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1.
Genes Dev ; 27(13): 1473-83, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-23796896

ABSTRACT

Regulated gene expression determines the intrinsic ability of neurons to extend axons, and loss of such ability is the major reason for the failed axon regeneration in the mature mammalian CNS. MicroRNAs and histone modifications are key epigenetic regulators of gene expression, but their roles in mammalian axon regeneration are not well explored. Here we report microRNA-138 (miR-138) as a novel suppressor of axon regeneration and show that SIRT1, the NAD-dependent histone deacetylase, is the functional target of miR-138. Importantly, we provide the first evidence that miR-138 and SIRT1 regulate mammalian axon regeneration in vivo. Moreover, we found that SIRT1 also acts as a transcriptional repressor to suppress the expression of miR-138 in adult sensory neurons in response to peripheral nerve injury. Therefore, miR-138 and SIRT1 form a mutual negative feedback regulatory loop, which provides a novel mechanism for controlling intrinsic axon regeneration ability.


Subject(s)
Axons/physiology , Feedback, Physiological , Gene Expression Regulation, Developmental , MicroRNAs/metabolism , Regeneration/genetics , Sirtuin 1/metabolism , Animals , Cells, Cultured , Mice , MicroRNAs/genetics , Sensory Receptor Cells/physiology , Signal Transduction , Sirtuin 1/genetics
2.
Biochem Biophys Res Commun ; 443(2): 743-8, 2014 Jan 10.
Article in English | MEDLINE | ID: mdl-24333443

ABSTRACT

Inactivation of glycogen synthase kinase 3 (GSK3) has been shown to mediate axon growth during development and regeneration. Phosphorylation of GSK3 by the kinase Akt is well known to be the major mechanism by which GSK3 is inactivated. However, whether such regulatory mechanism of GSK3 inactivation is used in neurons to control axon growth has not been directly studied. Here by using GSK3 mutant mice, in which GSK3 is insensitive to Akt-mediated inactivation, we show that sensory axons regenerate normally in vitro and in vivo after peripheral axotomy. We also find that GSK3 in sensory neurons of the mutant mice is still inactivated in response to peripheral axotomy and such inactivation is required for sensory axon regeneration. Lastly, we provide evidence that GSK3 activity is negatively regulated by PI3K signaling in the mutant mice upon peripheral axotomy, and the PI3K-GSK3 pathway is functionally required for sensory axon regeneration. Together, these results indicate that in response to peripheral nerve injury GSK3 inactivation, regulated by an alternative mechanism independent of Akt-mediated phosphorylation, controls sensory axon regeneration.


Subject(s)
Axons/metabolism , Glycogen Synthase Kinase 3/metabolism , Nerve Regeneration , Peripheral Nerve Injuries/metabolism , Peripheral Nerve Injuries/pathology , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Axons/ultrastructure , Enzyme Activation , Gene Expression Regulation , Mice , Mice, Knockout , Signal Transduction
3.
J Invest Dermatol ; 125(4): 805-17, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16185282

ABSTRACT

Transcription factor genes governing pigment cell development that are associated with spotting mutations in mice include members of several structural transcription factor classes but not members of the basic helix-loop-helix (bHLH) class, important for neurogenesis and myogenesis. To determine the effects of bHLH factor expression on pigment cell development, the neurogenic bHLH factor Mash1 was expressed early in pigment cell development in transgenic mice from the dopachrome tautomerase (Dct) promoter. Dct:Mash1 transgenic founders exhibit variable microphthalmia and patchy coat color hypopigmentation. Transgenic F1 mice exhibit microphthalmia with complete coat color dilution. Marker analysis demonstrates that Mash1 expression in the retinal pigmented epithelium (RPE) initiates neurogenesis in this cell layer, whereas expression in remaining neural crest-derived melanocytes alters their differentiation, in part by profoundly downregulating expression of the p (pink-eyed dilution) gene, while maintaining their cell fate. The effects of transcriptional perturbation of pigment cell precursors by Mash1 further highlight differences between pigment cells of distinct developmental origins, and suggest a mechanism for the alteration of melanogenesis to result in marked coat color dilution.


Subject(s)
Cell Differentiation , DNA-Binding Proteins/physiology , Melanocytes/cytology , Pigment Epithelium of Eye/embryology , Stem Cells/cytology , Transcription Factors/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Cell Line, Tumor , DNA-Binding Proteins/genetics , Embryonic Development , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/physiology , Mice , Mice, Transgenic , Neurons/cytology , Transcription Factors/genetics , Transgenes
4.
Neuroreport ; 15(8): 1225-9, 2004 Jun 07.
Article in English | MEDLINE | ID: mdl-15167538

ABSTRACT

We examined the effects of EPO on expression of suppressor of cytokine signaling 2 (SOCS2) and found that treatment of neural progenitor cells derived from the adult subventricular zone (SVZ) with recombinant human EPO (rhEPO) stimulated progenitor cell differentiation into neurons, but not astrocytes. Quantitative RT-PCR revealed that SOCS2 mRNA levels were increased in the progenitor cells treated with rhEPO. Immunostaining showed that neurons but not astrocytes were SOCS2 immunoreactive. Incubation of the progenitor cells with rhEPO in the presence of a neutralizing antibody against EPO abolished the effects of EPO on neuronal differentiation and expression of SOCS2. Our data suggest that up-regulation of SOCS2 in neuronal progenitor cells derived from the adult SVZ may regulate EPO enhanced neuronal differentiation.


Subject(s)
Brain/growth & development , Cell Differentiation/physiology , DNA-Binding Proteins/genetics , Erythropoietin/metabolism , Neurons/metabolism , Repressor Proteins/genetics , Stem Cells/metabolism , Trans-Activators/genetics , Animals , Antibodies/pharmacology , Astrocytes/cytology , Astrocytes/drug effects , Astrocytes/metabolism , Brain/cytology , Brain/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Erythropoietin/antagonists & inhibitors , Immunohistochemistry , Lateral Ventricles/cytology , Lateral Ventricles/growth & development , Male , Neurons/cytology , Neurons/drug effects , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , Rats, Wistar , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Spheroids, Cellular/metabolism , Stem Cells/cytology , Stem Cells/drug effects , Suppressor of Cytokine Signaling Proteins , Up-Regulation/drug effects , Up-Regulation/physiology
5.
Nat Commun ; 4: 2690, 2013.
Article in English | MEDLINE | ID: mdl-24162165

ABSTRACT

In contrast to neurons in the central nervous system, mature neurons in the mammalian peripheral nervous system (PNS) can regenerate axons after injury, in part, by enhancing intrinsic growth competence. However, the signalling pathways that enhance the growth potential and induce spontaneous axon regeneration remain poorly understood. Here we reveal that phosphatidylinositol 3-kinase (PI3K) signalling is activated in response to peripheral axotomy and that PI3K pathway is required for sensory axon regeneration. Moreover, we show that glycogen synthase kinase 3 (GSK3), rather than mammalian target of rapamycin, mediates PI3K-dependent augmentation of the growth potential in the PNS. Furthermore, we show that PI3K-GSK3 signal is conveyed by the induction of a transcription factor Smad1 and that acute depletion of Smad1 in adult mice prevents axon regeneration in vivo. Together, these results suggest PI3K-GSK3-Smad1 signalling as a central module for promoting sensory axon regeneration in the mammalian nervous system.


Subject(s)
Axons/metabolism , Gene Expression Regulation , Glycogen Synthase Kinase 3/metabolism , Nerve Regeneration/physiology , Phosphatidylinositol 3-Kinases/metabolism , Smad1 Protein/metabolism , Animals , Electroporation , Female , Ganglia, Spinal/metabolism , Mice , Neurons/metabolism , Sciatic Nerve/pathology , Signal Transduction
6.
PLoS One ; 7(12): e51895, 2012.
Article in English | MEDLINE | ID: mdl-23284807

ABSTRACT

Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3ß and that inhibition of GSK3ß is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3ß phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.


Subject(s)
Glycogen Synthase Kinase 3/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurites/metabolism , Signal Transduction , Animals , Cell Line , Dopaminergic Neurons/cytology , Dopaminergic Neurons/drug effects , Dopaminergic Neurons/metabolism , Enzyme Activation/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Intercellular Signaling Peptides and Proteins/pharmacology , Mice , Nerve Tissue Proteins/pharmacology , Neurites/drug effects , Phosphorylation/drug effects , Receptors, Immunologic/metabolism , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology
7.
Cancer Res ; 70(20): 8159-68, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-20889724

ABSTRACT

As a tumor marker for colorectal cancers, carcinoembryonic antigen (CEA) enhances the metastatic potential of cancer cells. CEA functions as an intercellular adhesion molecule and is upregulated in a wide variety of human cancers. However, the molecular mechanisms by which CEA mediates metastasis remain to be understood. Transforming growth factor-ß (TGF-ß) signaling regulates both tumor suppression and metastasis, and also contributes to the stimulation of CEA transcription and secretion in colorectal cancer cells. However, it remains unknown whether CEA, in turn, influences TGF-ß functions and if a regulatory cross-talk exists between CEA and the TGF-ß signaling pathway. Here, we report that CEA directly interacts with TGF-ß receptor and inhibits TGF-ß signaling. Targeting CEA with either CEA-specific antibody or siRNA rescues TGF-ß response in colorectal cancer cell lines with elevated CEA, thereby restoring the inhibitory effects of TGF-ß signaling on proliferation. CEA also enhances the survival of colorectal cancer cells in both local colonization and liver metastasis in animal study. Our study provides novel insights into the interaction between CEA and TGF-ß signaling pathway and establishes a negative feedback loop in amplifying the progression of colon cancer cells to more invasive phenotypes. These findings offer new therapeutic opportunities to inhibit colorectal cancer cell proliferation by cotargeting CEA in promoting tumor-inhibitory action of the TGF-ß pathway.


Subject(s)
Carcinoembryonic Antigen/metabolism , Carcinoembryonic Antigen/therapeutic use , Colorectal Neoplasms/drug therapy , Receptors, Transforming Growth Factor beta/metabolism , Receptors, Transforming Growth Factor beta/therapeutic use , Signal Transduction/drug effects , Cell Division , Cell Line, Tumor , Colorectal Neoplasms/pathology , Colorectal Neoplasms/physiopathology , DNA Primers , Genes, myc , Humans , Microsatellite Repeats/physiology , Microscopy, Confocal , Neoplasm Metastasis , Receptors, Transforming Growth Factor beta/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/antagonists & inhibitors , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/physiology
8.
J Biol Chem ; 282(44): 32462-70, 2007 Nov 02.
Article in English | MEDLINE | ID: mdl-17804404

ABSTRACT

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.


Subject(s)
Adult Stem Cells , Cell Differentiation , Cell Proliferation , Erythropoietin/analogs & derivatives , Hedgehog Proteins/metabolism , Signal Transduction , Animals , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Erythropoietin/metabolism , Male , Mice , Receptors, Erythropoietin/metabolism , Up-Regulation
9.
Dev Biol ; 296(2): 396-408, 2006 Aug 15.
Article in English | MEDLINE | ID: mdl-16857183

ABSTRACT

DOPAchrome tautomerase (Dct) functions downstream of tyrosinase in the biosynthetic pathway of eumelanin by catalyzing the conversion of dopachrome to 5,5-dihydroxyindole-2-carboxylic acid (DHICA) in pigment cells. Dct transcription is regulated directly or synergistically by Pax3, Sox10 and microphthalmia transcription factor (MITF). Using Dct-lacZ transgenic mice, we measured the spatial and temporal pattern of Dct expression in vivo during neocortical neurogenesis in the brain. Dct was expressed in all layers of the dorsal telencephalon in E10.5. At E15.5 and E17.5 when cortical neurogenesis occurs, expression of Dct was primarily localized to the ventricular zone (VZ) where neuronal stem cells reside. Blocking endogenous Dct by RNAi decreased proliferation of embryonic cortical neural progenitor cells (by 48%, P < 0.05), as determined by BrdU incorporation. In adult brain, Dct/Dct expression decreased in the subventricular zone (SVZ), dentate gyrus and olfactory bulb (OB). However, strong expression of Dct was observed in rostral migratory stream (RMS) and septum. Overexpression of Dct in SVZ cells derived from the adult mice significantly increased the number of cells by 260%, whereas silencing Dct by RNAi decreased cell numbers by 25.8% at 48 h post-nucleofection (P < 0.05). The results of RT-PCR analysis revealed that Dct in the brain lacks exon 7 and is identical to the form of Dct found in neural-crest-derived melanocytes. Our data indicate that Dct, previously known as a melanoblast marker, regulates neural progenitor cell proliferation.


Subject(s)
Cell Proliferation , Intramolecular Oxidoreductases/physiology , Neurons/enzymology , Stem Cells/enzymology , Animals , Cell Line, Tumor , Cells, Cultured , Intramolecular Oxidoreductases/genetics , Male , Melanoma, Experimental , Mice , Mice, Transgenic , NIH 3T3 Cells , Neocortex/cytology , Neocortex/embryology , Neocortex/enzymology , Neurons/cytology , Stem Cells/cytology
10.
Pigment Cell Res ; 17(4): 352-62, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15250937

ABSTRACT

The murine dopachrome tautomerase (Dct) gene is expressed early in melanocyte development during embryogenesis, prior to other members of the tyrosinase gene family important for regulating pigmentation. We have used deletion mutants of the Dct promoter, transfections with developmentally relevant transcription factors, and gel shift assays to define transcriptional determinants of Dct expression. Deletion mutagenesis studies show that sequences within the proximal 459 nucleotides are critical for high level expression in melanocytic cells. This region of the promoter contains candidate binding sites for the transcription factors Sox10 and Mitf. Transfections into 293T and NIH3T3 cells show that Sox10 and Mitf independently activate Dct expression, and, when co-transfected, synergistically activate Dct expression. To support the notion that Sox10 acts directly upon the Dct promoter to activate gene expression, direct interaction of Sox10 was demonstrated using gel shifts of oligonucleotide probes derived from promoter sequences within the region required for Sox10-dependent induction. These results suggest that a combinatorial transcription factor interaction is important for expression of Dct in neural crest-derived melanocytes, and support a model for sequential gene activation in melanocyte development whereby Mitf, a Sox10-dependent transcription factor, is expressed initially before an early melanocyte differentiation gene, Dct, is expressed.


Subject(s)
DNA-Binding Proteins/metabolism , High Mobility Group Proteins/metabolism , Intramolecular Oxidoreductases/genetics , Melanocytes/enzymology , Neoplasm Proteins/metabolism , Promoter Regions, Genetic/physiology , Transcription Factors/metabolism , Transcriptional Activation , Animals , Base Sequence , Cats , Cattle , Enzyme Activation , High Mobility Group Proteins/genetics , Intramolecular Oxidoreductases/metabolism , Mice , Microphthalmia-Associated Transcription Factor , Molecular Sequence Data , Mutation , Neoplasm Proteins/genetics , Rats , SOXE Transcription Factors , Sequence Alignment , Sequence Deletion
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