SummaryIn Drosophila, normal and transformed cells compete with each other for survival in a process called cell competition. However, it is not known whether comparable phenomena also occur in mammals. Scribble is a tumor suppressor protein in Drosophila and mammals. In this study we examine the interface between normal and Scribble-knockdown epithelial cells using Madin-Darby Canine Kidney (MDCK) cells expressing Scribble short hairpin RNA (shRNA) in a tetracycline-inducible manner. We observe that Scribbleknockdown cells undergo apoptosis and are apically extruded from the epithelium when surrounded by normal cells. Apoptosis does not occur when Scribble-knockdown cells are cultured alone, suggesting that the presence of surrounding normal cells induces the cell death. We also show that death of Scribble-knockdown cells occurs independently of apical extrusion. Finally, we demonstrate that apoptosis of Scribble-knockdown cells depends on activation of p38 mitogen-activated protein kinase (MAPK). This is the first demonstration that an oncogenic transformation within an epithelium induces cell competition in a mammalian cell culture system.
Expression of reelin, reelin receptors [apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (VldlR)] and the Disabled-1 (Dab1) protein was investigated in rat intestinal mucosa. Intestinal reelin and Dab1 mRNA levels were maximal in the early stages of life, reaching adult levels in 1-month-old rats. Expression of reelin mRNA was restricted to fibroblasts, whereas mRNAs of Dab1, ApoER2, VldlR and integrins α3 and β1 were observed in enterocytes, crypts and fibroblasts. Reelin protein was only observed in isolated intestinal fibroblasts and in a cell layer subjacent to the villus epithelium, which seems to be composed of myofibroblasts because it also reacted to α-smooth muscle actin. The Disabled-1 and VldlR protein signals were detected in the crypt and villus cells, and they were particularly abundant in the terminal web domain of the enterocytes. The ApoER2 protein signal was detected in the upper half of the villi but not in the crypts. This is the first report showing that rat intestinal mucosa expresses the reelin-Disabled-1 signalling system.
Intestinal myofibroblasts secrete substances that control organogenesis and wound repair of the intestine. The myofibroblasts of the rat small intestine express reelin and the present work explores whether reelin regulates crypt-villus unit homeostasis using normal mice and mice with the reelin gene disrupted (reeler). The results reveal that mouse small intestine expresses reelin, its receptors apolipoprotein E receptor 2 (ApoER2) and very low-density lipoprotein receptor (VldlR) and the reelin effector protein Disabled-1 (Dab1) and that reelin expression is restricted to myofibroblasts. The absence of reelin significantly reduces epithelial cell proliferation, migration, and apoptosis and the number of Paneth cells. These effects are observed during the suckling, weaning, and adult periods. The number of Goblet cells is increased in the 2-month-old reeler mice. The absence of reelin also expands the extracellular space of the adherens junctions and desmosomes without significantly affecting either the tight-junction structure or the epithelial paracellular permeability. In conclusion, this is the first in vivo work showing that the absence of reelin alters intestinal epithelium homeostasis.
The HIV-1 ribosomal frameshift element is highly structured, regulates translation of all virally encoded enzymes, and is a promising therapeutic target. The prior model for this motif contains two helices separated by a three-nucleotide bulge. Modifications to this model were suggested by SHAPE chemical probing of an entire HIV-1 RNA genome. Novel features of the SHAPE-directed model include alternate helical conformations and a larger, more complex structure. These structural elements also support the presence of a secondary frameshift site within the frameshift domain. Here, we use oligonucleotide-directed structure perturbation, probing in the presence of formamide, and in-virion experiments to examine these models. Our data support a model in which the frameshift domain is anchored by a stable helix outside the conventional domain. Less stable helices within the domain can switch from the SHAPE-predicted to the two-helix conformation. Translational frameshifting assays with frameshift domain mutants support a functional role for the interactions predicted by and specific to the SHAPE-directed model. These results reveal that the HIV-1 frameshift domain is a complex, dynamic structure and underscore the importance of analyzing folding in the context of full-length RNAs.
Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus-or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gagpolymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication. IMPORTANCEHIV, like many retroviruses, utilizes a ؊1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies. The genome of human immunodeficiency virus type 1 (HIV-1), like that of other retroviruses, has three genes that encode the structural proteins Gag, polymerase (Pol), and Env. The expression of the gag gene results in the synthesis of the Gag precursor protein, p55, which is subsequently processed by the viral protease to release the mature Gag proteins p17 (matrix protein), p24 (capsid), p15 (nucleocapsid), and p6 (late domain) and two so-called spacer peptides (SP) that flank p15, namely, p2 (SP1) and p1 (SP2), respectively (1). The synthesis of Gag precursor protein alone is sufficient for the assembly and release of noninfectious virus-like particles (VLPs) (2). The pol gene codes for the p160 polyprotein, which is su...
Human Immunodeficiency Virus (HIV) type 1 uses a −1 programmed ribosomal frameshift (−1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5–10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides.
Reelin is an extracellular matrix protein that plays a critical role in neuronal migration. Here we show that the mucosa of human colon expresses reelin, its receptors ApoER2 and VLDLR, and its effector protein Dab1. Immunohistochemical analyses reveal that reelin expression is restricted to pericryptal myofibroblasts; Dab1 is detected at myofibroblasts, the apical domain of surface epithelial and crypt cells, and a strong linear staining is observed at the basement membrane; VLDLR and ApoER2 are in the cytoplasm of surface epithelium and myofibroblasts, and VLDLR is also detected in the cytoplasm of the crypt cells. Human colorectal cancer downregulates reelin without change in vimentin or N-cadherin mRNA levels. Decreased Reelin mRNA expression is accompanied by decreased HIC1 mRNA levels, increased mRNA levels of ApoER2 and DNMT1, increased reelin hypermethylation and no change in either Cask or TGF-β1 mRNAs, suggesting that reelin repression results from a DNMT1-mediated hypermethylation of the reelin gene promoter. Decreased HIC1 expression may repress reelin transcription via increasing ApoER2 transcription. We conclude that the mucosa of human colon expresses the reelin-Dab1 signaling system and that reelin is repressed in colorectal cancer before epithelial-mesenchymal transition has occurred. The significant down-regulation of reelin expression makes this gene a promising biomarker for colorectal cancers. © 2016 Wiley Periodicals, Inc.
Reelin is an extracellular matrix protein first known for its key role in neuronal migration. Studies in rodent small intestine suggested that reelin protects the organism from intestinal pathology. Here we determined in mice colon, by real time-PCR and immunological assays, the expression of the reelin signalling system; its response to dextran sulphate sodium (DSS) and the response of wild-type and reeler mice to DSS-treatment. DNA methylation was determined by bisulfite modification and sequencing of genomic DNA. In the colon mucosa reelin expression is restricted to the myofibroblasts, whereas both epithelial cells and myofibroblasts express reelin receptors (ApoER2 and VLDLR) and its effector protein Dab1. The muscle layer also expresses reelin. DSS-treatment reduces reelin expression in the muscle but it is activated in the mucosa. Activation of mucosal reelin is greater in magnitude and is delayed until after the activation of the myofibroblasts marker, α-SMA. This indicates that the DSS-induced reelin up-regulation results from changes in the reelin gene expression rather than from myofibroblasts proliferation. DSS-treatment does not modify Sp1 or Tbr1 mRNA abundance, but increases that of TGF-β1 and ApoER2, decreases that of CASK and DNMT1 and it also decreases the reelin promoter methylation. Finally, the reeler mice exhibit higher inflammatory scores than wild-type mice, indicating that the mutation increases the susceptibility to DSS-colitis. In summary, this data are the first to demonstrate that mouse distal colon increases reelin production in response to DSS-colitis via a DNMT1-dependent hypo-methylation of the gene promoter region and that reelin provides protection against colitis.
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