During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

• MeSH
• aktivinové receptory typu I chemie   metabolismus   MeSH
• aktiviny metabolismus   MeSH
• benzamidy farmakologie   MeSH
• buněčná diferenciace * účinky léků   MeSH
• buněčný rodokmen účinky léků   MeSH
• dioxoly farmakologie   MeSH
• embryonální kmenové buňky cytologie   účinky léků   enzymologie   MeSH
• endoderm cytologie   účinky léků   metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• mezoderm cytologie   účinky léků   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• neurony cytologie   účinky léků   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• protein Smad2 metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * účinky léků   MeSH
• tyrosinfosfatasa nereceptorového typu 1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The inevitable accumulation of chromosomal abnormalities in human embryonic stem cells (hESCs) during in vitro expansion represents a considerable obstacle for cell replacement therapies. To determine the source of chromosomal abnormalities, we examined hESCs maintained in culture for over 55 months for defects in telomere maintenance and DNA repair. Although prolonged culture affected neither telomerase activity nor nonhomologous end joining, the efficiency of base excision repair (BER) was significantly decreased and correlated with reduced expression of apurinic/apyrimidinic endonuclease 1 (APE1), the major nuclease required for BER. Interestingly, the expression of other BER enzymes was unchanged. Addition of human recombinant APE1 protein to nuclear extracts from late passage hESCs increased BER efficiency to the level typical of early passage hESCs. The link between BER and double-strand breaks (DSB) was demonstrated by decreased DSB release after downregulation of APE1 in early passage hESCs via siRNA. Correspondingly lower APE1 level in late passage hESC resulted in slower and less intensive but long lasting DSB release upon ionizing radiation (IR). Downregulation of APE1 in early passage hESCs also led to approximately 30% decrease in γ-H2AX signaling following IR, similar to that in late passage hESCs. We suggest that downregulation of APE1 significantly contributes to the failure of BER during long-term culture of hESCs, and further that BER failure is one of the factors affecting the genomic instability of hESCs by altering BER-dependent DSB release and cell cycle/checkpoint signaling.

• MeSH
• buněčné linie MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA MeSH
• embryonální kmenové buňky enzymologie   metabolismus   MeSH
• imunohistochemie MeSH
• karyotypizace MeSH
• lidé MeSH
• oprava DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cyclin-dependent kinase two (Cdk2) is the major regulator of the G1/S transition and the target of an activated G1 checkpoint in somatic cells. In the presence of DNA damage, Cdk2 kinase activity is abrogated by a deficiency of Cdc25A phosphatase, which is marked by Chk1/Chk2 for proteasomal degradation. Embryonic stem cells (ESCs) lack a G1 checkpoint response. In this study, we analyzed the G1 checkpoint pathways in mouse ESCs (mESCs) in the presence of DNA double-strand breaks evoked by ionizing radiation (IR). We show that checkpoint pathways, which operate during G1 phase in somatic cells, are activated in mESCs after IR; however, Cdk2 activity is not abolished. We demonstrate that Cdc25A is degraded in mESCs, but this degradation is not regulated by Chk1 and Chk2 kinases because they are sequestered to the centrosome. Instead, Cdc25A degradation is governed by glycogen synthase kinase-3beta kinase. We hypothesize that Cdc25A degradation does not inhibit Cdk2 activity because a considerable proportion of Cdk2 molecules localize to the cytoplasm and centrosomes in mESCs, where they may be sheltered from regulation by nuclear Cdc25A. Finally, we show that a high Cdk2 activity, which is irresponsive to DNA damage, is the driving force of the rapid escape of mESCs from G1 phase after DNA damage.

• MeSH
• aktivace enzymů genetika   MeSH
• buněčné linie MeSH
• buněčný cyklus genetika   MeSH
• CDC geny fyziologie   MeSH
• centrozom enzymologie   MeSH
• cyklin-dependentní kinasa 2 genetika   MeSH
• cytoplazma enzymologie   MeSH
• DNA genetika   účinky záření   MeSH
• embryonální kmenové buňky cytologie   enzymologie   MeSH
• fosfatasy cdc25 genetika   MeSH
• G1 fáze genetika   MeSH
• ionizující záření MeSH
• kinasa 3 glykogensynthasy metabolismus   MeSH
• myši MeSH
• oprava DNA MeSH
• poškození DNA genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• proteinkinasy genetika   MeSH
• signální transdukce genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH