMP‐10 CPLX. In this model, the type II receptor binding site: 405GVVTYKF411 on BMP‐10 GF (light green) is masked by the α2‐helix, while ALK‐1 binding residues on the GF remain accessible. The BMP‐10 binding sites within FUN are labeled in black and yellow, and the fibrillin‐1 binding site in BMP‐10 PD is labeled in orange. PD residues are labeled in purple and GF residues in dark green.