Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells

The past few years have seen remarkable progress in our understanding of embryonic stem cell (ES cell) biology. The necessity of examining human ES cells in culture, coupled with the wealth of genomic data and the multiplicity of cell lines available, has enabled researchers to identify critical conserved pathways regulating self-renewal and identify markers that tightly correlate with the ES cell state. Comparison across species has suggested additional pathways likely to be important in long-term self-renewal of ES cells including heterochronic genes, microRNAs, genes involved in telomeric regulation, and polycomb repressors. In this review, we have discussed information on molecules known to be important in ES cell self-renewal or blastocyst development and highlighted known differences between mouse and human ES cells. We suggest that several additional pathways required for self-renewal remain to be discovered and these likely include genes involved in antisense regulation, microRNAs, as well as additional global repressive pathways and novel genes. We suggest that cross species comparisons using large-scale genomic analysis tools are likely to reveal conserved and divergent paths required for ES cell self-renewal and will allow us to derive ES lines from species and strains where this has been difficult.     

Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells

Although basic fibroblast growth factor (FGF2) is generally included in the media for maintenance of human embryonic stem cells (hESCs), the action of FGF2 in these cells has not been well defined. Here, we determined the roles of FGF2 in maintaining hESC self-renewal. Withdrawal of FGF2 from the media led to acquisition of typical differentiated characteristics in hESCs. In the presence of FGF2, which is normally required for proliferation in an undifferentiated state, inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt/PKB signal stimulated differentiation and attenuated the expression of extracellular matrix (ECM) molecules. We suggest that FGF2 maintains hESC self-renewal by supporting stable expression of ECM molecules through activation of the PI3K/Akt/PKB pathway.     

PI3K/Akt and CREB regulate adult neural hippocampal progenitor proliferation and differentiation

The phosphoinositide 3-OH kinase (PI3K)/Akt pathway has been implicated in regulating several important cellular processes, including apoptosis, survival, proliferation, and metabolism. Using both pharmacological and genetic means, we demonstrate here that PI3K/Akt plays a crucial role in the proliferation of adult hippocampal neural progenitor cells. PI3K/Akt transduces intracellular signals from multiple mitogens, including basic fibroblast growth factor (FGF-2), Sonic hedgehog (Shh), and insulin-like growth factor 1 (IGF-1). In addition, retroviral vector-mediated over-expression of wild type Akt increased cell proliferation, while a dominant negative Akt inhibited proliferation. Furthermore, wild type Akt over-expression reduced glial (GFAP) and neuronal (beta-tubulin III) marker expression during differentiation, indicating that it inhibits cell differentiation. We also show that activation of the cAMP response element binding protein (CREB), which occurs in cells stimulated by FGF-2, is limited when Akt signaling is inhibited, demonstrating a link between Akt and CREB. Over-expression of wild type CREB increases progenitor proliferation, whereas dominant negative CREB only slightly decreases proliferation. These results indicate that PI3K/Akt signaling integrates extracellular signaling information to promote cellular proliferation and inhibit differentiation in adult neural progenitors.     

microRNA-873 inhibits self-renewal and proliferation of pancreatic cancer stem cells through pleckstrin-2-dependent PI3K/AKT pathway

Recent studies have emphasized microRNAs (miRs) as crucial regulators in the occurrence and development of pancreatic cancer that continues to be one of the deadliest malignancies with few effective therapies. The study aimed to investigate the functional role of miR-873 and its associated mechanism to unravel the biological characteristics of pancreatic cancer stem cells in tumor growth. The expression patterns of pleckstrin-2 (PLEK2) and miR-873 were detected in the pancreatic cancer tissues. Then to further investigate specific role of miR-873, the pancreatic cancer stem cells were treated with miR-873 mimic, PLEK2, small interfering RNA against PLEK2, LY294002 (inhibitor of phosphatidylinositol 3-kinase/protein kinase B [PI3K/AKT] pathway) to detect the relative gene expression as well as their effects on cell self-renewal, proliferation and apoptosis. Finally, the tumor formation in nude mice was measured to verify the preceding results in vivo. Pancreatic cancer tissues exhibited a decline of miR-873 expression and an enhancement of PLEK2 expression. miR-873 targeted PLEK2 and downregulated its expression, leading to inhibition of PI3K/AKT pathway. Overexpressed miR-873 or silenced PLEK2 inhibited the self-renewal and proliferation while promoting the apoptosis of pancreatic cancer stem cells. Tumor formation was inhibited by overexpressed miR-873 or silenced PLEK2 in nude mice. Overall, miR-873 can suppress the self-renewal and proliferation of pancreatic cancer stem cells by blocking PLEK2-dependent PI3K/AKT pathway. Hence, this study contributes to understanding the role of miR-873 in pancreatic cancer stem cells and its underlying molecular mechanisms to aid in the development of effective pancreatic cancer therapeutics.     

高浓度七氟醚通过抑制VEGF/PI3K/AKT信号通路诱导神经干细胞凋亡的研究

目的探讨妊娠中期的七氟醚暴露对神经干细胞凋亡过程的影响和作用机制。 方法将孕中期大鼠随机分为3组,每组48只孕鼠：对照组,低浓度七氟醚组,高浓度七氟醚组。在妊娠第14天,以2﹪或3.5﹪七氟醚麻醉怀孕大鼠2?h。通过免疫荧光检查神经干细胞凋亡,收集麻醉后6、24和48?h以及出生后第0天（P?0）,第14天（P?14）和第28天（P?28）的脑组织进行Nestin-TUNEL免疫荧光双标染色以及Nestin、血管内皮生长因子（VEGF）和磷酸肌醇3-激酶（PI3K）AKT通路相关蛋白的免疫印迹检测。采用单因素方差分析和Bonferroni事后检验进行统计学分析。 结果麻醉后6、24和48?h以及P?0,P?14和P?28,脑组织中Nestin和TUNEL阳性细胞的百分比增加[6?h：对照组0.91±0.07,低浓度组1.01±0.08,高浓度组2.62±0.21（F?=?399,P?相似文献   

A novel GSK3 inhibitor that promotes self-renewal in mouse embryonic stem cells

ABSTRACT

Small molecules that regulate cell stemness have the potential to make a major contribution to regenerative medicine. In the course of screening for small molecules that affect stemness in mouse embryonic stem cells (mESCs), we discovered that NPD13432, an aurone derivative, promoted self-renewal of mESCs. Normally, mESCs start to differentiate upon withdrawal of 2i/LIF. However, cells treated with the compound continued to express endogenous Nanog, a pluripotency marker protein essential for sustaining the undifferentiated state, even in the absence of 2i/LIF. Biochemical characterization revealed that NPD13432 inhibited GSK3α and GSK3β with IC₅₀ values of 92 nM and 310 nM, respectively, suggesting that the compound promotes self-renewal in mESCs by inhibiting GSK3. The chemical structure of the compound is unique among known molecules with this activity, providing an opportunity to develop new inhibitors of GSK3, as well as chemical tools for investigating cell stemness.     

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures

ABSTRACT: The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up-regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up-regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes.     

PI3K activates negative and positive signals to regulate TRB3 expression in hepatic cells

TRB3 is a pseudokinase whose expression is regulated during stress response and changing of nutrient status. TRB3 negatively regulates Akt activation and noticeably, TRB3 expression is induced by insulin. Here, we sought to determine the dynamic relationship between TRB3 expression and Akt activation. We find that insulin induces TRB3 expression in cell type dependent manner such that in hepatic cells and adipocytes but not Beta cells and muscle cells. In Fao hepatoma cells, induction of TRB3 expression by insulin restrains Akt activation and renders Akt refractory to further activation. In addition, we have also analyzed the roles of PI3K and its downstream kinases Akt and atypical PKC in TRB3 expression. Induction of TRB3 expression by insulin requires PI3K. However, inactivation of Akt enhances TRB3 expression whereas inhibition of PKCzeta expression impairs TRB3 expression induced by insulin. Our data demonstrated that PI3K conveys both negative and positive signals to TRB3 expression. We suggest that insulin-induced TRB3 expression functions as an indicator how multiple insulin-induced signal transduction pathways are balanced.     

The RasGAP N-terminal fragment generated by caspase cleavage protects cells in a Ras/PI3K/Akt-dependent manner that does not rely on NFkappa B activation

RasGAP, a regulator of Ras GTPase family members, is cleaved at low levels of caspase activity into an N-terminal fragment (fragment N) that generates potent anti-apoptotic signals. At higher levels of caspase activity, fragment N is further cleaved into two fragments that strongly potentiate apoptosis. RasGAP could thus function as a sensor of caspase activity to determine whether a cell should survive or not. Here we show that fragment N protects cells by activating the Ras-PI3K-Akt pathway. Surprisingly, even though nuclear factor kappaB (NFkappaB) can be activated by Akt, it plays no role in the anti-apoptotic functions of fragment N. This indicates that Akt effectors are differentially regulated when fragment N is generated.     

PI3K/AKT and ERK regulate retinoic acid-induced neuroblastoma cellular differentiation

Thymoquinone (TQ), a bioactive component of black caraway seed (Nigella sativa) oil, is reported to have antineoplastic properties. In this study we investigated the effect of TQ on a panel of human breast cancer cell lines. Cell viability assays showed that TQ killed T-47D, MDA-MB-231, and MDA-MB-468 cells via p53-independent induction of apoptosis; however, MCF-7 cells were refractory to the cytotoxic action of TQ. Western Blot analysis showed that MCF-7 cells expressed high levels of cytoprotective NADPH quinone oxidoreductase 1 (NQO1), which was responsible for TQ-resistance since inhibition of NQO1 with dicoumarol rendered MCF-7 cells TQ-sensitive. These findings may be clinically important when considering TQ as a possible adjunct treatment for breast cancer since a high percentage of breast tumors express NQO1.     

Characteristics of the PI3K/AKT and MAPK/ERK pathways involved in the maintenance of self-renewal in lung cancer stem-like cells

Lung cancer is the leading cause of cancer-related mortality worldwide due to its early asymptomatic and late metastasis. While cancer stem cells (CSCs) may play a vital role in oncogenesis and development of lung cancer, mechanisms underlying CSCs self‐renewal remain less clear. In the present study, we constructed a clinically relevant CSCs enrichment recognition model and evaluated the potential functions of phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT) and mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) pathways in lung cancer via bioinformatic analysis, providing the basis for in depth mechanistic inquisition. Experimentally, we confirmed that PI3K/AKT pathway predominantly promotes proliferation through anti-apoptosis in lung adenocarcinoma cells, while MAPK/ERK pathway has an overwhelming superiority in regulating the proliferation in lung CSCs. Further, utilizing stemness score model, LLC-Symmetric Division (LLC-SD) cells and mouse orthotopic lung transplantation model, we elucidated an intricate cross-talk between the oncogenic pathway and the stem cell reprograming pathway that impact stem cell characteristics as well as cancer biology features of lung CSCs both in vitro and in vivo. In summary, our findings uncovered a new insight that PI3K/AKT and MAPK/ERK pathways as oncogenic signaling pathway and/or stem cell signaling pathway act distinctively and synergistically to regulate lung CSCs self-renewal.     

Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.     

Makorin-2 is a neurogenesis inhibitor downstream of phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signal

Makorin-2 belongs to the makorin RING zinc finger gene family, which encodes putative ribonucleoproteins. Here we cloned the Xenopus makorin-2 (mkrn2) and characterized its function in Xenopus neurogenesis. Forced overexpression of mkrn2 produced tadpoles with dorso-posterior deficiencies and small-head/short-tail phenotype, whereas knockdown of mkrn2 by morpholino antisense oligonucleotides induced double axis in tadpoles. In Xenopus animal cap explant assay, mkrn2 inhibited activin, and retinoic acid induced animal cap neuralization, as evident from the suppression of a pan neural marker, neural cell adhesion molecule. Surprisingly, the anti-neurogenic activity of mkrn2 is independent of the two major neurogenesis signaling cascades, BMP-4 and Wnt8 pathways. Instead, mkrn2 works specifically through the phosphatidylinositol 3-kinase (PI3K) and Akt-mediated neurogenesis pathway. Overexpression of mkrn2 completely abrogated constitutively active PI3K- and Akt-induced, but not dominant negative glycogen synthase kinase-3beta (GSK-3beta)-induced, neural cell adhesion molecule expression, indicating that mkrn2 acts downstream of PI3K and Akt and upstream of GSK-3beta. Moreover, mkrn2 up-regulated the mRNA and protein levels of GSK-3beta. These results revealed for the first time the important role of mkrn2 as a new player in PI3K/Akt-mediated neurogenesis during Xenopus embryonic development.     

Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells

Neuroepithelial cells undergoing differentiation efficiently remodel their cytoskeleton and shape in an energy-consuming process. The capacity of autophagy to recycle cellular components and provide energy could fulfill these requirements, thus supporting differentiation. However, little is known regarding the role of basal autophagy in neural differentiation. Here we report an increase in the expression of the autophagy genes Atg7, Becn1, Ambra1 and LC3 in vivo in the mouse embryonic olfactory bulb (OB) during the initial period of neuronal differentiation at E15.5, along with a parallel increase in neuronal markers. In addition, we observed an increase in LC3 lipidation and autophagic flux during neuronal differentiation in cultured OB-derived stem/progenitor cells. Pharmacological inhibition of autophagy with 3-MA or wortmannin markedly decreased neurogenesis. These observations were supported by similar findings in two autophagy-deficient genetic models. In Ambra1 loss-of-function homozygous mice (gt/gt) the expression of several neural markers was decreased in the OB at E13.5 in vivo. In vitro, Ambra1 haploinsufficient cells developed as small neurospheres with an impaired capacity for neuronal generation. The addition of methylpyruvate during stem/progenitor cell differentiation in culture largely reversed the inhibition of neurogenesis induced by either 3-MA or Ambra1 haploinsufficiency, suggesting that neural stem/progenitor cells activate autophagy to fulfill their high energy demands. Further supporting the role of autophagy for neuronal differentiation Atg5-null OB cells differentiating in culture displayed decreased TuJ1 levels and lower number of cells with neurites. These results reveal new roles for autophagy-related molecules Atg5 and Ambra1 during early neuronal differentiation of stem/progenitor cells.