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[Cancer Research 66, 1363-1370, February 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

Transcriptional Repression of Telomerase RNA Gene Expression by c-Jun-NH2-Kinase and Sp1/Sp3

Alan E. Bilsland1,3, Katrina Stevenson1,3, Stuart Atkinson1,3, Walter Kolch2,3 and W. Nicol Keith1,3

1 Centre for Oncology and Applied Pharmacology and 2 Institute of Biomedical and Life Sciences, University of Glasgow; and 3 Cancer Research UK Beatson Laboratories, Glasgow, United Kingdom

Requests for reprints: W. Nicol Keith, Cancer Research UK Beatson Laboratories, Alexander Stone Building, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom. Phone: 44-141-330-4811; Fax: 44-141-330-4127; E-mail: n.keith{at}beatson.gla.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Telomerase is essential for immortalization of most human cancer cells. Expression of the core telomerase RNA (hTR) and reverse transcriptase (hTERT) subunits is mainly regulated by transcription. However, hTR transcriptional regulation remains poorly understood. We previously showed that the core hTR promoter is activated by Sp1 and is repressed by Sp3. Here, we show that the mitogen-activated protein kinase kinase kinase 1 (MEKK1)/c-Jun-NH2-kinase (JNK) pathway represses hTR expression by a mechanism that involves Sp1 and Sp3. Promoter activity was induced by the JNK inhibitor SP600125 and was repressed by activated MEKK1. Repression by MEKK1 was blocked by SP600125 or enhanced by coexpression of wild-type but not phosphoacceptor mutated JNK. SP600125 treatment also increased levels of endogenous hTR. Mutations in the hTR promoter Sp1/Sp3 binding sites attenuated SP600125-mediated promoter induction, whereas coexpression of MEKK1 with Sp3 enhanced hTR promoter repression. Chromatin immunoprecipitation showed that levels of immunoreactive Sp1 associated with the hTR promoter were low in comparison with Sp3 in control cells but increased after JNK inhibition with a reciprocal decrease in Sp3 levels. No corresponding changes in Sp1/Sp3 protein levels were detected. Thus, JNK represses hTR promoter activity and expression, apparently by enhancing repression through Sp3. (Cancer Res 2006; 66(3): 1363-70)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Telomerase is a ribonucleoprotein reverse transcriptase minimally composed of core RNA (hTR) and catalytic (hTERT) subunits, which stabilizes the telomeres of linear chromosomes (1, 2). Although telomerase activity is present during human embryonic development, its expression and activity are repressed in most adult tissues. In contrast, it is essential for long-term proliferation of most human cancer cell lines and most human tumors express high levels of telomerase (3–6).

Levels of telomerase in cancer cells are primarily determined by transcriptional activity of the hTR and hTERT genes. Both transcripts are either absent or at very low levels in most normal human cell types but are readily detectable in most cancer cell lines (7). Interestingly, ectopic delivery of hTR and hTERT promoter constructs results in selective activity in cancer cells as shown by recent preclinical models of transcriptionally targeted gene therapy (7–9). Because of the potential for exploitation of telomerase as a relatively specific therapeutic target in a broad range of tumor types, expanded understanding of the regulation of hTR and hTERT expression is of immediate interest.

Recently, key promoter elements contributing to hTR promoter regulation have begun to emerge. The core promoter is essential for hTR expression in vivo because its methylation in some cell lines is sufficient to silence hTR expression (10). In the 5637 bladder cancer cell line, basal activity of transfected core promoter constructs (nucleotides –107/+69) is dependent on NF-Y binding to a CCAAT box sequence. The core promoter also contains four Sp1/Sp3 binding sites, which serve as sites of positive regulation by Sp1 and negative regulation by Sp3 (11–13).

We have recently identified, in archived DNA from the buccal smear of a paroxysmal nocturnal hemoglobinuria patient, a mutation in one of these sites that disrupts Sp1/Sp3 binding and increases the activity of a minimal hTR promoter in transfection assays in 5637 cells (14). In the same cells, NF-Y, Sp1, and TFIIB interact directly with hTR promoter chromatin (12). hTR promoter activity can also be regulated by overexpression of pRb and MDM2, which act, respectively, as positive and negative regulators (15). Notably, upstream signaling events that might regulate transcription factors at the hTR promoter are not well characterized.

Accumulating evidence indicates that multiple kinases, including mitogen-activated protein kinases (MAPK), directly phosphorylate Sp1 and modulate its DNA binding and/or transactivation activity (16–25). MAPK pathways are evolutionarily conserved signaling cascades with a minimal core structure in which an upstream MAPK kinase kinase (MAP3K) activates a MAPK kinase, which in turn activates a terminal effector MAPK (26). Because the hTR promoter is regulated in vitro by Sp1, MAPK pathways might be involved in hTR regulation (Fig. 1). This hypothesis was tested in this study.


Figure 1
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  		Figure 1. Rationale of the study. The hTR promoter is positively and negatively regulated in vitro by Sp1 and Sp3, respectively. MAPK pathways are known to phosphorylate and regulate DNA binding of Sp1. We tested whether the JNK pathway regulates hTR promoter activity and expression using overexpression of an activated mutant of MEKK1, wild-type JNK1, phosphoacceptor mutated JNK2, and the JNK-specific inhibitor SP600125.

 
Indeed, Sp1 sites are required for stimulation of several promoters by the extracellular signal–regulated kinase (ERK) and c-Jun-NH2-kinase (JNK) MAPK pathways, including the urokinase plasminogen activator and p21waf1/cip1 promoters (22, 25). Epidermal growth factor stimulation of gastrin promoter activity in gastric adenocarcinoma cells also involves Sp1 and ERK although the direct Sp1 kinase activity in these cells seems to be an ERK effector rather than ERK itself (24). Nevertheless, Sp1 can be directly phosphorylated by ERK in vitro, a modification that stimulates Sp1 DNA binding activity in gel shift assays (19). Furthermore, endogenous Sp1 and ERK coimmunoprecipitate in fibroblasts and, in these cells, Raf induction leads to direct phosphorylation by ERK of Sp1 residues Thr453 and Thr739. These modifications are required for efficient stimulation of vascular endothelial growth factor expression (20). In this study, we show that JNK signaling represses the hTR promoter and endogenous hTR levels and provide evidence for a JNK regulated transcriptional switch that enhances binding and/or activity of the transrepressor Sp3 at the hTR promoter and may inhibit Sp1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines, plasmids, and inhibitors. A2780 (ovarian adenocarcinoma) cells were used throughout. The other cell lines used were 5637 bladder carcinoma cells, C33A cervical carcinoma cells, HT29 colon carcinoma cells, A549 lung adenocarcinoma cells, and HCT116 colon carcinoma cells. Construction of both the 176 bp hTR core promoter reporter (nucleotides –107/+69) and the derived Sp1 mutant construct from pGL3-Basic (Promega Ltd., Madison, WI) has previously been reported in detail (11–13). The Sp1 site–mutated reporter was constructed by PCR mutagenesis of the wild-type promoter and carries mutations in all Sp1 sites that disrupt binding and regulation by Sp1 and Sp3 but not regulation by other key transcription factors. Plasmid pCMV-Sp3 was provided by Dr. G. Suske (Institut fur Molekularbiologie und Tumorforschung, Marburg, Germany). SP600125 was obtained from Merck Biosciences Ltd. (Nottingham, United Kingdom). SP600125 was either titrated as indicated or used at a concentration of 25 µmol/L in medium containing 0.1% DMSO per 10 µmol/L SP600125.

Transfections and luciferase assay. All transfections were done using superfect transfection reagent according to the instructions of the manufacturer (Qiagen Ltd., West Sussex, United Kingdom). A 1:2.5 ratio of DNA/superfect was used and 250 ng hTR reporter plasmid per well was transfected in 96-well luminometer plates (Fisher Scientific UK, Leicestershire, United Kingdom) together with varying amounts of pCMV empty vector or expression vectors encoding constitutively active MEKK1, wild-type or dominant-negative JNK, and Sp3. 30 ng pSV40-Renilla luciferase expression plasmid (Promega) was also cotransfected in each well for normalization of hTR promoter activity. hTR activity was also normalized to protein equivalents using the Bio-Rad assay (Bio-Rad Laboratories Ltd., Hemel Hempstead, United Kingdom). Forty-eight hours posttransfection, cells were lysed and luciferase activities were determined using Dual Luciferase Assay reagents (Promega) according to the instructions of the manufacturer. All transfections were done in quadruplicate and all experiments were repeated a minimum of thrice.

Western blotting. Protein extracts were prepared in SDS lysis buffer (10% SDS, 500 mmol/L EDTA, and 1 mol/L Tris-HCl). Protein concentrations were estimated at A595 using the Bio-Rad protein assay (Bio-Rad Laboratories). Twenty-microgram protein equivalents were separated by SDS-PAGE then blotted onto polyvinylidene difluoride filter (Millipore, Watford, United Kingdom) and blocked overnight at 4°C in PBS-T containing 5% nonfat dried milk. Filters were probed for 2 hours with 1:500 to 1:2,000 dilutions of primary antibodies and then with a 1:3,000 dilution of horseradish peroxidase (HRP)–conjugated antirabbit or antimouse secondary antibody. HRP was detected using enhanced chemiluminescence HRP detection reagents (Amersham Pharmacia, Buckinghamshire, United Kingdom). Antibodies raised against RSK1, c-Jun, c-Jun phospho-Ser73, JNK, phospho-JNK, and phospho-ERK were all obtained from Upstate Ltd. (Buckingham, United Kingdom). Antibody against RSK phospho-Ser363 was obtained from Abcam (Cambridge, United Kingdom) and antibodies against ERK, Sp1, and Sp3 were obtained from Autogen Bioclear UK Ltd. (Wiltshire, United Kingdom).

Chromatin immunoprecipitation assays. Formaldehyde cross-linking and chromatin immunoprecipitation were done as described previously (12, 15). A2780 cell cultures were treated with formaldehyde for 10 minutes followed by the addition of glycine to a final concentration of 0.125 mol/L. Cells were then washed twice with cold PBS and were resuspended in lysis buffer [1% SDS, 10 mmol/L EDTA, and 50 mmol/L Tris-HCl (pH 8.1)] containing proteinase inhibitor. DNA was sonicated to an average fragment size of 500 bp and cross-linked proteins were enriched by immunoprecipitation with Sp1 and Sp3 antibodies. A "no Ab" sample was included as a negative control for the immunoprecipitation step. After reversal of the cross-links and DNA purification, the extent of enrichment was monitored by PCR amplification of the hTR promoter using the primers detailed below. The PCR product was analyzed both by gel electrophoresis and by real-time PCR. For real-time PCR analysis, the ratio of specifically precipitated versus input chromatin was calculated, with the background (no Ab) subtracted. The input sample was processed with the rest of the samples from the point at which the cross-links were reversed.

Quantitative real-time reverse transcription-PCR. Quantitative PCR was done using GRI Opticon monitor equipment and software (Genetic Research Instrumentation, Essex, United Kingdom). Sybr green was used as the fluorophore for detection of amplified DNA. Reactions were done in triplicate. The primers 5'-CTAACCCTAACTGAGAAGGGCGTA-3' and 5'-GGCGAACGGGCCAGCAGCTGACATT-3' were used for detection of endogenous hTR and the primers 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3' were used for detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers 5'-CCCGCCCGAGAGAGTGAC-3' and 5'-AAGTCAGCGAGAAAAACAGC'3' were used for detection of the hTR promoter following chromatin immunoprecipitation experiments. Primer dimers were excluded from the quantification by performing the optical read step at 81°C.

To analyze effects of JNK inhibition on endogenous hTR levels, exponentially growing cells were treated for 16 hours with 25 µmol/L SP600125 or with DMSO followed by RNA extraction, cDNA synthesis, and quantitative PCR. The mean value of the triplicate measurements of hTR levels was normalized to the mean value of the GAPDH triplicates.

To analyze the relative levels of Sp1 and Sp3 at the hTR promoter in vivo, cells were treated for 16 hours with DMSO or 25 µmol/L SP600125 followed by chromatin immunoprecipitation and quantitative PCR. Background (no Ab) was subtracted and immunoprecipitated samples were compared with the input sample. Additionally, Sp1 immunoprecipitate levels were compared with those of Sp3. Quantitative PCR was repeated twice for each experiment, and the experiments were repeated four times.

Statistical analysis. Statistical analysis of all experiments was done by ANOVA using the Microsoft Excel data analysis tool pack. Mean values from each independent experiment were included in the analyses. Differences were statistically significant with P < 0.05 or highly significant with P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The JNK pathway represses the hTR promoter in vitro. To determine whether the JNK pathway regulates the hTR promoter in vitro, we transfected A2780 ovarian adenocarcinoma cells with a 176 bp hTR promoter-luciferase reporter construct. Our previous studies using this cell line have indicated that the basal hTR promoter activity is very strong in these cells by comparison with other cell lines and they, therefore, provide a robust model for analysis of promoter regulation (8, 9). Thirty-two hours postransfection, cells were treated for 16 hours with a titration of the JNK inhibitor SP600125. Concentrations of SP600125 over 12.5 µmol/L increased hTR promoter activity (Fig. 2A). The activity of the hTR promoter was induced by 2-fold at 12.5 µmol/L (P < 0.05) and by 2.4-fold at 50 µmol/L (P < 0.01). Therefore, despite high-level basal hTR promoter activity in these cells, manipulation of signal transduction pathways can still lead to significant promoter up-regulation. These data also suggested that JNK signaling might repress the hTR promoter.


Figure 2
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  		Figure 2. The JNK pathway represses the hTR promoter in vitro. A, a luciferase reporter driven by a 176 bp fragment of the wild-type hTR core promoter (nucleotides –107/+69) was transiently transfected into A2780 ovarian cancer cells. Thirty-two hours after transfection, the cells were exposed to a titration of the JNK inhibitor SP600125 for a further 16 hours before luciferase assay. B, the hTR core promoter was cotransfected in the cells indicated at a 1:1 ratio with either empty vector or pCMV-MEKK1, encoding a fusion protein comprising the constitutively active kinase domain of MEKK1. Luciferase activities were quantified 48 hours later. Relative luciferase activities were normalized using both an internal control vector and by measurement of luciferase activity in protein equivalents. All experiments were done in quadruplicate and repeated thrice. Columns, means derived from three independent experiments and represented as fold reporter activity relative to control; bars, SE. Results were analyzed by ANOVA (*, P < 0.05, **, P < 0.01).

 
To verify these results, expression vector encoding the constitutively active kinase domain of MEKK1, a major MAP3K for the JNK pathway, was used to test whether activation of the JNK pathway would repress hTR promoter activity. Figure 2B shows that MEKK1 led to strong repression of the hTR promoter. Using a 1:1 ratio of hTR/MEKK1 vectors, hTR promoter activity was reduced to 1.8% of basal levels in A2780 cells. We also did this experiment using five other cancer cell lines (5637 bladder carcinoma cells, C33A cervical carcinoma cells, HT29 colon carcinoma cells, HCT116 colon carcinoma cells, and A549 lung adenocarcinoma cells). MEKK1 transfection led to strong repression of the hTR promoter in all cells tested (P < 0.01). Together, these data suggested that the canonical JNK pathway might repress the hTR promoter.

To confirm that JNK is the effector of MEKK1-mediated hTR promoter repression, we examined the effect of blocking or augmenting activated MEKK1 signaling in A2780 and 5637 cells. As shown in Fig. 3A, a 10:1 ratio of hTR reporter/MEKK1 repressed hTR promoter activity to 42% of basal levels in A2780 cells. MEKK1-mediated repression was completely blocked by 25 µmol/L SP600125 (P < 0.05) and hTR promoter activity was even activated relative to basal levels in the presence of both MEKK1 and SP600125. In 5637 bladder cancer cells, a 2.5:1 ratio of hTR reporter/MEKK1 repressed hTR promoter activity to 55% of basal levels. In cells treated with both MEKK1 and 25 µmol/L SP600125, this effect was completely blocked (P < 0.05) and hTR promoter activity was strongly elevated relative to control. Thus, chemical inhibition of JNK blocks MEKK1-mediated repression of the hTR promoter.


Figure 3
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  		Figure 3. JNK activation mediates repression of the hTR promoter by MEKK1. A, a luciferase reporter driven by the wild-type hTR core promoter (nucleotides –107/+69) was transiently cotransfected into A2780 ovarian cancer cells or 5637 bladder cancer cells with empty vector or with pCMV-MEKK1 at a 10:1 ratio (reporter/MEKK1) in A2780 cells or a 2.5:1 ratio on 5637 cells. Thirty-two hours after transfection, MEKK1-transfected cells were treated with 25 µmol/L SP600125 or DMSO for a further 16 hours before luciferase assay. B, a luciferase reporter driven by the wild-type hTR core promoter was transiently cotransfected into A2780 ovarian cancer cells or 5637 bladder cancer cells with empty vector or with pCMV-MEKK1 alone, or in combination with wild-type pCMV-JNK1 or pCMV-JNK2APF at a 10:4:1 ratio (reporter/JNK/MEKK1). JNK2APF encodes an inactive JNK2 mutant in which the activating Thr-Pro-Tyr phosphoacceptor residues are mutated to Ala-Pro-Phe. Forty-eight hours posttransfection, luciferase activities were determined. Relative luciferase activities were normalized using both an internal control vector and by measurement of luciferase activity in protein equivalents. All experiments were done in quadruplicate and repeated thrice. Columns, means derived from three independent experiments and represented as fold reporter activity relative to control; bars, SE. Results were analyzed by ANOVA (*, P < 0.05).

 
We also hypothesized that repression of hTR promoter activity by MEKK1 could be enhanced by additional overexpression of JNK (Fig. 3B). A2780 or 5637 cells were transfected with a 10:1:4 ratio of hTR reporter/MEKK1/wild-type JNK1 or JNK2APF, which harbors mutated phosopho-acceptor residues and, therefore, cannot be activated by upstream MAPK kinase. In A2780 cells, MEKK1 alone reduced hTR promoter activity to 22% of basal levels. As predicted, repression of the hTR promoter by MEKK1 was further enhanced in the presence of wild-type JNK1 (10% of basal levels, P < 0.05), whereas JNK2APF was not able to enhance MEKK1-mediated repression. In 5637 cells, this dilution of MEKK1 only had a mild effect on hTR promoter activity, which was not significant (83% of basal levels). However, the addition of wild-type JNK1 enhanced repression to 62% of basal levels (P < 0.05 compared with control). Additionally, the phosphoacceptor mutant JNK2APF blocked mild repression by MEKK1 in these cells. These results confirm that JNK activation can mediate repression of the hTR promoter by MEKK1.

The JNK pathway represses endogenous hTR expression. To determine whether the JNK pathway affects the endogenous expression of hTR, exponentially growing A2780 cells, 5637 cells, or C33A cervical cancer cells were incubated with 25 µmol/L SP600125 or DMSO control. After 16-hour treatment, RNA was extracted from the cells and the levels of hTR were analyzed by quantitative PCR and compared with those of GAPDH. The results from three independent paired treatments were analyzed twice by quantitative PCR. The pooled data, shown in Fig. 4A, show that SP600125 treatment led to a 63% increase in endogenous hTR levels in A2780 cells and increases of 88% and 94% in 5637 and C33A cells, respectively. GAPDH levels were not affected by the treatment (data not shown), whereas induction of hTR in the presence of SP600125 was statistically significant as determined by ANOVA (P < 0.05 in A2780 cells, P < 0.01 in C33A and 5637 cells). These data indicate that JNK inhibition can substantially increase the steady-state hTR level in diverse cell lines even where hTR expression levels are already high (10).


Figure 4
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  		Figure 4. JNK inhibition increases endogenous levels of hTR. A, up-regulation of endogenous hTR levels. Exponentially growing A2780 cells, 5637 cells, or C33A cervical cancer cells were treated for 16 hours with 25 µmol/L SP600125 or with DMSO control. After treatment, cDNA was synthesized from RNA extracted from treated and untreated cells. Levels of endogenous hTR in control and treated samples were analyzed by quantitative PCR using hTR-specific primers and normalized to levels of GAPDH. The experiment was repeated thrice and quantitative PCR was done twice on each independent experiment. Columns, means of pooled data from all independent experiments; bars, SE. Results were analyzed by ANOVA (*, P < 0.05, **, P < 0.01). B, inhibition of JNK activity. Exponentially growing A2780 cells were treated for 16 hours with 25 µmol/L SP600125 or DMSO control. After treatment, protein extracts were made and probed for inhibition of the JNK pathway by Western blotting 20 µg extract for total and phosphorylated levels of ERK and its substrate RSK1 and for JNK and its substrate c-Jun. Experiments were repeated at least twice. Representative blots are shown.

 
To confirm that JNK activity was specifically inhibited by SP600125, exponentially growing A2780 cells were treated for 16 hours with 25 µmol/L SP600125. After treatment, protein was extracted and Western blot analysis was done for total expression and phosphorylation of ERK and the ERK-specific substrate p90-RSK1, and for JNK and the major JNK substrate c-Jun. As shown in Fig. 4B, SP600125 did not significantly affect the levels or phosphorylation of ERK, p90-RSK1, JNK, or the level of c-Jun protein; however, c-Jun Ser73 phosphorylation was thoroughly inhibited, demonstrating that SP600125 does not interfere with the activating phosphorylation of JNK but with its downstream signaling as expected. In contrast, SP600125 neither inhibited the phosphorylation of ERK or its downstream substrate RSK. Thus, under conditions of JNK inhibition, hTR expression is increased. Taken together, Figs. 2 to 4 suggest that the JNK pathway is a genuine repressor of hTR levels and that elements in the core promoter are likely to participate in JNK-mediated repression.

Repression of the hTR promoter involves Sp1 and Sp3. Several studies have indicated that MAPK pathways may regulate gene expression partly through Sp1/Sp3 binding elements (19, 20, 24, 25). To determine whether the hTR core promoter Sp1/Sp3 sites are involved in JNK-mediated repression of hTR promoter activity, we used an hTR reporter construct carrying functional mutations in all Sp1/Sp3 sites (construct no Sp1). Previous characterization determined that binding and regulation by Sp1 and Sp3 but not by NF-Y or MDM2 are specifically ablated in this construct (11, 12).

A2780 and 5637 cells were transfected in parallel with the wild-type or no-Sp1 reporters and were treated with DMSO or 25 µmol/L SP600125 (Fig. 5A). In these experiments, 25 µmol/L SP600125 resulted in 2.2-fold induction of wild-type promoter activity in A2780 cells but induction of the reporter lacking Sp1/Sp3 sites was attenuated, reaching only 1.5-fold induction (P < 0.05). In 5637 cells, SP600125 induced the wild-type hTR promoter by 1.61-fold but the mutant promoter was induced by only 1.26-fold (P < 0.05). These results suggested that part of the mechanism for induction of hTR promoter activity by JNK inhibition involves Sp1 and/or Sp3 proteins, although the incomplete attenuation indicates that other factors may also be involved. Our previous studies have shown that Sp1 activates the hTR promoter, whereas Sp3 is a repressor. We, therefore, sought to investigate whether Sp3 is involved in repression of hTR by JNK.


Figure 5
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  		Figure 5. Repression of the hTR promoter JNK involves Sp1 and Sp3. A, induction of promoter activity by SP600125 requires Sp1 binding sites. Luciferase reporters driven by either the wild-type hTR core promoter (nucleotides –107/+69) or a mutation construct in which all the Sp1 binding sites are ablated (construct no Sp1) were transiently transfected into A2780 ovarian cancer cells or 5637 bladder cancer cells. Thirty-two hours after transfection, cells were treated with 25 µmol/L SP600125 or DMSO for a further 16 hours before luciferase assay. B, MEKK1 enhances Sp3-dependent repression of the hTR promoter. Wild-type hTR core promoter reporter was transiently cotransfected into A2780 ovarian cancer cells or 5637 bladder cancer cells with vector or MEKK1 and vector or Sp3. Forty-eight hours posttransfection, luciferase activities were determined. The expected repression by MEKK1 + Sp3 assuming ideal additive effects was calculated in each experiment from the product of the fold repressions by Sp3 and MEKK1 alone. All experiments were done in quadruplicate and repeated thrice. Columns, means derived from three independent experiments and are represented as fold reporter activity relative to control; bars, SE. Results were analyzed by ANOVA (*, P < 0.05).

 
A2780 or 5637 cells were cotransfected the wild-type hTR promoter along with vector or MEKK1 and vector or Sp3 in a 5:1:2 ratio (reporter:MEKK1/vector:Sp3/vector). For each experiment, we also calculated the product of the repression mediated by MEKK1 and Sp3 individually as a representation of the expected promoter activity assuming additive effects between MEKK1 and Sp3. As shown in Fig. 5B, MEKK1 alone repressed the hTR to 30% of basal levels in A2780 cells, whereas Sp3 alone repressed the hTR to 68% of basal levels. The calculated expected promoter activity (exp column), assuming only additive effects between Sp3 and MEKK1, was 18% of basal levels. However, cotransfection of both MEKK1 and Sp3 repressed hTR promoter activity to 9% of basal levels (P < 0.05).

In 5637 cells, MEKK1 transfection alone resulted in 50% reduction of hTR promoter activity, whereas Sp3 alone decreased promoter activity to 55% of basal levels. Across all experiments, the expected promoter activity assuming perfect additive effects between MEKK1 and Sp3 (Fig. 5B, exp column), would have been 27% of basal levels. Instead, in the presence of both MEKK1 and Sp3, hTR was reduced to only 11.5% of basal levels (P < 0.05). Therefore, in both 5637 and A2780 cells, Sp3 and MEKK1 seem to act cooperatively to repress the hTR promoter.

JNK regulates Sp1/Sp3 binding at the hTR promoter in vivo. Several studies have suggested that regulated Sp1/Sp3-dependent promoter activation may be mediated by a change in the ratio of Sp1 and Sp3 expression (27–29). To determine whether our results reflect altered expression of Sp1 or Sp3 (e.g., repression of Sp1 expression by JNK), we did Western blots for total cellular levels of Sp1 and Sp3 in control cells or cells treated for 16 hours with SP600125. As shown in Fig. 6A, 16-hour treatment with 25 µmol/L SP600125 had no significant effect on the levels of Sp1 or Sp3 protein. Therefore, any JNK-dependent regulation of Sp1 or Sp3 is likely to be mediated through a posttranslational mechanism.


Figure 6
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  		Figure 6. The JNK pathway modulates Sp1 and Sp3 binding to the hTR promoter in vivo. A, cellular levels of Sp1 and Sp3 are not affected by SP600125. Exponentially growing A2780 cells were treated for 16 hours with 25 µmol/L SP600125 or DMSO control. After treatment, protein extracts were made and probed for total cellular levels of Sp1 and Sp3 protein. Sp1 p95 was the main product; Sp3 p100 (full-length) and p60 (alternative translation initiation site) isoforms were detected. The experiment was repeated twice and representative blots are shown. B and C, SP600125 increases detection of Sp1 in chromatin immunoprecipitates. Exponentially growing A2780 cells were treated either with DMSO or with 25 µmol/L SP600125 for 16 hours. After the treatment periods, proteins were cross-linked to DNA using formaldehyde and chromatin was immunoprecipitated using antibodies specific for Sp1 or Sp3. The presence of the hTR promoter in precipitated chromatin was monitored by conventional PCR (B) and quantitative PCR (C) using primers specific for the hTR promoter. One representative experiment. Input DNA samples were diluted 1:10. D, increased ratio of immunoreactive Sp1 relative to Sp3 detected at the hTR promoter in vivo after SP600125 treatment. After subtraction of background, Sp1 and Sp3 levels were compared with the total amount of Sp1 + Sp3 detected in chromatin immunoprecipitation. Chromatin immunoprecipitation experiments were repeated four times and each experiment was analyzed twice by quantitative PCR. Pooled data for all experiments. The relative shifts in Sp1 or Sp3 levels were analyzed by ANOVA (*, P < 0.05).

 
To investigate the hypothesis that the JNK pathway enhances repression of the hTR promoter by Sp3 and/or constrains Sp1-mediated activation in these cells, we did chromatin immunoprecipitation experiments, pulling down either Sp1 or Sp3 from control cells or from cells treated for 16 hours with 25 µmol/L SP600125. The presence of the hTR promoter in chromatin immunoprecipitates was assessed using both conventional and quantitative PCR.

Sp1-specific products seemed weak compared with the Sp3-specific products in control A2780 cells (Fig. 6B and C). This may simply reflect the different affinities of the two antibodies for their targets or it may indicate that Sp3 occupancy of the promoter is greater than Sp1. More importantly, after 16-hour treatment with SP600125, the intensity of the Sp1-specific product had increased, whereas that of Sp3 was reduced. As shown in Fig. 6D, pooled data from all experiments showed that Sp1 represented 33% of the total Sp1/Sp3 associated with the hTR promoter in control cells but increased to 55% in JNK-inhibited cells (P < 0.05). In contrast, Sp3 constituted 67% of the total Sp1/Sp3 protein pulled down in control cells but the proportion fell to 45% in JNK-inhibited cells (P < 0.05). Thus, JNK inhibition results in a shift in the ratio of immunoreactive Sp1 and Sp3 at the hTR promoter in vivo, favoring an increase in the levels of the Sp1 epitope.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Significant interest in recent years has focused on hTERT gene regulation as a mechanism controlling telomerase in cancer cells. However, recent findings in mice and humans have indicated that control of hTR levels may also contribute to overall regulation of telomerase. In the mTR knockout mouse, telomerase activity is abrogated resulting in telomere shortening and late-generation phenotypic abnormalities in multiple organs (30, 31). Interestingly, and, more recently, it has been shown that haploinsufficiency of mTR in mTR+/– heterozygotes results in defects of telomere elongation independent of telomerase activity detected by telomere repeat amplification protocol assay (32). In humans, hTR haploinsufficiency has been documented in autosomal dominant dyskeratosis patients (33). Indeed, several mutations that have the effect of reducing the stability of hTR are documented in autosomal dominant dyskeratosis (34).

Together, these data indicate that hTR levels are essential for telomerase holoenzyme function in vivo, although sparse data exist regarding signal transduction pathways affecting hTR expression. In situ hybridization analysis of 800 tumor biopsy samples show clearly that hTR levels are up-regulated specifically in cancer cells (35–40). It is, therefore, critical to begin to define pathways affecting hTR.

In this report, we provide evidence that JNK represses the hTR core promoter in transfection assays and also represses endogenous hTR levels partly via regulation of Sp1 and Sp3 at the hTR promoter. We found that the JNK inhibitor SP600125 up-regulated activity of a transiently transfected hTR core promoter reporter in a dose-dependent manner and also increased endogenous levels of hTR at 25 µmol/L. At this concentration, SP600125 also abrogated phosphorylation of c-Jun but not of RSK1, a major ERK specific effector, suggesting that the JNK pathway was indeed specifically inhibited at SP600125 concentrations leading to elevation of endogenous hTR.

Conversely, overexpression of active MEKK1, a major MAP3K for the JNK pathway, strongly repressed promoter activity. Further, repression by MEKK1 could be completely blocked by 25 µmol/L SP600125 or enhanced by coexpression of wild-type JNK1 but not the phosphoacceptor mutant JNK2APF. Together, these data suggest strongly that JNK is a major effector of MEKK1-mediated repression of the hTR promoter and that elements in the core promoter may be involved in induction of hTR following JNK inhibition.

We next assessed whether repression by the JNK pathway could be attributed to Sp1/Sp3-dependent mechanisms. Induction of the core promoter by SP600125 was partly dependent on intact, functional Sp1 binding sites, indicating that Sp1/Sp3 could indeed have a role in integrating JNK signaling at the hTR core promoter. Overexpression of MEKK1 enhanced repression by Sp3, suggesting that MEKK1 might selectively favor binding or activity of Sp3 at the hTR promoter.

Finally, we showed by chromatin immunoprecipitation that SP600125 treatment increases detection of Sp1 at the hTR promoter in vivo with a concomitant relative decrease in detectable Sp3. These observations are consistent with a model in which the JNK pathway enhances binding and/or transrepression by Sp3 and suppresses Sp1-dependent transcription of hTR. It is important to note that altered Sp1/Sp3 immunoreactivity does not necessarily reflect overall levels of Sp1/Sp3 binding. It may instead reflect other changes within promoter bound complexes that unmask the Sp1 epitope. However, in light of our other findings reported here, there remains a strong possibility that Sp1 epitope unmasking in this context might reflect relief from inhibition. In context of our finding that MEKK1 cooperates with Sp3 to repress the hTR promoter, and based on our previous analyses of positive hTR promoter regulation by Sp1, these observations provide initial evidence of a transcriptional switching mechanism underlying hTR repression by the MEKK1/JNK pathway. In totality, our data argue for a model in which the JNK pathway cooperates with Sp3 to repress the hTR promoter in vitro and in vivo.

The mechanism underlying the apparent differential regulation of Sp1 and Sp3 at this promoter is unclear at present. JNK signaling may directly modify Sp3, specifically enhancing its DNA binding activity and allowing it to compete more effectively with Sp1 for the same binding sites. Alternatively, JNK signaling may directly inhibit Sp1 in this instance, which would also be expected to enhance the activity of Sp3. It should be noted, however, that JNK signaling has generally been reported to result in activation of Sp1 DNA binding and transactivation (25, 41). It is also conceivable, however, that the overall architecture of individual promoters and/or expression of cell-specific factors influence the outcome of JNK activation at individual genes with respect to Sp1/Sp3. In this cell line, hTR promoter–bound Sp1 may simply be inaccessible for JNK or its effectors, leading to preferential signaling to Sp3.

Because both Sp1 and Sp3 contain potential docking sequences for JNK in the first zinc finger and the distribution of minimal consensus MAPK phosphoacceptor residues relative to the functional domains of Sp1 and Sp3 is also broadly similar, such a mechanism seems most likely. However, Sp3 lacks an equivalent residue to the Sp1 ERK phosphoacceptor Thr739. Given that the requisite docking domains for ERK and JNK reside in the zinc fingers and are, therefore, likely to be proximal in the tertiary structure of Sp1, it is possible that JNK binding to its putative domain might mask the ERK docking site, thereby inhibiting phosphorylation on Thr739 and, thus, DNA binding. Such a mechanism would not be expected to affect Sp3 due to the absence of an equivalent residue, although it is uncertain whether a JNK active site inhibitor could unmask such an effect.

In conclusion, this report provides the first evidence that Sp1 and Sp3 may act to integrate stress and/or growth signals at the hTR promoter. It will be of particular interest to investigate whether JNK also represses hTR in cells with low hTR levels, such as normal cell strains, and, if so, what its relative contribution to hTR repression is. From a therapeutic perspective, improved understanding of the signal transduction pathways that regulate the hTR gene promoter should allow for the development of lead drug candidates targeting pathways involved in hTR transcription. Several clinically relevant cytotoxic drugs in widespread use, including cisplatin, are known to activate the JNK pathway as part of their mechanism of action (42). Interestingly, some ovarian cancer cells acquire drug resistance through down-regulation of DNA damage pathways while retaining competent but compromised JNK activation (43, 44). It will also be of interest to determine whether long-term subtoxic schedules of such drugs might have a beneficial effect resulting from inhibition of telomerase even in cells that have acquired resistance to apoptosis.


   Acknowledgments
 
Grant support: Scottish Enterprise, Cancer Research UK, and Glasgow University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/ 3/05. Revised 10/14/05. Accepted 11/17/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Autexier C, Pruzan R, Funk WD, Greider CW. Reconstitution of human telomerase activity and identification of a minimal functional region of the human telomerase RNA. EMBO J 1996;15:5928–35.[Medline]
  2. Bachand F, Autexier C. Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions. Mol Cell Biol 2001;21:1888–97.[Abstract/Free Full Text]
  3. Nicol Keith W, Jeffry Evans TR, Glasspool RM. Telomerase and cancer: time to move from a promising target to a clinical reality. J Pathol 2001;195:404–14.[CrossRef][Medline]
  4. Keith WN, Bilsland A, Evans TR, Glasspool RM. Telomerase-directed molecular therapeutics. Expert Rev Mol Med 2002;2002:1–25.[Medline]
  5. Lavelle F, Riou JF, Laoui A, Mailliet P. Telomerase: a therapeutic target for the third millennium? Crit Rev Oncol Hematol 2000;34:111–26.[Medline]
  6. White LK, Wright WE, Shay JW. Telomerase inhibitors. Trends Biotechnol 2001;19:114–20.[CrossRef][Medline]
  7. Keith WN, Bilsland A, Hardie M, Evans TRJ. Cancer cell immortality—telomerase as a target for novel cancer gene therapies. Nat Clin Pract Oncol 2004;1:88–96.[CrossRef][Medline]
  8. Bilsland AE, Anderson CJ, Fletcher-Monaghan AJ, et al. Selective ablation of human cancer cells by telomerase-specific adenoviral suicide gene therapy vectors expressing bacterial nitroreductase. Oncogene 2003;22:370–80.[CrossRef][Medline]
  9. Plumb JA, Bilsland A, Kakani R, et al. Telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954. Oncogene 2001;20:7797–803.[CrossRef][Medline]
  10. Hoare SF, Bryce LA, Wisman GB, et al. Lack of telomerase RNA gene hTERC expression in alternative lengthening of telomeres cells is associated with methylation of the hTERC promoter. Cancer Res 2001;61:27–32.[Abstract/Free Full Text]
  11. Zhao JQ, Glasspool RM, Hoare SF, Bilsland A, Szatmari I, Keith WN. Activation of telomerase RNA gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3. Neoplasia 2000;2:531–9.[CrossRef][Medline]
  12. Zhao J, Bilsland A, Hoare SF, Keith WN. Involvement of NF-Y and Sp1 binding sequences in basal transcription of the human telomerase RNA gene. FEBS Lett 2003;536:111–9.[CrossRef][Medline]
  13. Zhao JQ, Hoare SF, McFarlane R, et al. Cloning and characterization of human and mouse telomerase RNA gene promoter sequences. Oncogene 1998;16:1345–50.[CrossRef][Medline]
  14. Keith WN, Vulliamy T, Zhao J, et al. A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC) promoter in a patient with paroxysmal nocturnal haemoglobinuria. BMC Blood Disord 2004;4:3.[CrossRef][Medline]
  15. Zhao J, Bilsland A, Jackson K, Keith WN. MDM2 negatively regulates the human telomerase RNA gene promoter. BMC Cancer 2005;5:6.[CrossRef][Medline]
  16. Black AR, Jensen D, Lin SY, Azizkhan JC. Growth/cell cycle regulation of Sp1 phosphorylation. J Biol Chem 1999;274:1207–15.[Abstract/Free Full Text]
  17. Armstrong SA, Barry DA, Leggett RW, Mueller CR. Casein kinase II-mediated phosphorylation of the C terminus of Sp1 decreases its DNA binding activity. J Biol Chem 1997;272:13489–95.[Abstract/Free Full Text]
  18. Daniel S, Zhang S, DePaoli-Roach AA, Kim KH. Dephosphorylation of Sp1 by protein phosphatase 1 is involved in the glucose-mediated activation of the acetyl-CoA carboxylase gene. J Biol Chem 1996;271:14692–7.[Abstract/Free Full Text]
  19. Merchant JL, Du M, Todisco A. Sp1 phosphorylation by Erk 2 stimulates DNA binding. Biochem Biophys Res Commun 1999;254:454–61.[CrossRef][Medline]
  20. Milanini-Mongiat J, Pouyssegur J, Pages G. Identification of two Sp1 phosphorylation sites for p42/p44 mitogen-activated protein kinases: their implication in vascular endothelial growth factor gene transcription. J Biol Chem 2002;277:20631–9.[Abstract/Free Full Text]
  21. Pal S, Claffey KP, Cohen HT, Mukhopadhyay D. Activation of Sp1-mediated vascular permeability factor/vascular endothelial growth factor transcription requires specific interaction with protein kinase C{zeta}. J Biol Chem 1998;273:26277–80.[Abstract/Free Full Text]
  22. De Siervi A, Marinissen M, Diggs J, Wang XF, Pages G, Senderowicz A. Transcriptional activation of p21(waf1/cip1) by alkylphospholipids: role of the mitogen-activated protein kinase pathway in the transactivation of the human p21(waf1/cip1) promoter by Sp1. Cancer Res 2004;64:743–50.[Abstract/Free Full Text]
  23. Jackson SP, MacDonald JJ, Lees-Miller S, Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell 1990;63:155–65.[CrossRef][Medline]
  24. Chupreta S, Du M, Todisco A, Merchant JL. EGF stimulates gastrin promoter through activation of Sp1 kinase activity. Am J Physiol Cell Physiol 2000;278:C697–708.[Abstract/Free Full Text]
  25. Benasciutti E, Pages G, Kenzior O, Folk W, Blasi F, Crippa MP. MAPK and JNK transduction pathways can phosphorylate Sp1 to activate the uPA minimal promoter element and endogenous gene transcription. Blood 2004;104:256–62.[Abstract/Free Full Text]
  26. Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997;9:180–6.[CrossRef][Medline]
  27. Discher DJ, Bishopric NH, Wu X, Peterson CA, Webster KA. Hypoxia regulates ß-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element. J Biol Chem 1998;273:26087–93.[Abstract/Free Full Text]
  28. Krikun G, Schatz F, Mackman N, Guller S, Demopoulos R, Lockwood CJ. Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3. Mol Endocrinol 2000;14:393–400.[Abstract/Free Full Text]
  29. Xu Q, Ji YS, Schmedtje JF, Jr. Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium. Implications for the mechanisms of aortic aneurysm and heart failure. J Biol Chem 2000;275:24583–9.[Abstract/Free Full Text]
  30. Lee HW, Blasco MA, Gottlieb GJ, Horner JW II, Greider CW, DePinho RA. Essential role of mouse telomerase in highly proliferative organs. Nature 1998;392:569–74.[CrossRef][Medline]
  31. Blasco MA, Lee HW, Hande MP, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 1997;91:25–34.[CrossRef][Medline]
  32. Hathcock KS, Hemann MT, Opperman KK, Strong MA, Greider CW, Hodes RJ. Haploinsufficiency of mTR results in defects in telomere elongation. Proc Natl Acad Sci U S A 2002;99:3591–6.[Abstract/Free Full Text]
  33. Vulliamy T, Marrone A, Goldman F, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature 2001;413:432–5.[CrossRef][Medline]
  34. Marrone A, Stevens D, Vulliamy T, Dokal I, Mason PJ. Heterozygous telomerase RNA mutations found in dyskeratosis congenita and aplastic anemia reduce telomerase activity via haploinsufficiency. Blood 2004;104:3936–42.[Abstract/Free Full Text]
  35. Keith WN, Sarvesvaran J, Downey M. Analysis of telomerase RNA gene expression by in situ hybridization. Methods Mol Biol 2002;191:65–81.[Medline]
  36. Downey MG, Going JJ, Stuart RC, Keith WN. Expression of telomerase RNA in oesophageal and oral cancer. J Oral Pathol Med 2001;30:577–81.[CrossRef][Medline]
  37. Wisman GB, De Jong S, Meersma GJ, et al. Telomerase in (pre)neoplastic cervical disease. Hum Pathol 2000;31:1304–12.[CrossRef][Medline]
  38. Sarvesvaran J, Going JJ, Milroy R, Kaye SB, Keith WN. Is small cell lung cancer the perfect target for anti-telomerase treatment? Carcinogenesis 1999;20:1649–51.[Abstract/Free Full Text]
  39. Soder AI, Going JJ, Kaye SB, Keith WN. Tumour specific regulation of telomerase RNA gene expression visualized by in situ hybridization. Oncogene 1998;16:979–83.[CrossRef][Medline]
  40. Soder AI, Hoare SF, Muir S, Going JJ, Parkinson EK, Keith WN. Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer. Oncogene 1997;14:1013–21.[CrossRef][Medline]
  41. Higuchi H, Grambihler A, Canbay A, Bronk SF, Gores GJ. Bile acids up-regulate death receptor 5/TRAIL-receptor 2 expression via a c-Jun N-terminal kinase-dependent pathway involving Sp1. J Biol Chem 2004;279:51–60.[Abstract/Free Full Text]
  42. Sanchez-Perez I, Murguia JR, Perona R. Cisplatin induces a persistent activation of JNK that is related to cell death. Oncogene 1998;16:533–40.[CrossRef][Medline]
  43. Brown R, Hirst GL, Gallagher WM, et al. hMLH1 expression and cellular responses of ovarian tumour cells to treatment with cytotoxic anticancer agents. Oncogene 1997;15:45–52.[CrossRef][Medline]
  44. Cui W, Yazlovitskaya EM, Mayo MS, Pelling JC, Persons DL. Cisplatin-induced response of c-jun N-terminal kinase 1 and extracellular signal-regulated protein kinases 1 and 2 in a series of cisplatin-resistant ovarian carcinoma cell lines. Mol Carcinog 2000;29:219–28.[CrossRef][Medline]



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