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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. Ho...

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Autores principales: Kinehara, Masaki, Kawamura, Suguru, Tateyama, Daiki, Suga, Mika, Matsumura, Hiroko, Mimura, Sumiyo, Hirayama, Noriko, Hirata, Mitsuhi, Uchio-Yamada, Kozue, Kohara, Arihiro, Yanagihara, Kana, Furue, Miho K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549959/
https://www.ncbi.nlm.nih.gov/pubmed/23349801
http://dx.doi.org/10.1371/journal.pone.0054122
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author Kinehara, Masaki
Kawamura, Suguru
Tateyama, Daiki
Suga, Mika
Matsumura, Hiroko
Mimura, Sumiyo
Hirayama, Noriko
Hirata, Mitsuhi
Uchio-Yamada, Kozue
Kohara, Arihiro
Yanagihara, Kana
Furue, Miho K.
author_facet Kinehara, Masaki
Kawamura, Suguru
Tateyama, Daiki
Suga, Mika
Matsumura, Hiroko
Mimura, Sumiyo
Hirayama, Noriko
Hirata, Mitsuhi
Uchio-Yamada, Kozue
Kohara, Arihiro
Yanagihara, Kana
Furue, Miho K.
author_sort Kinehara, Masaki
collection PubMed
description 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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.
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spelling pubmed-35499592013-01-24 Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal Kinehara, Masaki Kawamura, Suguru Tateyama, Daiki Suga, Mika Matsumura, Hiroko Mimura, Sumiyo Hirayama, Noriko Hirata, Mitsuhi Uchio-Yamada, Kozue Kohara, Arihiro Yanagihara, Kana Furue, Miho K. PLoS One Research Article 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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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. Public Library of Science 2013-01-21 /pmc/articles/PMC3549959/ /pubmed/23349801 http://dx.doi.org/10.1371/journal.pone.0054122 Text en © 2013 Kinehara et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kinehara, Masaki
Kawamura, Suguru
Tateyama, Daiki
Suga, Mika
Matsumura, Hiroko
Mimura, Sumiyo
Hirayama, Noriko
Hirata, Mitsuhi
Uchio-Yamada, Kozue
Kohara, Arihiro
Yanagihara, Kana
Furue, Miho K.
Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title_full Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title_fullStr Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title_full_unstemmed Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title_short Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal
title_sort protein kinase c regulates human pluripotent stem cell self-renewal
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549959/
https://www.ncbi.nlm.nih.gov/pubmed/23349801
http://dx.doi.org/10.1371/journal.pone.0054122
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