Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal

Abstract

BackgroundThe 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.Methodology/Principal FindingsIn 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/SignificanceOur 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.

Protein Kinase C-Induced Early Growth Response Protein-1 Binding to SNAIL Promoter in Epithelial–Mesenchymal Transition of Human Embryonic Stem Cells

“…T he balance between self-renewal and differentiation of human pluripotent stem (hPS) cells, including embryonic stem (hES) and induced pluripotent stem cells, is controlled by various signal pathways [1][2][3][4][5][6][7][8], including transforming growth factor-b/activin A/Nodal [9][10][11], sphingosine-1-phosphate/platelet-derived growth factor [12], insulin growth factor (IGF)/insulin [13], and fibroblast growth factor-2 (FGF-2) [14][15][16][17]. In particular, FGF-2 signaling appears indispensable to maintain the undifferentiated state of hPS cells [18][19][20].…”

“…On the other hand, the ERK-1/2 pathway has reported to have diverse roles in hPS cells, such as self-renewal, cell attachment, and cell differentiation [2,[25][26][27]. FGF-2 also induces inactive phosphorylation of glycogen synthase kinase-3 (GSK-3) [1,3,4,28] of which function has been reported to be controversial for self-renewal of hPS cells [29,30]. Moreover, we have recently reported that FGF-2 induces protein kinase C (PKC), which leads to phosphorylation of GSK3b [4].…”

“…FGF-2 also induces inactive phosphorylation of glycogen synthase kinase-3 (GSK-3) [1,3,4,28] of which function has been reported to be controversial for self-renewal of hPS cells [29,30]. Moreover, we have recently reported that FGF-2 induces protein kinase C (PKC), which leads to phosphorylation of GSK3b [4]. An activator of PKC, phorbol 12-myristate 13-acetate (PMA) changes the sharp-edged, flat, and tightly packed colonies of hPS cells to scattered cells, indicating that PKC induces the epithelial-mesenchymal transition (EMT) in hPS cells [4].…”

Bone morphogenetic protein 4 promotes craniofacial neural crest induction from human pluripotent stem cells

“…The human ES cell line, H9 (WA09, WISC Bank, WiCell Research Institute, Madison, WI, USA) was routinely maintained on mouse embryo fibroblast feeder cells inactivated by g-irradiation (MEF CF-1, Applied StemCell, Inc., CA, USA) in a hESC expansion medium (KSR-based medium) consisting of DMEM/F12 medium (Life Technologies, CA, USA) supplemented with 20% KSR (Life Technologies Ltd, Paisley, UK), 0.1 mM 2-mercaptoethanol (Sigma, St. Louis, MO, USA), 2 mM L-glutamine (Gibco, NY, USA), 0.1 mM non-essential amino acids (Gibco), and 5 ng/ ml human recombinant FGF-2 (Katayama Kagaku Kogyo, Japan) as previously described (Kinehara et al, 2013). The cells were split at a ratio of 1:8-1:10 every 6-7 days.…”

“…Trunk NC cells migrate along the dorsal and lateral pathway and ously reported that culture in the presence of BMP4 in combination with FGF-2 induces the up-regulation of NC marker genes, including AP2a and P0 protein, during the differentiation of mouse embryonic stem cells (ESCs) into NC cells under chemically defined conditions (Aihara et al, 2010, Furue, 2012. Because culture systems for the stable expansion of human pluripotent stem cells had been developed (Amit et al, 2000, Furue et al, 2008, Kinehara et al, 2013, in vitro studies producing NC cells from human pluripotent stem cells have been reported (Fukuta et al, 2014, Lee et al, 2010, Menendez et al, 2013, Menendez et al, 2011. Dalton et al (Menendez et al, 2013, Menendez et al, 2011 have demonstrated that a combination of Wnt activation and Smad inhibition under serum-free culture conditions directs the differentiation of human ESCs (hESCs) and induced pluripotent stem cells into NC stem cells (NCSCs).…”