Regulation of prostaglandin endoperoxide H synthase-2 induction by dioxin in rat hepatocytes: possible c-Src-mediated pathway
Christoph Vogel,
Anne-Marie J.F. Boerboom,
Claudia Baechle,
Claudia El-Bahay1,
Regine Kahl1,
Gisela H. Degen2 and
Josef Abel3
Department of Experimental Toxicology, Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, 40225 Duesseldorf,
1 Institute of Toxicology, Heinrich-Heine-University, 40225 Duesseldorf and
2 Institute of Occupational Physiology at the University of Dortmund, 44139 Dortmund, Germany
 |
Abstract
|
---|
The tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to increase the expression of prostaglandin endoperoxide H synthase (PGHS)-2. This study focused on the regulatory mechanism of TCDD-mediated transcriptional activation of PGHS-2. Treatment of rat hepatocytes with TCDD led to a dose-dependent induction of PGHS-2 mRNA levels associated with an increased synthesis of prostaglandin E2, whereas expression of PGHS-1 was not affected. In vitro experiments with c-Src inhibitors, such as herbimycin A and geldanamycin, and in vivo studies with c-Src-deficient mice indicated that up-regulation of PGHS-2 but not the cytochrome P450 gene CYP1A1 by TCDD is mediated via a c-Src-dependent pathway. Transient transfection studies with different reporter constructs of the murine PGHS-2 promoter mutated in the xenobiotic-responsive element (XRE) or CCAAT/enhancer binding protein (C/EBP) element revealed that a C/EBP-binding site is an important regulatory cis-acting factor for trans-activation of the PGHS-2 gene by TCDD. Consistent with transfection studies, gel mobility shift assays showed that TCDD led to an enhanced DNA-binding activity of C/EBPß transcription factor. The experimental data presented in this article reveal a XRE-independent and c-Src-mediated activation of the PGHS-2 gene by TCDD through the C/EBP response element located in its promoter region.
Abbreviations: AhR, aromatic hydrocarbon receptor; ATF/CRE, activating transcription factor/cyclic AMP response element; C/EBP, CCAAT/enhancer binding protein; CYP1A1, mammalian cytochrome P450; PGE2, prostaglandin E2; PGHS, prostaglandin endoperoxide H synthase; c-Src, tyrosine kinase pp60src; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin); XRE, xenobiotic-responsive element
 |
Introduction
|
---|
Arachidonic acid metabolites, such as prostaglandins, thromboxanes, and other bioactive lipids are important mediators of a wide variety of biological effects including cell growth, differentiation and adhesion. Prostanoids are considered to be involved in several pathophysiological and disease states, such as inflammation, pain, cardiovascular diseases and cancer (13). The rate-limiting step in formation of prostanoids is catalysed by cyclooxygenases (COXs) or prostaglandin endoperoxide H synthases (PGHSs). There are two structurally related isoforms of PGHS, which are encoded by separate genes (PGHS-1 and PGHS-2) and have very similar kinetic properties (4), but differ in regard to expression and regulation (2). PGHS-1 is expressed constitutively in most tissues and cells whereas PGHS-2 is usually not expressed in most resting tissues, but is readily induced in several cell types by various stimuli (2). DNA-binding sites for NF-
B, CCAAT/enhancer binding protein (C/EBP), ATF/CRE and E-box sequences in the 5'-flanking region of the PGHS-2 gene (57) mediate the rapid induction of PGHS-2 after stimulation of cells with, for example, cytokines, hormones and lipopolysaccharide (813). In addition to endogenous stimuli, the PGHS-2 gene can be activated by xenobiotics, e.g. phorbol esters, benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (7,1418). The molecular mechanism by which TCDD stimulates PGHS-2 expression is still unclear.
In contrast, the mechanism of transcriptional activation of genes for drug-metabolizing enzymes like those encoding CYP1A1, CYP1B1, glutathione S-transferase Ya and NAD(P)H:menadione oxidoreductase by TCDD is well documented (19,20). TCDD-dependent activation of these genes involves binding of aromatic hydrocarbon receptor (AhR)AhR nuclear translocator (Arnt) dimer to xenobiotic-responsive elements (XREs) located in the 5'-flanking regions of the respective genes. In the absence of an activating ligand the AhR resides in the cytosol as a complex with heat-shock protein 90 (Hsp90). In addition to Hsp90 other proteins seem to be involved in the formation of high-affinity ligand-binding form(s) of the AhR (21,22). The co-chaperone p23 plays a part in the activation process of AhR to the DNA-binding complex (23) and an immunophilin like protein (24,25) as well as the c-Src tyrosine kinase pp60src are apparently associated with cytosolic AhR (26). The precise role of these proteins in the activation process of AhR-regulated genes is not known.
XRE sequences have been identified in the promoter region of the mouse, rat and human PGHS-2 genes (5,27,28), but so far it is unclear whether recruitment of AhR/Arnt pathway is necessary for the transcriptional activation of PGHS-2 by TCDD. Transfection studies with reporter plasmids containing XRE elements of the PGHS-2 promoter are inconsistent regarding the functional activity of these XRE elements: in transfected Hepa 1c1c7 cells, the AhR failed to activate the transcription of the PGHS-2 gene (15), whereas in thymocytes the XRE element appears to be necessary for down-regulation of PGHS-2 (29). Results from animal studies indicated the involvement of the AhR in activation of PGHS-2. In C57BL/6J mice bearing the Ah b-1 allele, which codes for a high-affinity receptor (30,31), TCDD led to a stronger increase in PGHS-2 mRNA levels in lung compared with the DBA/2J mice strain, in which the Ah d allele codes for a low-affinity receptor. In AhR-deficient animals, TCDD failed to induce PGHS-2 mRNA expression (18). Although these reports suggest that PGHS-2 induction by TCDD may occur through AhR-mediated signalling, they do not afford direct insight into the molecular mechanisms involved. Specifically, it is not clear whether AhR/Arnt signalling is important for TCDD-mediated PGHS-2 induction. Therefore, in this study we have examined the enhancer activity of the XRE motif located on the promoter of the PGHS-2 gene in response to the AhR ligand TCDD. Furthermore, we have investigated the role of the tyrosine kinase c-Src in TCDD-mediated induction of PGHS-2.
Our results suggest that transcriptional activation of the PGHS-2 gene by TCDD can be regulated by the tyrosine kinase c-Src pathway which involves the cis-acting element C/EBP rather than DNA binding of the AhR on the XRE motif of the 5'-flanking region of the PGHS-2 gene.
 |
Materials and methods
|
---|
Cell cultures and treatment
Primary rat hepatocytes were isolated from female SpragueDawley rats (HarlanWinkelmann) with a body weight of 180220 g. The procedures of cell preparation and cultivation were carried out as described previously (32). The cells were grown in William's medium E supplemented with 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.5% IST, a medium containing insulin, transferrin and selenium (Becton Dickinson). Rat hepatocytes (viability >90% as determined by trypan blue exclusion test) were seeded at a density of 1 x 106/well in collagen-coated six-well culture plates. After 2 h of incubation, adherent cells were washed with phosphate-buffered saline (PBS) and fresh culture medium was added. Unless stated otherwise, rat hepatocytes were treated with the various chemicals for the indicated periods. The stock solutions of TCDD (50 µM, Oekometric), herbimycin A (0.5 mM, Calbiochem) and geldanamycin (3 mM, Sigma) were prepared in dimethylsulfoxide (DMSO). Cells treated with vehicle alone (0.1% DMSO) were used as controls. The mouse Hepa-1c1c7 cell line was obtained from the European Collection of Cell Cultures (ECACC) and was grown in
-minimal essential medium (Seromed) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum.
Reverse transcriptionpolymerase chain reaction (RTPCR)
Total RNA was isolated from primary rat hepatocytes or, in the in vivo studies, from organs of rats using TRI Reagent total RNA isolation kit (Sigma) and subjected to RNase-free DNase I digestion. RTPCR analysis was carried out as previously described (33). Briefly, reverse transcription was performed with 1 µg of total RNA, 1 µg of oligo(dT15) (Roche), 1 mM dithiothreitol (DTT) and 400 U murine leukaemia virus reverse transcriptase (Gibco-BRL) in a 40 µl-reaction volume according to the manufacturer's instructions. PCR amplifications were performed in a 50 µl reaction volume containing 2.5 µl of the reverse transcriptase reaction, 10x PCR buffer, 200 µM deoxynucleoside triphosphates (Pharmacia), 1 µCi of [
-32P]dCTP (3000 Ci/mmol) (ICN), 1 U Taq DNA polymerase (Roche) and 200 nM of each primer. Rat-specific primers for detection of PGHS-2, PGHS-1, CYP1A1, and ß-actin genes were selected from published sources (3437). The conditions for PCR amplification were (i) 4 min at 94°C; n cycles of 1 min at 94°C for denaturation, 1 min at the stated temperature for primer annealing, and 1 min at 72°C for primer extension; and (iii) 7 min at 72°C after the last cycle. The following annealing temperatures and cycle numbers were used for amplification: PGHS-1: 63°C, 30 cycles; PGHS-2: 63°C, 32 cycles; CYP1A1: 57°C, 23 cycles; ß-actin: 57°C, 23 cycles. Linearity of amplification reactions was checked by performing three different numbers of cycles for each cDNA concentration. Primers used for RTPCR analysis of murine CYP1A1, PGHS-1, PGHS-2 and ß-actin mRNA expression in lungs of mice and respective PCR conditions were as described previously (18). PCR products were analysed on 10% polyacrylamide gels and visualized by autoradiography. For semiquantitative analysis, respective bands were quantified using an OmniMedia gel scanner (BioImage); the data were integrated, normalized to the amount of ß-actin signals and analysed using the manufacturer's software.
Prostaglandin E2 determination
To determine the concentration of prostaglandin E2 (PGE2) in cell supernatants, 2.5 x 106 rat hepatocytes were seeded in 25 cm2 culture flasks and incubated for 12 h. Thereafter the endogenous PGHS activities were blocked by incubation of cells with 500 µM acetylsalicylic acid (Sigma) for 2.5 h. The culture medium was renewed and cells were treated with 50 nM TCDD or 0.1% DMSO (control) for 4 h, 6 h and 24 h. At various time points the culture medium was removed and the cells were then incubated in fresh culture medium containing 30 µM arachidonic acid for 15 min. PGE2 was enriched from cell supernatants by a solid-phase extraction on a C-18 cartridge (Bakerbond). The amounts of PGE2 were determined using the PGE2 enzyme immunoassay kit (Cayman). The extraction procedure was carried out according to the manufacturer's instructions. The rate of recovery of PGE2 was 90%.
Transient transfection studies
Hepa1c1c7 cells (5 x 104/well) were plated in six-well culture plates. After 16 h, standard medium was replaced with 1 ml medium without fetal calf serum. Cells were transiently transfected for 3 h by the liposome method using 5 µg Transfectam (Promega) with 2 µg of respective luciferase reporter constructs of the mouse PGHS-2 promoter. To determine the transfection efficiency, cells were cotransfected with 0.1 µg pRL-TK (Promega). After transfection, cells were incubated in 3 ml standard medium for 6 h before treatment for 16 h with 10 nM TCDD or 0.1% DMSO (control). Cells were washed twice with PBS and lysed with 250 µl (Promega) lysis buffer. Luciferase and renilla activities were measured with the Dual Luciferase Reporter Assay System (Promega) using an automatic luminometer (Berthold Micro Lumat Plus). Relative light units were normalized to renilla luciferase activity and to protein concentration, using the Bradford dye assay (Bio-Rad). Experiments were repeated three times. Three wells of cells were analysed per experiment.
Plasmids and site-directed mutagenesis
The pTIS10S luciferase expression vector (kindly provided by H.Herschman) containing nucleotides 371 to +70 of the 5'-upstream regulatory sequence of the murine PGHS-2 promoter was cloned in a pXP2 plasmid (Promega). The pTIS10S plasmid with the mutated C/EBP binding site 5'-CGGTTCTTGAGCACCTCACTGAA-3' (A instead of C at 135 and C instead of A at 131) was kindly provided by C.Pilbeam. A site-directed mutation in the XRE-binding site of the PGHS-2 promoter was performed by a PCR-based technique using a site-directed mutagenesis kit (QuickChange, Stratagene). The original plasmid pTIS10S was used as template. Mutation of the core sequence to 5'-TGCGCG-3' (C instead of T at 165) was previously shown completely to impair AhRArnt binding (38). The presence of this mutation in our sample was confirmed by sequence analysis (Cy5 AutoRead Sequencing Kit, Pharmacia).
Gel mobility shift assays
Nuclear extracts were isolated from primary rat hepatocytes according to Dennler et al. (39). In brief, 5 x 106 cells were treated with 10 nM TCDD for 1, 2 or 3 h, and harvested in Dulbecco's PBS containing 1 mM PMSF and 0.05 µg/µl of aprotinin. After centrifugation the cell pellets were gently resuspended in 1 ml of hypotonic buffer (20 mM HEPES, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.13 µM okadaic acid, 1 mM DTT, pH 7.9, and 1 µg/ml each leupeptin, aprotinin and pepstatin). The cells were allowed to swell on ice for 15 min and then homogenized by 25 strokes of a Dounce homogenizer. After centrifugation for 1 min at 16 000 x g, nuclear pellets were resuspended in 300 µl ice-cold high-salt buffer (hypotonic buffer with 420 mM NaCl and 20% glycerol). The samples were passed through a 21-gauge needle and stirred for 30 min at 4°C. The nuclear lysates were microcentrifuged at 16 000 x g for 20 min, aliquoted and stored at 70°C. Protein concentrations were determined by the method of Bradford (Bio-Rad).
For gel mobility shift assays, double-stranded oligonucleotides were used containing consensus sequences (underlined) for the C/EBP (5'-GGTATTATGCAATTGGAAGCG-3') or the XRE (5'-GCTCATTTGCGTGAGTAAAGCCTG-3') binding sites located between nucleotides 144 to 124 and 176 to 153 of the rat PGHS-2 promoter, respectively (27,40). DNAprotein binding reactions were carried out in a total volume of 20 µl containing 15 µg nuclear protein, 40 000 c.p.m. of DNA oligonucleotide, 25 mM Tris buffer (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 5% glycerol and 1 µg poly(dIdC). The samples were incubated at room temperature for 20 min. Supershift analysis were performed by addition of 2 µg of polyclonal C/EBP
, C/EBPß or C/EBP
antibodies (Santa Cruz) to the reaction mixtures. Competition experiments were done in the presence of a 200-fold molar excess of unlabelled DNA fragments. ProteinDNA complexes were resolved on a 5% non-denaturing polyacrylamide gel and visualized by exposure of the dehydrated gels to X-ray films.
Animals and treatment
Heterozygous and homozygous c-Src-deficient C57BL/6 mice (810 weeks old) were generated by back breeding of the original B6/129-srctmlSor mouse strain (stock no. J2381, Jackson Laboratory) to the C57BL/6 genetic background (41). The animals were intraperitoneally injected with a single dose of TCDD (115 µg/kg body weight) dissolved in a mixture of corn oil and acetone (9:1). Animals were killed 24 h after treatment, the organs (lungs and spleens) were prepared and kept in a tissue storage reagent (RNAlater, Ambion) for isolation of total RNA. The animal experiments were performed by D.Y.Dunlap and F.Matsumura (University of California at Davis, CA, USA) according to the national animal care guidelines.
 |
Results
|
---|
PGHS expression and induction by TCDD in primary cultured rat hepatocytes
While PGHS-2 is not expressed constitutively in hepatocytes of intact rat liver, we found that PGHS-2 expression could be detected in primary cultured rat hepatocytes. Therefore, at first the constitutive expression of PGHS isoforms in this in vitro system was analysed. PGHS-2 mRNA was detectable 1 h after cell seeding and the level increased with the time of culturing (Figure 1
). The increase in PGHS-2 mRNA was maximal 6 h after cell inoculation; thereafter the level of PGHS-2 mRNA returned to the initial level. In contrast, PGHS-1 was the only isoform expressed in freshly isolated rat hepatocytes and its mRNA level decreased continuously with duration of cell culturing (Figure 1
). Exposure of cells to various concentrations of TCDD (0.0110 nM) for 24 h led to a dose-dependent increase in PGHS-2 expression. At the highest concentration (10 nM TCDD), the PGHS-2 mRNA content was three times greater than control values (Figure 2
). The expression of PGHS-1 mRNA remained unchanged by TCDD treatment (data not shown). The expression of CYP1A1 mRNA was also monitored in this study. A concentration of 10 nM TCDD led to a 20-fold increase in CYP1A1 mRNA content (Figure 2
).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1. Expression of PGHS isoenzymes in freshly isolated and primary cultured rat hepatocytes at various time points. mRNA expression was detected by RTPCR and the respective bands were scanned; quantification was based on densitometric evaluation normalized to ß-actin as described in Materials and methods. The values are given as arbitrary units. Results of three separate experiments are presented as mean values ± SD.
|
|


View larger version (41K):
[in this window]
[in a new window]
|
Fig. 2. Dose-dependence of PGHS-2 and CYP1A1 mRNA induction by TCDD. Primary cultured rat hepatocytes were treated for 24 h with 0.01, 0.1, 1, and 10 nM TCDD; control cells received only the vehicle solvent. (A) Autoradiograms of RTPCR analysis of PGHS-2 and CYP1A1 mRNA levels. (B) TCDD doseresponse. mRNA expression is given relative to that of the respective controls. Student's t-test was used to determine if differences between TCDD-treated cells and control cells were significant; *significantly different from control (P < 0.05).
|
|
Effect of TCDD on PGE2 production in rat hepatocytes
To evaluate whether the increased level of PGHS-2 mRNA by TCDD is associated with increased PGHS enzyme activity, the PGE2 contents of cell supernatants were measured. Endogenous PGHS activity was blocked with acetylsalicylic acid for 2.5 h and then cells were treated with TCDD for 4, 6 or 24 h. De novo synthesis of PGE2 was monitored by incubation of cells with 30 µM arachidonic acid for 15 min. The results (Figure 3
) show that TCDD stimulated PGE2 synthesis in a time-dependent manner. For example, cells treated with 50 nM TCDD for 24 h contained 5-fold more PGE2 than control cells (Figure 3
). This confirms that induction of PGHS-2 mRNA by TCDD is associated with increased PGE2 synthesis.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3. Effect of TCDD on PGE2 production by rat hepatocytes. After incubation for 12 h, hepatocytes were treated with 500 µM acetylsalicylic acid (AA) for 2.5 h to inactivate endogenous PGHS activity. Cells were then treated with 50 nM TCDD for 4, 6 and 24 h (T), and control cells (C) were treated with 0.1% DMSO. The media were changed and hepatocytes were incubated with arachidonic acid (30 µM) for 15 min. The PGE2 in the medium was purified and measured by enzyme immunoassay. The first value (M) is the endogenous PGHS activity before acetylsalicylic acid treatment. Mean values of two independent experiments are given for each time point.
|
|
Effect of c-Src tyrosine kinase inhibitors on TCDD-mediated PGHS-2 induction
The involvement of a tyrosine kinase pathway in regulation of PGHS-2 expression is well documented (13,42,43). To investigate whether this pathway could be responsible for TCDD-mediated PGHS-2 induction, rat hepatocytes were pretreated for 10 min with the c-Src-specific inhibitors herbimycin A (0.5 µM) or geldanamycin (3 µM) and then treated with 10 nM TCDD for 12 h. The results in Figure 4
show that pretreatment with herbimycin A blocked both basal and TCDD-stimulated PGHS-2 expression, whereas geldanamycin suppressed only the TCDD-mediated increase in PGHS-2 mRNA (Figure 4
). Neither inhibitor affected TCDD-mediated CYP1A1 induction, suggesting that the mechanisms of modulation of PGHS-2 mRNA and CYP1A1 mRNA expression by TCDD may be different.


View larger version (88K):
[in this window]
[in a new window]
|
Fig. 4. Effect of tyrosine kinase inhibitors herbimycin A and geldanamycin on TCDD-induced mRNA expression of PGHS-2 and CYP1A1 in rat hepatocytes. Cells were treated with 10 nM TCDD, 0.5 µM herbimycin A, 3 µM geldanamycin or DMSO vehicle for 12 h. For inhibition studies cells were preincubated with herbimycin A or geldanamycin for 10 min and then co-treated with 10 nM TCDD for 12 h. (A) RTPCR analysis of PGHS-2, CYP1A1 and ß-actin mRNA. (B and C) Densitometric evaluation of PGHS-2 (B) and CYP1A1 (C) mRNA band intensities. The values are given as percentages of the control. C, control; T, TCDD; H, herbimycin A; G, geldanamycin.
|
|
The C/EBP-binding site is required for TCDD-mediated PGHS-2 induction
To identify DNA motifs possibly responsible for TCDD-mediated PGHS-2 activation, transfection studies with different luciferase reporter constructs of the murine PGHS-2 promoter pTIS10S were performed in the murine Hepa-1c1c7 cells. The pTIS10S construct spans from nucleotide 317 to +70 and contains an XRE motif at positions 169 to 163 and a C/EBP-binding site at positions 135 to 130. Treatment of transiently transfected Hepa-1c1c7 cells with 10 nM TCDD for 16 h led to a 4-fold induction in luciferase activity, indicating a transcriptional activation of the PGHS-2 gene by TCDD (Figure 5
). To determine whether PGHS-2 induction is mediated by binding of the AhRArnt complex to the XRE element, a point mutation was introduced into this motif which was shown to abolish binding (38). Transfection studies revealed that mutation of the XRE motif led to a minor but insignificant decrease of TCDD-stimulated luciferase activity (from 4-fold to 3- fold). However, point mutations in the C/EBP-binding site significantly reduced (from 4-fold to 2-fold) the luciferase activity (Figure 5
), suggesting an important role for the C/EBP-binding site in the TCDD-induced activation of PGHS-2. To corroborate these results, gel shift analyses with 32P-radiolabelled XRE-containing oligonucleotides from rat CYP1A1 (44) and rat PGHS-2 promoter were performed. As depicted in Figure 6
, XRE-binding activity was detected upon activation of AhR by TCDD when the XRE oligonucleotide of CYP1A1 promoter was used as described previously (45). In contrast, no binding activity was seen in gel mobility shift assays performed with the respective PGHS-2 probe, even after prolonged exposure to X-ray film (Figure 6
).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5. TCDD-stimulated transcriptional activation of PGHS-2 in transiently transfected murine Hepa-1c1c7 cells. Hepa-1c1c7 cells were transiently transfected with a pTIS10S reporter plasmid of the murine PGHS-2 promoter. pTIS10SXRE and pTIS10SC/EBP are reporter constructs containing mutations in the XRE- or C/EBP-binding site, respectively. Cells were treated with 10 nM TCDD (T) or with DMSO vehicle (C) for 16 h. Luciferase activity is given as fold induction compared with control cells. Mean values of triplicates are shown as a result of three independent experiments.
|
|

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 6. Sequence-specific binding of the TCDD-activated AhR to XREs. Nuclear extracts were prepared from rat hepatocytes incubated for 1, 2 or 3 h with 10 nM TCDD. The activated AhR does not bind to the XRE sequence of the rat PGHS-2 promoter (5'-CAGGCTTTACTCACGCAAATGAGC-3') (lanes 2, 4 and 6). As a positive control, the same nuclear extracts were assayed for binding to the XRE of the rat CYP1A1 promoter (5'-CCAGGCTCTTCTCACGCAACTCCGGGGC-3') (lanes 814). The positions of two retarded bands of the DNA-bound AhR complex activated by TCDD (lanes 9, 11 and 13) are indicated by arrows. DNA binding of AhR was completed with a 200-fold molar excess of unlabeled XRE (lane 14).
|
|
PGHS-2 induction by TCDD is mediated by the transcription factor C/EBP ß
Different isoforms of the C/EBP transcription factor family (C/EBP
, ß,
and
) have been identified (46,47) which can bind to the same DNA recognition site. To identify the components responsible for TCDD-mediated PGHS-2 induction, gel mobility supershift assays were conducted. The C/EBP-binding site of the rat PGHS-2 promoter was used as the oligonucleotide probe (27). Nuclear proteins were isolated from rat hepatocytes treated with 10 nM TCDD for 1, 2 or 3 h, respectively. The gel shift pattern revealed two bands (complexes I and II) with different intensities between TCDD-treated (Figure 7
, lanes 2, 4 and 6) and control cells (lanes 1, 3 and 5). Although the band intensities of control cells increased with time, those in TCDD-treated cells were significantly higher (Figure 7B
). This time-dependent increase in C/EBP binding activity in the control cells may be attributed to the DMSO treatment. Incubation of nuclear extracts from rat hepatocytes with a C/EBP
-specific antibody led to a shift of complex I but not of complex II (Figure 7
, lanes 9 and 10). Antibody specific for C/EBPß shifted both complexes (Figure 7
, lanes 11 and 12). Incubation of nuclear extracts with C/EBP
antibody did not influence the gel shift pattern (Figure 7
, lanes 13 and 14). Apparently the upper band (complex I) is formed by C/EBP
C/EBPß heterodimers, whereas the lower band (complex II) is formed by C/EBPß homodimers. Non-immune rabbit serum had no effect on binding of C/EBP (data not shown). Excess of unlabelled oligonucleotide eliminated both bands (Figure 7
, lane 15). Supershift assays support the view that C/EBPß is the predominant transcription factor whose DNA-binding activity is enhanced by TCDD. To investigate whether tyrosine kinase inhibitors can influence C/EBP-binding activities, rat hepatocytes were preincubated with herbimycin A (0.5 µM) or geldanamycin (3 µM) for 10 min and then treated with 10 nM TCDD for 1 h. As seen in Figure 7
, both inhibitors reduced the TCDD-stimulated increase of band intensities; herbimycin A (Figure 7
, line 7) was more effective than geldanamycin (Figure 7
, lane 9). These data suggest that the c-Src kinase pathway may be involved in TCDD-stimulated C/EBP binding which is consistent with mRNA expression studies demonstrating lack of PGHS-2 induction upon incubation of cells with these c-Src kinase inhibitors.
PGHS-2 mRNA induction in c-Src-deficient mice
The in vitro data suggested an involvement of tyrosine kinase c-Src in TCDD-mediated PGHS-2 induction. To test this supposed c-Src-dependent effect in vivo, we investigated the TCDD-induced PGHS-2 expression in lungs of wild-type, heterozygous and homozygous c-Src-deficient C57BL/6 mice. Consistent with in vitro findings, the induction of CYP1A1 (positive control) was not affected by c-Src deficiency (Figure 8
). On the other hand, PGHS-2 was induced in wild-type and heterozygous c-Src-deficient mice, but not in the lungs of homozygous c-Src knockout mice (Figure 8
).

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 8. Effect of TCDD on PGHS-2 and CYP1A1 mRNA expression in lungs of wild-type, heterozygous and homozygous c-Src-deficient C57BL/6 mice. Animals were treated with 115 µg TCDD/kg for 24 h and mRNA expression was analyzed as described in Materials and methods. Each group consisted of two animals.
|
|
 |
Discussion
|
---|
The main purpose of this study was to investigate the mechanisms by which TCDD activates PGHS-2 gene expression. TCDD was shown to induce PGHS-2 mRNA expression in primary cultured rat hepatocytes in a dose-dependent manner, and this was accompanied by an increased synthesis of PGE2. Since PGHS-1 mRNA expression was not changed by TCDD, increased PGE2 levels must result from induction of PGHS-2 activity. A similar observation has been made in mouse hepatoma cells Hepa1c1c7: Puga et al. demonstrated that TCDD led to increased levels of PGHS-2 mRNA and increased secretion of 12-hydroxyheptadecatrienoic acid (12-HHT), but TCDD had no effect on PGHS-2 in the AhR deficient mutant cell line Hepa-c2 (17). These findings and results from our previous in vivo studies (18) suggest that the TCDD-mediated modulation of PGHS-2 expression is AhR-dependent. Therefore, we examined whether the XRE-binding motif plays a role in trans-activation of PGHS-2 mediated by AhR. Yet, analyses of the functionally mutated XRE core motif of the mouse PGHS-2 promoter resulted only in a non-significant decrease in TCDD-stimulated luciferase activation. Thus, we conclude that PGHS-2 induction after TCDD is not mediated by binding of AhRArnt to the XRE element. This view is supported by results of gel mobility shift assays with DNA fragments of the rat PGHS-2 promoter that contain the putative XRE element. Binding of the AhRArnt complex to this fragment was not detected. A comparison of the XRE motif of the rat PGHS-2 promoter with that of the rat CYP1A1 promoter revealed three differences in base pairs adjacent to the core sequence, supporting previous studies showing that flanking nucleotides of the XRE core sequence have an important function in formation of the AhRArnt XRE-binding complex (38,45,48). In essence, our data suggest that PGHS-2 induction by TCDD is probably not mediated by the classical AhRArnt pathway.
There is evidence for several other types of AhR action, for example activation of tyrosine kinase pp60src (26). Since Src is a well known regulator of PGHS-2 gene expression (2), we focused on the possible involvement of this pathway in induction of PGHS-2 by TCDD. Our results obtained from in vitro studies using different c-Src inhibitors indicate the importance of the c-Src tyrosine kinase pathway in TCDD-mediated PGHS-2 induction. This view is supported by in vivo findings showing that PGHS-2 is induced in the lungs of wild-type and heterozygous c-Src-deficient mice, but not in the lungs of homozygous c-Src knockout animals. However, it is difficult to compare the in vitro and in vivo data because other regulatory pathways for the induction of PGHS-2 by TCDD could exist for the two different systems. Activation of tyrosine kinases, including pp60src, by TCDD has been reported in other studies (4952). Recently, it was shown that TCDD triggers the translocation of c-Src from the cytosol to the membrane fraction in an AhR-dependent manner. The increase in c-Src activity in the membrane fraction did not require de novo protein synthesis (52). However, the molecular mechanism connecting ligand-dependent activation of AhR and activation of c-Src is still unclear. It has been proposed that c-Src is part of an AhRHsp90 complex and is released after ligand binding (26). On the other hand, cross-linking studies have failed to detect c-Src in the AhR complex (22,23). A close connection has been found between glucocorticoid receptorHsp90 and c-SrcHsp90 complexes (53), but no such connection has been described for AhRHsp90 and c-SrcHsp90. Nevertheless, it has been shown that AhR signalling on c-Src is independent of Arnt (49). Our data with geldanamycin, which interacts with the c-SrcHsp90 heteroprotein complex (54,55), favour the view that c-Src and PGHS-2 are activated independently of Arnt. Pretreatment of cells with geldanamycin abolishes the TCDD-mediated PGHS-2 activation without disrupting the AhRArnt pathway, as indicated by the unchanged responsiveness of CYP1A1 towards TCDD. Other studies have described the rapid degradation of AhR and disruption of the AhRArnt pathway by geldanamycin (52,56,57). These discrepancies may have been caused by differences in experimental conditions. In our study cells were pretreated with geldanamycin for 10 min, whereas in the reported studies cells were exposed to geldanamycin for
1 h. We observed that the effect of geldanamycin on AhRArnt was strongly time- and concentration-dependent (data not shown).
The Src-mediated signalling on PGHS-2 transcription involves several steps of signal transduction and activation of different transcription factors for which binding sites have been identified in the PGHS-2 promoter (43). For example, an ATF/CRE element has been shown to be important for activation of PGHS-2 in mouse cell lines by platelet derived growth factor (PDGF), serum factors and v-Src (11,43,58). However, it is unlikely that TCDD-mediated PGHS-2 activation in primary rat hepatocytes occurs via the ATF/CRE site, since the rat PGHS-2 promoter does not contain this ATF/CRE-binding motif (9). Our results from transfection experiments performed with a mutated mice C/EBP-binding motif suggest that this regulatory element is important for PGHS-2 activation after TCDD treatment. Consistent with these findings are results from supershift analysis with DNA fragments containing the C/EBP-binding element of the rat PGHS-2 gene showing an enhanced C/EBPß-binding activity after TCDD. Pretreatment of rat hepatocytes with herbimycin A and geldanamycin diminished C/EBPß-binding activity suggesting that c-Src kinase pathway is involved in phosphorylation of C/EBPß, resulting in enhanced nuclear accumulation (47). Thus, we assume that TCDD induces PGHS-2 expression by recruitment of c-Src from a putative c-SrcHsp90 complex which initiates cellular signaling to C/EBPß resulting in enhanced transcription of the PGHS-2 gene. The precise nature of this cellular signalling to the PGHS-2 gene remains to be elucidated.
The expression of PGHS-2 in untreated primary cultured rat hepatocytes is noteworthy, since the enzyme it encodes is not usually detectable in hepatocytes of healthy rat or mouse liver. However, weak expression of PGHS-2 has been observed in human liver (59,60), indicating that there are species-specific differences in constitutive PGHS-2 expression. The transient increase of PGHS-2 expression in cultured rat hepatocytes is not mediated by serum factors, since a similar effect has been observed in cells cultured in serum-free media (data not shown). Possibly, a lack of cellcell contact and/or extracellular matrix components are responsible for the transient increase in PGHS-2 expression in isolated cultured hepatocytes. This view is supported by the observation that the PGHS-2 mRNA level returns to near the initial low level when cellcell contacts are restored. Recently, it was shown that extracellular matrix components such as E-cadherin and ß-catenin can control PGHS expression (61). It has been suggested that elevated nuclear accumulation of ß-catenin-TcfLEF transcription complex is linked mechanistically to increased PGHS-2 expression (62).
Numerous studies are under way to understand the mechanisms of TCDD toxicity including tumour promotion; PGHS-2 is suggested to play a critical role in this pathogenic process (2,6366). Overexpression of PGHS-2 has been found in tumours of human (67) and various other animal species as well as in transformed cell lines (68,69). It has been suggested that increased expression of PGHS-2 in tumors is regulated by altered C/EBP protein levels (70) which is also observed after TCDD treatment in vivo and in vitro (33,71). Thus, functional analysis of this class of transcription factors in the context of toxic effects of TCDD could give insight into the mechanisms underlying the pleiotropic responses evoked by polyhalogenated compounds like TCDD.
 |
Notes
|
---|
3 To whom correspondence should be addressed Email: josef.abel{at}uni-duesseldorf.de 
 |
Acknowledgments
|
---|
We gratefully acknowledge Fumio Matsumura and Deborah Y.Dunlap for providing organs of c-Src knockout mice. We thank Harvey R.Herschman and Carol Pilbeam for kindly providing different pTIS10 deletion constructs and C/EBP mutation construct of murine PGHS-2 promoter. This work was supported by the Deutsche Forschungsgemeinschaft (DFG AB 38/3-1 and Sonderforschungsbereich 503, A5). A.-M.F.J.B. was supported by a fellowship from the Postgraduate College `Toxicology and Environmental Hygiene' given to Duesseldorf University by Deutsche Forschungsgemeinschaft.
 |
References
|
---|
-
DeWitt,D.L. and Smith,W.L. (1995) Yes, but do they still get headaches?. Cell, 83, 345348.[Medline]
-
Herschman,H.R. (1996) Prostaglandin synthase 2. Biochim. Biophys. Acta, 1299, 125140.[Medline]
-
Taketo,M.M. (1998) Cyclooxygenase-2 inhibitors in tumorigenesis (part I). J. Natl Cancer Inst., 90, 15291536.[Abstract]
-
Smith,W.L. and De Witt,D.L. (1995) Biochemistry of prostaglandin endoperoxide H synthase-1 and synthase-2 and their differential susceptibility to nonsteroidal anti-inflammatory drugs. Sem. Nephrol., 15, 179194.
-
Fletcher,B.S., Kujubu,D.A., Perrin,D.M. and Herschman,H.R. (1992) Structure of the mitogen-inducible TIS10 gene and demonstration that the TIS10-encoded protein is a functional prostaglandin G/H synthase. J. Biol. Chem., 267, 43384344.[Abstract]
-
Kosaka,T., Miyata,A., Ihara,H., Hara,S., Sugimoto,T. and Takeda,O. (1994) Characterization of the human gene PTGS2 encoding prostaglandin endoperoxide synthase-2. Eur. J. Biochem., 221, 889897.[Abstract]
-
Inoue,H., Yokoyama,C., Hara,S., Tone,Y. and Tanabe,T. (1995) Transcriptional regulation of human prostaglandin-endoperoxide synthase-2 gene by lipopolysaccharide and phorbol ester in vascular endothelial cells. J. Biol. Chem., 270, 2496524971.[Abstract/Full Text]
-
Jones,D.A., Carlton,D.P., McIntyre,T.M., Zimmerman,G.A. and Prescott,S.M. (1993) Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J. Biol. Chem., 268, 90499054.[Abstract]
-
Morris,J.K. and Richards,J.S. (1996) An E-box region within the prostaglandin endoperoxide synthase-2 (PGS-2) promoter is required for transcription in rat ovarian granulosa cells. J. Biol. Chem., 271, 1663316643.[Abstract/Full Text]
-
Reddy,S.T., Wadleigh,D.J. and Herschman,H.R. (2000) Transcriptional regulation of the cyclooxygenase-2 gene in activated mast cells. J. Biol. Chem., 275, 31073113.[Abstract/Full Text]
-
Xie,W., Fletcher,B.S., Andersen,R.D. and Herschman,H.R. (1994) v-src induction of the TIS10/PGS2 prostaglandin synthase gene is mediated by an ATF/CRE transcription response element. Mol. Cell. Biol., 14, 65316539.[Abstract]
-
Yamamoto,K., Arakawa,T., Ueda,N. and Yamamoto,S. (1995) Transcriptional roles of nuclear factor
B and nuclear factor-interleukin-6 in the tumor necrosis factor
-dependent induction of cyclooxygenase-2 in MC3T3-E1 cells. J. Biol. Chem., 270, 3131531320.[Abstract/Full Text]
-
Han,J.W., Sadowski,H., Young,D.A. and Macara,I.G. (1990) Persistent induction of cyclooxygenase in p60v-src-transformed 3T3 fibroblasts. Proc. Natl Acad. Sci. USA, 87, 33733377.[Abstract]
-
Kelley,D.J., Mestre,J.R., Subbaramaiah,K., Sacks,P.G., Schantz,S.P., Tanabe,T., Inoue,H., Ramonetti,J.T. and Dannenberg,A.J. (1997) Benzo[a]pyrene up-regulates cyclooxygenase-2 gene expression in oral epithelial cells. Carcinogenesis, 18, 795799.[Abstract]
-
Kraemer,S.A., Arthur,K.A., Denison,M.S., Smith,W.L. and De Witt,D.L. (1996) Regulation of prostaglandin endoperoxide H synthase-2 expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Arch. Biochem. Biophys., 330, 319328.[Medline]
-
Liu,Y., Levy,G.N. and Weber,W.W. (1997) Induction of human prostaglandin H synthase-2 (PGHS-2) mRNA by TCDD. Prostaglandins, 53, 110.[Medline]
-
Puga,A., Hoffer,A., Zhou,S., Bohm,J.M., Leikauf,G.D. and Shertzer,H.G. (1997) Sustained increase in intracellular free calcium and activation of cyclooxygenase-2 expression in mouse hepatoma cells treated with dioxin. Biochem. Pharmacol., 54, 12871296.[Medline]
-
Vogel,C., Schuhmacher,U.S., Degen,G.H., Bolt,H.M., Pineau,T. and Abel,J. (1998) Modulation of prostaglandin H synthase-2 mRNA by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice. Arch. Biochem. Biophys., 351, 265271.[Medline]
-
Landers,J.P. and BunceN.J. (1991) The Ah receptor and mechanism of dioxin toxicity. Biochem. J., 276, 273287.[Medline]
-
Nebert,D.W., Roe,A.L., Dieter,M.Z., Solis,W.A., Yang,Y. and Dalton,T.P. (2000) Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem. Pharmacol., 59, 6585.[Medline]
-
LaPres,J.J., Glover,E., Dunham,E.E., Bunger,M.K. and Bradfield,C.A. (2000) ARA9 modifies agonist signaling through an increase in cytosolic aryl hydrocarbon receptor. J. Biol. Chem., 275, 61536159.[Abstract/Full Text]
-
Meyer,B.K. and Perdew,G.H. (1999) Characterization of the AhRhsp90XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization. Biochemistry, 38, 89078917.[Medline]
-
Kazlauskas,A., Poellinger,L. and Pongratz,I. (1999) Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor. J. Biol. Chem., 274, 1351913524.[Abstract/Full Text]
-
Carver,L.A. and Bradfield,C.A. (1997) Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel immunophilin homolog in vivo. J. Biol. Chem., 72, 1145211456.
-
Ma,Q. and Whitlock,J.P. Jr (1997) A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem., 272, 88788884.[Abstract/Full Text]
-
Enan,E. and Matsumura,F. (1996) Identification of c-src as the integral component of the cytosolic Ah receptor complex, transducing the signal of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the protein phosphorylation pathway. Biochem. Pharmacol., 52, 15991612.[Medline]
-
Sirois,J., Levy,L.O., Simmons,D.L. and Richards,J.S. (1993) Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. J. Biol. Chem., 268, 1219912206.[Abstract]
-
Hla,T. and Neilson,K. (1992) Human cyclooxygenase-2 cDNA. Proc. Natl. Acad. Sci. USA, 89, 73847388.[Abstract]
-
Olnes,M.J., Verma,M. and Kurl,R.N. (1996) 2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates expression of the prostaglandin G/H synthase-2 gene in rat thymocytes. J. Pharmacol. Exp. Ther., 279, 15661573.[Abstract]
-
Chang,C., Smith,D.R., Prasad,V.S., Sidman,C.L., Nebert,D.W and Puga,A. (1993) Ten nucleotide differences, five of which cause amino acid changes, are associated with the Ah receptor locus polymorphism of C57BL/6 and DBA/2 mice. Pharmacogenetics, 3, 312321.[Medline]
-
Poland,A. and Glover,E. (1990) Characterization and strain distribution pattern in murine Ah receptor specified by the Ahd and Ahb-3 alleles. Mol. Pharmacol., 38, 306312.[Abstract]
-
Horbach,M., Gerber,E. and Kahl,R. (1997) Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-
from rat hepatocyte cultures. Toxicology, 121, 117126.[Medline]
-
Doehr,O., Sinning,R., Vogel,C. Muenzel,P. and Abel,J. (1997) Effect of transforming growth factor-ß1 on expression of aryl hydrocarbon receptor and genes of Ah gene battery: clues for independent down-regulation in A549 cells. Mol. Pharmacol., 51, 703710.[Abstract/Full Text]
-
Kennedy,B.P., Chan,C.C., Culp,S.A. and Cromlish,W.A. (1993) Cloning and expression of rat PGHS- (Cox-)2 cDNA. Biochem. Biophys. Res. Commun., 197, 494500.[Medline]
-
Nanji,A.A., Lili,M., Thomas,P., Rahemtulla,A., Shamsuddin,K., Zhao,S., Peters,D., Tahan,S.R. and Dannenberg,A.J. (1997) Enhanced cyclooxygenase-2 gene expression in alcoholic liver disease in the rat. Gastroenterology, 112, 943951.[Abstract]
-
Omiecinski,C.J., Redlich,C.A. and Costa,P. (1990) Induction and developmental expression of cytochrome P4501A1 messenger mRNA in rat and human tissues: detection by the polymerase chain reaction. Cancer Res., 50, 43154321.[Abstract]
-
Vanden Heuvel,J.P., Clark,G.C., Kohn,M.C., Tritscher,A.M., Greenlee,W.F., Lucier,G.W. and Bell,D.A. (1994) Dioxin-responsive genes: examination of doseresponse relationships using quantitative reverse transcriptasepolymerase chain reaction. Cancer Res., 54, 6268.[Abstract]
-
Shen,E.S. and Whitlock,J.P. Jr (1992) ProteinDNA interactions at a dioxin-responsive enhancer. Mutational analysis of the DNA-binding site for the liganded Ah receptor. J. Biol. Chem., 267, 68156819.[Abstract]
-
Dennler,S., Itoh,S., Vivien,D., ten Dijke,P., Huet,S. and Gauthier,J.M. (1998) Direct binding of Smad3 and Smad4 to critical TGFß-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J., 17, 30913100.[Abstract/Full Text]
-
Sirois,J. and Richards,J.S. (1993) Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. J. Biol. Chem., 268, 2193121938.[Abstract]
-
Dunlap,D.Y., Moreno-Aliaga,M.J., Wu,Z. and Matsumura,F. (1999) Differential toxicities of TCDD in vivo among normal, c-src knockout, geldanamycin- and quercetin-treated mice. Toxicology, 135, 95107.[Medline]
-
Chanmugam,P., Feng,L., Liou,S. et al. (1995) Radicicol, a protein tyrosine kinase inhibitor, supresses the expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide and in experimental glomerulonephritis. J. Biol. Chem., 270, 54185426.[Abstract/Full Text]
-
Xie,W. and Herschman,H.R. (1995) v-Src induces prostaglandin synthase 2 gene expression by activation of the c-Jun N-terminal kinase and the c-Jun transcription factor. J. Biol. Chem., 270, 76227628.
-
Hapgood,J., Cuthill,S., Denis,M., Poellinger,L. and Gustafsson,J.A. (1989) Specific proteinDNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity. Proc. Natl Acad. Sci. USA, 86, 6064.[Medline]
-
Denison,M.S., Fisher,J.M. and Whitlock,J.P. Jr (1988) The DNA recognition site for the dioxinAh receptor complex. Nucleotide sequence and functional analysis. J. Biol. Chem., 263, 1722117224.[Abstract]
-
Landschulz,W.H., Johnson,P.F. and McKnight,S.L. (1988) The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science, 240, 17591764.[Medline]
-
Takiguchi,M. (1998) The C/EBP family of transcription factors in the liver and other organs. Int. J. Exp. Pathol., 79, 369391.[Medline]
-
Denison,M.S., Fisher,J.M. and Whitlock,J.P. Jr (1989) ProteinDNA interactions at recognition sites for the dioxinAh receptor complex. J. Biol. Chem., 264, 1647816482.[Abstract]
-
Blankenship,A. and Matsumura,F. (1997) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes an Ah receptor-dependent increase in membrane levels and activity of p60Src. Environ. Toxicol. Pharmacol., 3, 211220.
-
Bombick,D.W., Jankun,J., Tullis,K. and Matsumura,F. (1988) 2,3,7,8-Tetrachlorodibenzo-p-dioxin causes increases in expression of c-erb-A and levels of protein-tyrosine kinases in selected tissues of responsive mouse strains. Proc. Natl Acad. Sci. USA, 85, 41284132.[Medline]
-
DeVito,M.J., Ma,X., Babish,J.G., Menache,M. and Birnbaum,L.S. (1994) Doseresponse relationships in mice following subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin: CYP1A1, CYP1A2, estrogen receptor, and protein tyrosine phosphorylation. Toxicol. Appl. Pharmacol., 124, 8290.[Medline]
-
Koehle,C., Gschaidmeier,H., Lauth,D., Topell,S., Zitzer,H. and Bock,K.W. (1999) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-mediated membrane translocation of c-Src protein kinase in liver WB-F344 cells. Arch. Toxicol., 73, 152158.[Medline]
-
Nathan,D.F., Vos,M.H. and Lindquist,S. (1999) Identification of SSF1, CNS1, and HCH1 as multicopy suppressors of a Saccharomyces cerevisiae Hsp90 loss-of-function mutation. Proc. Natl Acad. Sci. USA, 96, 14091414.[Abstract/Full Text]
-
Sakagami,M., Morrison,P. and Welch,W.J. (1999) Benzoquinoid ansamycins (herbimycin A and geldanamycin) interfere with the maturation of growth factor receptor tyrosine kinases. Cell Stress Chaperones, 4, 1928.[Medline]
-
Whitesell,L., Mimnaugh,E.G., De Costa,B., Myers,C. and Neckers,L.M. (1994) Inhibition of heat shock protein HSP90pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc. Natl Acad. Sci., 91, 83248328.[Abstract]
-
Chen,H.S., Singh,S.S. and Perdew,G.H. (1997) The Ah receptor is a sensitive target of geldanamycin-induced protein turnover. Arch. Biochem. Biophys., 348, 190198.[Medline]
-
Lees,M.J. and Whitelaw,M.L. (1999) Multiple roles of ligand in transforming the dioxin receptor to an active basic helixloophelix/PAS transcription factor complex with the nuclear protein Arnt. Mol. Cell. Biol., 19, 58115822.[Abstract/Full Text]
-
Xie,W. and Herschman,H.R. (1996) Transcriptional regulation of prostaglandin synthase 2 gene expression by platelet-derived growth factor and serum. J. Biol. Chem., 271, 3174231748.[Abstract/Full Text]
-
O'Neill,G.P. and Ford-Hutchinson,A.W. (1993) Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett., 330, 156160.[Medline]
-
Koga,H., Sakisaka,S., Ohishi,M. et al. (1999) Expression of cyclooxyagenase-2 in human hepatocellular carcinoma: relevance to tumor dedifferentiation. Hepatology, 29, 688696.[Abstract/Full Text]
-
Hsi,L.C., Angerman-Stewart,J. and Eling,T.E. (1999) Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level. Carcinogenesis, 20, 20452049.[Abstract/Full Text]
-
Mei,J.M., Hord,N.G., Winterstein,D.F., Donald,S.P. and Phang,J.M. (1999) Differential expression of prostaglandin endoperoxide H synthase-2 and formation of activated ß-cateninLEF-1 transcription complex in mouse colonic epithelial cells contrasting in APC. Carcinogenesis, 20, 737740.[Abstract/Full Text]
-
Du Bois,R.N., Shao,J., Tsujii,M., Sheng,H. and Beauchamp,R.D. (1996) G1 delay in cells overexpressing prostaglandin endoperoxide synthase-2. Cancer Res., 56, 733737.[Abstract]
-
Mitchell,J.A., Larkin,S. and Williams,T.J. (1995) Cyclooxygenase-2: regulation and relevance in inflammation. Biochem. Pharmacol., 50, 15351542.[Medline]
-
Tsujii,M. and DuBois,R.N. (1995). Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell, 83, 493501.[Medline]
-
Woelfle,D., Marotzki,S., Dartsch,D., Schaefer,W. and Marquardt,H. (2000) Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis, 21, 1521.[Abstract/Full Text]
-
Sano,H., Kawahito,Y., Wilder,R.L., Hashiramoto,A., Mukai,S., Asai,K., Kimura,S., Kato,H., Kondo,M. and Hla,T. (1995) Expression of cyclooxygenase-1 and -2 in human colorectal cancer. Cancer Res., 55, 37853789.[Abstract]
-
Muller-Decker,K., Scholz,K., Marks,F. and Furstenberger,G. (1995) Differential expression of prostaglandin H synthase isozymes during multistage carcinogenesis in mouse epidermis. Mol. Carcinogen., 12, 3141.
-
Subbaramaiah,K., Telang,N., Ramonetti,J.T., Araki,R., DeVito,B., Weksler,B.B. and Dannenberg, A.J. (1996) Transcription of cyclooxygenase-2 is enhanced in transformed mammary epithelial cells. Cancer Res., 56, 44244429.[Abstract]
-
Kim,Y. and Fischer,S.M. (1998) Transcriptional regulation of cyclooxygenase-2 in mouse skin carcinoma cells. J. Biol. Chem., 42, 2768627694.
-
Liu,P.C.C., Dunlap,D.Y. and Matsumura,F. (1998) Suppression of C/EBP
and induction of C/EBPß by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse adipose tissue and liver. Biochem. Pharmacol., 55, 16471655.[Medline]
Received June 12, 2000;
revised August 3, 2000;
accepted August 15, 2000.