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C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

ABSTRACT: Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27(Kip1) and p57(Kip2). These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBPdelta, but not DeltaNp63alpha, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBPdelta is recruited to the chromatin of positively (p27(Kip1) and p57(Kip2)) and negatively (p16(INK4A) and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

SUBMITTER: Barbaro V

PROVIDER: S-EPMC2064364 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

Barbaro Vanessa V Testa Anna A Di Iorio Enzo E Mavilio Fulvio F Pellegrini Graziella G De Luca Michele M

The Journal of cell biology 20070611 6

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein delta (C/EBPdelta), Bmi1, and DeltaNp63alpha identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBPdelta and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBPdelta inhibits the grow ...[more]

PMID: 17562792

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Project description: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.

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C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

Publications

C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells.

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