The BMP2 prodomain promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers in vivo.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus . We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

BMP5 contributes to hepcidin regulation and systemic iron homeostasis in mice.

Hepcidin is the master regulator of systemic iron homeostasis. The bone morphogenetic protein (BMP) signaling pathway is a critical regulator of hepcidin expression in response to iron and erythropoietic drive. Although endothelial-derived BMP6 and BMP2 ligands have key functional roles as endogenous hepcidin regulators, both iron and erythropoietic drives still regulate hepcidin in mice lacking either or both ligands. Here, we used mice with an inactivating Bmp5 mutation (Bmp5se), either alone or together with a global or endothelial Bmp6 knockout, to investigate the functional role of BMP5 in hepcidin and systemic iron homeostasis regulation. We showed that Bmp5se-mutant mice exhibit hepcidin deficiency at age 10 days, blunted hepcidin induction in response to oral iron gavage, and mild liver iron loading when fed on a low- or high-iron diet. Loss of 1 or 2 functional Bmp5 alleles also leads to increased iron loading in Bmp6-heterozygous mice and more profound hemochromatosis in global or endothelial Bmp6-knockout mice. Moreover, double Bmp5- and Bmp6-mutant mice fail to induce hepcidin in response to long-term dietary iron loading. Finally, erythroferrone binds directly to BMP5 and inhibits BMP5 induction of hepcidin in vitro. Although erythropoietin suppresses hepcidin in Bmp5se-mutant mice, it fails to suppress hepcidin in double Bmp5- and Bmp6-mutant males. Together, these data demonstrate that BMP5 plays a functional role in hepcidin and iron homeostasis regulation, particularly under conditions in which BMP6 is limited.

Same-day repeatability and Between-Sequence reproducibility of Mean ADC in PI-RADS lesions.

PURPOSE: This study aimed to assess repeatability after repositioning (inter-scan), intra-rater, inter-rater and inter-sequence variability of mean apparent diffusion coefficient (ADC) measurements in MRI-detected prostate lesions. METHOD: Forty-three patients with suspicion for prostate cancer were included and received a clinical prostate bi-/multiparametric MRI examination with repeat scans of the T2-weighted and two DWI-weighted sequences (ssEPI and rsEPI). Two raters (R1 and R2) performed single-slice, 2D regions of interest (2D-ROIs) and 3D-segmentation-ROIs (3D-ROIs). Mean bias, corresponding limits of agreement (LoA), mean absolute difference, within-subject coefficient of variation (CoV) and repeatability/reproducibility coefficient (RC/RDC) were calculated. Bradley & Blackwood test was used for variance comparison. Linear mixed models (LMM) were used to account for multiple lesions per patient. RESULTS: Inter-scan repeatability, intra-rater and inter-sequence reproducibility analysis of ADC showed no significant bias. 3D-ROIs demonstrated significantly less variability than 2D-ROIs (p < 0.01). Inter-rater comparison demonstrated small significant systematic bias of 57 × 10-6 mm2/s for 3D-ROIs (p < 0.001). Intra-rater RC, with the lowest variation, was 145 and 189 × 10-6 mm2/s for 3D- and 2D-ROIs, respectively. For 3D-ROIs of ssEPI, RCs and RDCs were 190-198 × 10-6 mm2/s for inter-scan, inter-rater and inter-sequence variation. No significant differences were found for inter-scan, inter-rater and inter-sequence variability. CONCLUSIONS: In a single-scanner setting, single-slice ADC measurements showed considerable variation, which may be lowered using 3D-ROIs. For 3D-ROIs, we propose a cut-off of âˆ¼ 200 × 10-6 mm2/s for differences introduced by repositioning, rater or sequence effects. The results suggest that follow-up measurements should be possible by different raters or sequences.

Lack of evidence that fibrillin1 regulates bone morphogenetic protein 4 activity in kidney or lung.

BACKGROUND: The Bone morphogenetic protein 4 (BMP4) precursor protein is cleaved at two sites to generate an active ligand and inactive prodomain. The ligand and prodomain form a noncovalent complex following the first cleavage, but dissociate after the second cleavage. Transient formation of this complex is essential to generate a stable ligand. Fibrillins (FBNs) bind to the prodomains of BMPs, and can regulate the activity of some ligands. Whether FBNs regulate BMP4 activity is unknown. RESULTS: Mice heterozygous for a null allele of Bmp4 showed incompletely penetrant kidney defects and females showed increased mortality between postnatal day 6 and 8. Removal of one copy of Fbn1 did not rescue or enhance kidney defects or lethality. The lungs of Fbn1+/- females had enlarged airspaces that were unchanged in Bmp4+/- ;Fbn1+/- mice. Additionally, removal of one or both alleles of Fbn1 had no effect on steady state levels of BMP4 ligand or on BMP activity in postnatal lungs. CONCLUSIONS: These findings do not support the hypothesis that FBN1 plays a role in promoting BMP4 ligand stability or signaling, nor do they support the alternative hypothesis that FBN1 sequesters BMP4 in a latent form, as is the case for other BMP family members.

Tril dampens Nodal signaling through Pellino2- and Traf6-mediated activation of Nedd4l.

Toll-like receptor 4 (Tlr) interactor with leucine-rich repeats (Tril) functions as a Tlr coreceptor to mediate innate immunity in adults. In Xenopus embryos, Tril triggers degradation of the transforming growth factor ß (Tgf-ß) family inhibitor, Smad7. This enhances bone morphogenetic protein (Bmp) signaling to enable ventral mesoderm to commit to a blood fate. Here, we show that Tril simultaneously dampens Nodal signaling by catalytically activating the ubiquitin ligase NEDD4 Like (Nedd4l). Nedd4l then targets Nodal receptors for degradation. How Tril signals are transduced in a nonimmune context is unknown. We identify the ubiquitin ligase Pellino2 as a protein that binds to the cytoplasmic tail of Tril and subsequently forms a complex with Nedd4l and another E3 ligase, TNF-receptor associated factor 6 (Traf6). Pellino2 and Traf6 are essential for catalytic activation of Nedd4l, both in Xenopus and in mammalian cells. Traf6 ubiquitinates Nedd4l, which is then recruited to membrane compartments where activation occurs. Collectively, our findings reveal that Tril initiates a noncanonical Tlr-like signaling cascade to activate Nedd4l, thereby coordinately regulating the Bmp and Nodal arms of the Tgf-ß superfamily during vertebrate development.

Analysis of Transforming Growth Factor ß Family Cleavage Products Secreted Into the Blastocoele of Xenopus laevis Embryos.

The two arms of the Transforming Growth Factor ß (Tgfß) superfamily, represented by Tgfß/Nodal or Bone morphogenetic protein (Bmp) ligands, respectively, play essential roles in embryonic development and adult homeostasis. Members of the Tgfß family are made as inactive precursors that dimerize and fold within the endoplasmic reticulum. The precursor is subsequently cleaved into ligand and prodomain fragments. Although only the dimeric ligand can engage Tgfß receptors and activate downstream signaling, there is growing recognition that the prodomain moiety contributes to ligand activity. This article describes a protocol that can be used to identify cleavage products generated during activation of Tgfß precursor proteins. RNA encoding Tgfß precursors are first microinjected into X. laevis embryos. The following day, cleavage products are collected from the blastocoele of gastrula stage embryos and analyzed on Western blots. This protocol can be completed relatively quickly, does not require expensive reagents and provides a source of concentrated Tgfß cleavage products under physiologic conditions.

BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis.

BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation (Bmp7R-GFlag) that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While Bmp7 null homozygotes are live born, Bmp7R-GFlag homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the Bmp7R-G allele together with a null allele of Bmp2 or Bmp4 die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either Bmp4 or Bmp7 lead to a similar spectrum of congenital defects in humans.

Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos.

Bone morphogenetic proteins (Bmps) are synthesized as inactive precursors that are cleaved to generate active ligands, along with prodomain fragments that can modulate growth factor activity. Here we provide three protocols that can be used to examine the process of proteolytic activation of Bmps. The first protocol describes how to generate radiolabeled Bmp precursor proteins in Xenopus oocytes and then analyze the time course of precursor cleavage by recombinant enzymes in vitro. The second protocol details how to analyze cleavage of radiolabeled precursor proteins in Xenopus oocytes over time using pulse-chase analysis and autoradiography. This protocol can also be used to analyze folding and cleavage of radiolabeled precursor proteins at steady state. Finally, the third protocol details methods for isolating Bmp cleavage products from the blastocoele of Xenopus embryos and then analyzing them on immunoblots.

Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling.

Toll-like receptor 4 interactor with leucine-rich repeats (Tril) functions as a coreceptor for Toll-like receptors (Tlrs) to mediate innate immune responses in adults. In embryos, Tril signals to promote degradation of the Bmp inhibitor, Smad7, to allow for blood formation. It is not known whether this function requires, or is independent of, Tlrs. In the current studies, we performed a structure-function analysis, which indicated that the fibronectin type III (FN) domain and the intracellular domain of Tril are required to trigger Smad7 degradation in Xenopus embryos. Furthermore, we found evidence suggesting that a Tril deletion mutant lacking the FN domain (Tril∆FN) can dominantly inhibit signaling by endogenous Tril when overexpressed. This finding raises the possibility that the FN domain functions to bind endogenous Tril ligands. We also show that Tril cycles between the cell surface and endosomes and that the Tril extracellular domain, as well as cadherin based cell-cell adhesion, are required for cell surface retention, while the intracellular domain is required for internalization in Xenopus ectodermal explants. Using a CHO cell aggregation assay, we show that, unlike other transmembrane proteins that contain leucine-rich repeats, Tril is not sufficient to mediate homophilic adhesion.

The TGFß superfamily in Lisbon: navigating through development and disease.

The 10th FASEB meeting 'The TGFß Superfamily: Signaling in Development and Disease' took place in Lisbon, Portugal, in July 2017. As we review here, the findings presented at the meeting highlighted the important contributions of TGFß family signaling to normal development, adult homeostasis and disease, and also revealed novel mechanisms by which TGFß signals are transduced.

Diverse Non-genetic, Allele-Specific Expression Effects Shape Genetic Architecture at the Cellular Level in the Mammalian Brain.

Interactions between genetic and epigenetic effects shape brain function, behavior, and the risk for mental illness. Random X inactivation and genomic imprinting are epigenetic allelic effects that are well known to influence genetic architecture and disease risk. Less is known about the nature, prevalence, and conservation of other potential epigenetic allelic effects in vivo in the mouse and primate brain. Here we devise genomics, in situ hybridization, and mouse genetics strategies to uncover diverse allelic effects in the brain that are not caused by imprinting or genetic variation. We found allelic effects that are developmental stage and cell type specific, that are prevalent in the neonatal brain, and that cause mosaics of monoallelic brain cells that differentially express wild-type and mutant alleles for heterozygous mutations. Finally, we show that diverse non-genetic allelic effects that impact mental illness risk genes exist in the macaque and human brain. Our findings have potential implications for mammalian brain genetics. VIDEO ABSTRACT.

Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos.

In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. Here, we identify tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. We show that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification.

Providing a navigable route for acute medicine nurses to advance their practice: a framework of ascending levels of practice.

This article conveys concerns raised by delegates at the International SAM Conference (Manchester, 2015) regarding how to advance nursing practice in acute medicine. It endeavors to capture the essence of 'how to advance practice' and 'how to integrate advanced practice' within the workforce structures of an acute medicine unit (AMU). It addresses the production of tacit knowledge and the recognition and integration of this to developing the nursing workforce. The current context of NHS efficiencies and recruitment issues emphasize the value of retaining tacit knowledge. Uniquely, this article offers an early conceptual framework through which levels of advancement and potential transition points to advance nursing practice in acute medicine are articulated. Determining how to advance requires identification of prior accomplishments such as, tacit knowledge, experiential learning, CPD, specialist courses and management experience. This requires nurses to make judicious decisions to advance their practice and the distinction between 'amassing experience' and 'career progression'. It aims to stimulate thinking around the practicalities of advancement, the value of tacit knowledge and potential realization through the framework trajectory.

Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development.

Primitive hematopoiesis generates red blood cells that deliver oxygen to the developing embryo. Mesodermal cells commit to a primitive blood cell fate during gastrulation and, in order to do so the mesoderm must receive non-cell autonomous signals transmitted from other germ layers. In Xenopus, the transcription factor Gata2 functions in ectodermal cells to generate or transmit the non-cell autonomous signals. Here we have identified Breast Cancer Antiestrogen Resistance 3 (bcar3) as a gene that is induced in ectodermal cells downstream of Gata2. Bcar3 and its binding partner Bcar1 function to transduce integrin signaling, leading to changes in cellular morphology, motility and adhesion. We show that gata2, bcar3 and bcar1 are co-expressed in ventral ectoderm from early gastrula to early tailbud stages. At later stages of development, bcar3 and bcar1 are co-expressed in the spinal cord, notochord, fin mesenchyme and pronephros but each shows additional unique sites of expression. These co-expression and unique expression patterns suggest that Bcar3 and Bcar1 may function together but also independently during Xenopus development.

GATA2 regulates Wnt signaling to promote primitive red blood cell fate.

Primitive erythropoiesis is regulated in a non cell-autonomous fashion across evolution from frogs to mammals. In Xenopus laevis, signals from the overlying ectoderm are required to induce the mesoderm to adopt an erythroid fate. Previous studies in our lab identified the transcription factor GATA2 as a key regulator of this ectodermal signal. To identify GATA2 target genes in the ectoderm required for red blood cell formation in the mesoderm, we used microarray analysis to compare gene expression in ectoderm from GATA2 depleted and wild type embryos. Our analysis identified components of the non-canonical and canonical Wnt pathways as being reciprocally up- and down-regulated downstream of GATA2 in both mesoderm and ectoderm. We show that up-regulation of canonical Wnt signaling during gastrulation blocks commitment to a hematopoietic fate while down-regulation of non-canonical Wnt signaling impairs erythroid differentiation. Our results are consistent with a model in which GATA2 contributes to inhibition of canonical Wnt signaling, thereby permitting progenitors to exit the cell cycle and commit to a hematopoietic fate. Subsequently, activation of non-canonical Wnt signaling plays a later role in enabling these progenitors to differentiate as mature red blood cells.

PKC/ROS-Mediated NLRP3 Inflammasome Activation Is Attenuated by Leishmania Zinc-Metalloprotease during Infection.

Parasites of the Leishmania genus infect and survive within macrophages by inhibiting several microbicidal molecules, such as nitric oxide and pro-inflammatory cytokines. In this context, various species of Leishmania have been reported to inhibit or reduce the production of IL-1ß both in vitro and in vivo. However, the mechanism whereby Leishmania parasites are able to affect IL-1ß production and secretion by macrophages is still not fully understood. Dependent on the stimulus at hand, the maturation of IL-1ß is facilitated by different inflammasome complexes. The NLRP3 inflammasome has been shown to be of pivotal importance in the detection of danger molecules such as inorganic crystals like asbestos, silica and malarial hemozoin, (HZ) as well as infectious agents. In the present work, we investigated whether Leishmania parasites modulate NLRP3 inflammasome activation. Using PMA-differentiated THP-1 cells, we demonstrate that Leishmania infection effectively inhibits macrophage IL-1ß production upon stimulation. In this context, the expression and activity of the metalloprotease GP63 - a critical virulence factor expressed by all infectious Leishmania species - is a prerequisite for a Leishmania-mediated reduction of IL-1ß secretion. Accordingly, L. mexicana, purified GP63 and GP63-containing exosomes, caused the inhibition of macrophage IL-1ß production. Leishmania-dependent suppression of IL-1ß secretion is accompanied by an inhibition of reactive oxygen species (ROS) production that has previously been shown to be associated with NLRP3 inflammasome activation. The observed loss of ROS production was due to an impaired PKC-mediated protein phosphorylation. Furthermore, ROS-independent inflammasome activation was inhibited, possibly due to an observed GP63-dependent cleavage of inflammasome and inflammasome-related proteins. Collectively for the first time, we herein provide evidence that the protozoan parasite Leishmania, through its surface metalloprotease GP63, can significantly inhibit NLRP3 inflammasome function and IL-1ß production.

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease.

Impact of Leishmania infection on host macrophage nuclear physiology and nucleopore complex integrity.

The protease GP63 is an important virulence factor of Leishmania parasites. We previously showed that GP63 reaches the perinuclear area of host macrophages and that it directly modifies nuclear translocation of the transcription factors NF-κB and AP-1. Here we describe for the first time, using molecular biology and in-depth proteomic analyses, that GP63 alters the host macrophage nuclear envelope, and impacts on nuclear processes. Our results suggest that GP63 does not appear to use a classical nuclear localization signal common between Leishmania species for import, but degrades nucleoporins, and is responsible for nuclear transport alterations. In the nucleoplasm, GP63 activity accounts for the degradation and mislocalization of proteins involved amongst others in gene expression and in translation. Collectively, our data indicates that Leishmania infection strongly affects nuclear physiology, suggesting that targeting of nuclear physiology may be a strategy beneficial for virulent Leishmania parasites.