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Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

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Abstract

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways.

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References

  • Beddington, R. S., Robertson, E. J. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development. 105: 733–7; 1989.

    PubMed  CAS  Google Scholar 

  • Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytic Biochemistry. 72: 248–54; 1976.

    Article  PubMed  CAS  Google Scholar 

  • Buck, J., Sinclair, M. L., Schapal, L., Cann, M. J., Levin, L. R. Cytosolic adenylyl cyclase defines a unique signaling molecule in mammals. Proc Natl Acad Sci U S A. 96: 79–84; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Chambers, I. The molecular basis of pluripotency in mouse embryonic stem cells. Cloning Stem Cells. 6: 386–91; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Conti, M., Jin, S. L. The molecular biology of cyclic nucleotide phosphodiesterases. Prog Nucleic Acid Res Mol Biol. 63: 1–38; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Dani, C., Chambers, I., Johnstone, S., Robertson, M., Ebrahimi, B., Saito, M., Taga, T., Li, M., Burdon, T., Nichols, J., Smith, A. Paracrine induction of stem cell renewal by LIF-deficient cells: a new ES cell regulatory pathway. Dev Biol. 203: 149–62; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Faherty, S., Kane, M. T., Quinlan, L. R. Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 gene expression: effects of LIF, serum-free medium, retinoic acid, and dbcAMP. In Vitro Cell Dev Biol Anim. 41: 356–63; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Globus, R., Strewler, G., Nissenson, R. Up-regulation of adenylyl cyclase signaling and Gsa expression during differentiation of embryonic stem cells. Endocrine Journal. 2: 419–27; 1994.

    CAS  Google Scholar 

  • Grover, A., Adamson, E. Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: modulation by cyclic AMP. Developmental Biology. 114: 492–503; 1986.

    Article  PubMed  CAS  Google Scholar 

  • Maye, P., Becker, S., Kasameyer, E., Byrd, N., Grabel, L. Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies. Mechanisms of Development. 94: 117–32; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Mountford, P., Zevnik, B., Duwel, 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. 91: 4303–7; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Niwa, H. Molecular mechanism to maintain stem cell renewal of ES cells. Cell Struct Funct. 26: 137–48; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Niwa, H., Burdon, T., Chambers, I., Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes and Development. 12: 2048–60; 1998.

    PubMed  CAS  Google Scholar 

  • Ogawa, K., Matsui, H., Ohtsuka, S., Niwa, H. A novel mechanism for regulating clonal propagation of mouse ES cells. Genes Cells. 9: 471–7; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Pan, G. J., Chang, Z. Y., Schöler, H. R., Pei, D. Stem cell pluripotency and transcription factor Oct4. Cell Res. 12: 321–9; 2002.

    Article  PubMed  Google Scholar 

  • Pesce, M., Gross, M. K., Scholer, H. R. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays. 20: 722–32; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Robertson, E. J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford; IRL Press; 1987.

    Google Scholar 

  • Smith, A., Nichols, J., Robertson, M., Rathjen, P. Differentiation inhibiting activity (DIA/LIF) and mouse development. Developmental Biology. 151: 339–51; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Soderling, S. H., Beavo, J. A. Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions. Curr Opin Cell Biol. 12: 174–9; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Strickland, S., Mahdavi, V. The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell. 15: 393–403; 1978.

    Article  PubMed  CAS  Google Scholar 

  • Wang, Z., Dillon, T. J., Pokala, V., Mishra, S., Labudda, K., Hunter, B., Stork, P. J. Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation. Mol Cell Biol. 26: 2130–45; 2006.

    Article  PubMed  CAS  Google Scholar 

  • Williams, R., Hilton, D., Pease, S., Willson, T., Stewart, C., Gearing, D., Wagner, E., Metcalf, D., Nicola, N., Gough, N. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 336: 684–7; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Yeom, Y. I., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., Hubner, K. and Scholer, H. R. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development. 122: 881–94; 1996.

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors wish to thank Dr. Austin Smith, Centre for Genome Research, Edinburgh, UK, for the generous gift of his ES cell lines. This work was partly funded by the Millenium Research Fund National University of Ireland Galway.

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Authors and Affiliations

  1. Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland

    S. Faherty, A. Fitzgerald, M. Keohan & L. R. Quinlan

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  1. S. Faherty
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  2. A. Fitzgerald
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  3. M. Keohan
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  4. L. R. Quinlan
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Correspondence to L. R. Quinlan.

Additional information

This work was partly funded by the Millennium Research Fund National University of Ireland Galway.

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Faherty, S., Fitzgerald, A., Keohan, M. et al. Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway. In Vitro Cell.Dev.Biol.-Animal 43, 37–47 (2007). https://doi.org/10.1007/s11626-006-9001-5

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  • DOI: https://doi.org/10.1007/s11626-006-9001-5

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