p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

MYCN activation, mainly by gene amplification, is one of the most frequent molecular events in neuroblastoma (NB) oncogenesis, and is associated with increased malignancy and decreased neuronal differentiation propensity. The frequency of concomitant loss of heterozygosity at the 1p36.3 locus, which harbours the p53 anti-oncogene homologue TP73, indicates that MYCN and p73 alterations may cooperate in the pathogenesis of NB. We have previously shown that p73 isoforms are deregulated in NB tumours and that TAp73 co-operates synergistically with p53 for apoptosis of NB cells, whereas ΔNp73 activates the expression of neuronal differentiation genes such as BTG2. Herein, using both ectopic expression and RNA interference-mediated silencing of p73 in MYCN amplified NB cells, we show that p73α isoforms inhibit MYCN expression at both transcript and protein levels, in spite of transactivator effects on MYCN promoter. To explain this paradox, we found that TAp73α exerts negative post-transcriptional effects leading to reduced MYCN mRNA stability. RNA immunoprecipitation experiments suggest that this dominant inhibitory post-transcriptional effect could be due to an interaction between the p73 protein and MYCN mRNA, a hypothesis also raised for the regulation of certain genes by the p53 protein

Context: An Information Medium for Dynamic and Collaborative Situations

International audienceWe introduce, in this article, a way for building a Virtual Environment for Training to manage dynamic and collaborative situations in team sport. In these situations, relevant information can not be totally known in advance. Autonomous agents can only reason with few pieces of information and explanations on the situation are numerous and not easily predictable. We introduce, in this paper, the usefulness of the notion of context, in a VET, to set up agent decision-making and to generate explanations in order to help the learner. The main purpose of this article is that the context is a medium facilitating information reuse

H19 controls reactivation of the imprinted gene network during muscle regeneration

International audienceThe H19 locus controls fetal growth by regulating expression of several genes from the imprinted gene network (IGN). H19 is fully repressed after birth, except in skeletal muscle. Using loss-of-function H19(Delta 3) mice, we investigated the function of H19 in adult muscle. Mutant muscles display hypertrophy and hyperplasia, with increased Igf2 and decreased myostatin (Mstn) expression. Many imprinted genes are expressed in muscle stem cells or satellite cells. Unexpectedly, the number of satellite cells was reduced by 50% in H19(Delta 3) muscle fibers. This reduction occurred after postnatal day 21, suggesting a link with their entry into quiescence. We investigated the biological function of these mutant satellite cells in vivo using a regeneration assay induced by multiple injections of cardiotoxin. Surprisingly, despite their reduced number, the self-renewal capacity of these cells is fully retained in the absence of H19. In addition, we observed a better regeneration potential of the mutant muscles, with enhanced expression of several IGN genes and genes from the IGF pathway

Effects of WNT5A isoforms on the β-catenin/TCF pathway and Disheveled proteins phosphorylation.

<p>Both WNT5A proteins block Wnt3a-activation of β-catenin/TCF-driven transcriptional activity in HEK 293 (TOP-Luciferase, <b>A</b>.) and MDA-MB-231 (TOP-GFP reporter, <b>B</b>.). Cells were treated with purified Wnt3a and an increasing concentration of WNT5A for 24 hours (A) or 48 hours (B). <b>C</b>. Upon transfection, both WNT5A isoforms interfere with endogenous (MDA-MB-231, left panel) and WNT1-induced (HeLa, right panel) β-catenin/TCF-driven transcription in MDA-MB-231 (left panel) and HeLa cells (right panel). Cells were transfected with control, WNT5A-L or WNT5A-S expression vectors alone, or together with a WNT1 expression vector and assayed for β-catenin/TCF-driven TOP-Luciferase reporter activity. An expression vector encoding a dominant-negative form of TCF4 (ΔNTCF4) was used to interfere with the endogenous reporter activity in MDA-MB-231 cells. <b>D</b>. Both WNT5A isoforms promote DVL phosphorylation. L cells (left panel) and C2C12 cells (right panel) were treated with WNT5A isoforms (10 nM, 2 hours) and whole cell lysates were immuno-blotted with the indicated antibodies. Both WNT5A isoforms, as well as Wnt3a (10 nM, 2 hours), led to a mobility shift of Dvl1 and Dvl2 proteins, suggesting that Dvl proteins are post-translationally modified by phosphorylation in response to both canonical (Wnt3a) and non-canonical Wnt (WNT5A) signaling. Only Wnt3a induced accumulation of the β-catenin protein. Note: β-catenin accumulation in response to Wnt3a in C2C12 cells is not detectable in these whole cell lysates because these cells contain large amounts of membrane/adherens junction associated β-catenin.</p

<i>WNT5A</i> Encodes Two Isoforms with Distinct Functions in Cancers

<div><p>WNT5A, a member of the WNT family of secreted lipid-modified glycoproteins, is a critical regulator of a host of developmental processes, including limb formation, lung morphogenesis, intestinal elongation and mammary gland development. Altered WNT5A expression has been associated with a number of cancers. Interestingly, in certain types of cancers, such as hematological malignancies and colorectal carcinoma, WNT5A is inactivated and exerts a tumor suppressive function, while in other cancers, such as melanoma and gastric carcinoma, WNT5A is overexpressed and promotes tumor progression. The mechanism by which WNT5A achieves these distinct activities in cancers is poorly understood. Here, we provide evidence that the <i>WNT5A</i> gene produces two protein isoforms, WNT5A-long (WNT5A-L) and WNT5A-short (WNT5A-S). Amino-terminal sequencing and a WNT5A-L specific antibody demonstrate that the mature and secreted isoforms are distinct, with WNT5A-L carrying an additional 18 N-terminal amino acids. Biochemical analysis indicates that both purified proteins are similar with respect to their stability, hydrophobicity and WNT/β-catenin signaling activity. Nonetheless, modulation of these two <i>WNT5A</i> isoforms, either through ectopic expression or knockdown, demonstrates that they exert distinct activities in cancer cell lines: while WNT5A-L inhibits proliferation of tumor cell lines, WNT5A-S promotes their growth. Finally, we show that expression of these two WNT5A isoforms is altered in breast and cervix carcinomas, as well as in the most aggressive neuroblastoma tumors. In these cancers, WNT5A-L is frequently down-regulated, whereas WNT5A-S is found overexpressed in a significant fraction of tumors. Altogether, our study provides evidence that the distinct activities of WNT5A in cancer can be attributed to the production of two <i>WNT5A</i> isoforms. </p> </div

Effects of WNT5A isoform expression on cancer cell line proliferation.

<p><b>A</b>. WNT5A isoform expression in three cancer cell lines. Transcript levels of <i>WNT5A</i> isoforms were determined in MDA-MB-231 (breast carcinoma), HeLa (cervix carcinoma), and SH-SY5Y (neuroblastoma) by qRT-PCR and normalized by <i>EF1-α</i> mRNA (normalization by <i>GAPDH</i> mRNA and <i>18S</i> rRNA produced similar results). Mean ratios (<i>WNT5A</i> isoforms/ <i>EF1-α</i>) ± SEM from independent measurements are shown. <b>B</b>. Effects of ectopic expression of WNT5A-L and WNT5A-S isoforms on proliferation. The indicated cell lines were transduced with expression vectors encoding WNT5A-L (blue line), WNT5A-S (red line) or no WNT (Black line) and cell numbers were determined at 3 and 6 days. WNT5A-S increases while WNT5A-L decreases proliferative rates. Data (mean ± SEM from triplicate determinations) from a representative experiment are shown. Each experiment was performed at least three independent times. <b>C</b>. Isoform specific siRNAs reduce expression of each WNT5A isoform. The efficiency of WNT5A-L and WNT5A-S isoform knockdown (KD) was evaluated in MDA-MB-231, HeLa and SH-SY5Y transfected with control siRNA and <i>WNT5A</i> isoform-specific siRNA (KD). <i>WNT5A</i> isoforms transcript levels were measured by quantitative RT-PCR (qRT-PCR) and normalized by <i>EF1-α</i> mRNA (normalization by <i>GAPDH</i> mRNA and <i>18S</i> rRNA produced similar results). <b>D</b>. Effects of siRNA-mediated knockdown (KD) of WNT5A-L and WNT5A-S isoforms on proliferation. Cells were transfected with siRNAs specific to WNT5A-L (blue line) or WNT5A-S (red line) and cell numbers were determined at 3 and 6 days. A scrambled siRNA served as control (black line). Co-transduction of cells with a WNT5A-S expression vector rescues the effect of the WNT5A-S-specific siRNA (dashed red line). Data (mean ± SEM from triplicate determinations) from a representative experiment are shown. Each experiment was performed at least three independent times. * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.005; **** <i>P</i> < 0.001. <b>E</b>. Differential regulation of <i>AXIN2</i> and <i>CDK8</i> expression by isoform-specific knockdown of WNT5A. Transcript levels of <i>AXIN2</i> and <i>CDK8</i> were determined by quantitative RT-PCR analysis (normalized by <i>EF1-α</i> mRNA) in control MDA-MB-231 (breast carcinoma), HeLa (cervix carcinoma) and SH-SY5Y (neuroblastoma) cells, and in response to siRNA-mediated knock-down (KD) of WNT5A-L or WNT5A-S. Mean ratios ± SEM from independent measurements are shown.</p

Selenoprotein T Deficiency Leads to Neurodevelopmental Abnormalities and Hyperactive Behavior in Mice

International audienc

Mitochondrial Protein UCP2 Controls Pancreas Development

International audienc