Bone morphogenetic proteins (BMPs) require proteolytic activation by members of the proprotein convertase (PC) family. Pro-BMP4 is initially cleaved at a site adjacent to the mature ligand domain (S1) and then at an upstream site (S2) within the prodomain. Cleavage at the S2 site, which appears to occur in a tissue-specific fashion, regulates the activity and signaling range of mature BMP4. To test the hypothesis that tissue-specific cleavage of pro-BMP4 is regulated by differential expression of a site-specific protease, we identified the PCs that cleave each site in vivo. In Xenopus oocytes, furin and PC6 function redundantly to cleave both the S1 and S2 sites of pro-BMP4, as evidenced by the results of antisense-mediated gene knockdown and the use of the furin-and PC6-selective inhibitor ␣ 1 -PDX. By contrast, ␣ 1 -PDX blocked cleavage of the S2 but not the S1 site of pro-BMP4 in embryos, suggesting the existence of a developmentally regulated S1 site-specific convertase. This protease is likely to be PC7 based on knowledge of its required substrate cleavage motif and resistance to ␣ 1 -PDX. Consistent with this prediction, an ␣ 1 -PDX variant engineered to target PC7, in addition to furin and PC6, completely inhibited cleavage of BMP4 in oocytes and embryos. Further studies showed that pc7 transcripts are expressed and polyadenylated, and that the PC7 precursor protein undergoes efficient autocatalytic activation in both oocytes and embryos. These results suggest that PC7, or a convertase with similar substrate specificity, functions to selectively cleave the S1 site of pro-BMP4 in a developmentally regulated fashion. BMP4 (bone morphogenetic protein 4) is a cell to cell signaling molecule that was originally isolated for its ability to induce ectopic bone formation (1). More recent studies demonstrate diverse roles for BMP4 during development of the skeleton and other organs and in bone homeostasis after birth (2).The bioactivity of BMP4 is regulated post-translationally, at the level of proteolytic activation. BMP4 originates as an inactive dimeric precursor that is cleaved by specific members of the proprotein convertase (PC) 2 family of endoproteases (3, 4) to yield prodomain fragments along with the carboxyl-terminal mature ligand. In mammals, seven members of the PC family have been characterized. Among these only furin (also known as PACE, SPC1, or PCSK3), PACE4 (also named SPC4 or PCSK6), PC6 (also called PC5, SPC6, or PCSK5), and PC7 (also known as PC8, LPC, SPC7, or PCSK7) are broadly expressed and active within the constitutive, as opposed to the regulated, secretory pathway, making them appropriate candidates for endogenous BMP4 convertases (5, 6). Furin prefers to cleave proproteins at the carboxyl-terminal side of the optimal consensus sequence -RX(R/K)R-, but can also cleave following the minimal sequence -RXXR-(7). PACE4 and PC6 (also commonly called PC5) recognize the same optimal and minimal furin consensus motifs, whereas PC7 has a strict requirement for a basic residue in the P2 position ...

Bone morphogenetic protein-4 (BMP-4) is synthesized as a large precursor protein, which undergoes proprotein convertase-mediated proteolytic maturation along the secretory pathway to release the active ligand. Pro-BMP-4 is initially cleaved at a consensus furin motif adjacent to the mature ligand domain (the S1 site), and this allows for subsequent cleavage at an upstream motif (the S2 site). This sequential cleavage liberates a small, evolutionarily conserved, prodomain fragment (the linker peptide) of unknown fate and function. Here we show that the linker domain is essential for proper folding, exit from the endoplasmic reticulum, and thus cleavage of the BMP-4 precursor when overexpressed in Xenopus oocytes and embryos but not in cultured mammalian cells. Mature BMP-4 synthesized from a precursor in which the S1 site is non-cleavable, such that the linker domain remains covalently attached to the ligand, has little or no activity in vivo. Finally, analysis of folding, cleavage, and bioactivity of chimeric precursors containing the BMP-7 prodomain and BMP-4 mature domain, or vice versa, with or without the BMP-4 linker domain revealed that the linker domain is only functional in the context of the BMP-4 prodomain, and that differential cleavage around this domain can regulate the activity of a heterologous ligand.Bone morphogenetic protein-4 (BMP-4), 3 a member of the transforming growth factor-␤ (TGF-␤) superfamily, was first isolated for its ability to induce ectopic cartilage and bone formation. Subsequently, BMP-4 was shown to be involved in a wide variety of biological processes including cell proliferation and differentiation, apoptosis, and cell fate determination. BMP-4 has been established as a morphogen that plays essential roles during embryonic development (1). The mature BMP-4 ligand is a functional dimer harboring seven cystine residues, six of which are involved in intramolecular disulfide linkage, and the seventh of which is responsible for dimer formation (2), BMP-4 is synthesized as a large inactive precursor that is proteolytically cleaved following a multibasic motif (-RXKR-, termed the S1 site) to generate the active C-terminal ligand (3). Cleavage is carried out by specific member(s) of the proprotein convertase (PC) family of serine proteases (4, 5). The best characterized member of this family, furin, recognizes the preferred consensus sequence, -RX(K/R)R-, but can also cleave following the minimal sequence -RXXR-(6).We have shown that BMP-4 undergoes a second cleavage at a minimal furin consensus motif (-RXXR-, the S2 site) located upstream of the S1 site within the prodomain (7). Paradoxically, although cleavage of pro-BMP-4 occurs sequentially (S1 and then S2), and the initial cleavage at the S1 site releases the mature ligand, subsequent cleavage at the S2 site regulates the activity and the signaling range of mature BMP-4 (7). In Xenopus embryos, BMP-4 synthesized from exogenous precursor in which the S2 site is non-cleavable is less active, signals over a shorter range and accumu...

In this Pro-Con commentary article, we debate the importance of anterior thigh block locations for analgesia following total knee arthroplasty. The debate is based on the current literature, our understanding of the relevant anatomy, and a clinical perspective. We review the anatomy of the different fascial compartments, the course of different nerves with respect to the fascia, and the anatomy of the nerve supply to the knee joint. The Pro side of the debate supports the view that more distal block locations in the anterior thigh increase the risk of excluding the medial and intermediate cutaneous nerves of the thigh and the nerve to the vastus medialis, while increasing the risk of spread to the popliteal fossa, making distal femoral triangle block the preferred location. The Con side of the debate adopts the view that while the exact location of local anesthetic injection appears anatomically important, it has not been proven to be clinically relevant.

Mandible shape in the mouse is a complex trait that is influenced by many genetic factors. However, little is known about the action of single genes on adult mandible shape so far, since most developmentally relevant genes are already required during embryogenesis, i.e. knockouts lead to embryonic death or severe deformations, before the mandible is fully formed. We employ here a geometric morphometrics approach to identify subtle phenotypic differences caused by dosage effects of candidate genes. We use mouse strains with specific gene modifications (knock-outs and knock-ins) to compare heterozygous animals with controls from the same stock, which is expected to be equivalent to a change of gene expression of the respective locus. Such differences in expression level are also likely to occur as part of the natural variation. We focus on Bmp pathway genes (Bmp4, its antagonist Noggin and combinations of Bmp5–7 genotypes), but include also two other developmental control genes suspected to affect mandible development in some way (Egfr and Irf6). In addition, we study effects of Hoxd13, as well as an extracellular matrix constituent (Col2a1). We find that subtle, but significant shape differences are caused by differences in gene dosage of several of these genes. The changes seen for Bmp4 and Noggin are partially compatible with the action of these genes known from birds and fish. We find significant shape changes also for Hoxd13, although this gene has so far only been implicated in skeletal patterning processes of the limbs. Comparing the effect sizes of gene dosage changes to the variation found in natural populations of mice as well as QTL effects on mandible shape, we find that the effect sizes caused by gene dosage changes are at the lower end of the spectrum of natural variation, but larger than the average additive effects found in QTL studies. We conclude that studying gene dosage effects have the potential to provide new insights into aspects of craniofacial development, variation and evolution.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo.