Integration of multiple instructive cues by neural crest stem cells reveals cell-intrinsic biases in relative growth factor responsiveness

Growth factors can influence lineage determination of neural crest stem cells (NCSCs) in an instructive manner, in vitro. Because NCSCs are likely exposed to multiple signals in vivo, these findings raise the question of how stem cells would integrate such combined influences. Bone morphogenetic protein 2 (BMP2) promotes neuronal differentiation and glial growth factor 2 (GGF2) promotes glial differentiation; if NCSCs are exposed to saturating concentrations of both factors, BMP2 appears dominant. By contrast, if the cells are exposed to saturating concentrations of both BMP2 and transforming growth factor β1 (which promotes smooth muscle differentiation), the two factors appear codominant. Sequential addition experiments indicate that NCSCs require 48–96 hrs in GGF2 before they commit to a glial fate, whereas the cells commit to a smooth muscle fate within 24 hr in transforming growth factor β1. The delayed response to GGF2 does not reflect a lack of functional receptors; however, because the growth factor induces rapid mitogen-activated protein kinase phosphorylation in naive cells. Furthermore, GGF2 can attenuate induction of the neurogenic transcription factor mammalian achaete-scute homolog 1, by low doses of BMP2. This short-term antineurogenic influence of GGF2 is not sufficient for glial lineage commitment, however. These data imply that NCSCs exhibit cell-intrinsic biases in the timing and relative dosage sensitivity of their responses to instructive factors that influence the outcome of lineage decisions in the presence of multiple factors. The relative delay in glial lineage commitment, moreover, apparently reflects successive short-term and longer-term actions of GGF2. Such a delay may help to explain why glia normally differentiate after neurons, in vivo.

Vascular endothelial growth factor: Crystal structure and functional mapping of the kinase domain receptor binding site

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor β2 (TGF-β). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-β. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional “hot spots” composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-β. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

A family of membrane-embedded metalloproteases involved in regulated proteolysis of membrane-associated transcription factors

We present evidence that the sporulation protein SpoIVFB of Bacillus subtilis is a member of a newly recognized family of metalloproteases that have catalytic centers adjacent to or within the membrane. SpoIVFB is required for converting the membrane-associated precursor protein, pro-σK, to the mature and active transcription factor σK by proteolytic removal of an N-terminal extension of 20 amino acids. SpoIVFB and other family members share the conserved sequence HEXXH, a hallmark of metalloproteases, as well as a second conserved motif NPDG, which is unique to the family. Both motifs, which are expected to form the catalytic center of the protease, overlap hydrophobic segments that are predicted to be separate transmembrane domains. The only other characterized member of this family of membrane-embedded metalloproteases is the mammalian Site-2 protease (S2P), which is required for the intramembrane cleavage of the eukaryotic transcription factor sterol regulatory element binding protein (SREBP). We report that amino acid substitutions in the two conserved motifs of SpoIVFB impair pro-σK processing and σK-directed gene expression during sporulation. These results and those from a similar analysis of S2P support the interpretation that both proteins are founding members of a family of metalloproteases involved in the activation of membrane-associated transcription factors. Thus, the pathways that govern the activation of the prokaryotic transcription factor pro-σK and the mammalian transcription factor SREBP not only are analogous but also use processing enzymes with strikingly homologous features.

Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G1

Transcription of ribosomal RNA genes by RNA polymerase (pol) I oscillates during the cell cycle, being maximal in S and G2 phase, repressed during mitosis, and gradually recovering during G1 progression. We have shown that transcription initiation factor (TIF)-IB/SL1 is inactivated during mitosis by cdc2/cyclin B-directed phosphorylation of TAFI110. In this study, we have monitored reactivation of transcription after exit from mitosis. We demonstrate that the pol I factor UBF is also inactivated by phosphorylation but recovers with different kinetics than TIF-IB/SL1. Whereas TIF-IB/SL1 activity is rapidly regained on entry into G1, UBF is reactivated later in G1, concomitant with the onset of pol I transcription. Repression of pol I transcription in mitosis and early G1 can be reproduced with either extracts from cells synchronized in M or G1 phase or with purified TIF-IB/SL1 and UBF isolated in the presence of phosphatase inhibitors. The results suggest that two basal transcription factors, e.g., TIF-IB/SL1 and UBF, are inactivated at mitosis and reactivated by dephosphorylation at the exit from mitosis and during G1 progression, respectively.

Identification of eIF2Bγ and eIF2γ as cofactors of hepatitis C virus internal ribosome entry site-mediated translation using a functional genomics approach

The 5′-untranslated region of hepatitis C virus (HCV) is highly conserved, folds into a complex secondary structure, and functions as an internal ribosome entry site (IRES) to initiate translation of HCV proteins. We have developed a selection system based on a randomized hairpin ribozyme gene library to identify cellular factors involved in HCV IRES function. A retroviral vector ribozyme library with randomized target recognition sequences was introduced into HeLa cells, stably expressing a bicistronic construct encoding the hygromycin B phosphotransferase gene and the herpes simplex virus thymidine kinase gene (HSV-tk). Translation of the HSV-tk gene was mediated by the HCV IRES. Cells expressing ribozymes that inhibit HCV IRES-mediated translation of HSV-tk were selected via their resistance to both ganciclovir and hygromycin B. Two ribozymes reproducibly conferred the ganciclovir-resistant phenotype and were shown to inhibit IRES-mediated translation of HCV core protein but did not inhibit cap-dependent protein translation or cell growth. The functional targets of these ribozymes were identified as the gamma subunits of human eukaryotic initiation factors 2B (eIF2Bγ) and 2 (eIF2γ), respectively. The involvement of eIF2Bγ and eIF2γ in HCV IRES-mediated translation was further validated by ribozymes directed against additional sites within the mRNAs of these genes. In addition to leading to the identification of cellular IRES cofactors, ribozymes obtained from this cellular selection system could be directly used to specifically inhibit HCV viral translation, thereby facilitating the development of new antiviral strategies for HCV infection.

Vascular endothelial growth factor: Direct neuroprotective effect in in vitro ischemia

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an in vitro model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.

TFIIH action in transcription initiation and promoter escape requires distinct regions of downstream promoter DNA

TFIIH is a multifunctional RNA polymerase II general initiation factor that includes two DNA helicases encoded by the Xeroderma pigmentosum complementation group B (XPB) and D (XPD) genes and a cyclin-dependent protein kinase encoded by the CDK7 gene. Previous studies have shown that the TFIIH XPB DNA helicase plays critical roles not only in transcription initiation, where it catalyzes ATP-dependent formation of the open complex, but also in efficient promoter escape, where it suppresses arrest of very early RNA polymerase II elongation intermediates. In this report, we present evidence that ATP-dependent TFIIH action in transcription initiation and promoter escape requires distinct regions of the DNA template; these regions are well separated from the promoter region unwound by the XPB DNA helicase and extend, respectively, ≈23–39 and ≈39–50 bp downstream from the transcriptional start site. Taken together, our findings bring to light a role for promoter DNA in TFIIH action and are consistent with the model that TFIIH translocates along promoter DNA ahead of the RNA polymerase II elongation complex until polymerase has escaped the promoter.

FGF-2 regulation of neurogenesis in adult hippocampus after brain injury

Fibroblast growth factor-2 (FGF-2) promotes proliferation of neuroprogenitor cells in culture and is up-regulated within brain after injury. Using mice genetically deficient in FGF-2 (FGF-2−/− mice), we addressed the importance of endogenously generated FGF-2 on neurogenesis within the hippocampus, a structure involved in spatial, declarative, and contextual memory, after seizures or ischemic injury. BrdUrd incorporation was used to mark dividing neuroprogenitor cells and NeuN expression to monitor their differentiation into neurons. In the wild-type strain, hippocampal FGF-2 increased after either kainic acid injection or middle cerebral artery occlusion, and the numbers of BrdUrd/NeuN-positive cells significantly increased on days 9 and 16 as compared with the controls. In FGF-2−/− mice, BrdUrd labeling was attenuated after kainic acid or middle cerebral artery occlusion, as was the number of neural cells colabeled with both BrdUrd and NeuN. After FGF-2−/− mice were injected intraventricularly with a herpes simplex virus-1 amplicon vector carrying FGF-2 gene, the number of BrdUrd-labeled cells increased significantly to values equivalent to wild-type littermates after kainate seizures. These results indicate that endogenously synthesized FGF-2 is necessary and sufficient to stimulate proliferation and differentiation of neuroprogenitor cells in the adult hippocampus after brain insult.

Considerations of transcriptional control mechanisms: Do TFIID–core promoter complexes recapitulate nucleosome-like functions?

The general transcription initiation factor TFIID was originally identified, purified, and characterized with a biochemical assay in which accurate transcription initiation is reconstituted with multiple, chromatographically separable activities. Biochemical analyses have demonstrated that TFIID is a multiprotein complex that directs preinitiation complex assembly on both TATA box-containing and TATA-less promoters, and some TFIID subunits have been shown to be molecular targets for activation domains in DNA-binding regulatory proteins. These findings have most commonly been interpreted to support the view that transcriptional activation by upstream factors is the result of enhanced TFIID recruitment to the core promoter. Recent insights into the architecture and cell-cycle regulation of the multiprotein TFIID complex prompt both a reassessment of the functional role of TFIID in gene activation and a review of some of the less well-appreciated literature on TFIID. We present a speculative model for diverse functional roles of TFIID in the cell, explore the merits of the model in the context of published data, and suggest experimental approaches to resolve unanswered questions. Finally, we point out how the proposed functional roles of TFIID in eukaryotic class II transcription fit into a model for promoter recognition and activation that applies to both eubacteria and eukaryotes.

Molecular mechanisms of translation initiation in eukaryotes

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2/GTP/Met-tRNAi and eIF3) to the 5′ end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A/4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.

FRAG1, a gene that potently activates fibroblast growth factor receptor by C-terminal fusion through chromosomal rearrangement.

A constitutively active form of fibroblast growth factor 2 (FGFR2) was identified in rat osteosarcoma (ROS) cells by an expression cloning strategy. Unlike other tyrosine kinase receptors activated by N-terminal truncation in tumors, this receptor, FGFR2-ROS, contains an altered C terminus generated from chromosomal rearrangement with a novel gene, designated FGFR activating gene 1 (FRAG1). While the removal of the C terminus slightly activates FGFR2, the presence of the FRAG1 sequence drastically stimulates the transforming activity and autophosphorylation of the receptor. FGFR2-ROS is expressed as a unusually large protein and is highly phosphorylated in NIH 3T3 transfectants. FRAG1 is ubiquitously expressed and encodes a predicted protein of 28 kDa lacking significant structural similarity to known proteins. Epitope-tagged FRAG1 protein showed a perinuclear localization by immunofluorescence staining. The highly activated state of FGFR2-ROS appears to be attributed to constitutive dimer formation and higher phosphorylation level as well as possibly altered subcellular localization. These results indicate a unique mechanism of receptor activation by a C terminus alteration through a chromosomal fusion with FRAG1.

Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles.

The crystal structure of Pseudomonas aeruginosa exotoxin domain III with nicotinamide and AMP: conformational differences with the intact exotoxin.

Domain III of Pseudomonas aeruginosa exotoxin A catalyses the transfer of ADP-ribose from NAD to a modified histidine residue of elongation factor 2 in eukaryotic cells, thus inactivating elongation factor 2. This domain III is inactive in the intact toxin but is active in the isolated form. We report here the 2.5-A crystal structure of this isolated domain crystallized in the presence of NAD and compare it with the corresponding structure in the intact Pseudomonas aeruginosa exotoxin A. We observe a significant conformational difference in the active site region from Arg-458 to Asp-463. Contacts with part of domain II in the intact toxin prevent the adoption of the isolated domain conformation and provide a structural explanation for the observed inactivity. Additional electron density in the active site region corresponds to separate AMP and nicotinamide and indicates that the NAD has been hydrolyzed. The structure has been compared with the catalytic domain of the diphtheria toxin, which was crystallized with ApUp.