Tumor Induction and Tissue Atrophy in Mice Lacking E2F-1

AbstractThe retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor that regulates gene expression by physically associating with transcription factors such as E2F family members. Although pRB and its upstream regulators are commonly mutated in human cancer, the physiological role of the pRB–E2F pathway is unknown. To address the function of E2F-1 and pRB/E2F-1 complexes in vivo, we have produced mice homozygous for a nonfunctional E2F-1 allele. Mice lacking E2F-1 are viable and fertile, yet experience testicular atrophy and exocrine gland dysplasia. Surprisingly, mice lacking E2F-1 develop a broad and unusual spectrum of tumors. Although overexpression of E2F-1 in tissue culture cells can stimulate cell proliferation and be oncogenic, loss of E2F-1 in mice results in tumorigenesis, demonstrating that E2F-1 also functions as a tumor suppressor

Prestressed cells are prone to cytoskeleton failures under localized shear strain: an experimental demonstration on muscle precursor cells

International audienceWe report on a wavelet based space-scale decomposition method for analyzing the response of living muscle precursor cells (C2C12 myoblasts and myotubes) upon sharp indentation with an AFM cantilever and quantifying their aptitude to sustain such a local shear strain. Beyond global mechanical parameters which are currently used as markers of cell contractility, we emphasize the necessity of characterizing more closely the local fluctuations of the shear relaxation modulus as they carry important clues about the mechanisms of cytoskeleton strain release. Rupture events encountered during fixed velocity shear strain are interpreted as local disruptions of the actin cytoskeleton structures, the strongest (brittle) ones being produced by the tighter and stiffer stress fibers or actin agglomerates. These local strain induced failures are important characteristics of the resilience of these cells, and their aptitude to maintain their shape via a quick recovery from local strains. This study focuses on the perinuclear region because it can be considered as a master mechanical organizing center of these muscle precursor cells. Using this wavelet-based method, we combine the global and local approaches for a comparative analysis of the mechanical parameters of normal myoblasts, myotubes and myoblasts treated with actomyosin cytoskeleton disruptive agents (ATP depletion, blebbistatin)

Lack of muscle mTOR kinase activity causes early onset myopathy and compromises whole‐body homeostasis

Abstract Background The protein kinase mechanistic target of rapamycin (mTOR) controls cellular growth and metabolism. Although balanced mTOR signalling is required for proper muscle homeostasis, partial mTOR inhibition by rapamycin has beneficial effects on various muscle disorders and age‐related pathologies. Besides, more potent mTOR inhibitors targeting mTOR catalytic activity have been developed and are in clinical trials. However, the physiological impact of loss of mTOR catalytic activity in skeletal muscle is currently unknown. Methods We have generated the mTORmKOKI mouse model in which conditional loss of mTOR is concomitant with expression of kinase inactive mTOR in skeletal muscle. We performed a comparative phenotypic and biochemical analysis of mTORmKOKI mutant animals with muscle‐specific mTOR knockout (mTORmKO) littermates. Results In striking contrast with mTORmKO littermates, mTORmKOKI mice developed an early onset rapidly progressive myopathy causing juvenile lethality. More than 50% mTORmKOKI mice died before 8 weeks of age, and none survived more than 12 weeks, while mTORmKO mice died around 7 months of age. The growth rate of mTORmKOKI mice declined beyond 1 week of age, and the animals showed profound alterations in body composition at 4 weeks of age. At this age, their body weight was 64% that of mTORmKO mice (P < 0.001) due to significant reduction in lean and fat mass. The mass of isolated muscles from mTORmKOKI mice was remarkably decreased by 38–56% (P < 0.001) as compared with that from mTORmKO mice. Histopathological analysis further revealed exacerbated dystrophic features and metabolic alterations in both slow/oxidative and fast/glycolytic muscles from mTORmKOKI mice. We show that the severity of the mTORmKOKI as compared with the mild mTORmKO phenotype is due to more robust suppression of muscle mTORC1 signalling leading to stronger alterations in protein synthesis, oxidative metabolism, and autophagy. This was accompanied with stronger feedback activation of PKB/Akt and dramatic down‐regulation of glycogen phosphorylase expression (0.16‐fold in tibialis anterior muscle, P < 0.01), thus causing features of glycogen storage disease type V. Conclusions Our study demonstrates a critical role for muscle mTOR catalytic activity in the regulation of whole‐body growth and homeostasis. We suggest that skeletal muscle targeting with mTOR catalytic inhibitors may have detrimental effects. The mTORmKOKI mutant mouse provides an animal model for the pathophysiological understanding of muscle mTOR activity inhibition as well as for mechanistic investigation of the influence of skeletal muscle perturbations on whole‐body homeostasis

Interactome Mapping of the Phosphatidylinositol 3-Kinase-Mammalian Target of Rapamycin Pathway Identifies Deformed Epidermal Autoregulatory Factor-1 as a New Glycogen Synthase Kinase-3 Interactor

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression

LSD1 Controls Timely MyoD Expression via MyoD Core Enhancer Transcription

International audienceMyoD is a master regulator of myogenesis. Chromatin modifications required to trigger MyoD expression are still poorly described. Here, we demonstrate that the histone demethylase LSD1/KDM1a is recruited on the MyoD core enhancer upon muscle differentiation. Depletion of Lsd1 in myoblasts precludes the removal of H3K9 methylation and the recruitment of RNA polymerase II on the core enhancer, thereby preventing transcription of the non-coding enhancer RNA required for MyoD expression (CEeRNA). Consistently, Lsd1 conditional inactivation in muscle progenitor cells during embryogenesis prevented transcription of the CEeRNA and delayed MyoD expression. Our results demonstrate that LSD1 is required for the timely expression of MyoD in limb buds and identify a new biological function for LSD1 by showing that it can activate RNA polymerase II-dependent transcription of enhancers

Wnt4 expression during neuromuscular junction development.

<p>(<b>A</b>) Table showing results of Affymetrix microarrays data comparing relative Wnt4 mRNA expression during myotube differentiation, between stages T1/T2 and T2/T3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029976#s4" target="_blank">Materials and Methods</a>). Relative Wnt4 mRNA is upregulated more than three fold between stage T1/T2 and downregulated more than one fold between stages T2/T3. (<b>B and C</b>) Real time RT-PCR quantification of relative Wnt4 mRNA expression during myotube differentiation (B, stages T1, T2 and T3) and hind limb development (C, embryonic stages E13.5, E14, E16 and newborn mice P0, N = 6 embryos tested for each stage). Relative Wnt4 mRNA expression is significantly increased between stages T1/T2 and further downregulated between stages T2/T3 and decreases as the limb developed. (<b>D</b>) Real time RT-PCR quantification of relative MuSK and Wnt4 mRNA expression in synaptic and extrasynaptic regions of diaphragms from stage E18.5 embryos. Relative MuSK and Wnt4 expression are three and two fold increased in synaptic compared to extrasynaptic regions respectively. Results are represented as relative expression (2^{−Δ<i>C</i>t} versus reference gene ×100, N = 3). (<b>E and F</b>) In situ hybridization with probes for Wnt4 mRNAs in E11.5 and E13.5 spinal cord sections (thoracic level) of wild type mice embryos (N = 3 embryos tested for each condition). Wnt4 mRNA is expressed in the floor plate and dorsal spinal cord but not in motoneurons (MN). Error bars show means ± SEM from three independent experiments. *<i>P</i><0.05; **<i>P</i><0.001; Mann-Whitney <i>U</i> test. Scale bar: in E, 20 µm for E and F.</p