Protein kinase C regulates human pluripotent stem cell self-renewal.
BACKGROUND
The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells.


RESULTS
In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, β, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells.


CONCLUSIONS
Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3β. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.
Laboratory of Stem Cell Cultures, Department of Disease Bioresources Research, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan.
Masaki
M
Kinehara
Suguru
S
Kawamura
Daiki
D
Tateyama
Mika
M
Suga
Hiroko
H
Matsumura
Sumiyo
S
Mimura
Noriko
N
Hirayama
Mitsuhi
M
Hirata
Kozue
K
Uchio-Yamada
Arihiro
A
Kohara
Kana
K
Yanagihara
Miho K
MK
Furue
D028341:Activins / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000378:metabolism; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation; D002867:Chromones / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099221:Protein Kinase C zeta
D028341:Activins; D000469:Alkaline Phosphatase; D002227:Carbazoles; D002867:Chromones; D048049:Extracellular Signal-Regulated MAP Kinases; D016222:Fibroblast Growth Factor 2; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D007211:Indoles; D008301:Maleimides; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D051057:Proto-Oncogene Proteins c-akt; D013755:Tetradecanoylphorbol Acetate; D000099221:Protein Kinase C zeta; C422405:activin A; C081021:Go 6976; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C000605506:GSK3B protein, human; C070515:bisindolylmaleimide I