Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain (NICD) that functions directly in signal transduction. The Notch receptor is locked down in a proteaseresistant state by a negative regulatory region (NRR) that protects an ADAM (a disintegrin and metalloprotease) cleavage site. Engagement with ligand-bearing cells induces global conformational movements in Notch that unfold the NRR structure to expose the ADAM cleavage site and initiate proteolytic activation. Although both ADAM10 and ADAM17 have been reported to cleave Notch to facilitate NICD release by ␥-secretase, the relevant ADAM has remained controversial. Our study provides new insight into this conflict, as we find that although Notch1 (N1) is a substrate for both ADAM10 and ADAM17, the particular ADAM required for receptor activation is context dependent. Specifically, ADAM10 was absolutely required for N1 signaling induced by ligands, while signaling independent of ligands required ADAM17. In contrast to the strict and differential use of ADAM10 and ADAM17 in normal and dysregulated signaling, respectively, both proteases participated in signaling intrinsic to N1 mutations associated with leukemia. We propose that in addition to exposing the ADAM cleavage site, activating N1 conformational changes facilitate selective cleavage by specific proteases.
Background Supravalvular aortic stenosis (SVAS) is caused by mutations in the elastin (ELN) gene and is characterized by abnormal proliferation of vascular smooth muscle cells (SMCs) that can lead to narrowing or blockage of the ascending aorta and other arterial vessels. Availability of patient-specific SMCs may facilitate studying disease mechanisms and developing novel therapeutic interventions. Methods and Results Here, we report the development of a human induced pluripotent stem cell (iPSC) line from a patient with SVAS caused by the premature termination in exon 10 of the ELN gene due to an exon 9 4-nucleotide insertion. We showed that SVAS iPSC-derived SMCs (iPSC-SMCs) had significantly fewer organized networks of smooth muscle alpha actin (SM α-actin) filament bundles, a hallmark of mature contractile SMCs, compared to control iPSC-SMCs. Addition of elastin recombinant protein or enhancement of small GTPase RhoA signaling was able to rescue the formation of SM α-actin filament bundles in SVAS iPSC-SMCs. Cell counts and BrdU analysis revealed a significantly higher proliferation rate in SVAS iPSC-SMCs than control iPSC-SMCs. Furthermore, SVAS iPSC-SMCs migrated at a markedly higher rate to the chemotactic agent platelet-derived growth factor (PDGF) in comparison with the control iPSC-SMCs. We also provided evidence that elevated activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is required for hyper-proliferation of SVAS iPSC-SMCs. The phenotype was confirmed in iPSC-SMCs generated from a patient with deletion of elastin due to Williams-Beuren syndrome (WBS). Conclusions Thus, SVAS iPSC-SMCs recapitulate key pathological features of patients with SVAS and may provide a promising strategy to study disease mechanisms and to develop novel therapies.
Rationale The emergence of lymphatic endothelial cells (LECs) seems to be highly regulated during development. Although several factors that promote the differentiation of LECs in embryonic development have been identified, those that negatively regulate this process are largely unknown. Objective Our aim was to delineate the role of bone morphogenetic protein (BMP) 2 signaling in lymphatic development. Methods and Results BMP2 signaling negatively regulates the formation of LECs. Developing LECs lack any detectable BMP signaling activity in both zebrafish and mouse embryos, and excess BMP2 signaling in zebrafish embryos and mouse embryonic stem cell–derived embryoid bodies substantially decrease the emergence of LECs. Mechanistically, BMP2 signaling induces expression of miR-31 and miR-181a in a SMAD-dependent mechanism, which in turn results in attenuated expression of prospero homeobox protein 1 during development. Conclusions Our data identify BMP2 as a key negative regulator for the emergence of the lymphatic lineage during vertebrate development.
The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5 half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTBmediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.A common mechanism of the regulation of gene expression in metazoans is the alternative splicing of pre-mRNA (6,11,22,23,34). Through alternative splicing, multiple mRNAs are generated from the same primary RNA transcript. Changes in splice site choice are regulated by proteins that bind to the pre-mRNA and affect spliceosome assembly. One well-studied family of splicing regulatory factors is the SR proteins that function in both constitutive and alternative splicing and can act at various stages of spliceosome assembly (3,10,24,48). These proteins are perhaps best known for binding exonic splicing enhancer (ESE) elements and thus stimulating spliceosome assembly at the adjacent splice sites. Some exons bind multiple SR proteins, each of which can activate splicing. Other exons are dependent on a single SR protein. Two of the best-characterized SR protein family members that bind ESEs and activate splicing are SF2/ASF and SC35.Another large group of proteins that bind to nascent premRNAs are the heterogeneous nuclear ribonucleoproteins (hnRNPs) (19,30). At least in vitro, spliceosome assembly occurs after hnRNP binding and some hnRNPs are implicated in splicing regulation. Two of these, hnRNP A1 and polypyrimidine tract binding protein (PTB or hnRNP I) bind to exoni...
Heart disease is one of the leading causes of death worldwide and the number of patients with the disease is likely to grow with the continual decline in health for most of the developed world. Heart transplantation is one of the only treatment options for heart failure due to an acute myocardial infarction, but limited donor supply and organ rejection limit its widespread use. Cellular cardiomyoplasty, or cellular implantation, combined with various tissue-engineering methods aims to regenerate functional heart tissue. This review highlights the numerous cell sources that have been used to regenerate the heart as well as cover the wide range of tissue-engineering strategies that have been devised to optimize the delivery of these cells. It will probably be a long time before an effective regenerative therapy can make a serious impact at the bedside.
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