Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway.
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 muM IBMX without LIF, 10 muM 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.
Faherty
S
S
Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland.
Fitzgerald
A
A
Keohan
M
M
Quinlan
L R
LR
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000032:analysis* / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D015398:Signal Transduction; D014212:Tretinoin / Q000494:pharmacology
D050814:Octamer Transcription Factor-3; D010726:Phosphodiesterase Inhibitors; C494174:Pou5f1 protein, mouse; D005576:Colforsin; D014212:Tretinoin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015056:1-Methyl-3-isobutylxanthine