Skeletal muscle is formed via fusion of myoblasts, a well-studied process in Drosophila. In vertebrates however, this process is less well understood, and whether there is evolutionary conservation with the proteins studied in flies is under investigation. Sticks and stones (Sns), a cell surface protein found on Drosophila myoblasts, has structural homology to nephrin. Nephrin is a protein expressed in kidney that is part of the filtration barrier formed by podocytes. No previous study has established any role for nephrin in skeletal muscle. We show, using two models, zebrafish and mice, that the absence of nephrin results in poorly developed muscles and incompletely fused myotubes, respectively. Although nephrinknockout (nephrin KO ) myoblasts exhibit prolonged activation of MAPK/ERK pathway during myogenic differentiation, expression of myogenin does not seem to be altered. Nevertheless, MAPK pathway blockade does not rescue myoblast fusion. Co-cultures of unaffected human fetal myoblasts with nephrin KO myoblasts or myotubes restore the formation of mature myotubes; however, the contribution of nephrin KO myoblasts is minimal. These studies suggest that nephrin plays a role in secondary fusion of myoblasts into nascent myotubes, thus establishing a possible functional conservation with Drosophila Sns. myoblast fusion ͉ sticks and stones
Glycogen synthase kinase-3 (Gsk-3) activity regulates multiple signal transduction pathways, and is also a key component of the network responsible for maintaining stem cell pluripotency. Genetic deletion of Gsk-3α and Gsk-3β or inhibition of Gsk-3 activity via small molecules promotes stem cell pluripotency, yet the mechanism underlying the role for Gsk-3 in this process remains ambiguous. Another cellular process that has been shown to affect stem cell pluripotency is mRNA methylation (m 6 A). Here, we describe an intersection between these components -the regulation of m 6 A by Gsk-3. We find that protein levels for the RNA demethylase, FTO (fat mass and obesity-associated protein), are elevated in Gsk-3α;Gsk-3β-deficient mouse embryonic stem cells (ESCs). FTO is normally phosphorylated by Gsk-3, and mass spectrometry identified the sites on FTO that are phosphorylated in a Gsk-3-dependent fashion. Gsk-3 phosphorylation of FTO leads to polyubiquitination, but in Gsk-3 knockout ESCs, that process is impaired, resulting in elevated levels of FTO protein. As a consequence of altered FTO protein levels, mRNAs in Gsk-3 knockout ESCs have 50% less m 6 A than wild-type ESCs, and m 6 A-seq analysis reveals the specific mRNAs that have reduced m 6 A modifications. Taken together, we provide the first evidence for how m 6 A demethylation is regulated in mammalian cells, and sheds light onto a possible novel mechanism by which Gsk-3 activity regulates stem cell pluripotency.Glycogen synthase kinase-3 (Gsk-3) activity is an important regulator of numerous signal transduction pathways (1). Gsk-3 activity is the sum of two largely redundant proteins, Gsk-3α and Gsk-3β, and in general, Gsk-3 is a negative regulator of cellular signaling (2). Rare among kinases, Gsk-3 is active at a basal state, while pathway activation from upstream signaling cascades results in the inhibition of Gsk-3 activity (2). Gsk-3α and Gsk-3β together regulate signal transduction pathways such as Wnt, protein kinase A (PKA), Hedgehog, transforming growth factor-β (TGF-β), nuclear factor of activated T-cells (NF-AT) and phosphatidylinositol 3-kinase (PI3K)-dependent insulin signaling in a variety of biological settings (3)(4)(5).Gsk-3 activity can be inhibited through the use of small molecule inhibitors, such as SB- (6)(7)(8), and the clinically relevant mood-stabilizer lithium (9,10); however, a drawback to the use of small molecules to study Gsk-3 function is the potential for off-target effects (11). Cells in which Gsk-3α and Gsk-3β have been genetically deleted allows for a more confident assessment of Gsk-3-specific functions. Therefore, we utilize mouse embryonic stem cells (ESCs) deficient in both Gsk-3α and Gsk-3β (Gsk-3α -/-; Gsk-3β -/-), i.e., Gsk-3 double knockout (DKO), to assess Gsk-3-specific functions (12,13). One prominent phenotype of Gsk-3 DKO ESCs is their persistent pluripotency, assessed by their inability to differentiate into all three germs layers in a teratoma assay, as well as by analysis of global gene expression ...
Introduction-We report a boy who received two allogeneic stem cell transplantations from umbilical cord donors to treat chronic granulomatous disease (CGD). The CGD was cured after the second transplantation, but 2½ years later, he was diagnosed with Duchenne muscular dystrophy (DMD).
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