PKA and cAMP stimulate proliferation of mouse embryonic stem cells by elevating GLUT1 expression mediated by the NF-κB and CREB/CBP signaling pathways.
BACKGROUND
Regulation of glucose transporter (GLUT) expression and activity plays a vital role in the supply of glucose to embryonic stem (ES) cells.


METHODS
To observe the effect of 6-phenyl cyclic monophosphate (cAMP) on glucose uptake and cell proliferation, 2-deoxyglucose (2-DG) uptake, immunohistochemistry, Western blotting, and immunoprecipitation were carried out.


RESULTS
Among GLUT isoforms in mouse ES cells, GLUT1 was predominantly expressed and 6-phenyl cAMP increased GLUT mRNA levels. Among cAMP agonists, 6-phenyl cAMP increased 2-DG uptake more than that of 8-p-chlorophenylthio-2'-O-methyl-cAMP. 6-Phenyl cAMP increased GLUT1 expression and translocation from the cytosol to the plasma membrane. 6-Phenyl cAMP increased 2-DG uptake in a time- and concentration-dependent manner due to an increase in V(max) but not K(m). 6-Phenyl cAMP increased phosphorylation of nuclear factor-κB (NF-κB) and cAMP response element binding (CREB) and expression of the CREB protein (CBP) and transducer of regulated CREB activity 2 (TORC2) in sequence. 6-Phenyl cAMP induced complex formation of NF-κB/CREB/CBP/TORC2, which are involved in the increase of gluconeogenic enzyme expression. 6-Phenyl cAMP also increased cell cycle regulatory protein expression levels, the proportion of S-phase cells, and proto-oncogene expression via protein kinase A (PKA)-dependent NF-κB signaling. Finally, GLUT1 siRNA blocked the 6-phenyl cAMP-induced increase in ES cell proliferation. We conclude that PKA stimulated the complex formation of CREB/CBP/TORC2 via NF-κB, which induced effective coordination of glucose uptake as well as proliferation in ES cells.


CONCLUSIONS
6-Phenyl cAMP-induced PKA activation modified the proliferation, which may be beneficial for expanding ES cell use to cell therapy.
Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea.
Mi Ok
MO
Kim
Yu Jin
YJ
Lee
Jae Hong
JH
Park
Jung Min
JM
Ryu
Seung Pil
SP
Yun
Ho Jae
HJ
Han
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation / Q000187:drug effects; D051272:Glucose Transporter Type 1 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D051272:Glucose Transporter Type 1; D008565:Membrane Proteins; C483758:N(6)-phenyl-cAMP; D016328:NF-kappa B; C498709:Pag1 protein, mouse; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases