AICAR induces astroglial differentiation of neural stem cells via activating the JAK/STAT3 pathway independently of AMP-activated protein kinase

Neural stem cell differentiation and the determination of lineage decision between neuronal and glial fates have important implications in the study of developmental, pathological, and regenerative processes. Although small molecule chemicals with the ability to control neural stem cell fate are considered extremely useful tools in this field, few were reported. AICAR is an adenosine analog and extensively used to activate AMP-activated protein kinase (AMPK), a metabolic "fuel gauge" of the biological system. In the present study, we found an unrecognized astrogliogenic activity of AICAR on not only immortalized neural stem cell line C17.2 (C17.2-NSC), but also primary neural stem cells (NSCs) derived from post-natal (P0) rat hippocampus (P0-NSC) and embryonic day 14 (E14) rat embryonic cortex (E14-NSC). However, another AMPK activator, Metformin, did not alter either the C17.2-NSC or E14-NSC undifferentiated state although both Metformin and AICAR can activate the AMPK pathway in NSC. Furthermore, overexpression of dominant-negative mutants of AMPK in C17.2-NSC was unable to block the gliogenic effects of AICAR. We also found AICAR could activate the Janus kinase (JAK) STAT3 pathway in both C17.2-NSC and E14-NSC but Metformin fails. JAK inhibitor I abolished the gliogenic effects of AICAR. Taken together, these results suggest that the astroglial differentiation effect of AICAR on neural stem cells was acting independently of AMPK and that the JAK-STAT3 pathway is essential for the gliogenic effect of AICAR.     

A novel tobacco mitogen-activated protein (MAP) kinase kinase, NtMEK1, activates the cell cycle-regulated p43Ntf6 MAP kinase

Two-hybrid screening of a tobacco BY-2 cell suspension cDNA library using the p43(Ntf6) mitogen-activated protein (MAP) kinase as bait resulted in the isolation of a cDNA encoding a protein with features characteristic of a MAP kinase kinase (MEK), which has been called NtMEK1. Two-hybrid interaction analysis and pull-down experiments showed a physical interaction between NtMEK1 and the tobacco MAP kinases p43(Ntf6) and p45(Ntf4), but not p43(Ntf3). In kinase assays NtMEK1 preferentially phosphorylated p43(Ntf6). Functional studies in yeast showed that p43(Ntf6) could complement the yeast MAP kinase mutant mpk1 when co-expressed with NtMEK1, and that this complementation depended on the kinase activity of p43(Ntf6). Expression analysis showed that the NtMEK1 and ntf6 genes are co-expressed both in plant tissues and following the induction of cell division in leaf pieces. These data suggest that NtMEK1 is an MEK for the p43(Ntf6) MAP kinase.     

In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen

BACKGROUND INFORMATION: MAPKs (mitogen-activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization. RESULTS: In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. CONCLUSIONS: The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.     

RhoGDIβ Inhibits Bone Morphogenetic Protein 4 (BMP4)-induced Adipocyte Lineage Commitment and Favors Smooth Muscle-like Cell Differentiation

The integration of signals involved in deciding the fate of mesenchymal stem cells is largely unknown. We used proteomics profiling to identify RhoGDIβ, an inhibitor of the small G-protein Rho family, as a component that regulates commitment of C3H10T1/2 mesenchymal stem cells to the adipocyte or smooth muscle cell lineage in response to bone morphogenetic protein 4 (BMP4). RhoGDIβ is notably down-regulated during BMP4-induced adipocytic lineage commitment of C3H10T1/2 mesenchymal stem cells, and this involves the cytoskeleton-associated protein lysyl oxidase. Excess RhoGDIβ completely prevents BMP4-induced commitment to the adipocyte lineage and simultaneously stimulates smooth muscle cell commitment by suppressing the activation of Rac1. Overexpression of RhoGDIβ induces stress fibers of F-actin by a process involving phosphomyosin light chain, indicating that cytoskeletal tension regulated by RhoGDIβ contributes to determining adipocyte versus myocyte commitment. Furthermore, the overexpression of RacV12 (constitutively active form of Rac1) totally rescues the inhibition of adipocyte commitment by RhoGDIβ, simultaneously preventing formation of the smooth muscle-like phenotype and disrupting the stress fibers in cells overexpressing RhoGDIβ. Collectively, these results indicate that RhoGDIβ functions as a novel BMP4 signaling target that regulates adipogenesis and myogensis.     

Dok-1 is a positive regulator of IL-4 signalling and IgE response

Interleukin-4 (IL-4) plays an essential role in the control of humoral immunity by regulating lymphocyte proliferation and differentiation, including the T helper type 2 lineage commitment of CD4(+) T cells as well as the isotype switching to IgE in B cells. The adaptor protein Dok-1 is known to have an essential role in the negative regulation of a variety of cytokine signalling events. However, here we have found that the loss of Dok-1 impaired the proliferative response of CD4(+) T cells and B cells to IL-4. Conversely, the forced expression of Dok-1 in the myeloid cell line 32D augmented the IL-4-induced proliferation, indicating a positive role for Dok-1. Tyrosine phosphorylation, and thereby the activation of Stat6 and IRS-2, is critical for IL-4 signalling; however, only the activation of Stat6, not the IRS-2-dependent phosphorylation of Akt, was perturbed in Dok-1-deficient cells stimulated with IL-4. Furthermore, mice lacking Dok-1 showed an impaired IgE response to thymus-dependent antigen. Thus, Dok-1 is a positive regulator of IL-4 signalling and IgE response.     

FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment

Pluripotent embryonic stem (ES) cells must select between alternative fates of self-replication and lineage commitment during continuous proliferation. Here, we delineate the role of autocrine production of fibroblast growth factor 4 (Fgf4) and associated activation of the Erk1/2 (Mapk3/1) signalling cascade. Fgf4 is the major stimulus activating Erk in mouse ES cells. Interference with FGF or Erk activity using chemical inhibitors or genetic ablations does not impede propagation of undifferentiated ES cells. Instead, such manipulations restrict the ability of ES cells to commit to differentiation. ES cells lacking Fgf4 or treated with FGF receptor inhibitors resist neural and mesodermal induction, and are refractory to BMP-induced non-neural differentiation. Lineage commitment potential of Fgf4-null cells is restored by provision of FGF protein. Thus, FGF enables rather than antagonises the differentiation activity of BMP. The key downstream role of Erk signalling is revealed by examination of Erk2-null ES cells, which fail to undergo either neural or mesodermal differentiation in adherent culture, and retain expression of pluripotency markers Oct4, Nanog and Rex1. These findings establish that Fgf4 stimulation of Erk1/2 is an autoinductive stimulus for na?ve ES cells to exit the self-renewal programme. We propose that the Erk cascade directs transition to a state that is responsive to inductive cues for germ layer segregation. Consideration of Erk signalling as a primary trigger that potentiates lineage commitment provides a context for reconciling disparate views on the contribution of FGF and BMP pathways during germ layer specification in vertebrate embryos.     

SRY directly regulates the neurotrophin 3 promoter during male sex determination and testis development in rats

Neurotrophin 3 (Ntf3) is expressed in Sertoli cells and acts as a chemo-attractant for cell migration from the mesonephros into the developing testis, a process critical to the early morphological events of testis cord formation. The male sex-determining gene Sry initiates the process of testicular development. Sox9 is a key regulator of male sex determination and is directly regulated by SRY. Information on other downstream target genes of SRY is limited. The current study demonstrates an interaction of SRY with the Ntf3 promoter both in vitro and in vivo. The Ntf3 promoter in both rat and mouse contains at least one putative SRY binding site in the -0.6 kb promoter region. In a luciferase reporter assay system, both SRY and SOX9 stimulated the Ntf3 promoter in vitro through an interaction with this SRY-binding motif. In an immunoprecipitation-based pull-down assay, recombinant SRY protein bound the Ntf3 promoter fragment containing an intact SRY binding site, whereas the same protein did not interact with the fragment containing a mutated SRY motif. Specific antibodies against SRY were used in a chromatin immunoprecipitation (ChIP) assay of embryonic testis and were found to precipitate the Ntf3 promoter region. The SRY ChIP assay confirmed the direct interaction between SRY and the Ntf3 promoter in vivo during male sex determination. Observations suggest that SRY physically interacts with the Ntf3 promoter during male sex determination to coordinate cell migration in the testis to form testis cords.     

Development of the CD4 and CD8 lineage of T cells: instruction versus selection

T cells bearing the alpha beta T cell receptor (TCR) can be divided into CD4+8- and CD4-8+ subsets which develop in the thymus from CD4+8+ precursors. The commitment to the CD4 and CD8 lineage depends on the binding of the alpha beta TCR to thymic major histocompatibility complex (MHC) coded class II and class I molecules, respectively. In an instructive model of lineage commitment, the binding of the alpha beta TCR, for instance to class I MHC molecules, would generate a specific signal instructing the CD4+8+ precursors to switch off the expression of the CD4 gene. In a selective model, the initial commitment, i.e. switching off the expression of either the CD4 or the CD8 gene would be a stochastic event which is then followed by a selective step rescuing only CD4+ class II and CD8+ class I specific T cells while CD4+ class I and CD8+ class II specific cells would have a very short lifespan. The selective model predicts that a CD8 transgene which is expressed in all immature and mature T cells should rescue CD4+ class I MHC specific T cells from cell death. We have performed experiments in CD8 transgenic mice which fail to support a selective model and we present data which show that the binding of the alpha beta TCR to thymic class I MHC molecules results in up-regulation of the TCR in the CD4+8+ population. Therefore, these experiments are consistent with an instructive model of lineage commitment.     

人乳头瘤病毒16型E6和E7基因及其突变体转化活性的研究

为筛选出可用于研制HPV治疗性疫苗的HPV16型E6和E7基因突变体,故将HPV16型原型株(德国株)E6和E7基因及其各种突变体分别转染Balb/c3T3细胞,观察转染后的细胞在软琼脂培养中的集落形成能力和在裸鼠体内的成瘤能力.结果表明,单独转染和共转染HPV16野生型E6和E7基因的Balb/c3T3细胞系,在软琼脂中呈集落样生长,并在裸鼠体内成瘤;而转染E6基因突变体mE6(50G)、E7基因的两种突变体mE7-1(24G26G)和mE7-3(24G26G67R)以及共转染mE6和mE7-1的Balb/c3T3细胞,在软琼脂培养中极少形成集落,也不能在裸鼠体内成瘤.提示经结构改造后的HPV16 E6和E7基因已失去了对Balb/c3T3细胞的转化活性,而保留了免疫原性,可用于HPV16相关肿瘤治疗性疫苗的构建.     

Modulation of subfamily B/R4 RGS protein function by 14-3-3 proteins

Regulator of G protein signalling (RGS) proteins are primarily known for their ability to act as GTPase activating proteins (GAPs) and thus attenuate G protein function within G protein-coupled receptor (GPCR) signalling pathways. However, RGS proteins have been found to interact with additional binding partners, and this has introduced more complexity to our understanding of their potential role in vivo. Here, we identify a novel interaction between RGS proteins (RGS4, RGS5, RGS16) and the multifunctional protein 14-3-3. Two isoforms, 14-3-3β and 14-3-3ε, directly interact with all three purified RGS proteins and data from in vitro steady state GTP hydrolysis assays show that 14-3-3 inhibits the GTPase activity of RGS4 and RGS16, but has limited effects on RGS5 under comparable conditions. Moreover in a competitive pull-down experiment, 14-3-3ε competes with Go for RGS4, but not for RGS5. This mechanism is further reinforced in living cells, where 14-3-3ε sequesters RGS4 in the cytoplasm and impedes its recruitment to the plasma membrane by G protein. Thus, 14-3-3 might act as a molecular chelator, preventing RGS proteins from interacting with G, and ultimately prolonging the signal transduction pathway. In conclusion, our findings suggest that 14-3-3 proteins may indirectly promote GPCR signalling via their inhibitory effects on RGS GAP function.     

Duration of calcineurin and Erk signals regulates CD4/CD8 lineage commitment of thymocytes

CD4/CD8 lineage commitment of thymocytes is controlled by the T cell receptor-mediated signals and is mimicked in vitro by a long-pulse stimulation of isolated CD4(+)CD8(+) thymocytes with proper combinations of phorbol myristate acetate and the calcium ionophore ionomycin. CD4 lineage commitment required higher intracellular Ca(2+) levels than CD8 lineage commitment in this culture system. The calcineurin inhibitor FK506 at 1nM inhibited the development of thymocytes to either lineage, but 0.3nM FK506 significantly switched the development from the CD4 cell fate to the CD8 cell fate. The switch in lineage commitment was also observed when 1nM FK506 was added 8h after the start of the culture. Delayed addition of 20microM U0126, an Mek (Erk kinase) inhibitor, also induced the switch. These results suggest that the intensity of calcineurin activity and the duration of both calcineurin and Erk pathway activation are crucial for thymocyte lineage commitment.     

VEGFR1 (Flt1) Regulates Rab4 Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear. Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports αvβ3 integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and αvβ3 integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.     

Notch promotes neural lineage entry by pluripotent embryonic stem cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.     

Sonic hedgehog signalling in T-cell development and activation

The production of mature functional T cells in the thymus requires signals from the thymic epithelium. Here, we review recent experiments showing that one way in which the epithelium controls the production of mature T cells is by the secretion of sonic hedgehog (SHH). We consider the increasing evidence that SHH-induced signalling is not only important for the differentiation and proliferation of early thymocyte progenitors, but also for modulating T-cell receptor signalling during repertoire selection, with implications for positive selection, CD4 versus CD8 lineage commitment, and clonal deletion of autoreactive cells. We also review the influence of hedgehog signalling in peripheral T-cell activation.     

Activation of Ntf4, a tobacco mitogen-activated protein kinase, during plant defense response and its involvement in hypersensitive response-like cell death

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells. They function downstream of sensors/receptors and regulate cellular responses to external and endogenous stimuli. Recent studies demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, are involved in signaling plant defense responses to various pathogens. Ntf4, another tobacco MAPK that shares 93.6% and 72.3% identity with SIPK and WIPK, respectively, was reported to be developmentally regulated and function in pollen germination. We found that Ntf4 is also expressed in leaves and suspension-cultured cells. Genomic analysis excluded the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. In vitro and in vivo phosphorylation and activation assays revealed that Ntf4 shares the same upstream MAPK kinase, NtMEK2, with SIPK and WIPK. Similar to SIPK and WIPK, Ntf4 is also stress responsive and can be activated by cryptogein, a proteinaceous elicitin from oomycetic pathogen Phytophthora cryptogea. Tobacco recognition of cryptogein induces rapid hypersensitive response (HR) cell death in tobacco. Transgenic Ntf4 plants with elevated levels of Ntf4 protein showed accelerated HR cell death when treated with cryptogein. In addition, conditional overexpression of Ntf4, which results in high cellular Ntf4 activity, is sufficient to induce HR-like cell death. Based on these results, we concluded that Ntf4 is multifunctional. In addition to its role in pollen germination, Ntf4 is also a component downstream of NtMEK2 in the MAPK cascade that regulates pathogen-induced HR cell death in tobacco.