DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1⋅IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⋅IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⋅IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.

Activation of iron regulatory protein-1 by oxidative stress in vitro

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-γS and GTP-γS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.

Novel role of phosphorylation in Fe–S cluster stability revealed by phosphomimetic mutations at Ser-138 of iron regulatory protein 1

Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to iron-responsive elements (IRE) in the 5′ or 3′ untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly/disassembly of its [4Fe–4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe–4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe–S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe–4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3′→5′ exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Acceleration of atherogenesis by COX-1-dependent prostanoid formation in low density lipoprotein receptor knockout mice

The cyclooxygenase (COX) product, prostacyclin (PGI2), inhibits platelet activation and vascular smooth-muscle cell migration and proliferation. Biochemically selective inhibition of COX-2 reduces PGI2 biosynthesis substantially in humans. Because deletion of the PGI2 receptor accelerates atherogenesis in the fat-fed low density lipoprotein receptor knockout mouse, we wished to determine whether selective inhibition of COX-2 would accelerate atherogenesis in this model. To address this hypothesis, we used dosing with nimesulide, which inhibited COX-2 ex vivo, depressed urinary 2,3 dinor 6-keto PGF1α by approximately 60% but had no effect on thromboxane formation by platelets, which only express COX-1. By contrast, the isoform nonspecific inhibitor, indomethacin, suppressed platelet function and thromboxane formation ex vivo and in vivo, coincident with effects on PGI2 biosynthesis indistinguishable from nimesulide. Indomethacin reduced the extent of atherosclerosis by 55 ± 4%, whereas nimesulide failed to increase the rate of atherogenesis. Despite their divergent effects on atherogenesis, both drugs depressed two indices of systemic inflammation, soluble intracellular adhesion molecule-1, and monocyte chemoattractant protein-1 to a similar but incomplete degree. Neither drug altered serum lipids and the marked increase in vascular expression of COX-2 during atherogenesis. Accelerated progression of atherosclerosis is unlikely during chronic intake of specific COX-2 inhibitors. Furthermore, evidence that COX-1-derived prostanoids contribute to atherogenesis suggests that controlled evaluation of the effects of nonsteroidal anti-inflammatory drugs and/or aspirin on plaque progression in humans is timely.

The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

Epstein–Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors

Epstein–Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-κB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.

Induction of cyclooxygenase-2 by Epstein–Barr virus latent membrane protein 1 is involved in vascular endothelial growth factor production in nasopharyngeal carcinoma cells

Cyclooxygenase-2 (COX-2) is an inducible form of COX and is overexpressed in diverse tumors, raising the possibility of a role for COX-2 in carcinogenesis. In addition, COX-2 contributes to angiogenesis. The Epstein–Barr virus (EBV) oncoprotein, latent membrane protein 1 (LMP1), is detected in at least 70% of nasopharyngeal carcinoma (NPC) and all EBV-infected preinvasive nasopharyngeal lesions. We found that in specimens of LMP1-positive NPC, COX-2 is frequently expressed, whereas LMP1-negative NPC rarely express the enzyme. We next found that expression of LMP1 in EBV-negative nasopharyngeal epithelial cells induced COX-2 expression. Coexpression of IκBα(S32A/S36A), which is not phosphorylated and prevents NF-κB activation, with LMP1 showed that NF-κB is essential for induction of COX-2 by LMP1. We also demonstrate that NF-κB is involved in LMP1-induced cox-2 promoter activity with the use of reporter assays. Two major regions of LMP1, designated CTAR1 and CTAR2, are signal-transducing domains of LMP1. Constructs expressing either CTAR1 or CTAR2 induce COX-2 but to a lesser extent than wild-type LMP1, consistent with the ability of both regions to activate NF-κB. Furthermore, we demonstrate that LMP1-induced COX-2 is functional because LMP1 increased production of prostaglandin E2 in a COX-2-dependent manner. Finally, we demonstrate that LMP1 increased production of vascular endothelial growth factor (VEGF). Treatment of LMP1-expressing cells with the COX-2-specific inhibitor (NS-398) dramatically decreased production of VEGF, suggesting that LMP1-induced VEGF production is mediated, at least in part, by COX-2. These results suggest that COX-2 induction by LMP1 may play a role in angiogenesis in NPC.

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

c-myc activation renders proliferation of Epstein-Barr virus (EBV)-transformed cells independent of EBV nuclear antigen 2 and latent membrane protein 1.

Two genetic events contribute to the development of endemic Burkitt lymphoma (BL) infection of B lymphocytes with Epstein-Barr virus (EBV) and the activation of the protooncogene c-myc through chromosomal translocation. The viral genes EBV nuclear antigen 2 (EBNA2) and latent membrane protein 1 (LMP1) are essential for transformation of primary human B cells by EBV in vitro; however, these genes are not expressed in BL cells in vivo. To address the question whether c-myc activation might abrogate the requirement of the EBNA2 and LMP1 function, we have introduced an activated c-myc gene into an EBV-transformed cell line in which EBNA2 was rendered estrogen-dependent through fusion with the hormone binding domain of the estrogen receptor. The c-myc gene was placed under the control of regulatory elements of the immunoglobulin kappa locus composed a matrix attachment region, the intron enhancer, and the 3' enhancer. We show here that transfection of a c-myc expression plasmid followed by selection for high MYC expression is capable of inducing continuous proliferation of these cells in the absence of functional EBNA2 and LMP1. c-myc-induced hormone-independent proliferation was associated with a dramatic change in the growth behavior as well as cell surface marker expression of these cells. The typical lymphoblastoid morphology and phenotype of EBV-transformed cells completely changed into that of BL cells in vivo. We conclude that the phenotype of BL cells reflects the expression pattern of viral and cellular genes rather than its germinal center origin.

Induction of nephrogenic mesenchyme by osteogenic protein 1 (bone morphogenetic protein 7).

The definitive mammalian kidney forms as the result of reciprocal interactions between the ureteric bud epithelium and metanephric mesenchyme. As osteogenic protein 1 (OP-1/bone morphogenetic protein 7), a member of the TGF-beta superfamily of proteins, is expressed predominantly in the kidney, we examined its involvement during metanephric induction and kidney differentiation. We found that OP-1 mRNA is expressed in the ureteric bud epithelium before mesenchymal condensation and is subsequently seen in the condensing mesenchyme and during glomerulogenesis. Mouse kidney metanephric rudiments cultured without ureteric bud epithelium failed to undergo mesenchymal condensation and further epithelialization, while exogenously added recombinant OP-1 was able to substitute for ureteric bud epithelium in restoring the induction of metanephric mesenchyme. This OP-1-induced nephrogenic mesenchyme differentiation follows a developmental pattern similar to that observed in the presence of the spinal cord, a metanephric inducer. Blocking OP-1 activity using either neutralizing antibodies or antisense oligonucleotides in mouse embryonic day 11.5 mesenchyme, cultured in the presence of metanephric inducers or in intact embryonic day 11.5 kidney rudiment, greatly reduced metanephric differentiation. These results demonstrate that OP-1 is required for metanephric mesenchyme differentiation and plays a functional role during kidney development.