Activity In Nuclear Extracts Research Articles

Human papillomavirus type 16 E7 impairs the activation of the interferon regulatory factor-1.

We had previously observed that HPV-16 E7 disturbs the Guanylate Binding Protein (GBP)-ISRE reporter activation by IFN-gamma thus suggesting an alteration of the IRF-1 function. In this study we examined the mechanism by which E7 affects the IFN-gamma signals driving the activation of gene transcription. Using 14/2 BRK cells containing dexamethasone-inducible HPV-16 E7 gene, we observed a large inhibition of the IRF-1 DNA binding activity upon E7 induction. Concomitantly, there was no significant change in the levels of IRF-1, indicating that this was not due to reduced levels of IRF-1 expression. Likewise, in vitro translated E7 did not affect the IRF-1 DNA binding activity in nuclear extracts derived from IFN-induced cells, thus indicating that the effects of E7 are upstream of IRF-1's binding to its DNA recognition site. Finally, NFkappaB DNA binding activity was also inhibited under conditional expression of E7. These data indicate that HPV-16 E7 inhibits the IRF-1 and NFkappaB function and this could lead to the impairment of the IFN response in HPV-infected cells. Furthermore, the findings suggest that different events of the IFN inducible signal cascade seem to be target for HPV-16 E7.

Functional interactions between an atypical NF-kappaB site from the rat CYP2B1 promoter and the transcriptional repressor RBP-Jkappa/CBF1.

The phenobarbital-inducible rat cytochrome P450 (CYP) 2B1 and 2B2 proteins are encoded by homologous genes whose promoters contain a mammalian-apparent long terminal repeat retrotransposon (MaLR). An NF-kappaB-like site within the MaLR forms multiple protein-DNA complexes with rat liver and HeLa cell nuclear extracts. Using antibody supershift assays, we have identified these complexes as NF-kappaB and RPB-Jkappa/CBF1. Competition assays using a series of single site mutant oligonucleotides reveal that the recognition sites for these two factors overlap. We also show that the CYP2B1/2 NF-kappaB element, but not the Igkappa NF-kappaB element, can repress transcription in vitro when positioned upstream of the heterologous adenovirus major late core promoter. In addition, RBP-Jkappa over-expressed in COS-7 cells repressed expression in vivo from an SV40-luciferase reporter construct that contained the CYP2B1/2 NF-kappaB element. Finally, we observe similar levels of NF-kappaB and RBP-Jkappa binding activities in nuclear extracts prepared from control and phenobarbital-induced rat livers. The results suggest that RBP-Jkappa/CBF1 binds an atypical NF-kappaB site in the CYP2B1/2 promoters and may help to maintain a low level of expression in the absence of inducer.

The Acute Phase Response Is Associated with Retinoid X Receptor Repression in Rodent Liver

The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRalpha, RXRbeta, and RXRgamma proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRalpha (62%) and RXRbeta (50%) and at 2 h for RXRgamma (61%). These decreases were associated with a marked reduction in RXRalpha, RXRbeta, and RXRgamma mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decrease RXRbeta and RXRgamma transcription and only marginally inhibited (38%) RXRalpha transcription. RXR repression was associated with decreased LXRalpha and PPARalpha mRNA levels and reduced RXR x RXR, RXR x PPAR and RXR x LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARalpha target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR.

Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia.

Nuclear factor-kappa B (NF-kappaB) is a multisubunit transcription factor that when activated induces the expression of genes encoding acute-phase proteins, cell adhesion molecules, cell surface receptors, and cytokines. NF-kappaB is composed of a variety of protein subunits of which p50-and p65-kDa (RelA) are the most widely studied. Under resting conditions, these subunits reside in the cytoplasm as an inactive complex bound by inhibitor proteins, IkappaB alpha and IkappaB beta. On activation, IkappaB is phosphorylated by IkappaB kinase and ubiquitinated and degraded by the proteasome; simultaneously, the active heterodimer translocates to the nucleus where it can initiate gene transcription. In the periphery, NF-kappaB is involved in inflammation through stimulation of the production of inflammatory mediators. The role of NF-kappaB in the brain is unclear. In vitro, NF-kappaB activation can be either protective or deleterious. The role of NF-kappaB in ischemic neuronal cell death in vivo was investigated. Adult male rats were subjected to 2 hours of focal ischemia induced by middle cerebral artery occlusion (MCAO). At 2, 6, and 12 hours after reperfusion, the expression and transactivation of NF-kappaB in ischemic versus nonischemic cortex and striatum were determined by immunocytochemistry and by electrophoretic mobility gel-shift analysis. At all time points studied, p50 and p65 immunoreactivity was found exclusively in the nuclei of cortical and striatal neurons in the ischemic hemisphere. The contralateral nonischemic hemisphere showed no evidence of nuclear NF-kappaB immunoreactivity. Double immunofluorescence confirmed expression of p50 in nuclei of neurons. Increased NF-kappaB DNA-binding activity in nuclear extracts prepared from the ischemic hemisphere was further substantiated by electrophoretic mobility gel-shift analysis. Because the activation of NF-kappaB by many stimuli can be blocked by antioxidants in vitro, the effect of the antioxidant, LY341122, previously shown to be neuroprotective, on NF-kappaB activation in the MCAO model was evaluated. No significant activation of NF-kappaB was found by electrophoretic mobility gel-shift analysis in animals treated with LY341122. These results demonstrate that transient focal cerebral ischemia results in activation of NF-kappaB in neurons and supports previous observations that neuroprotective antioxidants may inhibit neuronal death by preventing the activation of NF-kappaB.

Up-regulation of base excision repair activity for 8-hydroxy-2'-deoxyguanosine in the mouse brain after forebrain ischemia-reperfusion.

The repair enzyme 8-oxoguanine glycosylase/ apyrimidinic/apurinic lyase (OGG) removes 8-hydroxy-2'deoxyguanosine (oh8dG) in human cells. Our goal was to examine oh8dG-removing activity in the cell nuclei of male C57BL/6 mouse brains treated with either forebrain ischemia-reperfusion (FblR) or sham operations. We found that the OGG activity in nuclear extracts, under the condition in which other nucleases did not destroy the oligodeoxynucleotide duplex, excised oh8dG with the greatest efficiency on the oligodeoxynucleotide duplex containing oh8dG/dC and with less efficiency on the heteroduplex containing oh8dG/dT, oh8dG/dG, or oh8dG/dA. This specificity was the same as for the recombinant type 1 OGG (OGG1) of humans. We observed that the OGG1 peptide and its activity in the mouse brain were significantly increased after 90 min of ischemia and 20-30 min of reperfusion. The increase in the protein level and in the activity of brain OGG1 correlated positively with the elevation of FblR-induced DNA lesions in an indicator gene (the c-fos gene) of the brain. The data suggest a possibility that the OGG1 protein may excise oh8dG in the mouse brain and that the activity of OGG1 may have a functional role in reducing oxidative gene damage in the brain after FblR.

Regulation of human 12/15-lipoxygenase by Stat6-dependent transcription.

Human 12/15-lipoxygenase is a lipid-peroxidating enzyme implicated in the pathophysiology of atherosclerosis and airway inflammation. Interleukin (IL)-4 specifically induces 12/15-lipoxygenase messenger RNA, protein, and enzymatic activity in primary cultures of human monocytes and airway epithelial cells. The induction of the human 12/15-lipoxygenase by IL-4 suggests that the signal transducer and activator of transcription (Stat)-6 protein is critical for its expression. Several putative Stat6 response elements are located in the proximal 1.8 kb of 12/15-lipoxygenase 5'-flanking region. In this study we use BEAS-2B human airway epithelial cells as a model to demonstrate the dependence of 12/15-lipoxygenase expression on the IL-4/Stat6 signal transduction pathway. Transient transfections of human 12/15-lipoxygenase promoter/luciferase reporter genes indicate that this induction occurs through direct transcriptional mechanisms mediated by a specific Stat6 response element located 952 base pairs upstream of the translational start codon. Using this Stat6 response element as a probe, electrophoretic mobility shift assays show an IL-4-dependent binding activity in nuclear extracts. Supershift assays confirm that Stat6 participates in this binding complex. These data indicate that the human 12/15-lipoxygenase gene is induced in airway epithelial cells through Stat6-dependent transcriptional mechanisms mediated by a specific Stat6 response element in the 5'-flanking region.

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H2O2-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H2O2-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H2O2-mediated DNA damage may be related to the carcinogenic effects of quercetin.

Differential signaling pathways of HO-1 gene expression in pulmonary and systemic vascular cells.

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-induced HO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B. -H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375-5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1 gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither induced HO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction of HO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5'-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or the HO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-induced HO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation of HO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-induced HO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.

Characterization of the Human Very-Long-Chain Acyl-CoA Dehydrogenase Gene Promoter Region: A Role for Activator Protein 2

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of a family of nuclear-encoded enzymes that catalyze the initial step in mitochondrial fatty acid β-oxidation (FAO). Previous studies have indicated that two other members of the AD gene family (medium-chain AD and long-chain AD) are controlled at the transcriptional level by nuclear hormone receptors. In this study, we have cloned and characterized the human VLCAD gene promoter region to identify cis-acting elements involved in its transcriptional control. VLCAD gene promoter-luciferase reporter (VLCAD-Luc) constructs were found to be transcriptionally active in a variety of mammalian cell lines and in primary rat cardiomyocytes when driven by varying lengths of the VLCAD promoter region. Removal of a 20-bp DNA segment of the proximal VLCAD gene promoter markedly reduced the transcriptional activity of VLCAD-Luc constructs. Gel mobility shift assays identified a DNA-binding activity in nuclear extracts prepared from human hepatoma G2 cells that interacted with the 20-bp regulatory region. Competition studies revealed that this DNA-binding activity could be abolished by a molar excess of unlabeled specific oligonucleotide as well as a DNA fragment containing an activator protein 2 (AP-2)-binding site but not by an unrelated nonspecific DNA fragment. These results provide an initial characterization of the human VLCAD gene promoter, identify AP-2 as a candidate activator of VLCAD gene transcription, and suggest that VLCAD gene transcription may be regulated by pathways distinct from that of other AD genes.

Identification of core sequences involved in metabolism-dependent nuclear protein binding to the rat insulin-like growth factor I gene.

In the liver, most insulin-like growth factor I (IGF-I) transcripts originate in exon 1, where important cis-regulatory regions are located downstream from the major transcription initiation sites. Within these regions, we have attempted to identify sequences which are involved in the decrease in IGF-I gene transcription associated with diabetes mellitus. The function of different genomic templates was assessed by in vitro transcription, which revealed a consistent 50-80% decrease in the activity of nuclear extracts from streptozotocin-diabetic as compared with normal rats. The disparity in transcriptional activity between normal and diabetic nuclear extracts was reduced with templates containing 11-bp mutations within DNase I protected regions III or V (+42 and +129 bp, respectively, from the major transcription initiation site), but a mutation between regions IV and V had little effect. Within region III, gel mobility shift analysis and methylation interference studies indicated that DNA-protein interactions involve a GCGC core sequence. In region V, gel mobility shift studies and uracil interference analysis revealed interactions involving a TTAT core. While gel mobility shift analysis and transient transfection studies indicate that the GCGC core sequence in region III recognizes C/EBP, the AT-rich sequence in region V is likely to recognize a protein with homeodomain characteristics. Identification of the nuclear factor(s) interacting with regions III and V, downstream from exon 1 initiation sites, will be important for understanding the mechanism of reduced IGF-I gene transcription due to diabetes mellitus.

Salicylate-enhanced activation of transcription factors induced by interferon-γ

Salicylate enhanced the interferon-gamma-dependent activation of two transcription factors in a murine macrophage cell line: signal transducer and activator of transcription (STAT)1 and interferon-gamma-responsive factor 1. Salicylate alone did not activate these transcription factors. This enhancement was reflected by increased DNA-binding activities and was the consequence of prolonged tyrosine phosphorylation of these transcription factors following interferon-gamma treatment. However, salicylate did not directly inhibit protein-tyrosine phosphatase activity in nuclear extracts of interferon-gamma-treated cells. The enhanced activation of STAT1 resulted in increased induction of mRNA encoding interferon regulatory factor-1. These results not only demonstrate that aspirin and its metabolite salicylate may have pro-inflammatory as well as anti-inflammatory effects but also raise the possibility that new cellular targets may be identified for modulating the Janus kinase-STAT signalling pathway.

Abnormal NF-κB Activity in T Lymphocytes from Patients with Systemic Lupus Erythematosus Is Associated with Decreased p65-RelA Protein Expression

AbstractNumerous cellular and biochemical abnormalities in immune regulation have been described in patients with systemic lupus erythematosus (SLE), including surface Ag receptor-initiated signaling events and lymphokine production. Because NF-κB contributes to the transcription of numerous inflammatory genes and has been shown to be a molecular target of antiinflammatory drugs, we sought to characterize the functional role of the NF-κB protein complex in lupus T cells. Freshly isolated T cells from lupus patients, rheumatoid arthritis (RA) patients, and normal individuals were activated physiologically via the TCR with anti-CD3 and anti-CD28 Abs to assess proximal membrane signaling, and with PMA and a calcium ionophore (A23187) to bypass membrane-mediated signaling events. We measured the NF-κB binding activity in nuclear extracts by gel shift analysis. When compared with normal cells, the activation of NF-κB activity in SLE patients was significantly decreased in SLE, but not in RA, patients. NF-κB binding activity was absent in several SLE patients who were not receiving any medication, including corticosteroids. Also, NF-κB activity remained absent in follow-up studies. In supershift experiments using specific Abs, we showed that, in the group of SLE patients who displayed undetectable NF-κB activity, p65 complexes were not formed. Finally, immunoblot analysis of nuclear extracts showed decreased or absent p65 protein levels. As p65 complexes are transcriptionally active in comparison to the p50 homodimer, this novel finding may provide insight on the origin of abnormal cytokine or other gene transcription in SLE patients.

Functional interaction between human topoisomerase IIalpha and retinoblastoma protein.

DNA topoisomerase II-an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression-is also a target of clinically useful anticancer drugs. Preliminary observations of a positive correlation between the expression of topoisomerase (topo) IIalpha and the retinoblastoma protein (Rb) in a series of rhabdomyosarcoma cells prompted us to ask whether these two proteins interact in vivo. Using human rhabdomyosarcoma and leukemic cell lines, we found a physical association between topo IIalpha and Rb protein by reciprocal immunoprecipitation and immunoblotting, in which topo IIalpha appeared to interact primarily with the underphosphorylated form of Rb. Experiments with truncated glutathione S-transferase-Rb fusion proteins and nuclear extracts of Rh1 rhabdomyosarcoma cells indicated that topo IIalpha binds avidly to the A/B pocket domain of Rb, which contains the intact spacer amino acid sequence. To determine whether this interaction has functional consequences in vivo, we expressed wild-type and mutant Rb in human cervical carcinoma cells lacking functional Rb. Wild-type, but not mutant, Rb inhibited topo II activity in nuclear extracts of these transfected cells. Moreover, purified wild-type Rb inhibited the activity of purified human topo IIalpha, indicating a direct interaction between these two proteins. We conclude that topo IIalpha associates physically with Rb in interactions that appear to have functional significance.

Transforming Growth Factor-β1 Induces Interleukin-6 Expression via Activating Protein-1 Consisting of JunD Homodimers in Primary Human Lung Fibroblasts

Transforming growth factor (TGF)-beta1 induces extracellular matrix deposition and proliferation of mesenchymal cells. We recently reported that interleukin (IL)-6 is an essential mediator of growth factor-induced proliferation of lung fibroblasts. Here, we demonstrate by reverse transcriptase polymerase chain reaction and enzyme-linked immunoassay that TGF-beta1 is a potent inducer of IL-6 mRNA and protein in primary human lung fibroblasts. Transient transfections of fibroblasts with a luciferase reporter gene construct containing nucleotides -651 to +1 of the human IL-6 promoter revealed that TGF-beta1 also potently activated IL-6 promoter activity. Progressive 5'-deletions and site-directed mutagenesis of the parental construct located the TGF-beta1-responsive cis-regulatory element to a known activating protein-1 (AP-1) sequence (nucleotides -284 to -276). Gel shift analyses revealed that AP-1 DNA binding activity in nuclear extracts was increased 30 min after stimulation with TGF-beta1. In contrast, neither CCAAT enhancer-binding protein-beta, NF-kappaB, nor Sp1 were activated by TGF-beta1. Supershift analyses demonstrated that the AP-1 complex induced by TGF-beta1 was composed of Jun isoforms and absent of Fos isoforms. Moreover, this complex was found to be a JunD homodimer. Our data thus demonstrate that TGF-beta1 is a potent inducer of IL-6 in primary human lung fibroblasts. The TGF-beta1-activated JunD homodimer may be essential for a majority of the biological effects induced by TGF-beta1 in this cell type, such as proliferation and extracellular matrix synthesis.

Phosphorylation of GATA-1 increases its DNA-binding affinity and is correlated with induction of human K562 erythroleukaemia cells.

We have investigated by electrophoretic mobility shift assay (EMSA) the level of GATA-1 DNA-binding activity in nuclear extracts prepared from the human erythroleukaemic cell line, K562, after erythroid induction by hemin, sodium butyrate (NaB) or Trichostatin A or treatment with N -acetylcysteine (NAC). Relative to extract from untreated cells, GATA-1 binding activity increased markedly in all cases. However, immunoblot analysis revealed unchanged levels of GATA-1 protein after induction. Incubation of induced but not uninduced K562 extracts with phosphatase prior to EMSA weakened the binding activity, suggesting that the increase in GATA-1 binding following induction of K562 cells was a consequence of phosphorylation. When the mouse erythroleukaemic cell line MEL was induced with dimethylsulphoxide (DMSO), NaB or NAC, GATA-1 binding activity fell with DMSO, rose significantly with NaB and remained at about the same level in NAC-induced cells. In this case immunoblotting revealed that GATA-1 protein levels were in accord with the EMSA data. The DNA-binding activities of induced and uninduced MEL cell nuclear extracts were decreased by incubation with phosphatase, showing that phosphoryl-ation and DNA binding of GATA-1 are already optimalin these cells. The DNA-binding activity of affinity-purified GATA-1 from MEL cells was also reduced by phosphatase treatment, showing that phosphorylation/dephosphorylation is directly affecting the factor. Furthermore, when a comparison was made by EMSA of nuclear extracts prepared from K562 and MEL cells untreated or incubated with okadaic acid, a phosphatase inhibitor, GATA-1 binding was seen to increase with K562 cells, whereas with MEL cells there was no change in GATA-1 binding. Overall the results suggest that the level of GATA-1 phosphorylation increases after the induction of K562, but not MEL cells, where GATA-1 is already highly phosphorylated. Furthermore, phosphorylation increases the binding affinity of GATA-1 for a canonical binding site.

Cationic lipids employed for antisense oligodeoxynucleotide transport may inhibit vascular cell adhesion molecule-1 expression in human endothelial cells: a word of caution.

Antisense oligodeoxynucleotides (ODN) have become a powerful tool to achieve specific gene inhibition in various cell types, including endothelial cells. The low spontaneous cellular uptake of ODN, however, usually requires the employment of transmembrane carriers, such as the positively charged liposome formulation dioleyloxypropyltrimethyl ammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE). In the present study, we observed that DOTMA/DOPE per se interferes with the inducible expression of vascular cell adhesion molecule-1 (VCAM-1) in human pulmonary artery endothelial cells (HPAEC). By RT-PCR analysis, a dose-dependent suppression of VCAM-1 but not intracellular adhesion molecule-1 (ICAM-1) mRNA levels in tumor necrosis factor-alpha (TNF-alpha)-challenged HPAEC pretreated with DOTMA/DOPE (5-20 microg/ml) was demonstrated. Correspondingly, a strong reduction of TNF-alpha-induced VCAM-1 but not ICAM-1 cell surface expression on HPAEC was observed. These DOTMA/DOPE-induced changes were not due to alterations in VCAM-1 mRNA stability, nor did DOTMA/DOPE inhibit TNF-alpha-induced NF-kappaB-like binding activity in nuclear extracts of HPAEC, as analyzed by electrophoretic mobility shift assay. In contrast, DOTMA/DOPE effected a dose-dependent increase in AP-1-like binding activity in nuclear extracts of HPAEC, as analyzed by Western blotting and EMSA. We conclude that positively charged liposome preparations may per se inhibit TNF-alpha-induced endothelial VCAM-1 expression, and this may be related to changes in AP-1 but not NF-kappaB-dependent transcriptional control. Notably, when used at concentrations below 5 microg/ml, DOTMA/DOPE may be employed for specific antisense-mediated downregulation of VCAM-1 in the absence of vehicle-related side effects on adhesion molecule transcription.

Lysophosphatidic Acid Activates NF-κB in Fibroblasts: A REQUIREMENT FOR MULTIPLE INPUTS

Lysophosphatidic acid (LPA) is a growth factor that exerts a number of well characterized biological actions on fibroblasts and other cells. In the present study, we investigated the possibility that LPA activates the transcription factor NF-kappaB. NF-kappaB is a target of cytokines, but its activation by other classes of agonists has raised considerable interest in the control of processes such as inflammation and wound healing through varied mechanisms. We find that LPA causes a marked activation of NF-kappaB in Swiss 3T3 fibroblasts as determined by the degradation of IkappaB-alpha in the cytosol and the emergence of kappaB binding activity in nuclear extracts. The EC50 for activation of NF-kappaB is 1-5 microM, a range similar to that reported for reinitiation of DNA synthesis and activation of the serum response element. Activation of NF-kappaB is attenuated by pertussis toxin and inhibitors of protein kinase C, and it is completely blocked by the Ca2+ chelator BAPTA-AM. The combination of phorbol ester and thapsigargin promotes an activation comparable with that of LPA. Activation by LPA is additionally inhibited by tyrphostin A25 but not genistein or AG1478, indicating a selective utilization of protein-tyrosine kinases, and by certain antioxidants, implying a role for reactive oxygen species. The activation is also inhibited by tricyclodecan-9-yl-xanthogenate (D609), implying a requirement for hydrolysis of phosphatidylcholine. The data demonstrate the utilization of multiple pathways in the activation of NF-kappaB by LPA, not inconsistent with the relevance of several families of GTP-binding regulatory proteins.

Transcription termination and 3'-End processing of the spliced leader RNA in kinetoplastids.

Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3' end of all kinetoplastid SL RNA genes, and that more than six T's are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3' ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T's. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3' end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3'-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

Evidence for factors regulating transfer cell-specific expression in maize endosperm.

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (< 100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.

NF-κB Activity Is Induced by Neural Cell Adhesion Molecule Binding to Neurons and Astrocytes

The neural cell adhesion molecule, N-CAM, is expressed on the surface of astrocytes and neurons, and N-CAM homophilic binding has been shown to alter gene expression in both of these cell types. To determine mechanisms by which N-CAM regulates gene expression, we have analyzed DNA binding of and transcriptional activation by NF-kappaB after N-CAM binding to the cell surface. Addition of purified N-CAM, the recombinant third immunoglobulin domain of N-CAM, or N-CAM antibodies either to neonatal rat forebrain astrocytes or to cerebellar granule neurons increased NF-kappaB/DNA binding activity in nuclear extracts as measured by electrophoretic mobility shift assays. Analysis using supershifting antibodies indicated that, in both cell types, p50 and p65 but not p52, c-Rel, or Rel B were contained in the NF-kappaB-binding complex. NF-kappaB-mediated transcription was increased after N-CAM binding to astrocytes and neurons as demonstrated by the activation of two different luciferase reporter constructs containing NF-kappaB-binding sites. N-CAM binding also resulted in degradation of IkappaB-alpha protein followed by an increase in the levels of IkappaB-alpha mRNA and protein. These results indicate that N-CAM homophilic binding at the cell membrane leads to increased NF-kappaB binding to DNA and transcriptional activation in both neurons and astrocytes. Activation of NF-kappaB, however, did not influence the previously reported ability of N-CAM to inhibit astrocyte proliferation. These observations together support the hypothesis that N-CAM binding activates multiple pathways leading to changes in gene expression in both astrocytes and neurons.