All-trans-retinoic acid (atRA) is an essential signaling molecule in embryonic development. It regulates cell differentiation by activating nuclear retinoic acid receptors (RAR) and retinoid-X receptors (RXR), which both control gene expression. In addition, atRA could act in the cytoplasm by modulating the activity of mitogen-activated protein kinases (MAPK) ERK and p38, which also have a role in cell differentiation. AtRA can induce the differentiation of P19 embryonic carcinoma stem cells into adipocytes, cardiomyocytes, and skeletal muscle cells, concurrently, in the same culture. We postulated that combinations of atRA, atRA analogs exhibiting selectivity for RAR or RXR, and inhibitors of ERK and p38 signaling (ERKi and p38i) could be used to favor one mesodermal fate over the others in the P19 model. In a first series of experiments, we replaced atRA by an agonist of RXR (LG100268) or RAR (TTNPB) to preferentially stimulate one group of receptors over the other. LG100268 was as adipogenic and myogenic as atRA, whereas TTNPB strongly induced adipogenesis, but not myogenesis. ERKi enhanced the myogenic action of atRA, and p38i increased both adipogenesis and myogenesis. In a second series of experiments, we combined atRA with an RAR or RXR antagonist (RARatg or RXRatg) to preferentially deactivate each receptor group in turn. The combinations atRA+RXRatg and atRA+RARatg, including or not ERKi, had similar mesodermal actions as atRA. In contrast, there was no myogenesis with atRA+RXRatg+p38i treatment, and there were no myogenesis and no adipogenesis with the atRA+RARatg+p38i combination. Overall, the results indicate that p38 has a role in mesodermal differentiation that depends on the retinoid context. Indeed, p38 in conjunction with RXR is important in myogenesis, and p38 and RAR in adipogenesis. Under the conditions tested, it was possible to stimulate adipogenesis with a block on myogenesis, whereas increased myogenesis was accompanied by adipogenesis.
Get full access to this article
View all access options for this article.
References
1.
RossSA, McCafferyPJ, DragerUC, De LucaLM. 2000. Retinoids in embryonal development. Physiol Rev, 80:1021–1054.
2.
ZileMH. 2004. Vitamin a requirement for early cardiovascular morphogenesis specification in the vertebrate embryo: insights from the avian embryo. Exp Biol Med (Maywood), 229:598–606.
3.
KastnerP, MarkM, GhyselinckN, KrezelW, DupeV, GrondonaJM, ChambonP. 1997. Genetic evidence that the retinoid signal is transduced by heterodimeric RXR/RAR functional units during mouse development. Development, 124:313–326.
4.
ChambonP. 1996. A decade of molecular biology of retinoic acid receptors. FASEB J, 10:940–954.
5.
AhujaHS, SzantoA, NagyL, DaviesPJ. 2003. The retinoid X receptor and its ligands: versatile regulators of metabolic function, cell differentiation and cell death. J Biol Regul Homeost Agents, 17:29–45.
6.
SucovHM, EvansRM. 1995. Retinoic acid and retinoic acid receptors in development. Mol Neurobiol, 10:169–184.
7.
AltucciL, LeibowitzMD, OgilvieKM, de LeraAR, GronemeyerH. 2007. RAR and RXR modulation in cancer and metabolic disease. Nat Rev Drug Discov, 6:793–810.
8.
BrtkoJ. 2007. Retinoids, rexinoids and their cognate nuclear receptors: character and their role in chemoprevention of selected malignant diseases. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 151:187–194.
McBurneyMW. 1993. P19 embryonal carcinoma cells. Int J Dev Biol, 37:135–140.
11.
RudnickiMA, McBurneyMW. 1987. Cell cultre methods and induction of differenciation of embryonal carcinoma cell lines. Dans Teratocarcinomas and embryonic stem cells: a practical approach. IRL: Oxford.
12.
BouchardF, PaquinJ. 2009. Skeletal and cardiac myogenesis accompany adipogenesis in P19 embryonal stem cells. Stem Cells Dev, 18:1023–1032.
13.
HondaM, HamazakiTS, KomazakiS, KagechikaH, ShudoK, AsashimaM. 2005. RXR agonist enhances the differentiation of cardiomyocytes derived from embryonic stem cells in serum-free conditions. Biochem Biophys Res Commun, 333:1334–1340.
14.
Le MayM, MachH, LacroixN, HouC, ChenJ, LiQ. 2011. Contribution of retinoid X receptor signaling to the specification of skeletal muscle lineage. J Biol Chem, 286:26806–26812.
15.
MonteiroMC, WdziekonskiB, VillageoisP, VernochetC, IehleC, BillonN, DaniC. 2009. Commitment of mouse embryonic stem cells to the adipocyte lineage requires retinoic acid receptor beta and active GSK3. Stem Cells Dev, 18:457–463.
16.
AouadiM, BinetruyB, CaronL, Le Marchand-BrustelY, BostF. 2006. Role of MAPKs in development and differentiation: lessons from knockout mice. Biochimie, 88:1091–1098.
17.
BarruetE, HadadehO, PeirettiF, RenaultVM, HadjalY, BernotD, TournaireR, NegreD, Juhan-VagueI, AlessiMC, BinetruyB. 2011. p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells. Stem Cells Dev, 20:1233–1246.
18.
DuvalD, MalaiseM, ReinhardtB, KedingerC, BoeufH. 2004. A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells. Cell Death Differ, 11:331–341.
19.
DuvalD, TrouillasM, ThibaultC, DembeleD, DiemunschF, ReinhardtB, MertzAL, DierichA, BoeufH. 2006. Apoptosis and differentiation commitment: novel insights revealed by gene profiling studies in mouse embryonic stem cells. Cell Death Differ, 13:564–575.
20.
RoseBA, ForceT, WangY. 2010. Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale. Physiol Rev, 90:1507–1546.
21.
AouadiM, LaurentK, ProtM, Le Marchand-BrustelY, BinetruyB, BostF. 2006. Inhibition of p38MAPK increases adipogenesis from embryonic to adult stages. Diabetes, 55:281–289.
22.
AouadiM, BostF, CaronL, LaurentK, Le Marchand BrustelY, BinetruyB. 2006. p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis. Stem Cells, 24:1399–1406.
23.
DavidsonSM, MorangeM. 2000. Hsp25 and the p38 MAPK pathway are involved in differentiation of cardiomyocytes. Dev Biol, 218:146–160.
24.
ErikssonM, LeppaS. 2002. Mitogen-activated protein kinases and activator protein 1 are required for proliferation and cardiomyocyte differentiation of P19 embryonal carcinoma cells. J Biol Chem, 277:15992–16001.
25.
KerenA, TamirY, BengalE. 2006. The p38 MAPK signaling pathway: a major regulator of skeletal muscle development. Mol Cell Endocrinol, 252:224–230.
26.
WuJ, KubotaJ, HirayamaJ, NagaiY, NishinaS, YokoiT, AsaokaY, SeoJ, ShimizuNet al.2010. p38 Mitogen-activated protein kinase controls a switch between cardiomyocyte and neuronal commitment of murine embryonic stem cells by activating myocyte enhancer factor 2C-dependent bone morphogenetic protein 2 transcription. Stem Cells Dev, 19:1723–1734.
27.
GaurM, RitnerC, SieversR, PedersenA, PrasadM, BernsteinHS, YeghiazariansY. 2010. Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes. Cytotherapy, 12:807–817.
28.
GraichenR, XuX, BraamSR, BalakrishnanT, NorfizaS, SiehS, SooSY, ThamSC, MummeryCet al.2008. Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK. Differentiation, 76:357–370.
29.
XuXQ, GraichenR, SooSY, BalakrishnanT, RahmatSN, SiehS, ThamSC, FreundC, MooreJet al.2008. Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells. Differentiation, 76:958–970.
30.
BinetruyB, HeasleyL, BostF, CaronL, AouadiM. 2007. Concise review: regulation of embryonic stem cell lineage commitment by mitogen-activated protein kinases. Stem Cells, 25:1090–1095.
31.
BostF, AouadiM, CaronL, BinetruyB. 2005. The role of MAPKs in adipocyte differentiation and obesity. Biochimie, 87:51–56.
32.
BostF, AouadiM, CaronL, EvenP, BelmonteN, ProtM, DaniC, HofmanP, PagesGet al.2005. The extracellular signal-regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes, 54:402–411.
33.
BostF, CaronL, MarchettiI, DaniC, Le Marchand-BrustelY, BinetruyB. 2002. Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage. Biochem J, 361:621–627.
34.
BruckN, VitouxD, FerryC, DuongV, BauerA, de TheH, Rochette-EglyC. 2009. A coordinated phosphorylation cascade initiated by p38MAPK/MSK1 directs RARalpha to target promoters. Embo J, 28:34–47.
35.
ZassadowskiF, Rochette-EglyC, ChomienneC, CassinatB. 2012. Regulation of the transcriptional activity of nuclear receptors by the MEK/ERK1/2 pathway. Cell Signal, 24:2369–2377.
36.
PiskunovA, Rochette-EglyC. 2011. A retinoic acid receptor RARalpha pool present in membrane lipid rafts forms complexes with G protein alphaQ to activate p38MAPK. Oncogene.
37.
GianniM, BauerA, GarattiniE, ChambonP, Rochette-EglyC. 2002. Phosphorylation by p38MAPK and recruitment of SUG-1 are required for RA-induced RAR gamma degradation and transactivation. EMBO J, 21:3760–3769.
38.
TasseffR, NayakS, SongSO, YenA, VarnerJD. 2011. Modeling and analysis of retinoic acid induced differentiation of uncommitted precursor cells. Integr Biol (Camb), 3:578–591.
39.
GianniM, PevianiM, BruckN, RambaldiA, BorleriG, TeraoM, KurosakiM, ParoniG, Rochette-EglyC, GarattiniE. 2012. p38alphaMAPK interacts with and inhibits RARalpha: suppression of the kinase enhances the therapeutic activity of retinoids in acute myeloid leukemia cells. Leukemia, 26:1850–1861.
40.
MilellaM, KonoplevaM, PrecupanuCM, TabeY, RicciardiMR, GregorjC, CollinsSJ, CarterBZ, D'AngeloCet al.2007. MEK blockade converts AML differentiating response to retinoids into extensive apoptosis. Blood, 109:2121–2129.
41.
MooreJC, SpijkerR, MartensAC, de BoerT, RookMB, van der HeydenMA, TertoolenLG, MummeryCL. 2004. A P19Cl6 GFP reporter line to quantify cardiomyocyte differentiation of stem cells. Int J Dev Biol, 48:47–55.
42.
PaquinJ, DanalacheBA, JankowskiM, McCannSM, GutkowskaJ. 2002. Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes. Proc Natl Acad Sci U S A, 99:9550–9555.
43.
DucharmeP, MaltaisD, DesrochesD, MateescuMA, PaquinJ. 2010. Ceruloplasmin-induced aggregation of P19 neurons involves a serine protease activity and is accompanied by reelin cleavage. Neuroscience, 167:633–643.
44.
BoehmMF, ZhangL, ZhiL, McClurgMR, BergerE, WagonerM, MaisDE, SutoCM, DaviesJAet al.1995. Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J Med Chem, 38:3146–155.
45.
MouL, Lankford-TurnerP, LeanderMV, BissonnetteRP, DonahoeRM, RoyalW. 2004. RXR-induced TNF-alpha suppression is reversed by morphine in activated U937 cells. J Neuroimmunol, 147:99–105.
46.
PignatelloMA, KauffmanFC, LevinAA. 1997. Multiple factors contribute to the toxicity of the aromatic retinoid, TTNPB (Ro 13–7410): binding affinities and disposition. Toxicol Appl Pharmacol, 142:319–327.
47.
CrettazM, BaronA, SiegenthalerG, HunzikerW. 1990. Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta. Biochem J, 272:391–397.
48.
VuligondaV, LinY, ChandraratnaRAS. 1996. Synthesis of highly potent RXR-specific retinoids: The use of a cyclopropyl group as a double bond isostere. Bioorganic & Medicinal Chemistry Letters, 6:213–218.
49.
ChenY, DokmanovicM, SteinWD, ArdeckyRJ, RoninsonIB. 2006. Agonist and antagonist of retinoic acid receptors cause similar changes in gene expression and induce senescence-like growth arrest in MCF-7 breast carcinoma cells. Cancer Res, 66:8749–8761.
50.
PalmieriSL, PeterW, HessH, ScholerHR. 1994. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol, 166:259–267.
51.
ScholerHR, BallingR, HatzopoulosAK, SuzukiN, GrussP. 1989. Octamer binding proteins confer transcriptional activity in early mouse embryogenesis. Embo J, 8:2551–2557.
52.
SamarutE, Rochette-EglyC. 2012. Nuclear retinoic acid receptors: conductors of the retinoic acid symphony during development. Mol Cell Endocrinol, 348:348–360.
53.
DaniC, SmithAG, DessolinS, LeroyP, StacciniL, VillageoisP, DarimontC, AilhaudG. 1997. Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci, 110,Pt 11:1279–1285.
54.
HillelAT, VargheseS, PetscheJ, ShamblottMJ, ElisseeffJH. 2009. Embryonic germ cells are capable of adipogenic differentiation in vitro and in vivo. Tissue Eng Part A, 15:479–486.
55.
AusoniS, CampioneM, PicardA, MorettiP, VitadelloM, De NardiC, SchiaffinoS. 1994. Structure and regulation of the mouse cardiac troponin I gene. J Biol Chem, 269:339–346.
56.
GorzaL, AusoniS, MerciaiN, HastingsKE, SchiaffinoS. 1993. Regional differences in troponin I isoform switching during rat heart development. Dev Biol, 156:253–264.
57.
DupinE, CalloniGW, Le DouarinNM. 2010. The cephalic neural crest of amniote vertebrates is composed of a large majority of precursors endowed with neural, melanocytic, chondrogenic and osteogenic potentialities. Cell Cycle, 9:238–249.
58.
PignatelloMA, KauffmanFC, LevinAA. 1999. Multiple factors contribute to the toxicity of the aromatic retinoid TTNPB (Ro 13–7410): interactions with the retinoic acid receptors. Toxicol Appl Pharmacol, 159:109–116.
59.
ChibaH, CliffordJ, MetzgerD, ChambonP. 1997. Specific and redundant functions of retinoid X Receptor/Retinoic acid receptor heterodimers in differentiation, proliferation, and apoptosis of F9 embryonal carcinoma cells. J Cell Biol, 139:735–747.
60.
SmithTK, BaderDM. 2007. Signals from both sides: Control of cardiac development by the endocardium and epicardium. Semin Cell Dev Biol, 18:84–89.
61.
TianY, MorriseyEE. 2012. Importance of myocyte-nonmyocyte interactions in cardiac development and disease. Circ Res, 110:1023–1034.
62.
BillonN, KoldeR, ReimandJ, MonteiroMC, KullM, PetersonH, TretyakovK, AdlerP, WdziekonskiB, ViloJ, DaniC. 2010. Comprehensive transcriptome analysis of mouse embryonic stem cell adipogenesis unravels new processes of adipocyte development. Genome Biol, 11:R80.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
