Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Feb 1;361(Pt 3):621–627. doi: 10.1042/0264-6021:3610621

Retinoic acid activation of the ERK pathway is required for embryonic stem cell commitment into the adipocyte lineage.

Frédéric Bost 1, Leslie Caron 1, Irène Marchetti 1, Christian Dani 1, Yannick Le Marchand-Brustel 1, Bernard Binétruy 1
PMCID: PMC1222345  PMID: 11802792

Abstract

Mouse embryonic stem (ES) cells are pluripotent cells that differentiate into multiple cell lineages. The commitment of ES cells into the adipocyte lineage is dependent on an early 3-day treatment with all-trans retinoic acid (RA). To characterize the molecular mechanisms underlying this process, we examined the contribution of the extracellular-signal-regulated kinase (ERK) pathway. Treatment of ES cell-derived embryoid bodies with RA resulted in a prolonged activation of the ERK pathway, but not the c-Jun N-terminal kinase, p38 mitogen-activated protein kinase or phosphoinositide 3-kinase pathways. To investigate the role of ERK activation, co-treatment of RA with PD98059, a specific inhibitor of the ERK signalling pathway, prevented both adipocyte formation and expression of the adipogenic markers, adipocyte lipid-binding protein and peroxisome-proliferator-activated receptor gamma. Furthermore, we show that ERK activation is required only during RA treatment. PD98059 does not interfere with the commitment of ES cells into other lineages, such as neurogenesis, myogenesis and cardiomyogenesis. As opposed to the controversial role of the ERK pathway in terminal differentiation, our results clearly demonstrate that this pathway is specifically required at an early stage of adipogenesis, corresponding to the RA-dependent commitment of ES cells.

Full Text

The Full Text of this article is available as a PDF (200.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agadir A., Chen G. q., Bost F., Li Y., Mercola D., Zhang X. Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines. J Biol Chem. 1999 Oct 15;274(42):29779–29785. doi: 10.1074/jbc.274.42.29779. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  3. Aubert J., Dessolin S., Belmonte N., Li M., McKenzie F. R., Staccini L., Villageois P., Barhanin B., Vernallis A., Smith A. G. Leukemia inhibitory factor and its receptor promote adipocyte differentiation via the mitogen-activated protein kinase cascade. J Biol Chem. 1999 Aug 27;274(35):24965–24972. doi: 10.1074/jbc.274.35.24965. [DOI] [PubMed] [Google Scholar]
  4. Bain G., Kitchens D., Yao M., Huettner J. E., Gottlieb D. I. Embryonic stem cells express neuronal properties in vitro. Dev Biol. 1995 Apr;168(2):342–357. doi: 10.1006/dbio.1995.1085. [DOI] [PubMed] [Google Scholar]
  5. Barak Y., Nelson M. C., Ong E. S., Jones Y. Z., Ruiz-Lozano P., Chien K. R., Koder A., Evans R. M. PPAR gamma is required for placental, cardiac, and adipose tissue development. Mol Cell. 1999 Oct;4(4):585–595. doi: 10.1016/s1097-2765(00)80209-9. [DOI] [PubMed] [Google Scholar]
  6. Bost F., McKay R., Dean N., Mercola D. The JUN kinase/stress-activated protein kinase pathway is required for epidermal growth factor stimulation of growth of human A549 lung carcinoma cells. J Biol Chem. 1997 Dec 26;272(52):33422–33429. doi: 10.1074/jbc.272.52.33422. [DOI] [PubMed] [Google Scholar]
  7. Camp H. S., Tafuri S. R. Regulation of peroxisome proliferator-activated receptor gamma activity by mitogen-activated protein kinase. J Biol Chem. 1997 Apr 18;272(16):10811–10816. doi: 10.1074/jbc.272.16.10811. [DOI] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Dani C. Embryonic stem cell-derived adipogenesis. Cells Tissues Organs. 1999;165(3-4):173–180. doi: 10.1159/000016697. [DOI] [PubMed] [Google Scholar]
  10. Dani C., Smith A. G., Dessolin S., Leroy P., Staccini L., Villageois P., Darimont C., Ailhaud G. Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci. 1997 Jun;110(Pt 11):1279–1285. doi: 10.1242/jcs.110.11.1279. [DOI] [PubMed] [Google Scholar]
  11. Dudley D. T., Pang L., Decker S. J., Bridges A. J., Saltiel A. R. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7686–7689. doi: 10.1073/pnas.92.17.7686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Favata M. F., Horiuchi K. Y., Manos E. J., Daulerio A. J., Stradley D. A., Feeser W. S., Van Dyk D. E., Pitts W. J., Earl R. A., Hobbs F. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998 Jul 17;273(29):18623–18632. doi: 10.1074/jbc.273.29.18623. [DOI] [PubMed] [Google Scholar]
  13. Font de Mora J., Porras A., Ahn N., Santos E. Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation. Mol Cell Biol. 1997 Oct;17(10):6068–6075. doi: 10.1128/mcb.17.10.6068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. González-Billault C., Avila J. Molecular genetic approaches to microtubule-associated protein function. Histol Histopathol. 2000 Oct;15(4):1177–1183. doi: 10.14670/HH-15.1177. [DOI] [PubMed] [Google Scholar]
  15. Gottlieb D. I., Huettner J. E. An in vitro pathway from embryonic stem cells to neurons and glia. Cells Tissues Organs. 1999;165(3-4):165–172. doi: 10.1159/000016696. [DOI] [PubMed] [Google Scholar]
  16. Hescheler J., Fleischmann B. K., Wartenberg M., Bloch W., Kolossov E., Ji G., Addicks K., Sauer H. Establishment of ionic channels and signalling cascades in the embryonic stem cell-derived primitive endoderm and cardiovascular system. Cells Tissues Organs. 1999;165(3-4):153–164. doi: 10.1159/000016695. [DOI] [PubMed] [Google Scholar]
  17. Heydrick S. J., Jullien D., Gautier N., Tanti J. F., Giorgetti S., Van Obberghen E., Le Marchand-Brustel Y. Defect in skeletal muscle phosphatidylinositol-3-kinase in obese insulin-resistant mice. J Clin Invest. 1993 Apr;91(4):1358–1366. doi: 10.1172/JCI116337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  19. Hu E., Kim J. B., Sarraf P., Spiegelman B. M. Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARgamma. Science. 1996 Dec 20;274(5295):2100–2103. doi: 10.1126/science.274.5295.2100. [DOI] [PubMed] [Google Scholar]
  20. Hu E., Tontonoz P., Spiegelman B. M. Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9856–9860. doi: 10.1073/pnas.92.21.9856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jaiswal R. K., Jaiswal N., Bruder S. P., Mbalaviele G., Marshak D. R., Pittenger M. F. Adult human mesenchymal stem cell differentiation to the osteogenic or adipogenic lineage is regulated by mitogen-activated protein kinase. J Biol Chem. 2000 Mar 31;275(13):9645–9652. doi: 10.1074/jbc.275.13.9645. [DOI] [PubMed] [Google Scholar]
  22. Jho E. H., Davis R. J., Malbon C. C. c-Jun amino-terminal kinase is regulated by Galpha12/Galpha13 and obligate for differentiation of P19 embryonal carcinoma cells by retinoic acid. J Biol Chem. 1997 Sep 26;272(39):24468–24474. doi: 10.1074/jbc.272.39.24468. [DOI] [PubMed] [Google Scholar]
  23. Kanungo J., Potapova I., Malbon C. C., Wang H. y. MEKK4 mediates differentiation in response to retinoic acid via activation of c-Jun N-terminal kinase in rat embryonal carcinoma P19 cells. J Biol Chem. 2000 Aug 4;275(31):24032–24039. doi: 10.1074/jbc.M002747200. [DOI] [PubMed] [Google Scholar]
  24. Kastner P., Mark M., Chambon P. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell. 1995 Dec 15;83(6):859–869. doi: 10.1016/0092-8674(95)90202-3. [DOI] [PubMed] [Google Scholar]
  25. Kubota N., Terauchi Y., Miki H., Tamemoto H., Yamauchi T., Komeda K., Satoh S., Nakano R., Ishii C., Sugiyama T. PPAR gamma mediates high-fat diet-induced adipocyte hypertrophy and insulin resistance. Mol Cell. 1999 Oct;4(4):597–609. doi: 10.1016/s1097-2765(00)80210-5. [DOI] [PubMed] [Google Scholar]
  26. Lee H. Y., Sueoka N., Hong W. K., Mangelsdorf D. J., Claret F. X., Kurie J. M. All-trans-retinoic acid inhibits Jun N-terminal kinase by increasing dual-specificity phosphatase activity. Mol Cell Biol. 1999 Mar;19(3):1973–1980. doi: 10.1128/mcb.19.3.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lewis T. S., Shapiro P. S., Ahn N. G. Signal transduction through MAP kinase cascades. Adv Cancer Res. 1998;74:49–139. doi: 10.1016/s0065-230x(08)60765-4. [DOI] [PubMed] [Google Scholar]
  28. Lohnes D., Mark M., Mendelsohn C., Dollé P., Dierich A., Gorry P., Gansmuller A., Chambon P. Function of the retinoic acid receptors (RARs) during development (I). Craniofacial and skeletal abnormalities in RAR double mutants. Development. 1994 Oct;120(10):2723–2748. doi: 10.1242/dev.120.10.2723. [DOI] [PubMed] [Google Scholar]
  29. Mangelsdorf D. J., Evans R. M. The RXR heterodimers and orphan receptors. Cell. 1995 Dec 15;83(6):841–850. doi: 10.1016/0092-8674(95)90200-7. [DOI] [PubMed] [Google Scholar]
  30. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  31. Mountford P., Zevnik B., Düwel A., Nichols J., Li M., Dani C., Robertson M., Chambers I., Smith A. Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4303–4307. doi: 10.1073/pnas.91.10.4303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ricort J. M., Tanti J. F., Van Obberghen E., Le Marchand-Brustel Y. Cross-talk between the platelet-derived growth factor and the insulin signaling pathways in 3T3-L1 adipocytes. J Biol Chem. 1997 Aug 8;272(32):19814–19818. doi: 10.1074/jbc.272.32.19814. [DOI] [PubMed] [Google Scholar]
  33. Rohwedel J., Guan K., Wobus A. M. Induction of cellular differentiation by retinoic acid in vitro. Cells Tissues Organs. 1999;165(3-4):190–202. doi: 10.1159/000016699. [DOI] [PubMed] [Google Scholar]
  34. Rohwedel J., Guan K., Zuschratter W., Jin S., Ahnert-Hilger G., Fürst D., Fässler R., Wobus A. M. Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro. Dev Biol. 1998 Sep 15;201(2):167–184. doi: 10.1006/dbio.1998.9002. [DOI] [PubMed] [Google Scholar]
  35. Rosen E. D., Sarraf P., Troy A. E., Bradwin G., Moore K., Milstone D. S., Spiegelman B. M., Mortensen R. M. PPAR gamma is required for the differentiation of adipose tissue in vivo and in vitro. Mol Cell. 1999 Oct;4(4):611–617. doi: 10.1016/s1097-2765(00)80211-7. [DOI] [PubMed] [Google Scholar]
  36. Sale E. M., Atkinson P. G., Sale G. J. Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J. 1995 Feb 15;14(4):674–684. doi: 10.1002/j.1460-2075.1995.tb07046.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shao D., Lazar M. A. Peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha, and cell cycle status regulate the commitment to adipocyte differentiation. J Biol Chem. 1997 Aug 22;272(34):21473–21478. doi: 10.1074/jbc.272.34.21473. [DOI] [PubMed] [Google Scholar]
  38. Smith A. G., Heath J. K., Donaldson D. D., Wong G. G., Moreau J., Stahl M., Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688–690. doi: 10.1038/336688a0. [DOI] [PubMed] [Google Scholar]
  39. Tong Q., Dalgin G., Xu H., Ting C. N., Leiden J. M., Hotamisligil G. S. Function of GATA transcription factors in preadipocyte-adipocyte transition. Science. 2000 Oct 6;290(5489):134–138. doi: 10.1126/science.290.5489.134. [DOI] [PubMed] [Google Scholar]
  40. Tontonoz P., Hu E., Graves R. A., Budavari A. I., Spiegelman B. M. mPPAR gamma 2: tissue-specific regulator of an adipocyte enhancer. Genes Dev. 1994 May 15;8(10):1224–1234. doi: 10.1101/gad.8.10.1224. [DOI] [PubMed] [Google Scholar]
  41. Tontonoz P., Hu E., Spiegelman B. M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994 Dec 30;79(7):1147–1156. doi: 10.1016/0092-8674(94)90006-x. [DOI] [PubMed] [Google Scholar]
  42. Williams R. L., Hilton D. J., Pease S., Willson T. A., Stewart C. L., Gearing D. P., Wagner E. F., Metcalf D., Nicola N. A., Gough N. M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988 Dec 15;336(6200):684–687. doi: 10.1038/336684a0. [DOI] [PubMed] [Google Scholar]
  43. Wu Z., Wu J., Jacinto E., Karin M. Molecular cloning and characterization of human JNKK2, a novel Jun NH2-terminal kinase-specific kinase. Mol Cell Biol. 1997 Dec;17(12):7407–7416. doi: 10.1128/mcb.17.12.7407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yen A., Roberson M. S., Varvayanis S. Retinoic acid selectively activates the ERK2 but not JNK/SAPK or p38 MAP kinases when inducing myeloid differentiation. In Vitro Cell Dev Biol Anim. 1999 Oct;35(9):527–532. doi: 10.1007/s11626-999-0063-z. [DOI] [PubMed] [Google Scholar]
  45. Yen A., Varvayanis S. Retinoic acid increases amount of phosphorylated RAF; ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced GO arrest is. In Vitro Cell Dev Biol Anim. 2000 Apr;36(4):249–255. doi: 10.1290/1071-2690(2000)036<0249:raiaop>2.0.co;2. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES