Maturation of bone morphogenetic proteins (BMPs) requires cleavage of their precursor proteins by furin-type proprotein convertases. Here, we find that cleavage sites of the BMP2/4/decapentaplegic (DPP) subfamily have been evolutionary diversified and can be categorized into 4 different types. Cnidaria BMP2/4/DPP is considered to be a prototype containing only 1 furin site. Bilateria BMP2/4/DPP acquired an additional cleavage site with either the combination of minimal-optimal or optimal-optimal furin sites. DPPs belonging to Diptera, such as Drosophila and mosquito, and Lepidoptera of silkworm contain a third cleavage site between the 2 optimal furin sites. We studied how the 3 furin sites (FSI-III) of Drosophila DPP coordinate maturation of ligands and contribute to signals in vivo. Combining mutational analysis of furin-recognition sites and RNAi experiments, we found that the Drosophila DPP precursor is initially cleaved at an upstream furin-recognition site (FSII), with consequent cleavages at 2 furin sites (FSI and FSIII). Both Dfurin1 and Dfurin2 are involved in the processing of DPP proproteins. Biochemical and genetic analyses using cleavage mutants of DPP suggest the first cleavage at FSII to be critical and sufficient for long-range DPP signaling. Our data suggest that the Drosophila DPP precursor is cleaved in a different manner from vertebrate BMP4 even though they are functional orthologs. This indicates that the furin-cleavage sites in BMP2/4/DPP precursors are tolerant to mutations acquired through evolution and have adapted to different systems in diversified species.bone morphogenetic protein (BMP) ͉ decapentaplegic ͉ proprotein convertase
Therapeutic angiogenesis provides a potential alternative for the treatment of cardiovascular ischemic diseases. Vascular endothelial growth factor (VEGF) is an important component of the angiogenic response to ischemia. Here we used adeno-associated virus (AAV) gene delivery to skeletal muscle to examine the effects of VEGF vs. a stabilized form of hypoxia-inducible factor-1alpha (HIF-1alpha). The recombinant AAVs were injected into mouse tibialis anterior muscle, and their effects were analyzed by immunohistochemistry and functional assays. These analyses showed that stabilized HIF-1alpha markedly increase capillary sprouting and proliferation, whereas VEGF164 or VEGF120 induced only proliferation of endothelial cells without formation of proper capillary structures. The Evans Blue permeability assay indicated that, unlike VEGF, HIF-1alpha overexpression did not increase vascular leakiness in the transduced muscle. Doppler ultrasound imaging showed that vascular perfusion in the HIF-1alpha treated muscles was significantly enhanced when compared to the controls and not further improved by co-expression of the arteriogenic growth factors angiopoietin-1 or platelet-derived growth factor-B. Our results show that AAV-mediated transduction of a stabilized form of HIF-1alpha can circumvent the problems associated with overexpression of individual angiogenic growth factors. HIF-1alpha should thus offer a potent alternative for pro-angiogenic gene therapy.
Specific proteolytic cleavages turn on, modify, or turn off the activity of vascular endothelial growth factors (VEGFs). Proteolysis is most prominent among the lymphangiogenic VEGF-C and VEGF-D, which are synthesized as precursors that need to undergo enzymatic removal of their C- and N-terminal propeptides before they can activate their receptors. At least five different proteases mediate the activating cleavage of VEGF-C: plasmin, ADAMTS3, prostate-specific antigen, cathepsin D, and thrombin. All of these proteases except for ADAMTS3 can also activate VEGF-D. Processing by different proteases results in distinct forms of the “mature” growth factors, which differ in affinity and receptor activation potential. The “default” VEGF-C-activating enzyme ADAMTS3 does not activate VEGF-D, and therefore, VEGF-C and VEGF-D do function in different contexts. VEGF-C itself is also regulated in different contexts by distinct proteases. During embryonic development, ADAMTS3 activates VEGF-C. The other activating proteases are likely important for non-developmental lymphangiogenesis during, e.g., tissue regeneration, inflammation, immune response, and pathological tumor-associated lymphangiogenesis. The better we understand these events at the molecular level, the greater our chances of developing successful therapies targeting VEGF-C and VEGF-D for diseases involving the lymphatics such as lymphedema or cancer.
Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scw(E1) encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scw(E1) contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that Scw(E1) preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scw(E1) alleles. The unprocessed prodomain of Scw(E1) remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.
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