Search

In vertebrates, specification of anteroposterior (A/P) and left–right (L/R) axes depends on TGFβ-related signals, including Nodal, Lefty, and BMPs. Endoproteolytic maturation of these proteins is probably mediated by the proprotein convertase SPC1/Furin. In addition, precursor processing may be regulated by related activities such as SPC4 (also known as PACE4). Here, we show that a proportion of embryos lacking SPC4 develop situs ambiguus combined with left pulmonary isomerism or complex craniofacial malformations including cyclopia, or both. Gene expression analysis during early somite stages indicates that spc4 is genetically upstream of nodal, pitx2, lefty1, and lefty2 and perhaps maintains the balance between Nodal and BMP signaling in the lateral plate that is critical for L/R axis formation. Furthermore, genetic interactions betweennodal and spc4, together with our analysis of chimeric embryos, strongly suggest that during A/P axis formation, SPC4 acts primarily in the foregut. These findings establish an important role for SPC4 in patterning the early mouse embryo.

Transforming growth factor-beta (TGF beta) is a potent immunosuppressive cytokine which inhibits the antigen (Ag)-dependent expansion of T cells both in vitro and in vivo by mechanisms not well defined yet. Here we report that exposure of interleukin (IL)-2-dependent T cell lines to TGF beta 2 results in apoptosis defined by morphology, nucleosomal size DNA fragmentation and in situ DNA end labeling. TGF beta 2-induced T cell apoptosis showed the following characteristics: (1) in contrast to the rapid evolution of apoptosis following IL-2 deprivation, apoptosis of T cells triggered by TGF beta 2 was delayed; (2) cycloheximide prevented TGF beta 2-induced apoptosis of CTLL-2 but not of OVA-7 T helper cells; (3) in contrast to apoptosis following IL-2 deprivation, TGF beta 2-mediated T cell apoptosis was not associated with decreased expression of the proto-oncogenes, bcl-2 or c-myc; (4) TGF beta 2-induced apoptosis was not restricted to IL-2-dependent T cell lines since the IL-4-dependent T cell line, CT.4S, as well as EL4 lymphoma cells, which grow independently of exogenous IL-2, were also susceptible to TGF beta 2-mediated apoptosis. Taken together, these data may present a novel mechanism of TGF beta 2-mediated suppression of T cell expansion in response to Ag and IL-2, the activation of the endogenous death program of apoptosis, which appears to operate independently of direct interactions of TGF beta 2 with the IL-2/IL-2 receptor system.

The beta transforming growth factors (TGF-beta) are suggested to regulate developmental processes since they are distinctly expressed during embryogenesis and exert pleiotropic effects on cell growth and differentiation, In the present study the expression of TGF-beta isoforms was investigated in the postnatal and adult mouse brain. As shown by in situ hybridization, TGF-beta 2 was expressed in the choroid plexus, hippocampus, dentate gyrus and cerebellar Purkinje neurons, both postnatally and in adults. Furthermore, TGF-beta 2 expression was observed postnatally in immature cerebellar neurons of both the external and internal granule cell layers. In the external granule cell layer, the frequency of TGF-beta2 transcripts increased until postnatal day 10 and declined thereafter. In contrast to TGF-beta 2, no TGF-beta 1 mRNA was detected in cerebellar granule cells. TGF-beta 3 expression was widely distributed in postnatal brains although at very low levels. The significance of TGF-beta 2 production by cerebellar granule cells was further investigated using cultures of small cerebellar neurons. In these cultures reverse polymerase chain reaction analysis revealed expression of TGF-beta 2 but low or almost undetectable levels of TGF-beta 1 or -beta 3 mRNAs. Likewise, only TGF-beta 2 protein in its latent form was identified in the culture supernatant; the release of TGF-beta 2 was maximal during the second day in vitro. Furthermore, TGF-beta was found to inhibit the proliferation of cultured small cerebellar neurons. Taken together, these data suggest that TGF-beta 2 is involved in the regulation of postnatal development of the cerebellum.

Secreted TGFβ proteins of the Nodal family pattern the vertebrate body axes and induce mesoderm and endoderm [1]. Nodal proteins can act as morphogens [2], but the mechanisms regulating their activity and signaling range are poorly understood. In particular, it has been unclear how inefficient processing or rapid turnover of the Nodal protein influences autocrine and paracrine signaling properties [3, 4]. Here, we evaluate the role of Nodal processing and stability in tissue culture and zebrafish embryos. Removal of the pro domain potentiates autocrine signaling but reduces Nodal stability and signaling range. Insertion of an N-glycosylation site present in several related TGFβ proteins increases the stability of mature Nodal. The stabilized form of Nodal acts at a longer range than the wild-type form. These results suggest that increased proteolytic maturation of Nodal potentiates autocrine signaling, whereas increased Nodal stability extends paracrine signaling.

The fate of pluripotent cells in early mouse embryos is controlled by graded Nodal signals that are activated by the endoproteases Furin and Pace4. Soluble forms of Furin and Pace4 cleave proNodal in vitro and after secretion in transfected cells, but direct evidence for paracrine activity in vivo is elusive. Here, we show that Furin and Pace4 are released by the extraembryonic microenvironment, and that they cleave a membrane-bound reporter substrate in adjacent epiblast cells and activate Nodal to maintain pluripotency. Secreted Pace4 and Furin also stimulated mesoderm formation, whereas endoderm was only induced by Pace4, correlating with a difference in the spatiotemporal distribution of these proteolytic activities. Our analysis of paracrine Furin and Pace4 activities and their in vivo functions significantly advances our understanding of how the epiblast is patterned by its microenvironment. Adding cell–cell communication to the pleiotropic portfolio of these proteases provides a new framework to study proprotein processing also in other relevant contexts.

We have examined the role of Furin in postimplantation-stage mouse embryos by analyzing both the expression pattern of fur mRNA and the developmental consequences of a loss-of-function mutation at the fur locus. At early stages (day 7.5), fur mRNA is abundant in extraembryonic endoderm and mesoderm, anterior visceral endoderm, and in precardiac mesoderm. 1 day later fur is expressed throughout the heart tube and in the lateral plate mesoderm, notochordal plate and definitive gut endoderm. Embryos lacking Furin die between days 10.5 and 11.5, presumably due to hemodynamic insufficiency associated with severe ventral closure defects and the failure of the heart tube to fuse and undergo looping morphogenesis. Morphogenesis of the yolk sac vasculature is also abnormal, although blood islands and endothelial precursors form. Analysis of cardiac and endodermal marker genes shows that while both myocardial precursors and definitive endoderm cells are specified, their numbers and migratory properties are compromised. Notably, mutant embryos fail to undergo axial rotation, even though Nodal and eHand, two molecular markers of left-right asymmetry, are appropriately expressed. Overall, the present data identify Furin as an important activator of signals responsible for ventral closure and embryonic turning.

Among the molecular mechanisms that control the cell division cycle, proteolysis has emerged as a key regulatory process enabling cells to pass critical check points. Such proteolysis involves a cascade of enzymes including a multisubunit complex termed 26S proteasome. Here we report on the analysis of a novel mouse cDNA encoding the puromycin-sensitive aminopeptidase (PSA) and on its expression in COS cells and 3T3 fibroblasts. PSA is 27-40% homologous to several known Zn(2+)-binding aminopeptidases including aminopeptidase N. Immunohistochemical analysis revealed that PSA is localized to the cytoplasm and to the nucleus and associates with microtubules of the spindle apparatus during mitosis. Furthermore, puromycin and bestatin both arrested the cell cycle, leading to an accumulation of cells in G2/M phase, and ultimately induced cells to undergo apoptosis at concentrations that inhibit PSA. Control experiments including cycloheximide further suggested that the induction of apoptosis by puromycin was not attributable to inhibition of protein synthesis. Taken together, these data favor the novel idea that PSA participates in proteolytic events essential for cell growth and viability.

Secreted cytokines of the TGF beta family are found in all multicellular organisms and implicated in regulating fundamental cell behaviors such as proliferation, differentiation, migration and survival. Signal transduction involves complexes of specific type I and II receptor kinases that induce the nuclear translocation of Smad transcription factors to regulate target genes. Ligands of the BMP and Nodal subgroups act at a distance to specify distinct cell fates in a concentration-dependent manner. These signaling gradients are shaped by multiple factors, including proteases of the proprotein convertase (PC) family that hydrolyze one or several peptide bonds between an N-terminal prodomain and the C-terminal domain that forms the mature ligand. This review summarizes information on the proteolytic processing of TGF beta and related precursors, and its spatiotemporal regulation by PCs during development and various diseases, including cancer. Available evidence suggests that the unmasking of receptor binding epitopes of TGF beta is only one (and in some cases a non-essential) function of precursor processing. Future studies should consider the impact of proteolytic maturation on protein localization, trafficking and turnover in cells and in the extracellular space. (C) 2014 The Author. Published by Elsevier Ltd. All rights reserved.

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knockout (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by similar to 50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.

Targeted deletion of PC7 and the related proprotein convertases Furin and Pace4, combined with live imaging of their activities, unmasks their overlapping and complementary functions in morula compaction and ICM formation in mouse blastocysts and in E-cadherin precursor processing., The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell–cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell–cell adhesion and fate allocation.

A fundamental challenge in biology is to explain how progenitor cells in developing tissues acquire distinct fates according to their position. In vertebrates, the positional information that specifies the germ layers and the primary body axes is mediated by Nodal. Nodal meets the criteria of a morphogen since it can diffuse through tissues and signal far away from its source to directly specify multiple cell fates in a dosage-dependent manner. To consider the relationship between trafficking and graded Nodal signaling, this review summarizes recent findings how the spreading of Nodal activity is regulated by factors that promote long range signaling during left-right axis formation, and by structural features that affect Nodal protein stability and endosomal sorting.

Cripto is a glycosylphosphatidylinositol (GPI)-anchored co-receptor of Nodal and several other transforming growth factor-beta (TGF-beta) family ligands. It contains an epidermal growth factor (EGF)-like motif and a Cripto-FRL1-Cryptic (CFC) domain, which are conserved in a family of EGF-CFC proteins. The EGF domain is thought to recruit Nodal, whereas the CFC domain mediates binding to activin receptor-like kinase 4 (ALK4). We found that the EGF-like motif of Cripto was not essential for its binding to Nodal. However, through residues phenylalanine 78 and glycine 71, Cripto enriched Nodal at the limiting membrane of early endosomes. Similarly, residues in the CFC domain that mediate binding of Cripto to ALK4 were required to attenuate sequestration of Nodal in the endosomal lumen. Thus, we propose that Cripto stimulates Nodal activity by localizing it at the interface of endosomes with cytoplasmic effectors. To our knowledge, Cripto is the first GPI-anchored protein shown to control intraendosomal sorting of its associated cargo.

Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.

Background Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by numerous fluid-filled cysts that frequently result in end-stage renal disease. While promising treatment options are in advanced clinical development, early diagnosis and follow-up remain a major challenge. We therefore evaluated the diagnostic value of Fetuin-A as a new biomarker of ADPKD in human urine. Results We found that renal Fetuin-A levels are upregulated in both Pkd1 and Bicc1 mouse models of ADPKD. Measurement by ELISA revealed that urinary Fetuin-A levels were significantly higher in 66 ADPKD patients (17.5 ± 12.5 μg/mmol creatinine) compared to 17 healthy volunteers (8.5 ± 3.8 μg/mmol creatinine) or 50 control patients with renal diseases of other causes (6.2 ± 2.9 μg/mmol creatinine). Receiver operating characteristics (ROC) analysis of urinary Fetuin-A levels for ADPKD rendered an optimum cut-off value of 12.2 μg/mmol creatinine, corresponding to 94% of sensitivity and 60% of specificity (area under the curve 0.74 ; p = 0.0019). Furthermore, urinary Fetuin-A levels in ADPKD patients correlated with the degree of renal insufficiency and showed a significant increase in patients with preserved renal function followed for two years. Conclusions Our findings establish urinary Fetuin-A as a sensitive biomarker of the progression of ADPKD. Further studies are required to examine the pathogenic mechanisms of elevated renal and urinary Fetuin-A in ADPKD. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0463-7) contains supplementary material, which is available to authorized users.

Anteroposterior (AP) polarity in the mammalian embryo is specified during gastrulation when naive progenitor cells in the primitive ectoderm are recruited into the primitive streak to form mesoderm and endoderm. At the opposite pole, this process is inhibited by signals previously induced in distal visceral endoderm (DVE). Both DVE and primitive streak formation, and hence positioning of the AP axis, rely on the TGFβ family member Nodal and its proprotein convertases Furin and Pace4. Here, we show that Nodal and Furin are initially co-expressed in the primitive endoderm together with a subset of DVE markers such as Lefty1 and Hex. However, with the appearance of extra-embryonic ectoderm (ExE), DVE formation is transiently inhibited. During this stage, Nodal activity is essential to specify embryonic VE and restrict the expression of Furin to the extra-embryonic region. Activation of Nodal is also necessary to maintain determinants of pluripotency such as Oct4, Nanog and Foxd3 during implantation, and to stimulate elongation of the egg cylinder, before inducing DVE and germ layer formation. We conclude that Nodal is already activated in primitive endoderm, but induces a functional DVE only after promoting the expansion of embryonic VE and pluripotent progenitor cells in the epiblast.

The EGF-CFC gene cripto governs anterior–posterior (A–P) axis specification in the vertebrate embryo. Existing models suggest that Cripto facilitates binding of Nodal to an ActRII–activin-like kinase (ALK) 4 receptor complex. Cripto also has a crucial function in cellular transformation that is independent of Nodal and ALK4. However, how ALK4-independent Cripto pathways function in vivo has remained unclear. We have generated cripto mutants carrying the amino acid substitution F78A, which blocks the Nodal–ALK4–Smad2 signaling both in embryonic stem cells and cell-based assays. In criptoF78A/F78A mouse embryos, Nodal fails to expand its own expression domain and that of cripto, indicating that F78 is essential in vivo to stimulate Smad-dependent Nodal autoinduction. In sharp contrast to cripto-null mutants, criptoF78A/F78A embryos establish an A–P axis and initiate gastrulation movements. Our findings provide in vivo evidence that Cripto is required in the Nodal–Smad2 pathway to activate an autoinductive feedback loop, whereas it can promote A–P axis formation and initiate gastrulation movements independently of its stimulatory effect on the canonical Nodal–ALK4–Smad2 signaling pathway.