Many studies reported that FGF-2 activates both the MAPK/ERK, and PI3K/AKT pathways, which are important for maintaining pluripotency and viability in hPS cells 
. However, FGF-2 downstream signaling is not clearly understood in hPS cells. In this study using a minimum essential defined culture system 
, we showed that FGF-2 activated PI3K/AKT and MEK/ERK-1/2, but also PKCδ, ε and ζ isoforms in hPS cells ().
Model for the molecular mechanism of PKCs regulating self-renewal or differentiation in hPS cells.
The PKC family has been implicated as an intracellular mediator of several neurotransmitters, hormones, tumor promoters, α1-adrenergic agonists, and phorbol esters, and it is important in the regulation of growth, differentiation, cell death, and neurotransmission 
. The PKC family comprises classical (PKCα, β, and γ; activated by Ca2+
and phorbol esters), novel PKC (PKCδ, ε, η, and θ; activated by phorbol esters but not regulated by Ca2+
), and atypical PKC (PKCζ and PKCι/λ; not activated by Ca2+
or phorbol esters). Different isoforms may perform distinct functions, as suggested by their differential pattern of localization, differences in condition of activation, and some differences in substrate specificity 
. PKC has previously been implicated in GSK-3 regulation 
. Fang et al. 
showed that PKCα, βII, γ, η, and δ were capable of phosphorylating GSK-3β while PKCε and PKCζ did not phosphorylate GSK-3 by in vitro kinase assays; also, expression of constitutively active PKCα, βI, γ, η enhanced phosphorylation of cotransfected GSK-3β in HEK293 cells. On the other hand, Eng et al. 
reported that negative construct of PKCε isoform prevented phosphorylation of GSK-3 in migrating fibroblasts._These pieces of evidence suggested that specific isoforms of PKC have different roles in different types of cells. Shuibing et al. 
reported that activation of PKCα and/or β directs the pancreatic specification of hES cells. Recently, Feng et al. 
reported that activation of PKCδ induces extraembryonic endoderm differentiation of hES cells. These studies suggested that PKCs might be involved in differentiation of hPS cells. Our study showed that FGF-2 induced PKCδ, ε, and ζ, resulting in phosphorylation of GSK-3β, ERK-1/2, or AKT. Chou, et al. 
reported that the phosphorylation of PKCζ was regulated by PI3-kinase and PDK-1 in NIH 3T3 fibroblasts. Intriguingly, PKCζ can stimulate GSK-3 activity, by relieving PKB-imposed inhibition 
. In mouse ES cells, it has been shown that PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ–nuclear factor kappa-light-chain-enhancer of activated B cells signaling axis 
. However, PKC inhibition does not change phosphorylation of ERK-1/2 or GSK-3β. In view of the fact that LIF mainly regulates self-renewal in mouse ES cells, isoform specific function might be cross-regulated by other signaling in the cells. Further, our study showed that the combination effect by inhibition of PKCα, β, γ, δ, ε, and ζ was different from that by inhibition of PKCε and ζ, suggesting that each PKC might interact in different contexts and also PKCδ, ε, and ζ might have different activation mechanisms in hPS cells. It is needed further investigation in future.
GSK-3β is inhibited by phosphorylation stimulated by the canonical Wnt signal pathway, which is followed by the accumulation of β-catenin to the nucleus 
. From the above findings, it follows that FGF-2 may activate Wnt signaling through PKC leading to differentiation of hPS cells. This conclusion contradicts the findings of previous studies demonstrating that canonical Wnt signaling supports self-renewal of stem cells 
. However, it is consistent with a study showing that canonical Wnt signaling does not appear to promote stem cell maintenance, which prevents differentiation of stem cells 
. On the other hand, some studies have shown a dual function for Wnt signaling in hES cells in that the pathways of self-renewal or differentiation are dependent on the presence of hES cell supporting factors 
. Recently, Ding et al. 
showed that FGF-2 modulates Wnt signaling through AKT/GSK-3β signaling and suggested that the differences in the results could be due to the culture platform. Our findings suggest that GSK-3β activity is regulated by FGF-2 through both PI3K/AKT and PKC pathways. AKT/GSK-3β signaling may support self-renewal whereas PKC/GSK-3β may promote cell differentiation of hPS cells. However, GFX decreased the phosphorylation level of GSK-3β to lower level than non-treatment. GSK-3β signaling might be stimulated also by other signal pathway in hPS cells. Target genes of these pathways and further regulation mechanisms in GSK-3β signaling should be analyzed in future.
TGF-β/activin/nodal pathways are thought to crosstalk with FGF signaling in regulating hPS cells. Vallier et al. 
demonstrated that activin/nodal pathway in co-operation with FGF-2 is necessary for the maintenance of pluripotency in hES cells. We recently reported that activin A enhances FGF-2-induced ERK-1/2, which permits neural and mesendodermal differentiation of hES cells 
. In this study we showed that activin A enhanced FGF-2-induced phosphorylation of not only ERK-1/2 but also GSK-3β. Inhibition of these pathways provided stable culture of hPS cells for long-term. In this study, we used both GFX and U0126 to inhibit these pathways. GFX targeting all of PKCα, β, γ, δ, ε, and ζ had no inhibitory effect on ERK-1/2 pathway although siRNA targeting PKCε or PKCζ decreased it. If more specific inhibitor is developed in future, it would be more useful. To maintain undifferentiated state, balancing among ERK-1/2, PI3K, SMAD, and PKC signal pathways may be required in any culture conditions. KSR of which components are not disclosed in public is known to have BMP-4-like activity 
. Some components including BMP-4 in KSR together with secreting factors from mouse feeders might regulate PKC/ERK-1/2 signaling. Using our defined conditions, more molecules including growth factors would be screened to detect their accurate effects on hPS cells.
In conclusion, our study suggested that FGF-2 induced PI3K/AKT and MEK/ERK-1/2, but also PKCs in hPS cells. PI3K/AKT promotes cell self-renewal whereas the MEK/ERK-1/2, PKC/ERK-1/2 and PKC/GSK-3β pathways down-regulate hPS cell self-renewal. This study helps to untangle the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.