Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

<div><p>Background</p><p>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.</p><p>Methodology/Principal Findings</p><p>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.</p><p>Conclusions/Significance</p><p>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.</p></div

Specific-isoform of PKCs function in FGF-2 signaling.

<p>(<b>A</b>) RT-PCR analysis of PKC isoform expression. Total RNA was extracted from the undifferentiated 201B7 hiPS cells cultured on feeder cells (CF-1) with KSR-based medium or the feeder cells. Primers were listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054122#pone.0054122.s012" target="_blank">Table S3</a>. (<b>B</b>) Phosphorylation of PKCδ, ε, or ζ isoforms induced by FGF-2 (open square) with GFX (closed square). 201B7 hiPS cells were stimulated with FGF-2 (100 ng/ml) after overnight starvation and incubated with or without GFX (5 µM) for 180 minutes. The cells were lysed and followed by western blot analysis using an antibody detecting the phosphorylation or total protein amount of PKCδ, PKCε, or PKCζ. Protein content quantified from the gel blot images (n = 3). The values of the y-axis are the ratio of each phosphorylation to each total signal protein. (<b>C</b>) FGF-2 signaling in hPS cells with specific PKC isoforms-targeting siRNA. 201B7 iPS cells were transfected with specific PKCδ, ε, or ζ isoforms-targeting siRNA or non-targeting siRNA. The phosphorylation levels of the cells treated with FGF-2(100 ng/ml) after overnight starvation were measured by AlphaScreen® SureFire® assay kit. The values of the y-axis are the ratio of each phosphorylation to each total signal protein. The data are represented as means ± SE (n = 3). *P<0.05.</p

An ALP activity-based high-throughput screening assay of chemical library for PKC inhibitors.

<p>The ALP activity using 4-methylumbelliferyl phosphate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054122#pone.0054122-Okamoto1" target="_blank">[59]</a> in 201B7 hiPS cells in a 96-well plate was measured by fluorometry. Each dot on the graph represents the fluorescent intensity for each compound of the kinase inhibitor library. Dotted line indicates the level for DMSO as a control.</p

Model for the molecular mechanism of PKCs regulating self-renewal or differentiation in hPS cells.

<p>Our study suggested a model that FGF-2 activates PI3K/AKT, MEK/ERK-1/2, and PKCε/δ/ζ. PKCε, δ, and ζ inactivates directly or indirectly GSK-3β by phosphorylation which promotes differentiation of hPS cells. PKCε and ζ activates ERK-1/2 which promotes differentiation of hPS cells. Activin A activates SMAD-2/3 which controls self-renewal and differentiation while activin A together with FGF-2 activates both ERK-1/2 and PKCs. Inhibition of both ERK-1/2 and PKCs pathway provides a metastable undifferentiated state of hPS cells. Blue arrow indicated pathway promoting hPS cell self-renewal and black arrow indicated pathway promoting hPS cell differentiation.</p