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Congenital disorders of glycosylation (CDGs) constitute a rapidly growing family of human diseases resulting from heritable mutations in genes driving the production and modification of glycoproteins. The resulting symptomatic hypoglycosylation causes multisystemic defects that include severe neurological impairments, revealing a particularly critical requirement for tightly regulated glycosylation in the nervous system. The most common CDG, CDG-Ia (PMM2-CDG), arises from phosphomannomutase type 2 (PMM2) mutations. Here, we report the generation and characterization of the first Drosophila CDG-Ia model. CRISPR-generated pmm2-null Drosophila mutants display severely disrupted glycosylation and early lethality, whereas RNAi-targeted knockdown of neuronal PMM2 results in a strong shift in the abundance of pauci-mannose glycan, progressive incoordination and later lethality, closely paralleling human CDG-Ia symptoms of shortened lifespan, movement impairments and defective neural development. Analyses of the well-characterized Drosophila neuromuscular junction (NMJ) reveal synaptic glycosylation loss accompanied by defects in both structural architecture and functional neurotransmission. NMJ synaptogenesis is driven by intercellular signals that traverse an extracellular synaptomatrix and are co-regulated by glycosylation and matrix metalloproteinases (MMPs). Specifically, trans-synaptic signaling by the Wnt protein Wingless (Wg) depends on the heparan sulfate proteoglycan (HSPG) co-receptor Dally-like protein (Dlp), which is regulated by synaptic MMP activity. Loss of synaptic MMP2, Wg ligand, Dlp co-receptor and downstream trans-synaptic signaling occurs with PMM2 knockdown. Taken together, this Drosophila CDG disease model provides a new avenue for the dissection of cellular and molecular mechanisms underlying neurological impairments and is a means by which to discover and test novel therapeutic treatment strategies.
© 2016. Published by The Company of Biologists Ltd.
Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.
© 2016 AlphaMed Press.
Transcription factor expression fluctuates during β-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature β-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with β-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature β-cells has no functional consequences; in contrast, Prox1 overexpression in immature β-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal β-cells. Also, we uncovered substantial deficiency in β-cells that overexpress Prox1 of the key regulator of β-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-βH1 β-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal β-cells.
© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
BACKGROUND - Gastric cancer (GC) is a deadly malignancy worldwide. In the past, it has been shown that cellular signaling pathway alterations play a crucial role in the development of GC. In particular, deregulation of the PI3K/AKT/mTOR pathway seems to affect multiple GC functions including growth, proliferation, metabolism, motility and angiogenesis. Targeting alterations in this pathway by microRNAs (miRNAs) represents a potential therapeutic strategy, especially in inhibitor-resistant tumors. The objective of this study was to evaluate the expression of 3 pre-selected miRNAs, miR-101-2, miR-125b-2 and miR-451a, in a series of primary GC tissues and matched non-GC tissues and in several GC-derived cell lines, and to subsequently evaluate the functional role of these miRNAs.
METHODS - Twenty-five primary GC samples, 25 matched non-GC samples and 3 GC-derived cell lines, i.e., AGS, MKN28 and MKN45, were included in this study. miRNA and target gene expression levels were assessed by quantitative RT-PCR and western blotting, respectively. Subsequently, cell viability, clone formation, cell death, migration and invasion assays were performed on AGS cells.
RESULTS - miR-101-2, miR-125b-2 and miR-451a were found to be down-regulated in the primary GC tissues and the GC-derived cell lines tested. MiRNA mimic transfections significantly reduced cell viability and colony formation, increased cell death and reduced cell migration and invasion in AGS cells. We also found that exogenous expression of miR-101-2, miR-125b-2 and miR-451a decreased the expression of their putative targets MTOR, PIK3CB and TSC1, respectively.
CONCLUSIONS - Our expression analyses and in vitro functional assays suggest that miR-101-2, miR-125b-2 and miR-451a act as potential tumor suppressors in primary GCs as well as in GC-derived AGS cells.
The growing need for therapies to treat large cutaneous defects has driven recent interest in the design of scaffolds that stimulate regenerative wound healing. While many studies have investigated local delivery of biologics as a restorative approach, an increasing body of evidence highlights the contribution of the mechanical properties of implanted scaffolds to wound healing. In the present study, we designed poly(ester urethane) scaffolds using a templated-Fused Deposition Modeling (t-FDM) process to test the hypothesis that scaffolds with substrate modulus comparable to that of collagen fibers enhance a regenerative versus a fibrotic response. We fabricated t-FDM scaffolds with substrate moduli varying from 5 to 266 MPa to investigate the effects of substrate modulus on healing in a rat subcutaneous implant model. Angiogenesis, cellular infiltration, collagen deposition, and directional variance of collagen fibers were maximized for wounds treated with scaffolds having a substrate modulus (Ks = 24 MPa) comparable to that of collagen fibers. The enhanced regenerative response in these scaffolds was correlated with down-regulation of Wnt/β-catenin signaling in fibroblasts, as well as increased polarization of macrophages toward the restorative M2 phenotype. These observations highlight the substrate modulus of the scaffold as a key parameter regulating the regenerative versus scarring phenotype in wound healing. Our findings further point to the potential use of scaffolds with substrate moduli tuned to that of the native matrix as a therapeutic approach to improve cutaneous healing.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Using in vitro and in vivo models, we investigated the role of TFF1 in suppressing H. pylori-mediated activation of oncogenic β-catenin in gastric tumorigenesis. A reconstitution of TFF1 expression in gastric cancer cells decreased H. pylori-induced β-catenin nuclear translocation, as compared to control (p < 0.001). These cells exhibited significantly lower β-catenin transcriptional activity, measured by pTopFlash reporter, and induction of its target genes (CCND1 and c-MYC), as compared to control. Because of the role of AKT in regulating β-catenin, we performed Western blot analysis and demonstrated that TFF1 reconstitution abrogates H. pylori-induced p-AKT (Ser473), p-β-catenin (Ser552), c-MYC, and CCND1 protein levels. For in vivo validation, we utilized the Tff1-KO gastric neoplasm mouse model. Following infection with PMSS1 H. pylori strain, we detected an increase in the nuclear staining for β-catenin and Ki-67 with a significant induction in the levels of Ccnd1 and c-Myc in the stomach of the Tff1-KO, as compared to Tff1-WT mice (p < 0.05). Only 10% of uninfected Tff1-KO mice, as opposed to one-third of H. pylori-infected Tff1-KO mice, developed invasive adenocarcinoma (p = 0.03). These findings suggest that loss of TFF1 could be a critical step in promoting the H. pylori-mediated oncogenic activation of β-catenin and gastric tumorigenesis.
Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.
© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
BACKGROUND - The recurrent BRAF driver mutation V600E (BRAF (V600E)) is currently one of the most clinically relevant mutations in melanoma. However, the genome-wide transcriptional and epigenetic dysregulations induced by BRAF (V600E) are still unclear. The investigation of this driver mutation's functional consequences is critical to the understanding of tumorigenesis and the development of therapeutic strategies.
METHODS AND RESULTS - We performed an integrative analysis of transcriptomic and epigenomic changes disturbed by BRAF (V600E) by comparing the gene expression and methylation profiles of 34 primary cutaneous melanoma tumors harboring BRAF (V600E) with those of 27 BRAF (WT) samples available from The Cancer Genome Atlas (TCGA). A total of 711 significantly differentially expressed genes were identified as putative BRAF (V600E) target genes. Functional enrichment analyses revealed the transcription factor MITF (p < 3.6 × 10(-16)) and growth factor TGFB1 (p < 3.1 × 10(-9)) were the most significantly enriched up-regulators, with MITF being significantly up-regulated, whereas TGFB1 was significantly down-regulated in BRAF (V600E), suggesting that they may mediate tumorigenesis driven by BRAF (V600E). Further investigation using the MITF ChIP-Seq data confirmed that BRAF (V600E) led to an overall increased level of gene expression for the MITF targets. Furthermore, DNA methylation analysis revealed a global DNA methylation loss in BRAF (V600E) relative to BRAF (WT). This might be due to BRAF dysregulation of DNMT3A, which was identified as a potential target with significant down-regulation in BRAF (V600E). Finally, we demonstrated that BRAF (V600E) targets may play essential functional roles in cell growth and proliferation, measured by their effects on melanoma tumor growth using a short hairpin RNA silencing experimental dataset.
CONCLUSIONS - Our integrative analysis identified a set of BRAF (V600E) target genes. Further analyses suggested a complex mechanism driven by mutation BRAF (V600E) on melanoma tumorigenesis that disturbs specific cancer-related genes, pathways, and methylation modifications.
Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology.
When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA-mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.
© 2015 Aditi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).