Luciferase Reporter Gene Activity Research Articles

Glucocorticoid Receptor Nuclear Translocation in Airway Cells after Inhaled Combination Therapy

Clinical evidence is accumulating for the efficacy of adding inhaled long-acting beta(2)-agonists (LABAs) to corticosteroids in asthma. Corticosteroids bind to cytoplasmic glucocorticoid receptors (GRs), which then translocate to the nucleus where they regulate gene expression. This article reports the first evidence in vivo of an interaction between inhaled LABA and corticosteroid on GR nuclear translocation in human airway cells using immunocytochemistry. We initially demonstrated significant GR activation 60 minutes after inhalation of 800 microg beclomethasone dipropionate in six healthy subjects. Subsequently, we determined the effects of salmeterol and fluticasone propionate (FP) in seven steroid-naive patients with asthma. We observed dose-dependent GR activation with 100- and 500-microg doses of FP, and to a lesser extent with 50 microg salmeterol alone. However, combination therapy with 100 microg FP and salmeterol augmented the action of FP on GR nuclear localization. In vitro, salmeterol enhanced FP effects on GR nuclear translocation in epithelial and macrophage-like airway cell lines. In addition, salmeterol in combination with FP enhanced glucocorticoid response element (GRE)-luciferase reporter gene activity and mitogen-activated protein kinase phosphatase 1 (MKP-1) and secretory leuko-proteinase inhibitor (SLPI) gene induction. Together, our data confirm that GR nuclear translocation may underlie the complementary interactions between LABAs and corticosteroids, although the precise signal transduction mechanisms remain to be determined.

Dexamethasone Fails to Inhibit the Induction of Cytosolic Phospholipase A 2 Expression by Interleukin-1β in Cultured Primary Human Amnion Fibroblasts

To investigate the interaction of dexamethasone and interleukin-1beta (IL-1beta) on the expression of cytosolic phospholipase A(2) (cPLA(2)), the enzyme catalyzing the first reaction in the formation of prostaglandins, in cultured primary human amnion fibroblasts. Human amnion fibroblasts were prepared from fetal amnion collected at term and were treated with dexamethasone with or without interleukin-1beta for 24h. Prostaglandin E(2) (PGE(2)) output and cPLA(2) expression in cultured amnion fibroblasts were measured with radioimmunoassay, quantitative real-time polymerase chain reaction, Western blotting and cPLA(2) promoter-driven luciferase reporter gene activity. Both dexamethasone and IL-1beta caused a significant increase in prostaglandin E(2) output, cPLA(2) mRNA and protein expression in cultured human amnion fibroblasts. Both dexamethasone and IL-1beta stimulated cPLA(2) promoter-driven luciferase reporter gene activity. There was no obvious antagonistic or synergistic effect of combined treatment of dexamethasone and IL-1beta on PGE(2) output, cPLA(2) expression or cPLA(2) promoter-driven luciferase reporter gene activity in cultured human amnion fibroblasts. The above findings suggest that paradoxically dexamethasone is a stimulator for both prostaglandin synthesis and cPLA(2) expression in human amnion fibroblasts. The interaction between dexamethasone and IL-1beta on prostaglandin synthesis and cPLA(2) expression is neither synergistic nor conventionally antagonistic.

Heparanase Regulates Murine Hair Growth

Heparanase is an endoglycosidase that cleaves heparan sulfate, the main polysaccharide component of the extracellular matrix. Heparan sulfate moieties are responsible for the extracellular matrix barrier function, as well as for sequestration of heparin-binding growth factors in the extracellular matrix. Degradation of heparan sulfate by heparanase enables cell movement through extracellular barriers and releases growth factors from extracellular matrix depots, making them bioavailable. Here, we demonstrate a highly coordinated temporospatial pattern of heparanase expression and enzymatic activity during hair follicle cycling. This pattern paralleled the route and timing of follicular stem cell progeny migration and reconstitution of the lower part of the follicle, which is a prerequisite for hair shaft formation. By monitoring in vivo activation of luciferase reporter gene driven by heparanase promoter, we observed activation of heparanase gene transcription at a specific stage of the hair cycle. Heparanase was produced by rat vibrissa bulge keratinocytes, closely related to a follicular stem cell population. Heparanase contributed to the ability of the bulge-derived keratinocytes to migrate through the extracellular matrix barrier in vitro. In heparanase-overexpressing transgenic mice, increased levels of heparanase enhanced active hair growth and enabled faster hair recovery after chemotherapy-induced alopecia. Collectively, our results identify heparanase as an important regulator of hair growth and suggest that cellular mechanisms of its action involve facilitation of follicular stem cell progeny migration and release of extracellular matrix-resident, heparin-bound growth factors, thus regulating hair cycle.

Serum-dependent potentiation of lipopolysaccharide-induced nitric oxide production is mediated by the events after the transcription of inducible type of nitric oxide synthase

The mechanism of serum-dependent potentiation of lipopolysaccharide (LPS)-induced nitric oxide (NO) production was studied by incubating mouse macrophage cell line, RAW 264.7, in the presence of fetal bovine serum (FBS). The addition of FBS definitely enhanced LPS-induced NO production through augmented expression of inducible type NO synthase (iNOS) mRNA and protein. However, nuclear run-on analysis demonstrated only marginal enhancement in the rate of LPS-induced iNOS gene transcription in the presence of FBS. Further, there was no significant difference in the luciferase reporter gene activity linked to the iNOS promoter–enhancer gene in response to LPS between the presence and absence of FBS. FBS-dependent enhancement did not appear to involve the initial step for triggering iNOS transcription in LPS-induced NO production. Rather, FBS was suggested to affect the accumulation and stabilization of iNOS mRNA leading to iNOS protein and NO production by some post-transcriptional regulatory mechanism.

NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthésy, J. Biol. Chem. 269 (1994) 20691–20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin–proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-κB (NF-κB) in regulating muscle protein degradation and expression of the ubiquitin–proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C2C12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-κBα, an NF-κB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-κBα, nuclear accumulation of NF-κB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-κBα proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-κB. Proteolysis-inducing factor also induced increased expression of the ubiquitin–proteasome pathway, as determined by ‘chymotrypsin-like’ enzyme activity, the predominant proteolytic activity of the β-subunits of the proteasome, protein expression of 20S α-subunits and the 19S subunits MSS1 and p42, as well as the ubiquitin conjugating enzyme, E214k, in cells containing wild-type, but not mutant, I-κBα. The ability of mutant I-κBα to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin–proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-κB.

Assessment of Estrogenic Endocrine-Disrupting Chemical Actions in the Brain Using in Vivo Somatic Gene Transfer

Estrogenic endocrine-disrupting chemicals abnormally stimulate vitellogenin gene expression and production in the liver of many male aquatic vertebrates. However, very few studies demonstrate the effects of estrogenic pollutants on brain function. We have used polyethylenimine-mediated in vivo somatic gene transfer to introduce an estrogen response element–thymidine kinase–luciferase (ERE-TK-LUC) construct into the brain. To determine if waterborne estrogenic chemicals modulate gene transcription in the brain, we injected the estrogen-sensitive construct into the brains of Nieuwkoop-Faber stage 54 Xenopus laevis tadpoles. Both ethinylestradiol (EE2; p < 0.002) and bisphenol A (BPA; p < 0.03) increased luciferase activity by 1.9- and 1.5-fold, respectively. In contrast, low physiologic levels of 17β-estradiol had no effect (p > 0.05). The mixed antagonist/agonist tamoxifen was estrogenic in vivo and increased (p < 0.003) luciferase activity in the tadpole brain by 2.3-fold. There have been no previous reports of somatic gene transfer to the fish brain; therefore, it was necessary to optimize injection and transfection conditions for the adult goldfish (Carassius auratus). Following third brain ventricle injection of cytomegalovirus (CMV)-green fluorescent protein or CMV-LUC gene constructs, we established that cells in the telencephalon and optic tectum are transfected. Optimal transfections were achieved with 1 μg DNA complexed with 18 nmol 22 kDa polyethylenimine 4 days after brain injections. Exposure to EE2 increased brain luciferase activity by 2-fold in males (p < 0.05) but not in females. Activation of an ERE-dependent luciferase reporter gene in both tadpole and fish indicates that waterborne estrogens can directly modulate transcription of estrogen-responsive genes in the brain. We provide a method adaptable to aquatic organisms to study the direct regulation of estrogen-responsive genes in vivo.

Developments toward a microfluidic system for long-term monitoring of dynamic cellular events in immobilized human cells.

A microfluidic system for long-term real-time monitoring of dynamic cellular events of immobilized human cells was investigated. The luciferase reporter gene activity in the reporter cell line HFF11, based on HeLa cells, was used as the model system. The cells were immobilized on silicon flow-through microchips and continuously supplied with a cell medium at 2 microL/min while maintaining the chip at 37 degrees C. The HFF11 cell line was designed for high-throughput screening of ligands for seven-transmembrane receptors. When a ligand binds, the receptor is activated and a cascade of intracellular reactions starts, ending with the synthesis of the reporter protein Photinus luciferase. The major goal was to develop a microfluidic system for continuous long-term assaying of the intracellular reporter gene activity in real time and determine the conditions, which could minimize cells stress and hence unspecific expression of the reporter gene. In the resulting microfluidic system and assay protocol, the cell microchip could be kept and assayed for a period up to 30 h. The developed system and data outcome was compared with a corresponding microtiter plate performed with the same cell line to highlight the advantages obtained in the microfluidic format.

Activation of AP-1 and CRE-dependent gene expression via mu-opioid receptor.

Addiction to opiates depend on drug-induced neuroplastic changes and are underlain by alterations of gene expression. Transcription factors Ca2+/cAMP responsive element binding protein (CREB) and activator protein 1 (AP-1) may constitute a direct link between the opioid-regulated signal transduction pathways and modulation of gene expression. Acute treatment of Neuro2a MOR neuroblastoma cells with opioids stimulated CREB activity; prolonged treatment normalized it, while withdrawal from the drug again elicited an increase in phosphorylated CREB levels. Protein kinase C was responsible for the activation of transcription following acute opioid administration whereas the cAMP pathway activated similar mechanisms during withdrawal, making CREB a kind of 'a trigger' reacting to the presence or withdrawal of the opioid signal. Apart from the elevated CREB phosphorylation, CRE binding activity and expression of luciferase reporter gene regulated by CRE elements were increased after single administration and during withdrawal from the prolonged opioid treatment. Along with CREB, AP-1 binding activity and AP-1-directed transcription were stimulated after single administration and during withdrawal from the opioid. These results provide evidence that both single opioid administration and opioid withdrawal activate CREB and CRE-dependent transcriptional mechanisms via distinct intracellular signaling pathways.

Transactivation of the PPAR-responsive enhancer module in chemopreventive glutathione S-transferase gene by the peroxisome proliferator-activated receptor-gamma and retinoid X receptor heterodimer.

Cancer chemopreventive agents transcriptionally induce glutathione S-transferase (GST), which can protect cells from chemical-induced carcinogenesis. Activation of either NF-E2-related factor-2 (Nrf2) or the CCAAT/enhancer binding protein-beta (C/EBPbeta) contributes to GST induction. Peroxisome proliferator-activated receptor-gamma (PPARgamma) and the retinoic acid X receptor (RXR) play roles in regulating cell differentiation and chemoprevention. This study examined GSTA2 gene induction by the PPARgamma activator and 9-cis-retinoic acid (RA), a RXR ligand, and investigated the molecular basis of PPAR-RXR-mediated GSTA2 induction in the H4IIE hepatocytes. Either 15-deoxy-delta (12, 14)-prostaglandin J(2) (PGJ(2)) or RA induced GSTA2 with Nrf2 and C/EBPbeta activation. When compared with PGJ(2) or RA alone, PGJ(2) + RA enhanced GSTA2 induction, with increases in Nrf2 and C/EBPbeta activation. PGJ(2) + RA increased the luciferase reporter gene activity in the cells transfected with the -1.65-kb flanking region of the GSTA2 gene. Thiazolidinedione PPARgamma agonists, troglitazone, rosiglitazone, and pioglitazone, in combination with RA, potentiated GSTA2 induction, confirming that the activation of the PPARgamma and RXR heterodimer contributed to GSTA2 expression. Deletion of the antioxidant response element- or C/EBP-binding sites or the overexpression of dominant-negative mutant of C/EBP abolished the reporter gene expression. PGJ2 + RA increased the binding of the PPARgamma - RXR heterodimer to the putative PPAR-response elements (PPREs) in the GSTA2 promoter. Specific mutations of these multiple PPRE sites resulted in the complete loss of its responsiveness to PGJ2 + RA, which suggests that these binding sites function as a PPRE-responsive enhancer module (PPREM). Transactivation of PPREM by the PPARgamma - RXR heterodimer was verified by the effective GSTA2 induction in the cells treated with PGJ2 + RA after transfecting them with the plasmids encoding PPARgamma1 and RXRalpha. In conclusion, the PPARgamma - RXR heterodimer promotes GSTA2 induction by activating PPREM in the GSTA2 gene, as well as inducing Nrf2 and C/EBPbeta activation.

122. Expression of hCD40 in the Pulmonary Endothelium Targeted Via a Fiber-Modified Adenovirus Improves Gene Delivery to the Lung

The lung is an attractive target for gene therapy since many diseases such as cystic fibrosis, lung cancer, primary pulmonary hypertension and acute respiratory distress syndrome require novel therapeutic approaches. However, pulmonary gene transfer by adenovirus (Ad) vectors via the vascular route has proven to be limited with respect to efficiency due to low expression of human coxsackie adenovirus receptor (hCAR) in the lung. Conversely, the liver expresses high levels of hCAR leading to ectopic transduction and the risk of liver damage. Therefore, development of CAR-independent gene transfer strategy to the lung is needed. We recently constructed an Ad in which the fiber was replaced by human CD40 ligand (Ad5 Luc FF / CD40L) and showed the utility of this fiber replacement strategy for genetic targeting of the virus to human CD40 (Belousova et al. J. Virol 77:11367–77, 2003). In addition, we recently showed the promoter for vascular endothelial growth factor receptor 1 (fms-like tyrosine kinase-1; flt-1) has high activity in pulmonary endothelial cells, low activity in hepatocytes (Nicklin et al. Hypertension 38:65–70, 2001), and also provided transcriptional targeting to the lung vasculature (Reynolds et al. Nat. Biotechnol.19:838–42, 2001). We hypothesized that hCD40 expression, transcriptionally targeted to the vascular endothelium via the flt-1 promoter, could provide CD40-targeted gene delivery to the lung in a CAR-independent manner. We constructed an E1-deleted recombinant adenovirus expressing hCD40 driven by the flt-1 promoter (Ad5flt1-hCD40). We then analyzed hCD40 expression and flt-1 activity in several cell lines by luciferase assay and fluorescence-activated cell sorting (FACS) analysis. We selected NTERA-2 cells as low hCD40 and flt-1 positive cell line, and HeLa cells as low hCD40 and flt-1 negative cell line as control for further experiments. Forty-eight hours after the first infection with Adflt1-hCD40 and the control virus, we quantitated hCD40 expression on the cell surface by FACS analysis. Adflt1-hCD40 infected NTERA-2 cells had markedly increased hCD40 expression compared to either control virus infected cells or flt-1 negative HeLa cells. Further, we evaluated infectivity enhancement gained by increased hCD40 in the flt-1 positive cell line. Forty-eight hours after the first infection, cells were infected with Ad5 Luc FF / CD40L vectors at an MOI of 1000 viral particles / cell to determine infectivity enhancement accrued via CD40 targeting. Luciferase assays were performed 24 hours post second infection. Ad5 Luc FF / CD40L vector infectivity in Ad flt-1 hCD40 infected NTERA-2 cells showed 13-fold greater luciferase reporter gene activities versus negative control virus infected cells. These data suggest that expression of hCD40 in pulmonary endothelial cells using recombinant Ad vectors containing hCD40 gene under the control of the flt-1 promoter and targeted with a novel fiber modified Ad vector can increase the efficacy and specificity of adenoviral gene therapy for lung disease.

Pharmacological and genetic modulation of Wnt-targeted Cre-Lox-mediated gene expression in colorectal cancer cells.

Wnt-targeted gene therapy has been proposed as a treatment for human colorectal cancer (CRC). The Cre-Lox system consists of methodology for enhancing targeted expression from tissue-specific or cancer-specific promoters. We analyzed the efficiency of Wnt-specific promoters as drivers of the Cre-mediated activity of a luciferase reporter gene or cell death effector gene in CRC cell lines in the presence and absence of two modulators of Wnt activity, sodium butyrate and lithium chloride. Butyrate is present in the colonic lumen after digestion of fiber-rich foods, whereas the colonic lumen is readily accessible to lithium chloride. In both SW620 and HCT-116 CRC cells, a physiologically relevant concentration of butyrate upregulated reporter and effector activity and altered the Wnt-specific expression pattern. Lithium chloride markedly enhanced Cre-Lox-mediated Wnt-specific reporter expression only in APC wild-type CRC cells. Possibilities for genetic modulation of the proposed CRC therapy included Wnt-specific expression of a floxed Lef1-VP16 fusion that enhanced Wnt-specific cell death and of a floxed dominant-negative Tcf4 that specifically downregulated endogenous Wnt activity. These findings demonstrated that the Cre-Lox system, in combination with pharmacological and genetic modulators, represents effective methodology for enhancing Wnt-targeted gene therapy.

The angiotensin II type I receptor-associated protein, ATRAP, is a transmembrane protein and a modulator of angiotensin II signaling.

Our group identified angiotensin II type 1 (AT1) receptor-associated protein (ATRAP) in a yeast two-hybrid screen for proteins that bind to the carboxyl-terminal cytoplasmic domain of the AT1. In this work, we characterize ATRAP as a transmembrane protein localized in intracellular trafficking vesicles and plasma membrane that functions as a modulator of angiotensin II-induced signal transduction. ATRAP contains three hydrophobic domains at the amino-terminal end of the protein, encompassing the amino acid residues 14-36, 55-77, and 88-108 and a hydrophilic cytoplasmic carboxyl-terminal tail from residues 109-161. Endogenous and transfected ATRAP cDNA shows a particulate distribution; electron microscopy reveals the presence of ATRAP in prominent perinuclear vesicular membranes; and colocalization analysis by immunofluorescence shows that ATRAP colocalizes in an intracellular vesicular compartment corresponding to endoplasmic reticulum, Golgi, and endocytic vesicles. Real-time tracking of ATRAP vesicles shows constitutive translocation toward the plasma membrane. Using epitope-tagged forms of ATRAP at either the amino or carboxyl end of the molecule, we determined the orientation of the amino end as being outside the cell. Mutant forms of ATRAP lacking the carboxyl end are unable to bind to the AT1 receptor, leading to the formation of prominent perinuclear vesicle clusters. Functional analysis of the effects of ATRAP on angiotensin II-induced AT1 receptor signaling reveals a moderate decrease in the generation of inositol lipids, a marked decrease in the angiotensin II-stimulated transcriptional activity of the c-fos promoter luciferase reporter gene, and a decrease in cell proliferation.

Activation of TGF-beta-Smad signaling pathway following polyamine depletion in intestinal epithelial cells.

Smad proteins are transcription activators that are critical for transmitting transforming growth factor-beta (TGF-beta) superfamily signals from the cell surface receptors to the nucleus. Our previous studies have shown that cellular polyamines are essential for normal intestinal mucosal growth and that a decreased level of polyamines inhibits intestinal epithelial cell proliferation, at least partially, by increasing expression of TGF-beta/TGF-beta receptors. The current study went further to determine the possibility that Smads are the downstream intracellular effectors of activated TGF-beta/TGF-beta receptor signaling following polyamine depletion. Studies were conducted in IEC-6 cells derived from rat small intestinal crypts. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) increased basal levels of Smad3 and Smad4 proteins, induced their nuclear translocation, and stimulated Smad sequence-specific DNA-binding activity. Polyamine depletion-induced Smads were also associated with a significant increase in transcription activation as measured by luciferase reporter gene activity of Smad-dependent promoters. Inhibition of Smads by a dominant-negative mutant Smad4 in the DFMO-treated cells prevented the increased Smad transcription activation. Polyamine-deficient cells highly expressed TGF-beta and were growth-arrested at the G1 phase. Inhibition of TGF-beta by treatment with either immunoneutralizing anti-TGF-beta antibody or TGF-beta antisense oligodeoxyribonucleotides not only blocked the induction of Smad activity but also decreased the Smad-mediated transcriptional activation in polyamine-depleted cells. These findings suggest that Smads are involved in the downstream cellular processes mediated by cellular polyamines and that increased TGF-beta/TGF-beta receptor signaling following polyamine depletion activates Smads, thus resulting in the stimulation of Smad target gene expression.

Functional characterization of the human resistin promoter with adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element binding protein 1c and CCAAT enhancer binding protein-alpha.

Recent studies with murine models propose that resistin would be a possible mediator to link between obesity and insulin resistance. Although it has been reported that resistin is highly expressed and secreted by adipocytes, transcription factors that are involved in resistin gene expression have not been well characterized. To investigate the molecular mechanisms of resistin gene expression, we cloned and characterized the human resistin promoter. Sequence analysis of the resistin promoter revealed several putative binding sites for adipogenic transcription factors including adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element binding protein 1c (SREBP1c) and CCAAT enhancer binding protein-alpha (C/EBP alpha). EMSA and chromatin immunoprecipitation assays demonstrated that ADD1/SREBP1c binds to the human resistin promoter in vitro and in vivo. Expression of ADD1/SREBP1c transactivated the luciferase reporter gene activity, the promoter region of which contains a human resistin promoter in a sterol regulatory element (SRE)-dependent manner. Furthermore, ectopic expression of ADD1/SREBP1c by adenovirus significantly increased the expression of resistin mRNA in adipocytes. Human resistin promoter was also activated by C/EBP alpha expression, although ectopic expression of both transcription factors did not show any synergistic effects on the activation of resistin promoter. Together, these data suggest that ADD1/SREBP1c and C/EBP alpha may play discrete roles in the regulation of the resistin gene expression.

Hypertonic induction of COX-2 expression in renal medullary epithelial cells requires transactivation of the EGFR.

Hypertonic stress increases expression of cyclooxygenase-2 (COX-2) in renal medullary epithelial and interstitial cells. Because hypertonic COX-2 expression is, in part, sensitive to inhibition of the ERK MAPK, an effector of activated receptor tyrosine kinases such as the EGF receptor, we investigated a role for this receptor in signaling to COX-2 expression. Hypertonic stress increased COX-2 expression at the mRNA and protein levels at 6 and 24 h of hypertonic treatment. Two potent, specific inhibitors of the EGF receptor kinase, AG-1478 and PD-153035, abrogated this effect. These inhibitors also blocked the ability of hypertonic stress to increase PGE2 release; in addition, they partially blocked tonicity-dependent phosphorylation of ERK but not of the related MAPKs, JNK or p38. Pharmacological inhibition of ERK activation partially blocked tonicity-dependent COX-2 expression. Hypertonic induction of COX-2 was likely transcriptionally mediated, as NaCl stress increased luciferase reporter gene activity under control of the human COX-2 promoter, and this effect was also sensitive to inhibition of the EGF receptor kinase. Metalloproteinase action is required for transactivation of the EGF receptor. Pharmacological inhibition of metalloproteinase function blocked tonicity-inducible COX-2 expression. Furthermore, the effect of hypertonicity on COX-2 expression was also evident in the EGF-responsive Madin-Darby canine kidney and 3T3 cell lines but was virtually absent from the EGF-unresponsive (and EGF receptor null) Chinese hamster-derived CHO cell line. Taken together, these data indicate that hypertonicity-dependent COX-2 expression in medullary epithelial cells requires transactivation of the EGF receptor and, potentially, ectodomain cleavage of an EGF receptor ligand.

Activation of CCAAT/enhancer-binding protein beta by 2'-amino-3'-methoxyflavone (PD98059) leads to the induction of glutathione S-transferase A2.

The induction of glutathione S-transferases by flavonoids is associated with cancer chemopreventive effects. We reported that 2'-amino-3'-methoxyflavone (PD98059), an MKK1 inhibitor, induces glutathione S-transferase A2 (rGSTA2). This report comparatively examines the role of CCAAT/enhancer-binding protein (C/EBP) and Nrf2 in the induction of rGSTA2 by PD98059. We first assessed whether the MKK1/ERK1/2 pathway regulated rGSTA2 induction. Northern and western blot analyses showed that PD98059 at the concentrations effective for the inhibition of MKK1 increased the rGSTA2 protein and mRNA levels in H4IIE cells. PD98059 also induced rGSTA2 in cells stably transfected with dominant-negative mutant of MKK1(-), which provided evidence that the inhibition of MKK1/ERK1/2 by PD98059 was not responsible for rGSTA2 induction. Gel shift assay and immunoblot analysis of subcellular fractions revealed that PD98059 caused nuclear translocation of C/EBP beta and increased C/EBP DNA binding, which was super-shifted with anti-C/EBP beta antibody. Nrf2 was not activated by PD98059. PD98059 increased the luciferase reporter gene activity in cells transfected with the C/EBP-containing -1.65 kb flanking region of the rGSTA2 gene. Deletion of the C/EBP-binding site or over-expression of dominant-negative mutant of C/EBP abolished the reporter gene activity. Flavone, a backbone structure of PD98059, also induced nuclear translocation of C/EBP beta and C/EBP-mediated rGSTA2 gene induction. Inhibition of phosphatidylinositol 3-kinase abolished C/EBP beta-mediated rGSTA2 induction by PD98059. These results provide evidence that PD98059 and flavone induce nuclear translocation of C/EBP beta and activate the C/EBP-binding site in the rGSTA2 gene, which constitutes the distinct pathway for the enzyme induction irrespective of the inhibition of MKK1/ERK activity.

Oxidative stress‐mediated down‐regulation of bcl‐2 promoter in hippocampal neurons

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19-7) and in rat primary hippocampal neurons. Treatment of H19-7 cells with hydrogen peroxide (150 micro m) resulted in a 40% decrease in Bcl-2 protein and a parallel decrease in bcl-2 mRNA levels. H19-7 cells overexpressing bcl-2 were found to be resistant to ROS-induced apoptosis. We had previously shown that bcl-2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl-2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor-2-stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19-7 cells resulted in significant (p < 0.01) protection against ROS-induced apoptosis through up-regulation of Bcl-2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress-induced neuronal dysfunction.

Essential role of phosphatidylinositol 3-kinase-dependent CCAAT/enhancer binding protein beta activation in the induction of glutathione S-transferase by oltipraz.

Cancer chemopreventive agents transcriptionally induce genes whose protein products can protect cells from chemical-induced carcinogenesis. Oltipraz, a dithiolthione, exerts chemopreventive responses through glutathione S-transferase (GST) induction. We investigated the role of the CCAAT/enhancer binding protein (C/EBP) in the induction of the GSTA2 gene (alpha class) by oltipraz and identified the enhancer element(s) responsible for GSTA2 gene expression. H4IIE rat hepatocyte-derived cells were treated with oltipraz, and GSTA2 expression was determined by northern and immunoblot analyses. The activation of C/EBPbeta and alpha forms and NF-E2-related factor 2 (Nrf2) was assessed by immunochemical assays. C/EBPbeta-DNA binding activity was determined by subcellular fractionation and electrophoretic mobility shift assays. The role of the C/EBP binding site in the induction of the GSTA2 gene was assessed by luciferase reporter-gene activity. The role of phosphatidylinositol 3-kinase (PI3-kinase) and mitogen-activated protein (MAP) kinase signaling pathways in C/EBP-mediated GSTA2 induction was studied by using chemical inhibitors, overexpression vectors, and dominant-negative mutants. All statistical tests were two-sided. Oltipraz induced GSTA2 mRNA and protein expression. In oltipraz-treated cells, C/EBPbeta translocated to the nucleus and bound to the consensus sequence of C/EBP (TTGCGCAA). Oltipraz treatment increased luciferase reporter-gene activity in H4IIE cells transfected with the C/EBP-containing regulatory region of the GSTA2 gene. Deletion of the C/EBP binding site or overexpression of a dominant-negative mutant form of C/EBP (AC/EBP) abolished the reporter gene activity. PI3-kinase, but not MAP kinases, was required for C/EBPbeta-dependent induction of GSTA2 by oltipraz. Oltipraz-induced GSTA2 gene expression is dependent upon PI3-kinase-mediated nuclear translocation and binding of C/EBPbeta to the C/EBP response element in the GSTA2 gene promoter.

IKKα, IKKβ, and NEMO/IKKγ Are Each Required for the NF-κB-mediated Inflammatory Response Program

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.