Acute inhibition of fatty acid import inhibits GLUT4 transcription in adipose tissue, but not skeletal or cardiac muscle tissue, partly through liver X receptor (LXR) signaling.

OBJECTIVE: Insulin-mediated glucose uptake is highly sensitive to the levels of the facilitative GLUT protein GLUT4. Transcription of the GLUT4 gene is repressed in states of insulin deficiency and insulin resistance and can be induced by states of enhanced energy output, such as exercise. The cellular signals that regulate GLUT4 transcription are not well understood. We hypothesized that changes in energy substrate flux regulate GLUT4 transcription. RESEARCH DESIGN AND METHODS: To test this hypothesis, we used transgenic mice in which expression of the chloramphenicol acetyltransferase (CAT) gene is driven by a functional 895-bp fragment of the human GLUT4 promoter, thereby acting as a reporter for transcriptional activity. Mice were treated with a single dose of etomoxir, which inhibits the transport of long-chain fatty acids into mitochondria and increases basal, but not insulin-mediated, glucose flux. GLUT4 and transgenic CAT mRNA were measured. RESULTS: Etomoxir treatment significantly reduced CAT and GLUT4 mRNA transcription in adipose tissue, but did not change transcription in heart and skeletal muscle. Downregulation of GLUT4 transcription was cell autonomous, since etomoxir treatment of 3T3-L1 adipocytes resulted in a similar downregulation of GLUT4 mRNA. GLUT4 transcriptional downregulation required the putative liver X receptor (LXR) binding site in the human GLUT4 gene promoter in adipose tissue and 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the LXR agonist, TO901317, partially restored GLUT4 expression in etomoxir-treated cells. CONCLUSIONS: Our data suggest that long-chain fatty acid import into mitochondria in adipose tissue may produce ligands that regulate expression of metabolic genes.

Induction of MMP-1 (collagenase-1) by relaxin in fibrocartilaginous cells requires both the AP-1 and PEA-3 promoter sites.

OBJECTIVES - Relaxin induces the matrix metalloproteinase MMP-1 (collagenase-1) in TMJ fibrocartilaginous cells, and this response is potentiated by beta-estradiol. We identified the MMP-1 promoter sites and transcription factors that are induced by relaxin with or without beta-estradiol in fibrocartilaginous cells. MATERIAL AND METHODS - Early passage cells were transiently transfected with the pBLCAT2 plasmid containing specific segments of the human MMP-1 promoter regulating the chloramphenicol acyl transferase (CAT) gene and co-transfected with a plasmid containing the beta-galactosidase gene. The cells were cultured in serum-free medium alone or medium containing 0.1 ng/ml relaxin, or 20 ng/ml beta-estradiol or both hormones, and lysates assayed for CAT and beta-galactosidase activity. RESULTS - Cells transfected with the -1200/-42 or -139/-42 bp MMP-1 promoter-reporter constructs showed 1.5-fold and 3-fold induction of CAT by relaxin in the absence or presence of beta-estradiol, respectively. Relaxin failed to induce CAT in the absence of the -137/-69 region of the MMP-1 promoter, which contains the AP-1-and PEA3-binding sites. Using wild type or mutated minimal AP-1 and PEA-3 promoters we found that both these promoter sites are essential for the induction of MMP-1 by relaxin. The mRNAs for transcription factors c-fos and c-jun, which together form the AP-1 heterodimer, and Ets-1 that modulates the PEA-3 site, were upregulated by relaxin or beta-estradiol plus relaxin. CONCLUSION - These studies show that both the AP-1 and PEA-3 promoter sites are necessary for the induction of MMP-1 by relaxin in fibrocartilaginous cells.

Analysis of HSV-I ICP22 effects on HCMV major immediate-early promoter structure.

The human cytomegalovirus (HCMV) major immediate-early (MIE) promoter has strong transcriptional promoting capability. Its cis-acting regulatory elements form a special structure in this region that is repeated multiple times; the biological significance of these elements and their different compositions in the transcriptional promoting process remain unclear. Our results demonstrate that the HSV-I MIE protein ICP22 can generate strong repression of many viral and cellular promoters and enhancers. We further studied the transcriptional effects of ICP22 on structural elements and mutations in various HCMV MIE promoters by using a CAT assay. In spite of different transcriptional effects of all the elements in the presence of ICP22, the transcriptional efficiencies exhibited by mutations generated by different compositions and an entire HCMV promoter, are not the simple sum of the functions of these elements. Furthermore, the transcriptional activities of specific sequences were not affected by the presence of ICP22. Therefore, it is assumed that the HCMV MIE promoter co-regulates expression of downstream genes by using viral and cellular specific factors via a specific pathway.

Synthesis and biological properties of new stilbene derivatives of resveratrol as new selective aryl hydrocarbon modulators.

We developed new stilbene derivatives of resveratrol (E)-1-(4'-hydroxyphenyl)-2-(3,5-dihydroxyphenyl)ethene) selective for AhR and devoid of affinity for ER. Among the 24 stilbenes synthesized, all display a higher affinity than resveratrol for AhR. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-ditrifluoromethylphenyl)ethene (4e), (E)-1-(4'-methoxyphenyl)-2-(3,5-dichlorophenyl)ethene (4j), and (E)-1-(4'-chlorophenyl)-2-(3,5-dichlorophenyl)ethene (4b) are selective, high-affinity AhR antagonists with, respective, K(i)s of 2.1, 1.4, and 1.2 nM. (E)-1-(4'-Trifluoromethylphenyl)-2-(3,5-dichlorophenyl)ethene (4i) displays a K(i) of 0.2 nM and is a selective and high-affinity agonist on AhR.

3'-methoxy-4'-nitroflavone, a reported aryl hydrocarbon receptor antagonist, enhances Cyp1a1 transcription by a dioxin responsive element-dependent mechanism.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor, regulating expression of a group of specific genes including cytochrome P450 1A1 (Cyp1a1). Stably transfected luciferase with dioxin responsive elements (DRE) in its promoter region has been commonly used as a reporter gene to study the mechanism of AhR signaling and compare potencies of TCDD and related compounds. However, how these two genes might respond to structurally diverse AhR ligands was unknown. This study investigates their expression in the same cells in response to TCDD, the most potent agonist, and 3'M4'NF, a reported potent antagonist. Our data suggest that these two compounds appear to play different roles in regulating these genes. While TCDD enhanced transcription of both genes, 3'M4'NF induced the endogenous Cyp1a1, but not the reporter gene. Mechanistic studies indicated that the increase in induction of CYP1A1 protein by 3'M4'NF was mediated by AhR-dependent transcriptional activation. Further analysis of the Cyp1a1 promoter sequence did not reveal any 3'M4'NF-specific responsive elements other than DREs. Rather, the interaction between the 3'M4'NF-bound receptor complex and DREs was confirmed by the observation that a single nucleotide mutation in DRE core sequences obliterated AhR enhancer activity in response to both TCDD and 3'M4'NF. Together these data suggest that 3'M4'NF, a weak AhR agonist, activates the AhR to recognize and interact with the same DREs as TCDD. However, depending on its concentration as well as the promoter context of a particular gene, the ability of 3'M4'NF to act as an AhR antagonist or agonist may appear different for various genes.

Chlorpromazine inhibits the glucocorticoid receptor-mediated gene transcription in a calcium-dependent manner.

Antipsychotic drugs can modulate transcription factors and also nuclear receptors, but their action on glucocorticoid receptors (GR)-members of the steroid/thyroid hormone receptor family has not been studied so far. In the present study we investigated effects of various antipsychotics on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with a mouse mammary tumor virus promoter (LMCAT cells). Chlorpromazine (3-100 microM) inhibited the corticosterone-induced gene transcription in a concentration- and time-dependent manner. Clozapine showed a similar, but less potent effect, while haloperidol acted only in high concentrations, and other antipsychotic drugs (sulpiride, raclopride, remoxipride) were without any effect. It was also found that a phorbol ester (an activator of protein kinase C (PKC)) and A-23187 (Ca(2+)-ionophore) attenuated the inhibitory effect of chlorpromazine on the GR-induced gene transcription. An antagonist of the L-type Ca(2+) channel, as well as an inhibitor of phospholipase C (PLC) inhibited the corticosterone-induced gene transcription, but had no effect on the chlorpromazine-induced changes. The involvement of a PKC/PLC pathway in the chlorpromazine action was confirmed by Western blot analysis which showed that the drug in question decreased the PLC-beta(1) protein level, and to a lesser extent that of the PKC-alpha protein in LMCAT cells. The aforementioned data suggest that inhibition of the glucocorticosteroid-induced gene transcription by chlorpromazine and clozapine may be a mechanism by which these drugs block some effects induced by glucocorticoids. The inhibitory effect of chlorpromazine on the corticosterone-induced gene transcription seems to depend on the inhibition of Ca(2+) influx and/or the inhibition of some calcium-dependent enzymes, e.g. phospholipase beta(1).

Human aldolase C gene expression is regulated by adenosine 3',5'-cyclic monophosphate (cAMP) in PC12 cells.

We have examined the effects of an adenosine 3',5'-cyclic monophosphate (cAMP) analog on human aldolase C gene expression in the rat pheochromocytoma cell line PC12. Incubation for 4 h with 500 microM 8-Br-cAMP increased aldolase C mRNA expression 2.5-fold and the expression was still above basal level 24 h later. Using transient transfection experiments we demonstrate that the distal element D in the promoter region of the human aldolase C gene, which binds a transcriptional activator (NGFI-B), is involved in this regulation. NGFI-B mRNA and protein expression were promptly (15 min) increased after 8-Br-cAMP treatment and precedes aldolase C mRNA increase (30 min). After 4 h of 8-Br-cAMP treatment, the binding of NGFI-B protein to the distal element D in the distal promoter region was increased twofold and this correlates with the increased expression of the clone that contains distal element D. These results indicate that the distal element D in the promoter region of the human aldolase C gene is the target of a cAMP-dependent regulation pathway.

Human choriocarcinoma cell line JEG-3 produces and secretes active retinoids from retinol.

Vitamin A (retinol) and its active derivatives (the retinoids) are essential for growth and development of the mammalian fetus. Maternally-derived retinol has to pass through the placenta to reach the developing fetus. Despite its apparent importance, little is known about placental metabolism of retinol, and particularly placental production and/or secretion of active retinoids. It has been previously considered that retinoids are recruited from the uterine environment to influence placental development and function during gestation. We have studied retinoid metabolism in the human choriocarcinoma cell line JEG-3 and demonstrate, for the first time, that active retinoids are produced endogenously by the JEG-3 cell line from retinol. These retinoids induce gene expression from a retinoic acid-responsive enhancer element reporter plasmid and modulate placental transglutaminase activity. Furthermore, retinoids are secreted from JEG-3, as shown by the activation of retinoic acid-responsive beta lacZ reporter cells grown in conditioned media. These results suggest that there could be an active role for trophoblast-derived retinoids during human development.

[Effect of IFN-gamma and TNF-alpha on hepatitis B virus posttranscriptional regulatory elements].

OBJECTIVE: To explore the effect of IFN-gamma and TNF-alpha on hepatitis B virus posttranscriptional regulatory elements. METHODS: The objective eukaryotic expression vectors were constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identification was performed using PCR and sequencing analysis. CAT assays were performed by using ELISA kit. RESULTS: The eukaryotic expression vectors containing HPRE segment and mutating point were constructed successfully as confirmed by sequencing analysis. The normalized CAT activity from recombinant pDM138 and control produced in transfected cells incubated in the absence of cytokine were 896 +/- 214 and 37 +/- 11 pg/ml, respectively. After incubation with IFN-gamma, TNF-alpha and IFN-gamma + TNF-alpha, the normalized CAT activity from recombinant pDM138 produced in transfected cells were 324 +/- 57, 396 +/- 82 and 175 +/- 36 pg/ml, respectively (t = 5.19,4.39, 6.68, P < 0.01). The effect of IFN-gamma and TNF-alpha were dose dependent over a range of 0 to 1,000 U/ml. CONCLUSION: The expression of HBV gene may be regulated by IFN-gamma and TNF-alpha through inhibiting the function of HPRE.

Conserved as well as divergent regulatory elements account for expression of the human and rodent phenylalanine hydroxylase genes.

We have uncovered a fundamental difference in the regulation of the rodent and the human phenylalanine hydroxylase (PAH) genes: expression of human PAH is independent of glucocorticoids and/or cAMP in contrast to the mouse gene which is not only highly inducible but dependent upon hormones for expression. Nevertheless, the two genes do exhibit similarities: DNaseI hypersensitive sites are identically located in the regulatory regions, and the sequences around these sites are partially conserved and associated with regulatory elements sharing similar function. In transient transfections, the human proximal promoter is tissue-specific and presents significant activity compared to the extremely low and ubiquitous activity of the mouse promoter. DNA fragments corresponding to the two upstream hypersensitive sites of both genes have enhancer activity that depends upon the liver-enriched transcription factor binding sites for hepatocyte nuclear factor (HNF) 1 and/or CCAAT/enhancer binding protein (C/EBP). While expression of the rodent gene relies upon two modules in the HSIII enhancer, one activated by HNF1 and C/EBP and the other required for the hormone response, the human equivalent has conserved only the liver-specific transcription factor binding module. Even though the more proximal enhancer is not necessary for full reporter gene activity in transient transfection assays in Pah-expressing hepatoma cells, this enhancer could be required in both species for activation during development.

Transcriptional regulation of fatty acid synthase gene by somatotropin in 3T3-F442A adipocytes.

Somatotropin (ST) antagonizes insulin stimulation of fatty acid synthase (FAS) enzyme activity and gene transcription in adipocytes. Previous studies have shown that an insulin response element (IRE) is located in the proximal region of the FAS promoter (-71 to -50) and upstream stimulatory factor (USF) 1 binds to this IRE. The present study was conducted to initially evaluate whether there is a somatotropin response element (STRE) in the 5'flanking region of the FAS gene and to determine whether USF1 mediates the effect of ST on FAS gene transcription in 3T3-F442A adipocytes. Two 5' deletion FAS promoter constructs (pFAS-CATS4 and pFAS-CAT5), which contain the 5' flanking sequences of the rat FAS gene at -112 to +65 and -2195 to +65, respectively, were stably transfected into 3T3-F442A preadipocytes. Insulin stimulated chloramphenicol acetyltransferase (CAT) activity 1.7- and 4.7-fold (P < 0.05) in 3T3-F442A adipocytes transfected with pFAS-CATS4 and pFAS-CAT5, respectively. In contrast, bovine somatotropin (bST) attenuated the stimulatory effect of insulin on CAT activity by approximately 60% (P < 0.05) in both constructs. When 3T3-F442A adipocytes were treated with insulin (10 ng/mL) or insulin (10 ng/mL) plus bST (100 ng/mL) for 24, 48, or 72 h, neither insulin nor bST significantly affected USF1 mRNA levels. When human USF1 (hUSF1) cDNA probe was used, however, insulin increased the abundance of an unidentified transcript (named hUSF1-like mRNA) 11- to 25-fold (P < 0.05) and ST decreased the stimulatory effect of insulin on hUSF1-like mRNA levels by 50 to 90% (P < 0.05). Western blot analyses of nuclear extracts from cells treated with insulin (10 ng/mL) or insulin (10 ng/mL) plus bST (100 ng/mL) for 48 h demonstrated that the abundance of USF1 was not affected by insulin or ST. Furthermore, electrophoretic mobility shift analyses (EMSA) of nuclear extracts revealed that neither insulin nor ST had an effect on the binding of USF1 to the IRE. These results suggest that a STRE may be located within the first 112 bp of the FAS promoter and that USF1 does not directly mediate the effect of ST on transcription of the FAS gene in 3T3-F442A adipocytes.

Membrane-bound protein kinase A inhibits smooth muscle cell proliferation in vitro and in vivo by amplifying cAMP-protein kinase A signals.

cAMP-dependent protein kinase is anchored to discrete cellular compartments by a family of proteins, the A-kinase anchor proteins (AKAPs). We have investigated in vivo and in vitro the biological effects of the expression of a prototypic member of the family, AKAP75, on smooth muscle cells. In vitro expression of AKAP75 in smooth muscle cells stimulated cAMP-induced transcription, increased the levels of the cyclin-dependent kinase-2 inhibitor p27(kip1), and reduced cell proliferation. In vivo expression of exogenous AKAP75 in common carotid arteries, subjected to balloon injury, significantly increased the levels of p27(kip1) and inhibited neointimal hyperplasia. Both the effects in smooth muscle cells in vitro and in carotid arteries in vivo were specifically dependent on the amplification of cAMP-dependent protein kinase (PKA) signals by membrane-bound PKA, as indicated by selective loss of the AKAP75 biological effects in mutants defective in the PKA anchor domain or by suppression of AKAP effects by the PKA-specific protein kinase inhibitor. These data indicate that AKAP proteins selectively amplify cAMP-PKA signaling in vitro and in vivo and suggest a possible target for the inhibition of the neointimal hyperplasia after vascular injury.

Sp1 phosphorylation regulates inducible expression of platelet-derived growth factor B-chain gene via atypical protein kinase C-zeta.

Platelet-derived growth factor (PDGF) is a broadly expressed mitogenic and chemotactic factor with diverse roles in a number of physiologic and pathologic settings. The zinc finger transcription factors Sp1, Sp3 and Egr-1 bind to overlapping elements in the proximal PDGF B-chain promoter and activate transcription of this gene. The anthracycline nogalamycin has previously been reported to inhibit the capacity of Egr-1 to bind DNA in vitro. Here we used electrophoretic mobility shift assays to show that nogalamycin added to cells in culture did not alter the interaction of Egr-1 with the PDGF-B promoter. Instead, it enhanced the capacity of Sp1 to bind DNA. Nogalamycin increased PDGF-B mRNA expression at the level of transcription, which was abrogated by mutation of the Sp1 binding site in the PDGF-B promoter or overexpression of mutant Sp1. Rather than increasing total levels of Sp1, nogalamycin altered the phosphorylation state of the transcription factor. Overexpression of dominant-negative PKC-zeta blocked nogalamycin-inducible Sp1 phosphorylation and PDGF-B promoter-dependent expression. Nogalamycin stimulated the phosphorylation of PKC-zeta (on residue Thr(410)). These findings demonstrate for the first time that PKC-zeta and Sp1 phosphorylation mediate the inducible expression of this growth factor.

Inhibition of HIV-1 transcription by cyclopentenone prostaglandin A1 in Jurkat T lymphocytes.

Cyclopentenone prostaglandins inhibit virus replication in several DNA and RNA virus models. In this report we investigated the effect of prostaglandin A1 (PGA1) on HIV-1 transcription in human CD4+ Jurkat T lymphocyte cells. A dramatic reduction of HIV-1 RNA levels was detected up to seven days post infection in both unstimulated and phorbol 12-mystrate 13-acetate (PMA)-stimulated cells treated with PGA1- PGA1 treatment of cells was also effective in inhibiting the transcription of a chloramphenicol acetyltransferase (CAT) reporter gene, under the control of HIV-1 LTR, in Jurkat-Tat cells. We also show that PGA1 induced the synthesis of 70-kDa heat-shock protein (HSP70) in this cell system and the induction correlated with the drug-antiviral activity. PGA1 was also found to induce the loss of the tumor suppressor p53 protein, in the "proliferative" conformation, in a time correlation with the induction of the HSP70 As the "proliferative" p53 has been involved in the positive trans-activation of the HIV-1 LTR its depletion could contribute to the inhibitory mechanisms of PGA1 on virus transcription.

Antidepressant drugs inhibit glucocorticoid receptor-mediated gene transcription - a possible mechanism.

1. Antidepressant drugs are known to inhibit some changes evoked by glucocorticoids, as well as a hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis, often observed in depression. 2. The aim of present study was to investigate effects of various antidepressant drugs on the glucocorticoid-mediated gene transcription in fibroblast cells, stably transfected with an MMTV promoter (LMCAT cells). 3. The present study have shown that antidepressants (imipramine, amitriptyline, desipramine, fluoxetine, tianeptine, mianserin and moclobemide), but not cocaine, inhibit the corticosterone-induced gene transcription in a concentration- and a time-dependent manner. 4. Drugs which are known to augment clinical effects of medication in depressed patients (lithium chloride, amantadine, memantine), do not affect the inhibitory effects of imipramine on the glucocorticoid receptor (GR)-mediated gene transcription. 5. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca(2+)/calmodulin-dependent protein kinase (CaMK) and antagonists of the L-type Ca(2+) channel also inhibit the corticosterone-induced gene transcription. 6. Inhibitors of protein kinase A (PKA) and protein kinase G (PKG) are without effect on the GR-induced gene transcription. 7. Phorbol ester (an activator of PKC) attenuates the inhibitory effect of imipramine on the GR-induced gene transcription. 8. Imipramine decreases binding of corticosterone-receptor complex to DNA. 9. It is concluded that antidepressant drugs inhibit the corticosterone-induced gene transcription, and that the inhibitory effect of imipramine depends partly on the PLC/PKC pathway.

Estrogen and aryl hydrocarbon receptor expression and crosstalk in human Ishikawa endometrial cancer cells.

Ishikawa endometrial cancer cells express the estrogen receptor (ER), and this study investigates aryl hydrocarbon receptor (AhR) expression and inhibitory AhR-ER crosstalk in this cell line. Treatment of Ishikawa cells with the AhR agonist [3H]2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) gave a radiolabeled nuclear complex that sedimented at 6.0 S in sucrose density gradients, and Western blot analysis confirmed that Ishikawa cells expressed human AhR and AhR nuclear translocator (Arnt) proteins. Treatment of Ishikawa cells with 10 nM TCDD induced a 9.7-fold increase in CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity and a 10.5-fold increase in chloramphenicol acetyltransferase (CAT) activity in cells transfected with pRNH11c containing an Ah-responsive human CYP1A1 gene promoter insert (-1142 to +2434). Inhibitory AhR-ER crosstalk was investigated in Ishikawa cells using E2-induced cell proliferation and transcriptional activation assays in cells transfected with E2-responsive constructs containing promoter inserts from the progesterone receptor and vitellogenin A2 genes. AhR agonists including TCDD, benzo[a]pyrene (BaP) and 6-methyl-1,3,8-trichlorodibenzofuran, inhibited 32-47% of the E2-induced responses. In contrast, neither estrogen nor progesterone inhibited EROD activity induced by TCDD in Ishikawa cells, whereas inhibitory ER-AhR crosstalk was observed in ECC-1 endometrial cells suggesting that these interactions were cell context-dependent.

Photodynamic therapy-mediated oxidative stress as a molecular switch for the temporal expression of genes ligated to the human heat shock promoter.

Oxidative stress associated with photodynamic therapy (PDT) is a transcriptional inducer of genes encoding stress proteins, including those belonging to the heat shock protein (hsp) family. The efficiency of PDT to function as a molecular switch by initiating expression of heterologous genes ligated to the human hsp promoter was examined in the present study. Selective and temporal reporter gene expression was documented after PDT in mouse radiation-induced fibrosarcoma cells stably transfected with recombinant vectors containing an hsp promoter ligated to either the lac-z or CAT reporter genes and in transfected radiation-induced fibrosarcoma tumors grown in C3H mice. Hyperthermia treatments were included as a positive control for all experiments. Expression vectors containing either human p53 or tumor necrosis factor (TNF)-alpha cDNA under the control of an hsp promoter were also constructed and evaluated. A p53 null and TNF-alpha-resistant human ovarian carcinoma (SKOV-3) cell line was stably transfected with either the p53 or TNF-alpha constructs. Inducible expression and function of p53 as well as inducible expression, secretion, and biological activity of TNF-alpha were documented after PDT or hyperthermia in transfected SKOV cells. These results demonstrate that PDT-mediated oxidative stress can function as a molecular switch for the selective and temporal expression of heterologous genes in tumor cells containing expression vectors under the control of an hsp promoter.

[Ca(2+)](i) oscillation frequency regulates agonist-stimulated NF-kappaB transcriptional activity.

In nonexcitable cells, stimulation by high agonist concentrations typically produces a biphasic increase in cytosolic Ca(2+) ([Ca(2+)](i)). This response is characterized by a transient initial increase because of intracellular Ca(2+) release followed by a sustained elevation which varies in amplitude depending on the nature of the stimulus. In contrast, low-level stimulation often evokes oscillatory changes in [Ca(2+)](i). The specific information provided by repetitive [Ca(2+)](i) spikes appears to be encoded in the frequency rather than in the amplitude of [Ca(2+)](i) oscillations. The specific, membrane-permeable inositol 1,4, 5-trisphosphate (Ins-1,4,5-P(3)) receptor blocker Xestospongin C (XeC, 2-20 microM) was used to affect [Ca(2+)](i) signaling in human aortic endothelial cells (HAEC) during an established response to low-level (1 microM) histamine stimulation. XeC produced a dose-dependent decrease in the frequency of [Ca(2+)](i) oscillations during histamine stimulation without affecting oscillation amplitude. Histamine stimulated a 14-fold increase in NF-kappaB-chloramphenicol acetyltransferase reporter gene activity that was dose-dependently decreased by XeC. Thus, during low-level agonist stimulation, [Ca(2+)](i) oscillation frequency regulates nuclear transcription in HAEC.

Isolation and functional characterization of the human 90K promoter.

90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive synergism between different elements within it. Consistent with the proposed function of 90K, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small region around the minimal promoter (-99 --> -12) was highly homologous between human and mouse. While both human and mouse minimal promoters contained an interferon-responsive element (IRF-E), the human minimal promoter was not inducible by poly(I). poly(C) in contrast to that of the mouse. Point mutations 30 bp upstream of the IRF-E, however, conferred inducibility to the human minimal promoter, suggesting interaction between different promoter elements.

Inhibition of astroglial nitric oxide synthase type 2 expression by idazoxan.

Binding of idazoxan (IDA) to imidazoline receptors of the I2 subtype in astrocytes influences astroglial gene expression as evidenced by increased expression of glial fibrillary acidic protein and mRNA. To determine whether IDA affected glial inflammatory gene expression, we tested the effects of IDA on astroglial nitric oxide synthase type-2 (NOS-2) expression. NOS-2 was induced in primary rat astrocytes and C6 glioma cells by incubation with 1 microgram/ml lipopolysaccharide (LPS) plus three cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma) or three cytokines alone. Cells were incubated with 1-100 microM IDA, and at 24 h NOS-2 expression assessed. In astrocytes and C6 cells, preincubation with IDA dose-dependently inhibited nitrite accumulation (IC50 approximately 25 microM), accompanied by a reduction in NOS-2 protein levels and L-citrulline synthesis activity in cell lysates. IDA also inhibited nitrite production in LPS stimulated RAW 264.7 macrophages. In astrocytes, but not C6 cells, longer preincubation times with IDA yielded significantly greater suppression, and maximal suppression (>90%) was achieved after a 8 h preincubation in 100 microM IDA. The degree of inhibition was diminished whether IDA was added after LPS plus cytokine mixture. In contrast to NE, continuous incubation with IDA was required to achieve suppression. IDA reduced induction of NOS-2 protein levels, steady state NOS-2 mRNA levels, and activity of a NOS-2 promoter construct stably transfected in C6 cells. These results show that IDA inhibits NOS-2 activity and protein expression in glial cells and macrophages, and suggest that this occurs by decreasing transcription from the NOS-2 promoter.