Aberrant over-expression of a forkhead family member, FOXO1A, in a brain tumor cell line

<p>Abstract</p> <p>Background</p> <p>The mammalian FOXO (forkhead box, O subclass) proteins are a family of pleiotropic transcription factors involved in the regulation of a broad range of cellular processes critical for survival. Despite the essential and diverse roles of the FOXO family members in human cells and their involvement in tumor pathogenesis, the regulation of <it>FOXO </it>expression remains poorly understood. We have addressed the mechanisms underlying the high level of expression of the <it>FOXO1A </it>gene in a cell line, PER-453, derived from a primitive neuroectodermal tumor of the central nervous system (CNS-PNET).</p> <p>Methods</p> <p>The status of the <it>FOXO1A </it>locus in the PER-453 CNS-PNET cell line was investigated by Southern blotting and DNA sequence analysis of the proximal promoter, 5'-UTR, open reading frame and 3'-UTR. FOXO1A expression was assessed by conventional and quantitative RT-PCR, Northern and Western blotting.</p> <p>Results</p> <p>Quantitative real-time RT-PCR (qRT-PCR) data indicated that after normalization to <it>ACTB </it>mRNA levels, canonical <it>FOXO1A </it>mRNA expression in the PER-453 cell line was 124-fold higher than the average level of five other CNS-PNET cell lines tested, 24-fold higher than the level in whole fetal brain, and 3.5-fold higher than the level in fetal brain germinal matrix cells. No mutations within the <it>FOXO1A </it>open reading frame or gross rearrangements of the <it>FOXO1A </it>locus were detected. However, a single nucleotide change within the proximal promoter and several nucleotide changes within the 3'-UTR were identified. In addition, two novel <it>FOXO1A </it>transcripts were isolated that differ from the canonical transcript by alternative splicing within the 3'-UTR.</p> <p>Conclusion</p> <p>The CNS-PNET cell line, PER-453, expresses <it>FOXO1A </it>at very high levels relative to most normal and cancer cells from a broad range of tissues. The <it>FOXO1A </it>open reading frame is wild type in the PER-453 cell line and the abnormally high <it>FOXO1A </it>mRNA expression is not due to mutations affecting the 5'-UTR or proximal promoter. Over expression of <it>FOXO1A </it>may be the result of PER-453 specific epimutations or imbalances in regulatory factors acting at the promoter and/or 3'-UTR.</p

Structural mechanism of JH delivery in hemolymph by JHBP of silkworm, Bombyx mori

Juvenile hormone (JH) plays crucial roles in many aspects of the insect life. All the JH actions are initiated by transport of JH in the hemolymph as a complex with JH-binding protein (JHBP) to target tissues. Here, we report structural mechanism of JH delivery by JHBP based upon the crystal and solution structures of apo and JH-bound JHBP. In solution, apo-JHBP exists in equilibrium of multiple conformations with different orientations of the gate helix for the hormone-binding pocket ranging from closed to open forms. JH-binding to the gate-open form results in the fully closed JHBP-JH complex structure where the bound JH is completely buried inside the protein. JH-bound JHBP opens the gate helix to release the bound hormone likely by sensing the less polar environment at the membrane surface of target cells. This is the first report that provides structural insight into JH signaling

The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation

Determining PTEN Functional Status by Network Component Deduced Transcription Factor Activities

PTEN-controlled PI3K-AKT-mTOR pathway represents one of the most deregulated signaling pathways in human cancers. With many small molecule inhibitors that target PI3K-AKT-mTOR pathway being exploited clinically, sensitive and reliable ways of stratifying patients according to their PTEN functional status and determining treatment outcomes are urgently needed. Heterogeneous loss of PTEN is commonly associated with human cancers and yet PTEN can also be regulated on epigenetic, transcriptional or post-translational levels, which makes the use of simple protein or gene expression-based analyses in determining PTEN status less accurate. In this study, we used network component analysis to identify 20 transcription factors (TFs) whose activities deduced from their target gene expressions were immediately altered upon the re-expression of PTEN in a PTEN-inducible system. Interestingly, PTEN controls the activities (TFA) rather than the expression levels of majority of these TFs and these PTEN-controlled TFAs are substantially altered in prostate cancer mouse models. Importantly, the activities of these TFs can be used to predict PTEN status in human prostate, breast and brain tumor samples with enhanced reliability when compared to straightforward IHC-based or expression-based analysis. Furthermore, our analysis indicates that unique sets of PTEN-controlled TFAs significantly contribute to specific tumor types. Together, our findings reveal that TFAs may be used as “signatures” for predicting PTEN functional status and elucidate the transcriptional architectures underlying human cancers caused by PTEN loss

A Transcription Elongation Factor That Links Signals from the Reproductive System to Lifespan Extension in Caenorhabditis elegans

In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues

Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo

VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2(+/-);Vegfr3(+/-) compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts

Insulin-like growth factor-I induces the phosphorylation and nuclear exclusion of forkhead transcription factors in human neuroblastoma cells

Akt-mediated phosphorylation of forkhead transcription factors is linked to growth factor-stimulated cell survival. We investigated whether the survival activity of insulin-like growth factor-I (IGF-I) in SH-SY5Y human neuroblastoma (NBL) cells is associated with phosphorylation and/or localization changes in forkhead proteins. IGF-I induced phosphorylation of Erks (p42/p44), FKHR (FOXO1a) (Ser 253), FKHRL1 (FOXO3a) (Ser 256), and Akt (Ser 473). PI3-K inhibitor, LY294002, reduced IGF-I-stimulated phosphorylation of FKHR, FKHRL1, and Akt, but did not affect Erk phosphorylation. Using a GFP-FKHR construct, FKHR imported into the nucleus during growth factor withdrawal-induced apoptosis. In addition, IGF-I rescue from serum withdrawal-induced apoptosis is associated with a rapid export of GFP-FKHR into the cytoplasm. Leptomycin B, an inhibitor of Crm1-mediated nuclear export, decreased the level of FKHRL1 phosphorylation in the presence of IGF-I in vector and FKHR overexpressing cells, but had no effect on the phosphorylation status of FKHR. In addition, leptomycin B prevented IGF-I stimulated nuclear export of GFP-FKHR. These studies show IGF-I phosphorylation of FKHR and FKHRL1 via a PI3-K-dependent pathway in NBL cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44353/1/10495_2005_Article_429.pd