Nuclear Protein Binding Activity Research Articles

Recombination and purification of IL-23 gene and its effect on immune response to airway inflammation in asthma during pregnancy

Aim To investigate the immune regulation of IL-23 fusion gene and the correlation between its biological function and the airway inflammation in asthma during pregnancy. Methods The eukaryotic expression plasmid,which was constructed to express the IL-23p40 + p19 double subunit,was used to transform E.coilBL21 bacteria,and the expression products,which were induced by IPTG,were purified and detected by SDS-PAGE and Western blot. The transfection,mediated by recombinant protein,was identified by fluorescence microscopy and plasmid's Western blot,while DNA binding activity of nuclear proteins was measured by gel electrophoresis. To evaluate the impact of foreign protein in vivo,mouse serum inflammatory cytokines levels and inflammation injury in lung tissue were measured by ELISA method and HE staining. The CD4+T cell subset's proportion of mononuclear cells in peripheral blood was verified by flow cytometry. Results The results showed that soluble exogenous recombinant protein,which was obtained successfully,had the ability of trans-membrane transport and could effectively mediate EGFP into the target cells. Its stable expression and transcriptional activity were higher than those of simple plasmid's effect. The specific binding between GST/pCEP4-IL-23 and plasmid DNA formed macromolecular complexes,which could decrease the mobility of gel. The combined effect with LPS significantly increased the IL-17A levels in serum(P 0. 05) and the inflammation reaction in lung tissue of sensitized mice. Mean while,it also reduced the IFN-γ,Foxp3 level in serum,promoted the activation of IL-4 and IL-17A,and then increased the ratio of Th2/Th1and Th17/Treg(P 0. 05). Conclusions The recombinant IL-23 protein is pathogenic,and it adjusts the inflammation of asthma in pregnancy. The immune imbalance of CD4+T subsets affects the immune tolerance of pregnant body and the development of asthma during pregnancy.

Nickel differentially regulates NFAT and NF-κB activation in T cell signaling

Nickel is a potent hapten that induces contact hypersensitivity in human skin. While nickel induces the maturation of dendritic cells via NF-κB and p38 MAPK activation, it also exerts immunosuppressive effects on T cells through an unknown mechanism. To elucidate the molecular mechanisms of its effects on T cells, we examined the effects of NiCl 2 on mRNA expression in human CD3+ T cells stimulated with CD3 and CD28 antibodies. Using a DNA microarray and Gene Ontology, we identified 70 up-regulated (including IL-1β, IL-6 and IL-8) and 61 down-regulated (including IL-2, IL-4, IL-10 and IFN-γ) immune responsive genes in NiCl 2-treated T cells. The DNA microarray results were verified using real-time PCR and a Bio-Plex TM suspension protein array. Suppression of IL-2 and IFN-γ gene transcription by NiCl 2 was also confirmed using Jurkat T cells transfected with IL-2 or IFN-γ luciferase reporter genes. To explore the NiCl 2-regulated signaling pathway, we examined the binding activity of nuclear proteins to NFAT, AP-1, and NF-κB consensus sequences. NiCl 2 significantly and dose-dependently suppressed NFAT- and AP-1-binding activity, but augmented NF-κB-binding activity. Moreover, NiCl 2 decreased nuclear NFAT expression in stimulated T cells. Using Jurkat T cells stimulated with PMA/ionomycin, we demonstrated that NiCl 2 significantly suppressed stimulation-evoked cytosolic Ca 2+ increases, suggesting that NiCl 2 regulates NFAT signals by acting as a blocker of Ca 2+ release-activated Ca 2+ (CRAC) channels. These data showed that NiCl 2 decreases NFAT and increases NF-κB signaling in T cells. These results shed light on the effects of nickel on the molecular regulation of T cell signaling.

Protective effect of sulforaphane on indomethacin‐induced cytotoxicity via heme oxygenase‐1 expression in human intestinal Int 407 cells

Sulforaphane is known to be an indirect antioxidant that acts by inducing NF-E2-related factor 2 (Nrf2)-dependent phase II enzymes. In the present study, we investigated the effect of sulforaphane on the expression of heme oxygenase-1 (HO-1) in human intestinal Int 407 cells. RT-PCR and Western blot data revealed that sulforaphane induced an increase in HO-1 expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in HO-1 activity. Actinomycin D (an RNA synthesis inhibitor) and cycloheximide (a protein synthesis inhibitor) inhibited sulforaphane-responsive HO-1 mRNA expression, indicating that sulforaphane is a requirement for transcription and de novo protein synthesis. Moreover, sulforaphane increased the nuclear levels of Nrf2 and increased the binding activity of nuclear proteins to the antioxidant responsive element consensus sequence. We also found that U0126, an ERK kinase inhibitor, suppressed the sulforaphane-induced HO-1 expression and nuclear translocation of Nrf2. Moreover, the cytoprotective effect of sulforaphane on indomethancin-induced cytotoxicity was partially blocked by ERK and HO-1 inhibitors, further demonstrating that sulforaphane attenuated oxidative stress through a pathway that involved ERK and HO-1. Taken together, this study gives additional support to the possible use of sulforaphane as a dietary preventive agent against oxidative stress-induced intestinal injury.

The Conversion of Rapid TCCD Nongenomic Signals to Persistent Inflammatory Effects via Select Protein Kinases in MCF10A Cells

Previously we found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces rapid inflammatory cellular responses in MCF10A mammary epithelial cells through a distinct nongenomic pathway by activating cytosolic phospholipase A2 and Src kinase within 30 min. In the current study we investigated how such an initial, seemingly transient signaling induced by TCDD is subsequently converted into more stable long-term messages. We found that TCDD causes prolonged activation of the binding activity of nuclear proteins to the oligonucleotide probes representing consensus activator protein 1 and CCAAT enhancer binding protein response element sequences, followed by later induction of some diagnostic marker including cyclooxgenase-2, matrix metalloproteinase-2, colony stimulating factor-1, and cytochrome P450 19 (or aromatase). Blocking the early steps of the nongenomic pathway inhibits this action of TCDD. It was also found that Src kinase is mainly responsible for the increase of binding activity to the activator protein 1 probe, and another kinase, protein kinase A (PKA), is accountable for most of the increase of binding activity to the CCAAT enhancer binding protein probe. The induction of those diagnostic markers is also affected by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (a Src kinase inhibitor) or H89 (a PKA inhibitor). These results indicate that Src kinase and PKA act as the second messengers in propagating the initial nongenomic signaling of TCDD.

Sp1 is an essential transcription factor for LPS-induced tissue factor expression in THP-1 monocytic cells, and nobiletin represses the expression through inhibition of NF-κB, AP-1, and Sp1 activation

Nobiletin is a citrus polymethoxylated flavonoid extracted from Citrus depressa, and has several reported biological effects. In this study, we investigated the effect of nobiletin on bacterial lipopolysaccharide (LPS)-induced expression of tissue factor (TF), a trigger protein for the blood coagulation cascade, and studied the possible mechanism of TF transcriptional regulation. THP-1 monocytic cells stimulated with LPS showed an increased expression of both TF protein and mRNA levels. However, pretreatment with nobiletin resulted in inhibition of LPS-induced expression of both TF protein and mRNA in a dose-dependent manner. Electrophoretic mobility shift assays revealed that binding of nuclear proteins from LPS-stimulated THP-1 cells to the NF-κB or AP-1 binding motif was increased as compared to non-stimulated control cells. Such increased binding activities were significantly reduced by pretreatment with nobiletin. Binding activity of nuclear proteins to the Sp1 binding motif was observed irrespective of LPS stimulation, but Sp1 activation was inhibited by nobiletin treatment of the cells. Treatment of THP-1 cells with Sp1-specific small interfering RNA (Sp1 siRNA) abolished the ability of LPS to induce TF activity. A similar reduction in the level of TF mRNA was also observed upon treatment of cells with Sp1 siRNA. These studies reveal that constitutive Sp1 activation is an essential event for transcriptional activation of TF, and nobiletin prevents LPS-induced TF expression by inhibiting NF-κB, AP-1, and Sp1 activation.

Na+-Glucose Transporter-2 Messenger Ribonucleic Acid Expression in Kidney of Diabetic Rats Correlates with Glycemic Levels: Involvement of Hepatocyte Nuclear Factor-1α Expression and Activity

Mutations in Na(+)-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression (~50%) and binding of nuclear proteins to a HNF-1 consensus motif (~100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

NF-κB, MEF2A, MEF2D and HIF1-a involvement on insulin- and contraction-induced regulation of GLUT4 gene expression in soleus muscle

The GLUT4 gene transcriptional activity has a profound impact on the insulin-mediated glucose disposal and it is, therefore, important to understand the mechanisms underlying it. Insulin and exercise modulate GLUT4 expression in vivo, but the net control and involved mechanisms of each one have not been established yet. This paper sought to discriminate, in soleus muscle, the effects of insulin and muscle contraction on GLUT4 gene expression, and the involvement of transcriptional factors: myocite enhancer factor 2 (MEF2 A/C/D), hypoxia inducible factor 1-a (HIF1-a) and nuclear factor-kappa B (NF-κB). The GLUT4 mRNA was reduced by fasting (40%), and increased by in vitro incubation with insulin (25%) or insulin plus glucose (40%), which was accompanied by opposite regulations of NF-κB mRNA. Differently, in vitro, muscle contraction led to a rapid increase (35–80%) in GLUT4, MEF2A, MEF2D and HIF1-a mRNAs. Additionally, electrophoretic mobility shift assay confirmed changes in the binding activity of nuclear proteins to consensus NF-κB, GLUT4-Ebox and GLUT4-AT-rich element probes, parallel to the mRNA changes of their respective transcriptional factors NF-κB, HIF1-a and MEF2s. Concluding, insulin- and contraction-induced regulation of GLUT4 expression involves distinct transcriptional factors.

Ets transcriptional regulation of gastrin-releasing peptide receptor in neuroblastomas

BackgroundWe have demonstrated that gastrin-releasing peptide (GRP) binds specifically to its cell surface receptor, GRP-R, to act as an autocrine/paracrine growth factor for neuroblastomas (NBs); an increased expression of GRP-R was found in more advanced-stage NBs. Ets family proteins are nuclear targets for intracellular kinase pathways that can lead to cell proliferation; however, a potential role of Ets in the expression of GRP-R in NBs is unknown. Therefore, the purpose of our study was to determine whether Ets regulates transcriptional activity of GRP-R in NBs. MethodsWe identified multiple DNA-binding sites for various nuclear transcription factors in the proximal (ie, 263 bp) GRP-R promoter. Luciferase assay was performed to measure GRP-R promoter activity that contained site-specific mutations of various binding elements. Electrophoretic mobility shift assay was performed to determine transcription factor–binding activity. ResultsMutation of a consensus Ets-binding site in the GRP-R promoter significantly decreased GRP-R promoter activity. Electrophoretic mobility shift assay demonstrated a decrease in Ets nuclear protein–binding activity. Furthermore, overexpression of Ets1 resulted in upregulation of GRP-R promoter activity. ConclusionsOur results demonstrate that Ets is a transcription factor that significantly contributes to the GRP-R transcription in NBs. This finding may allow us to develop novel molecular tools to downregulate expression of GRP-R and hence inhibit mitogenic effects of GRP in NBs.

Conjugated Linoleic Acid Blocks Estrogen Signaling in Human Breast Cancer Cells

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid found in dairy products and meat from ruminants, has been widely shown to possess anticarcinogenic activity against breast cancer both in vitro and in animal models. However, little information is available concerning the mechanisms of the antitumor effects of these compounds. In this study, we investigated whether CLA has direct antiestrogenic activity in estrogen receptor positive (ER+) breast cancer cells. Treatment of the ER+ cell line, MCF-7, with 5 purified CLA isomers as well as "mixed" CLA showed a dose-dependent growth inhibition with the 9cis,11cis and 9cis,11trans being the most and least potent isomers, respectively. In assessing effects on a number of variables that play obligatory roles in the estrogen signaling pathway, we determined that CLA treatment downregulated ERalpha expression at both mRNA and protein levels and decreased binding activity of nuclear protein to a canonical estrogen response element (ERE(v)). Using a reporter gene construct (ERE(v)-tk-Luc) that was transiently transfected into MCF-7 cells, we also demonstrated inhibition of promoter activity by CLA that was directly mediated by blockage of activity through the ERE. The results indicated that the order of potency of the CLA isomers for inhibiting activation of ERE(v) was similar to that demonstrated for their antiproliferative activity on MCF-7 cells. Taken together, these findings demonstrate that CLA compounds possess potent antiestrogenic properties that may at least partly account for their antitumor activity on breast cancer cells.

AP-1 complexes mediate oxidized LDL-induced overproduction of TGF-beta(1) in rat mesangial cells.

Oxidized Low Density Lipoprotein (Ox-LDL)-induced overproduction of the prosclerotic cytokine transforming growth factor-beta1 (TGF-beta(1)) has been implicated in the pathogenesis of renal fibrosis and sclerosis. Because Ox-LDL increases TGF-beta(1) mRNA levels in rat mesangial cells, our investigation was designed to characterize these effects on the rat TGF-beta(1) promoter activity. We transfected luciferase reporter gene constructs containing TGF-beta(1) 5'-flanking sequence (from -1550 to +57 bp) into mesangial cells. By assaying progressively deleted mutations in the promoter, we found two regions that were responsible for the induction. One is a negative regulatory region (-422 to -629) which represses the transcription of the TGF-beta(1) gene, the other is a positive regulatory region (-845 to -1550) which enhances the transcription unit efficiently. There is an activating protein-1(AP-1) binding site in the latter region. Mutagenesis in the AP-1 binding sites abolished the Ox-LDL effect. Furthermore, addition of the AP-1 inhibitor curcumin obliterated the Ox-LDL response. The Ox-LDL-induced TGF-beta(1) promoter activation was also prevented by inhibitors of protein kinase C, but not by p38 mitogen-activated protein kinase. Electrophoretic mobility shift assays with oligonucleotides containing AP-1 binding sites showed that Ox-LDL treatment significantly enhanced the binding activity of nuclear proteins of mesangial cells. Supershift assays demonstrated that c-Jun was present in the protein-DNA complexes under stimulation of Ox-LDL. The functional and structural results show that Ox-LDL regulates rat TGF-beta(1) gene expression through AP-1 binding sites and gives rise to the involvement of protein kinase C in Ox-LDL-induced TGF-beta(1) gene expression.

The vitamin K–dependent γ-glutamyl carboxylase gene contains a TATA-less promoter with a novel upstream regulatory element

AbstractThe expression of the vitamin K–dependent γ-glutamyl carboxylase gene in liver is developmentally regulated. Since the gene product catalyzes an essential post-translational modification of the vitamin K–dependent blood coagulation proteins, the regulation of carboxylase expression is critical for hemostasis. We analyzed the activity of the rat carboxylase gene 5′-regulatory DNA sequences in rat hepatoma cell lines at different states of differentiation. These studies demonstrated that the 2.6-kb 5′-flanking sequence has differentiation-dependent transcriptional activity. Transient gene expression assays, examining the effects of nested deletions and site-directed mutagenesis of putative regulatory sequences, together with electrophoretic mobility shift assays (EMSAs) were used to identify sequences critical for the developmentally regulated transcription of the rat carboxylase gene. We identified a DNA sequence (–76 to –65; GTTCCGGCCTTC) not known to bind to transcription factors, yet which functions as an upstream promoter element. In vivo genomic DNA footprinting confirms the presence of nuclear protein–DNA interactions at this site in the endogenous carboxylase gene in differentiated hepatoma cells. Therefore, this DNA sequence has specific nuclear protein–binding activity and functional properties consistent with a regulatory element that plays a critical role in the developmental expression of the carboxylase gene, and hence the regulation of vitamin K–dependent blood coagulation protein synthesis.

Hydrogen peroxide induced down-regulation of CD28 expression of Jurkat cells is associated with a change of site α-specific nuclear factor binding activity and the activation of caspase-3

CD28 is the requisite co-stimulatory molecule in the activation of T cells and in the generation of immune responses. But expression of CD28 declined and oxidants accumulated in the elderly. Although accumulation of reactive oxygen species (ROS) during senescence has been reported extensively, the effect of oxidants on CD28-expression remains totally unknown. In this study, we tried to address the molecular mechanism underlying the decrease in CD28-expression of Jurkat T cells cultured in H 2O 2. Our results indicate that H 2O 2 could partially block the expression of CD28. This correlates well with a change of nuclear protein binding activity to the motif of site α of the CD28 gene, while the site β-binding activity remained unaltered. On the other hand, since caspase-3 is activated by H 2O 2, inhibitors of caspase-3 should increase the expression of CD28. What is more interesting is the fact that the site α-binding activity was mostly restored after caspase-3 inhibitors had being added. However, caspase-3 is not activated by caspase-8. Maybe it is activated by caspase-9, which is triggered by cytochrome c. We believe that the procaspase-3 is activated by ROS, and the active caspase-3 can induce the change of the site α-binding activity, causing a decrease in CD28 expression.

Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARβ-RXRα heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter

AbstractThe present work investigated the mechanism for down-regulation of thrombomodulin (TM), an anticoagulant glycoprotein, on cultured umbilical vein endothelial cells (HUVECs) exposed to lipid extracts from oxidized low-density lipoprotein (ox-LDL). HUVECs exposed to phospholipid extracts, but not to free cholesterol, triglyceride, or cholesterol ester, isolated from ox-LDL reduced TM mRNA levels to nearly the same extent as native ox-LDL. Oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), but not native PAPC or a reduced form of ox-PAPC, markedly decreased TM mRNA levels. The apparent half-life (t 1/2 = 2.7 hours) of TM mRNA in control cells was not significantly different from that in cells exposed to ox-LDL or ox-PAPC. TM mRNA levels were regulated by transcriptional activation via a retinoid receptor β (RARβ). The binding activities of nuclear proteins from HUVECs treated with ox-LDL or ox-PAPC to the DR4 or stimulatory protein 1 (Sp1) sequence in the TM promoter were significantly reduced with decreased expression of RARβ, retinoid X receptor α (RXRα), Sp1, and Sp3 in the nuclei. The promoter activity in HUVECs transfected with a reporter plasmid expressing the TM promoter with targeted deletions in the DR4 and Sp1 binding elements was decreased to about 20% of that with the wild-type construct. Treatment of the cells with ox-PAPC had no additional effect on the promoter activity. These results suggest that oxidized phospholipids in ox-LDL inhibit transcription of the TM gene in HUVECs by inhibiting the binding of RARβ-RXRα heterodimer and Sp, including Sp1 and Sp3, to the DR4 element and Sp1 binding element, respectively, in the TM promoter with reduced expression of RARβ, RXRα, and Sp1 and Sp3 in the nuclei.

Lipopolysaccharide represses cholesterol 7-alpha hydroxylase and induces binding activity to the bile acid response element II.

Inflammatory states such as hepatitis and sepsis are frequently associated with alterations of bile acid synthesis. These conditions are mediated by bacterial wall products like lipopolysaccharide (LPS). Cholesterol 7-alpha hydroxylase is the rate-limiting enzyme of bile acid synthesis. Hydrophobic bile acids repress cholesterol 7-alpha hydroxylase transcription via binding to the farnesoid X receptor and interaction with the bile acid response element II in the cholesterol 7-alpha hydroxylase promoter. We tested the effect of LPS on hepatic expression of cholesterol 7-alpha hydroxylase in C57BL/6 mice and Wistar rats. Further, we analyzed the binding activity of hepatic nuclear extracts to the bile acid response element II and the binding site for farnesoid X receptor heterodimers (ecdysone response element). Lipopolysaccharide caused a 100-fold reduction of cholesterol 7-alpha hydroxylase mRNA levels in mice and a 10-fold reduction in rats. Protein levels of cholesterol 7-alpha hydroxylase also decreased in both species. These changes inversely correlated with the increased binding activity of nuclear proteins to the bile acid response element II and the ecdysone response element. Lipopolysaccharide-induced repression of cholesterol 7-alpha hydroxylase occurs at the transcriptional level. The underlying mechanism involves an increased binding activity of nuclear proteins to the bile acid response element II and the ecdysone response element. In conclusion, the farnesoid and retinoid X receptors participate in LPS-induced cholesterol 7-alpha hydroxylase repression.

Vascular Smooth Muscle α-Actin Gene Transcription during Myofibroblast Differentiation Requires Sp1/3 Protein Binding Proximal to the MCAT Enhancer

The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.

CpR18, a novel SAP-domain plant transcription factor, binds to a promoter region necessary for ABA mediated expression of the CDeT27-45 gene from the resurrection plant Craterostigma plantagineum Hochst.

CDeT27-45 is a lea-like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29-bp cis-regulatory region of the promoter which is essential for transcriptional activation of the CDeT27-45 gene by ABA. Using a yeast one-hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis-regulatory element in the CDeT27-45 promoter. The protein contains an acidic region, a SAP-domain, a zinc finger of the C3H-type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix-loop-helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue-specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27-45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27-45 promoter-GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome.

15-Deoxy-Δ12,14-prostaglandin J2 inhibits IL-1β–induced cyclooxygenase-2 expression in mesangial cells

Cyclooxygenase-2 (COX-2), a key enzyme in the synthesis of prostaglandins, is induced in mesangial cells in response to proinflammatory cytokines. Recently, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), one of the natural ligands of peroxisome proliferator-activated receptor gamma (PPARgamma), has been reported to have an anti-inflammatory effect. Therefore, we examined the effect of 15d-PGJ2 on COX-2 expression in cultured rat mesangial cells. Mesangial cells were incubated with 15d-PGJ2 for 30 minutes and then exposed to interleukin-1beta (IL-1beta). The expression of COX-2 mRNA and proteins was determined by Northern blot and immunoblot analyses, respectively. Accumulation of prostaglandin E2 (PGE2) was measured by an enzyme-linked immunosorbent assay (ELISA). Activities of mitogen-activated protein kinases (MAPKs) were evaluated by an immunoblot analysis. DNA binding activities of activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) were examined by an electrophoretic mobility shift assay (EMSA). The activities of PPAR responsive elements (PPRE) and COX-2 promoter were measured by a luciferase reporter assay. 15D-PGJ2 significantly suppressed IL-1beta-induced COX-2 expression and PGE2 production, but thiazolidinediones, synthetic PPARgamma ligands, did not affect COX-2 expression. Moreover, the cells transfected with a PPRE luciferase reporter did not respond to 15d-PGJ2. IL-1beta rapidly activated extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), which were involved in the up-regulation of COX-2 induction, but 15d-PGJ2 inhibited the activation of these kinases. 15d-PGJ2 inhibited the IL-1beta-induced increase in binding activities of nuclear proteins to consensus AP-1 site and AP-1-like site of COX-2 promoter but not of NF-kappaB. IL-1beta was unable to activate the COX-2 promoter when the AP-1-like site was mutated. These data suggest that 15d-PGJ2 inhibits IL-1beta-induced COX-2 expression, independent of PPARgamma activation, by suppression of ERK and JNK pathways and AP-1 activation in mesangial cells. Thus, 15d-PGJ2 may play an important role in the negative feedback mechanism of COX-2 expression in renal inflammation and may be useful as an anti-inflammatory agent.

Promoter sequences involved in transforming growth factor beta1 gene induction in HaCaT keratinocytes after gamma irradiation.

Transforming growth factor beta 1 (TGFB1) is a cytokine involved in the development of both acute and late cutaneous radiation syndromes. We previously demonstrated that ionizing radiation induces TGFB1 expression in vivo in pig skin within a few hours. The purpose of the present study was to develop an in vitro human model to identify the mechanisms of this early activation. Accordingly, human HaCaT keratinocytes were irradiated with a single dose of 20 Gy. First, radiation-induced TGFB1 overexpression was checked at both the transcriptional and transductional levels in HaCaT cells. Then electrophoretic mobility shift assays (EMSA) and transient transfection with various TGFB1 promoter constructs were used to identify the sequences involved in regulating this promoter. EMSA analysis showed the induction of nuclear protein binding activity by gamma irradiation to the -365 AP1 sequence (TGTCTCA), suggesting the involvement of AP1 sequences in the regulation of TGFB1 transcription. In gene reporter assays, maximal TGFB1 promoter activation was found for the longest construct, which contains two AP1 sequences. However, assays with constructs including deletions showed that these two AP1 sequences were not sufficient to confer TGFB1 inducibility. These results showed for the first time, to our knowledge, that transcriptional regulation is involved in radiation-induced activation of TGFB1 gene expression.

Regulation and expression of progesterone receptor mRNA isoforms A and B in the male and female rat hypothalamus and pituitary following oestrogen treatment.

Progesterone receptors play a central role in neuroendocrine and behavioural regulation. To gain insight into the sex- and tissue-specific regulation of progesterone receptors, protein binding on a progesterone receptor-oestrogen response element and mRNA levels for progesterone receptor (PR)-A and PR-B were compared between female and male rats following oestradiol benzoate replacement treatment in hypothalamic and pituitary tissue. Both male and female pituitary protein extracts demonstrated an increase in nuclear protein binding activity to a progesterone receptor-oestrogen response element following oestradiol benzoate treatment. However, there was a greater difference in total binding activity seen in the female pituitary extracts compared to male pituitary protein extracts. In both cases, reflecting the binding data, oestradiol benzoate pretreatment led to an increase in pituitary PR-B messenger RNA, although this increase was significantly larger in females than in males. Oestradiol benzoate treatment also led to a significant increase in specific binding of hypothalamic nuclear proteins to the progesterone receptor oestrogen response element from both females and male hypothalamic extracts. In addition, PR-B messenger RNA was induced by oestradiol benzoate treatment in the female rat hypothalamus, under circumstances where no PR-A could be detected. The male also demonstrated an increase in PR-B messenger RNA following oestradiol benzoate treatment, with undetectable levels of PR-A, although to a lesser degree than that seen in the female. The predominance of PR-B over PR-A messenger RNA in rat hypothalamus and pituitary, and the quantitative differences between female and male rats, could both contribute to the greater responsiveness of female rats to progesterone with respect to control over luteinizing hormone release from the pituitary, and lordosis behaviour regulated by hypothalamic neurones.

C-Myc and Sp1/3 are required for transactivation of hamster telomerase catalytic subunit gene promoter

The hamster and human TERT promoters share common critical protein binding sites, such as the GC-box or E-box, which is known to be a binding site for Sp1/Sp3 transcriptional factors and c-Myc, respectively. Our previous data demonstrated that Sp1/Sp3 synergistically transactivate the hamster TERT (hamTERT) promoter. In this study, we determined the role of c-Myc in the regulation of hamTERT, and analyzed the relative significance of GC-boxes and the E-box for transcriptional activation of hamster TERT. Wild-type, mutated E-box or mutated GC-box hamTERT core promoter reporter was introduced into 293T cells in combination with murine or human Myc expression vectors. The promoter activity was determined using the luciferase assay, and the transfection efficiency was normalized with CAT activity. The electrophoretic mobility shift assay (EMSA) was done to prove the nuclear protein binding activity of the GC-box (region II) or E-box. Overexpression of murine or human Myc transactivated hamTERT core promoter activity. Inversion mutation in the E-box or substitution mutation in the GC-boxes abrogated endogenous or Myc induced hamTERT transactivation. Region II is the single most important Sp1/3 binding site in transcriptional activation, and multiple combined mutations in the GC-boxes abolished the hamTERT promoter activity. These results indicate that c-Myc and Sp1/3 are the major regulatory determinants of the hamTERT transcriptional activation. The mechanism of TERT gene activation during immortalization and carcinogenesis may be conserved among species.