Study of Muscle Cell Dedifferentiation after Skeletal Muscle Injury of Mice with a Cre-Lox System

Background: Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied. Methodology/Principal Findings: In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells. Conclusions/Significance: These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo. © 2011 Mu et al

Expression profile analysis of the inflammatory response regulated by hepatocyte nuclear factor 4α

<p>Abstract</p> <p>Background</p> <p>Hepatocyte nuclear factor 4α (HNF4α), a liver-specific transcription factor, plays a significant role in liver-specific functions. However, its functions are poorly understood in the regulation of the inflammatory response. In order to obtain a genomic view of HNF4α in this context, microarray analysis was used to probe the expression profile of an inflammatory response induced by cytokine stimulation in a model of HNF4α knock-down in HepG2 cells.</p> <p>Results</p> <p>The expression of over five thousand genes in HepG2 cells is significantly changed with the dramatic reduction of HNF4α concentration compared to the cells with native levels of HNF4α. Over two thirds (71%) of genes that exhibit differential expression in response to cytokine treatment also reveal differential expression in response to HNF4α knock-down. In addition, we found that a number of HNF4α target genes may be indirectly mediated by an ETS-domain transcription factor ELK1, a nuclear target of mitogen-activated protein kinase (MAPK).</p> <p>Conclusion</p> <p>The results indicate that HNF4α has an extensive impact on the regulation of a large number of the liver-specific genes. HNF4α may play a role in regulating the cytokine-induced inflammatory response. This study presents a novel function for HNF4α, acting not only as a global player in many cellular processes, but also as one of the components of inflammatory response in the liver.</p

Nuclear Receptor HNF4α Binding Sequences are Widespread in Alu Repeats

<p>Abstract</p> <p>Background</p> <p>Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression.</p> <p>Results</p> <p>Here, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes (<it>ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR</it>) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation <it>in vivo </it>(<it>APOM, PRODH2, TTR, APOA4</it>). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites.</p> <p>Conclusions</p> <p>Our findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.</p

The Cycad Genotoxin MAM Modulates Brain Cellular Pathways Involved in Neurodegenerative Disease and Cancer in a DNA Damage-Linked Manner

Methylazoxymethanol (MAM), the genotoxic metabolite of the cycad azoxyglucoside cycasin, induces genetic alterations in bacteria, yeast, plants, insects and mammalian cells, but adult nerve cells are thought to be unaffected. We show that the brains of adult C57BL6 wild-type mice treated with a single systemic dose of MAM acetate display DNA damage (O6-methyldeoxyguanosine lesions, O6-mG) that remains constant up to 7 days post-treatment. By contrast, MAM-treated mice lacking a functional gene encoding the DNA repair enzyme O6-mG DNA methyltransferase (MGMT) showed elevated O6-mG DNA damage starting at 48 hours post-treatment. The DNA damage was linked to changes in the expression of genes in cell-signaling pathways associated with cancer, human neurodegenerative disease, and neurodevelopmental disorders. These data are consistent with the established developmental neurotoxic and carcinogenic properties of MAM in rodents. They also support the hypothesis that early-life exposure to MAM-glucoside (cycasin) has an etiological association with a declining, prototypical neurodegenerative disease seen in Guam, Japan, and New Guinea populations that formerly used the neurotoxic cycad plant for food or medicine, or both. These findings suggest environmental genotoxins, specifically MAM, target common pathways involved in neurodegeneration and cancer, the outcome depending on whether the cell can divide (cancer) or not (neurodegeneration). Exposure to MAM-related environmental genotoxins may have relevance to the etiology of related tauopathies, notably, Alzheimer's disease

Role of IL-1β in experimental cystic fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftrtm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1-/-), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1-/- double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1-/- and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients

A Kir6.2 mutation causing neonatal diabetes impairs electrical activity and insulin secretion from INS-1 beta-cells.

ATP-sensitive K(+) channels (K(ATP) channels) couple beta-cell metabolism to electrical activity and thereby play an essential role in the control of insulin secretion. Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of this channel, cause neonatal diabetes. We investigated the effect of the most common neonatal diabetes mutation (R201H) on beta-cell electrical activity and insulin secretion by stable transfection in the INS-1 cell line. Expression was regulated by placing the gene under the control of a tetracycline promoter. Transfection with wild-type Kir6.2 had no effect on the ATP sensitivity of the K(ATP) channel, whole-cell K(ATP) current magnitude, or insulin secretion. However, induction of Kir6.2-R201H expression strongly reduced K(ATP) channel ATP sensitivity (the half-maximal inhibitory concentration increased from approximately 20 mumol/l to approximately 2 mmol/l), and the metabolic substrate methyl succinate failed to close K(ATP) channels or stimulate electrical activity and insulin secretion. Thus, these results directly demonstrate that Kir6.2 mutations prevent electrical activity and insulin release from INS-1 cells by increasing the K(ATP) current and hyperpolarizing the beta-cell membrane. This is consistent with the ability of the R201H mutation to cause neonatal diabetes in patients. The relationship between K(ATP) current and the membrane potential reveals that very small changes in current amplitude are sufficient to prevent hormone secretion

Distances needed to limit cross-fertilization between GM and conventional maize in Europe

This letter shows that unnecessary large isolation distances may restrict farmers' freedom of choice to cultivate GM varieties given the particularities of European landscapes. For the first time we perform a statistical meta-analyses of over 1000 observations on maize gene flow in Europe. Taking into account the results obtained by several statistical approaches, we conclude that separation distances of 20-30 m are enough to keep out-crossing below legal thresholds (0.9 % in the EU). We conclude that mandatory distances established by some European countries (up to 800 m) may be disproportionate to the desired policy objective.JRC.J.4-Agriculture and Life Sciences in the Econom