Stat3 signaling regulates embryonic stem cell fate in a dose-dependent manner.
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ABSTRACT:
SUBMITTER: Tai CI
PROVIDER: S-EPMC4197444 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1beta (TIF1beta), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4-dependent transcription in a phosphorylation-dependent manner. TIF1beta is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1beta is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcd1 [corrected], Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1beta specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1beta regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1beta provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.
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Project description:Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P2 expression. Inhibition of S1P2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.
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Project description:Signal transducers and activators of transcription 3 (STAT3) is a pleiotropic transcription factor involved in a variety of physiological processes. STAT3 acts as a key transcriptional determinant of mouse embryonic stem (ES) cell self-renewal and plays a pivotal function in early mammalian embryogenesis because the development of many organs requires STAT3 activation. However, little is known about the role of STAT3 function during ES cell differentiation. To answer this question, we built a lentiviral construct with 7-repeat STAT3-binding sequence (enhancer) and minimal TA (promoter) driving renilla luciferase and monomeric red fluorescence protein (Rluc-mRFP), followed by a constitutive cytomegalovirus promoter driving green fluorescent protein as a selection marker. The specificity of our custom-designed 7-repeat STAT3 reporter construct was first confirmed by cotransfection with constitutively active version of STAT3 (STAT3C) into human embryonic kidney 293T cells. Next, a mouse ES cell line stably transduced with STAT3 reporter construct was isolated. This ES cell line showed a tight response in reporter gene expression with leukemia inhibitory factor (LIF) induction and was chosen as a developmental model for the STAT3 functional study. Using serial noninvasive bioluminescence imaging, we showed that the onset of embryoid body (EB) formation involved inhibition of STAT3 activity. However, during differentiation, STAT3 activity steadily increased from day 5 to 14 and was reduced by day 21. STAT3 activity was also confirmed separately by Western blots. Finally, phosphorylation of STAT3 was also found to correspond with cardiomyocyte differentiation. In summary, this is the first study to monitor real-time STAT3 activity during ES cell differentiation. This genetically modified line can be used to study the biological role of STAT3 during ES cell differentiation into different derivatives.
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Project description:Research on human embryonic stem cells (hESCs) has attracted much attention given their great potential for tissue regenerative therapy and fundamental developmental biology studies. Yet, there is still limited understanding of how mechanical signals in the local cellular microenvironment of hESCs regulate their fate decisions. Here, we applied a microfabricated micromechanical platform to investigate the mechanoresponsive behaviors of hESCs. We demonstrated that hESCs are mechanosensitive, and they could increase their cytoskeleton contractility with matrix rigidity. Furthermore, rigid substrates supported maintenance of pluripotency of hESCs. Matrix mechanics-mediated cytoskeleton contractility might be functionally correlated with E-cadherin expressions in cell-cell contacts and thus involved in fate decisions of hESCs. Our results highlighted the important functional link between matrix rigidity, cellular mechanics, and pluripotency of hESCs and provided a novel approach to characterize and understand mechanotransduction and its involvement in hESC function.
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Project description:C3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-β1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation.
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Project description:An improved understanding of the pluripotency maintenance of embryonic stem (ES) cells is important for investigations of early embryo development and for cell replacement therapy, but the mechanism behind pluripotency is still incompletely understood. Recent findings show that zinc, an essential trace element in humans, is critically involved in regulating various signaling pathways and genes expression. However, its role in ES cell fate determination remains to be further explored. Here we showed that 2?M zinc chloride (ZnCl2) transiently maintained mouse ES cell pluripotency in vitro. The cultured mouse ES cells remained undifferentiated under 2?M ZnCl2 treatment in leukemia inhibitory factor (LIF) withdrawal, retinoic acid (RA) or embryoid bodies (EBs) differentiation assays. In addition, ZnCl2 increased pluripotency genes expression and inhibited differentiation genes expression. Further mechanistic studies revealed that ZnCl2 transiently activated signal transducers and activators of transcription 3 (Stat3) signaling through promoting Stat3 phosphorylation. Inhibition of Stat3 signaling abrogated the effects of ZnCl2 on mouse ES cell pluripotency. Taken together, this study demonstrated a critical role of zinc in the pluripotency maintenance of mouse ES cells, as well as an important regulator of Stat3 signaling.
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