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Identification of Cell Signaling Pathways involved in the repression of mouse ES Neuronal Differentiation by RAR Beta
Author(s) -
Joseph Serenthia,
Moses Diamond,
MartinezCeballos Eduardo
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.644.5
Subject(s) - retinoic acid , cellular differentiation , embryonic stem cell , microbiology and biotechnology , signal transduction , biology , transcription factor , beta (programming language) , cell culture , chemistry , biochemistry , gene , genetics , computer science , programming language
Embryonic Stem (ES) cells are pluripotent cells that can be induced to differentiate into cells of all three lineages: mesodermal, endodermal, and ectodermal. In culture, ES cells can be differentiated into mature neurons by treatment with Retinoic Acid (RA), a Vitamin A derivative. The effect of RA on cells is mediated mainly through the activation of the RA nuclear receptors of which there are three types: RAR alpha, RAR beta, and RAR gamma. As preliminary evidence from our laboratory indicates that active RAR beta acts as an inhibitor of neuronal differentiation and, since we also found that neuronal differentiation depends upon the RA‐induced activation of Hoxa1, a homeobox transcription factor, we then first sought to investigate whether RAR beta is an inhibitor of Hoxa1. We found that treatment of mouse ES cells with AC55649, an RAR beta agonist, prevented the pharmacological activation of Hoxa1 with RA. Next, we asked whether the pharmacological inhibition of RAR beta, together with RA treatment, would increase the efficiency of mouse ES cell differentiation into neurons. To test this hypothesis, mouse ES cells were treated with 1 micromolar RA plus/minus LE135, an RAR beta inhibitor, at 1 or 5 micromolar concentrations. Results from immunofluorescence analyses using a B‐Tubulin III antibody indicate that the highest efficiency of neuronal differentiation was obtained with a combination of RA and 5 micromolar LE135 for an 8 day treatment. Finally, we performed proteomic analyses on ES cells treated with RA vs RA plus AC55649 in order to identify the signaling pathways activated by the RAR beta agonist. Our proteomic analyses using antibody microarrays indicated that proteins such as p38, AKT, and Erk1 were upregulated in cells treated with RA plus the agonist as compared to cells treated wit RA alone. Thus, our results indicate that RAR beta functions as a repressor of neuronal differentiation by activating major cell signaling pathways, and its pharmacological inhibition may constitute a novel method to increase the efficiency of ES to neuronal differentiation in culture.