Regulation of Cyclooxgenase-2 mRNA Stability by Taxanes

Taxanes are widely used to treat malignancies and are known to modulate the transcription of several genes. We investigated the effects of taxanes (docetaxel and paclitaxel) on cyclooxygenase-2 (COX-2) transcription and mRNA stability in human mammary epithelial cells. As reported previously for paclitaxel, docetaxel stimulated COX-2 transcription by an AP-1-dependent mechanism. Treatment with taxanes also enhanced the stability of COX-2 mRNA. To define the mechanism by which taxanes stabilized COX-2 mRNA, transient transfections were carried out using luciferase expression constructs containing the COX-2 3′-untranslated region (3′-untranslated region (UTR)). The stabilizing effects of taxanes were localized to the AU-rich region of COX-2 3′-UTR. RNA binding studies indicated that taxanes stimulated the binding of HuR to the AU-rich region of the COX-2 3′-UTR. Overexpression of antisense HuR suppressed taxane-mediated induction of COX-2 3′-UTR activity. We next investigated the signal transduction pathway responsible for taxane-mediated induction of COX-2. Taxanes enhanced protein kinase C activity; overexpressing dominant negative PKC-α suppressed taxane-mediated stimulation of both COX-2 3′-UTR and 5′-promoter activities. Interestingly, ERK1/2, JNK, and p38 MAPKs were important for taxane-mediated activation of COX-2 transcription, but only p38 MAPK appeared to be responsible for the increase in COX-2 mRNA stability. MAPKAPK-2, a known target of p38 MAPK, contributed to increased COX-2 mRNA stability following taxane treatment. SB 202190, a selective p38 MAPK inhibitor, and dexamethasone suppressed taxane-mediated stimulation of the COX-2 3′-UTR and binding of HuR. Taken together, these data indicate that taxanes induce COX-2 by stimulating both transcription and mRNA stability. To the best of our knowledge, this is the first evidence that taxanes can promote stabilization of mRNA in addition to modulating gene transcription.

Taxanes are widely used for the treatment of cancer. The anti-cancer properties of taxanes are due at least in part to interference with microtubule assembly, impairment of mitosis, and changes in the cytoskeleton (41). There is growing evidence, however, that taxanes have multiple cellular effects. For example, taxanes stimulate mitogen-activated protein kinases (MAPKs) and the expression of numerous genes including COX-2 (38,42,43). However, there is limited knowledge about the effects of taxanes on the molecular mechanisms regulating immediate-early gene expression.
The concentration of mRNA is determined by both rates of synthesis and degradation (44). Although great emphasis has been placed on defining the mechanisms that control COX-2 transcription (13), there is growing evidence that post-transcriptional mechanisms are also important (45,46). It has become clear, for example, that an AU-rich element (ARE) within the 3Ј-untranslated region (3Ј-UTR) of COX-2 mRNA can affect both mRNA stability and protein translation (45)(46)(47). In this study, we investigated whether taxanes (docetaxel and paclitaxel) induced COX-2 via effects on both message stability and transcription. Evidence is presented that taxanes stimulated COX-2 transcription by enhancing the binding of AP-1 to the cyclic AMP-response element (CRE) of the COX-2 promoter. Additionally, taxanes stabilized COX-2 message by enhancing the binding of the mRNA stabilization factor HuR to the ARE of the COX-2 3Ј-UTR. Remarkably, different MAPK signaling pathways were important for mediating the effects of taxanes on COX-2 transcription versus message stability. In addition to modulating transcription, these findings provide the first evidence that taxanes can alter post-transcriptional mechanisms.
Western Blotting-Cell lysates were prepared by treating cells with lysis buffer (150 mM NaCl, 100 mM Tris (pH 8.0), 1% Tween 20, 50 mM diethyl dithiocarbamate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml trypsin inhibitor, and 10 g/ml leupeptin). Lysates were sonicated for 20 s on ice and centrifuged at 10,000 ϫ g for 10 min to sediment the particulate material. The protein concentration of the supernatant was measured by the method of Lowry et al. (49). SDS-PAGE was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (50). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (51). The nitrocellulose membrane was then incubated with primary antisera. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions.
Northern Blotting-Total cellular RNA was isolated from cell monolayers using an RNA isolation kit from Qiagen Inc. (Valencia, CA). 10 g of total cellular RNA per lane were electrophoresed in a formaldehyde-containing 1.2% agarose gel and transferred to nylon-supported membranes. After baking, membranes were prehybridized overnight in a solution containing 50% formamide, 5ϫ sodium chloride/sodium phosphate/EDTA buffer (SSPE), 5ϫ Denhardt's solution, 0.1% SDS, and 100 g/ml single-stranded salmon sperm DNA and then hybridized for 12 h at 42°C with radiolabeled cDNA probes. After hybridization, membranes were washed twice for 20 min at room temperature in 2ϫ SSPE, 0.1% SDS, twice for 20 min in the same solution at 55°C, and twice for 20 min in 0.1ϫ SSPE, 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography. COX-2 (4.3 kb), luciferase (1.6 kb), and 18 S rRNA (1.6 kb) probes were labeled with [ 32 P]CTP by random priming.
Analysis of Protein-RNA Interactions-The COX-2 ARE sequence cloned into pBlueScript (KSϩ) (45) was used in in vitro transcription reactions incorporating [ 32 P]UTP (50 Ci) in sense RNAs for COX-2. Unlabeled competitor RNAs were made using the Ribomax kit (Pro-mega). Cytoplasmic cell lysates were prepared as described previously (45). Cells were grown in T150 tissue culture dishes and washed twice with phosphate-buffered saline before 4 ml of lysis buffer (25 mM Tris-HCl (pH 7.5), 0.5% Nonidet P-40) was added. Thawed cells were scraped from the plate, vortexed briefly, and centrifuged at 14,000 ϫ g for 10 min. The supernatant was assayed for protein concentration using a Lowry protein assay with bovine serum albumin as standard and used immediately or snap-frozen at Ϫ70°C. For native gel mobility shift assay, 5 g of cytoplasmic lysate was incubated with radiolabeled RNA in binding buffer (20 mM HEPES (pH 7.5), 3 mM MgCl 2 , 40 mM KCl, 1 mM dithiothreitol, 5% glycerol) in a total volume of 20 l. The mixture was then incubated for 15 min at room temperature prior to the addition of heparin (5 mg/ml). Following incubation for an additional 20 min, samples were electrophoresed in 4% polyacrylamide gels (60:1 acrylamide/bisacrylamide) in 0.5ϫ TBE (Tris borate/EDTA) buffer containing 5% glycerol. The gel was dried and exposed overnight with Kodak Bio-Max MS film and an intensifying screen.
Nuclear Run-off Assay-2.5 ϫ 10 5 cells were plated in four T150 dishes for each condition. Cells were grown in growth medium until ϳ60% confluent. Nuclei were isolated and stored in liquid nitrogen. For the transcription assay, nuclei (1.0 ϫ 10 7 ) were thawed and incubated in reaction buffer (10 mM Tris (pH 8), 5 mM MgCl 2 , and 0.3 M KCl) containing 100 Ci of [ 32 P]UTP and 1 mM unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. The human COX-2 and 18 S rRNA cDNAs were immobilized onto nitrocellulose and prehybridized overnight in hybridization buffer. Hybridization was carried out at 42°C for 24 h using equal cpm/ml labeled nascent RNA transcripts for each treatment group. The membranes were washed twice with 2ϫ SSC buffer for 1 h at 55°C and then treated with 10 mg/ml RNase A in 2ϫ SSC at 37°C for 30 min, dried, and autoradiographed.
Transient Transfection Assays-Cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes and grown to 50 -60% confluence. For each well, 2 g of plasmid DNA were introduced into cells using 8 g of LipofectAMINE as per manufacturer's instructions. After 7 h of incubation, the medium was replaced with basal medium. Reporter activities were measured at the indicated times.
Protein Kinase C Assay-The activity of PKC was measured according to directions from Invitrogen. Briefly, cells were plated in 10-cm dishes at 10 6 cells/dish and grown to 60% confluence. Cells were then treated with fresh basal medium containing vehicle (0.2% Me 2 SO) or taxane (0 -10 M) for 6 h. Total PKC activity was measured in cell lysates as per the manufacturer's instructions. Briefly, PKC activity was measured by incubating partially purified PKC with [ 32 P]ATP (3000 -6000 Ci/mmol) and the substrate myelin basic protein for 20 min at room temperature. The activity of PKC is expressed as counts/min incorporated per g of protein.
Statistics-Comparisons between groups were made by the Student's t test. A difference between groups of p Ͻ 0.05 was considered significant.

Docetaxel
Stimulates the Transcription of COX-2-Treatment of the mammary epithelial cell line 184B5/HER with docetaxel caused dose-dependent induction of PGE 2 biosynthesis (Fig. 1A). 10 M docetaxel led to 6.3-fold induction of PGE 2 . Western blotting was carried out to determine whether the differences in PGE 2 production correlated with amounts of COX-2. Consistent with the observed increase in PG biosynthesis, docetaxel was a potent inducer of COX-2 protein (Fig.  1B). COX-1 was not detectable by immunoblotting in this cell line (data not shown). To elucidate further the mechanism responsible for the changes in amounts of COX-2 protein, we examined steady-state levels of COX-2 mRNA by Northern blotting. As shown in Fig. 1C, higher levels of COX-2 mRNA were also detected following treatment with docetaxel. To investigate whether docetaxel regulated the transcription of COX-2, nuclear run-offs were performed. Higher rates of synthesis of nascent COX-2 mRNA were observed after treatment with docetaxel (Fig. 1D).
We next were interested in identifying the region of the COX-2 promoter that was important for mediating the inductive effects of docetaxel. Transient transfections were performed with a series of human COX-2 5Ј-promoter-deletion constructs ( Fig. 2A). In agreement with the results shown in Fig. 1D, treatment of cells with docetaxel led to a doubling of COX-2 promoter activity when a Ϫ1432/ϩ59 COX-2 promoter construct was utilized (Fig. 2B). A stepwise decrease in basal COX-2 promoter activity was observed when shorter constructs were used. However, the magnitude of induction by docetaxel remained essentially constant with all promoter deletion constructs except the Ϫ52/ϩ59 construct (Fig. 2B). The Ϫ52/ϩ59 COX-2 promoter construct was not stimulated by docetaxel. This result implies that one or more promoter elements lying between Ϫ53 and Ϫ123 are necessary for docetaxel-mediated induction of COX-2. A CRE is present between nucleotides Ϫ59 and Ϫ53 raising the possibility that this element could be involved in mediating the inductive effects of docetaxel. To test this notion, transient transfections were performed utilizing COX-2 promoter constructs in which specific known enhancer elements including the CRE were mutagenized. As shown in Fig. 2C, mutagenizing the CRE site abrogated docetaxel-mediated stimulation of COX-2 promoter activity. By contrast, mutagenizing the NFB or NF-IL6 sites had no effect on COX-2 promoter function.
Electrophoretic mobility shift assays were performed to identify the transcription factor that contributed to docetaxel-mediated induction of COX-2. Increased binding of nuclear proteins to the CRE site of the COX-2 promoter was detected (Fig.  3, A and B). In contrast, docetaxel did not increase binding to either the NF-IL6 or NF-B sites of the COX-2 promoter (Fig.  3A). The increase in binding to the COX-2 CRE was competed by incubating nuclear extract from docetaxel-treated cells with an excess of CRE or AP-1 cold probes (data not shown) but not NF-B, NF-IL6, PEA3 or mutant CRE cold probes (Fig. 3C). Supershift analysis identified c-Jun, c-Fos, and ATF-2 in the binding complex (Fig. 3, D and E). A supershift was not observed with antibodies to NF-B p65, NF-IL6, or PEA3 (Fig.  3D). These findings are consistent with prior evidence that microtubule interfering agents including paclitaxel stimulate AP-1-mediated activation of COX-2 transcription via the CRE (38).
Taxanes Stabilize COX-2 mRNA-We also investigated whether docetaxel or paclitaxel stabilized COX-2 mRNA. Cells were treated with vehicle or taxane for 3 h to induce COX-2 transcription, and then transcription was stopped with the addition of actinomycin D. RNA was isolated at different time points after treatment with actinomycin D and subjected to Northern blot analysis (Fig. 4, A and B). As shown in Fig. 4C, treatment with either docetaxel or paclitaxel caused a significant increase in the half-life of COX-2 mRNA.
Recently, an ARE was identified in the proximal end of the 3Ј-UTR of COX-2 mRNA (45). Because the ARE is important for modulating COX-2 mRNA stability, experiments were carried out to determine whether it mediated the stabilizing effects of docetaxel. Transient transfections were carried out with expression constructs containing luciferase ligated to the fulllength COX-2 3Ј-UTR, the ARE region, or the ARE deleted from the full-length 3Ј-UTR (Fig. 5A). Treatment with docetaxel caused about a doubling of luciferase activity when either the full-length COX-2 3Ј-UTR or the ARE containing construct were used (Fig. 5B). In contrast, docetaxel did not stimulate luciferase activity when the ARE was deleted from the fulllength COX-2 3Ј-UTR. Corresponding changes in the amounts of luciferase mRNA were detected (Fig. 5C). Similar results were obtained with paclitaxel (data not shown). Taken together, these results suggest that the ARE of the COX-2 3Ј-UTR is responsible for mediating the increase in COX-2 message stability following treatment with taxane.
Post-transcriptional regulation mediated by AREs is facili-tated by trans-acting factors that bind to the ARE. Hence, we investigated whether treatment with taxanes altered binding of cytosolic RNA-binding proteins to the COX-2 ARE. To evaluate this, we incubated cytoplasmic lysates with in vitro transcribed 32 P-labeled RNA containing the COX-2 ARE. As shown in Fig. 5D, treatment with docetaxel caused a marked increase in binding of cytoplasmic factors to the COX-2 ARE. Importantly, the docetaxel-mediated increase in binding to the COX-2 ARE was competed by an excess of cold ARE but not by mutant ARE or other regions of the 3Ј-UTR (Fig. 5E). In addi- Localization of region of COX-2 promoter that mediates the effects of taxotere. A, shown is a schematic of the human COX-2 promoter. B, 184B5/HER cells were transfected with 1.8 g of a series of human COX-2 promoter deletion constructs ligated to luciferase (Ϫ1432/ϩ59, Ϫ327/ϩ59, Ϫ220/ϩ59, Ϫ124/ϩ59, and Ϫ52/ϩ59) and 0.2 g pSV␤gal. C, cells were transfected with 1.8 g of a series of human COX-2 promoter-luciferase constructs (Ϫ327/ϩ59; KBM; ILM; CRM) and 0.2 g pSV␤gal. KBM represents the Ϫ327/ϩ59 COX-2 promoter construct in which the NFB site was mutagenized; ILM represents the Ϫ327/ϩ59 COX-2 promoter construct in which the NF-IL6 site was mutagenized; CRM refers to the Ϫ327/ϩ59 COX-2 promoter construct in which the CRE was mutagenized. After transfection, cells were treated with vehicle or 10 M docetaxel. Reporter activities were measured in cellular extract 7 h later. Luciferase activity represents data that have been normalized with ␤-galactosidase. Columns, means; bars, S.D.; n ϭ 6. tion, docetaxel treatment did not augment binding to either mutant ARE or to other regions of the 3Ј-UTR (Fig. 5E), thereby confirming the specificity of the protein binding to the COX-2 ARE. We next attempted to identify the factors responsible for increased COX-2 ARE binding following treatment of cells with docetaxel. To accomplish this, supershift assays were performed, and the ARE-binding protein HuR was identified as the major protein in the binding complex that was induced by treatment with docetaxel (Fig. 6A). In contrast, normal IgG did not cause a supershift (Fig. 6B). To confirm the functional importance of HuR for mediating the stabilizing effects of docetaxel, additional experiments were performed. Overexpressing antisense HuR resulted in reduced levels of HuR (Fig. 6C) and inhibited docetaxel-mediated stimulation of the COX-2 3Ј-UTR (Fig. 6D). Corresponding changes in amounts of luciferase mRNA were detected (Fig. 6E). Comparable results were obtained with paclitaxel (data not shown).
Defining the Signaling Mechanism by Which Docetaxel Induces COX-2-Activation of PKC is known to induce COX-2 (13). Hence, it was of interest to determine whether treatment with docetaxel or paclitaxel enhanced PKC activity. Treatment with either taxane induced PKC activity. As shown in Fig. 7A, docetaxel caused a dose-dependent increase in PKC activity. Transient transfections were performed to determine whether taxane-mediated stimulation of PKC activity contributed to the induction of COX-2. Overexpressing a dominant negative form of PKC-␣ abrogated the activation of both the COX-2 5Ј-UTR and the COX-2 3Ј-UTR by docetaxel (Fig. 7B). Changes in amounts of luciferase mRNA corresponded to the effects on the COX-2 3Ј-UTR (Fig. 7C). Similar results were obtained when paclitaxel was used (data not shown). Previous studies have demonstrated that MAPKs can be important for regulating both COX-2 transcription and mRNA stability. Interestingly, docetaxel stimulated the activities of ERK1/2, JNK, and p38 MAPKs (Fig. 8, A-C). Subsequently, experiments were done to determine whether increased MAPK activity was linked to elevated levels of COX-2 in docetaxel-treated cells. In the first experiment, we utilized PD 98059, a specific inhibitor of MAPK kinase, which prevents activation of ERK1 and ERK2. Treatment with PD 98059 caused a decrease in docetaxel-mediated induction of COX-2 (Fig. 8D). Similarly, SB 202190, a selective inhibitor of p38 MAPK, suppressed the inductive effects of docetaxel (Fig. 8D). An inhibitor of JNK activity also suppressed docetaxel-mediated induction of COX-2 (Fig. 8E). For each inhibitor, changes in amounts of COX-2 correlated with changes in the activities of MAPKs (Fig. 8, F-H). These results clearly indicate that multiple MAPKs are important for docetaxel-mediated induction of COX-2. Because docetaxel induces COX-2 by stimulating transcription and stabilizing message, additional experiments were carried out to define the role of specific MAPKs in regulating these different effects. As shown in Fig. 9, dominant negative forms of ERK1 and JNK were effective in suppressing docetaxel-mediated activation of the COX-2 5Ј-UTR but not the COX-2 3Ј-UTR. By contrast, the dominant negative form of p38 MAPK abrogated the inductive effects of docetaxel on the COX-2 3Ј-UTR while having a much more modest inhibitory effect on the activation of the COX-2 5Ј-UTR. Changes in luciferase mRNA corresponded to the observed changes in COX-2 3Ј-UTR activity (Fig. 9C). The above results strongly suggest that p38 MAPK is very important for stabilizing COX-2 message following treatment with docetaxel. In comparable experiments, p38 MAPK also appeared to mediate enhanced COX-2 message stability following treatment with paclitaxel (data not shown).
Additional experiments were carried out to further investigate the importance of p38 MAPK signaling in regulating COX-2 message stability. p38 activates MK-2 (53). As shown in Fig. 10, docetaxel induced MK-2 activity (Fig. 10A), and a dominant negative form of MK-2 blocked docetaxel-mediated stimulation of COX-2 3Ј-UTR activity (Fig. 10B). Corresponding changes in amounts of luciferase mRNA were observed (Fig. 10C). Similar effects were observed when paclitaxel was used (data not shown). As shown in Fig. 11, A and B, the inductive effects of docetaxel on the COX-2 3Ј-UTR and levels of luciferase mRNA were suppressed by treatment with a selective inhibitor of p38 MAPK. Consistent with this finding, docetaxel-mediated induction of HuR binding to the COX-2 3Ј-UTR was also suppressed by the inhibitor of p38 MAPK (Fig.  11C). This effect was specific because PD 98059, a compound that blocks the activation of ERK1/2, failed to alter the increase in HuR binding mediated by docetaxel (Fig. 11C). Dexamethasone has been reported to destabilize COX-2 mRNA (45). Hence, it was also of interest to evaluate whether dexamethasone could alter the mRNA stabilizing effects of docetaxel. Dexamethasone blocked docetaxel-mediated activation of the COX-2 3Ј-UTR (Fig. 11A), caused a corresponding change in amounts of luciferase mRNA (Fig. 11B), and inhibited the binding of HuR to the COX-2 ARE (Fig. 11C).
Taxane-mediated stabilization of COX-2 mRNA could be mediated by effects on the cytoskeleton. To evaluate this concept further, it was important to investigate the effects of nocodazole, a prototypic microtubule interfering agent. As shown in Fig. 12, nocodazole stimulated binding to the ARE (Fig. 12A) and induced COX-2 3Ј-UTR activity via the ARE (Fig. 12B). A corresponding change in levels of luciferase mRNA was observed (Fig. 12C). Similar effects were observed when cytochalasin D, an inhibitor of actin polymerization, was used (data not shown).  1-4, A) or 0, 30, 60, and 180 min (lanes 1-4, B). Total cellular RNA was isolated. The decay of COX-2 mRNA was analyzed by Northern blotting. The blots shown are representative of three independent experiments. C, the results of three independent experiments for vehicle (control), 10 M docetaxel, and 10 M paclitaxel-treated cells were quantified. Band density was quantified with a scanning densitometer. Amounts of COX-2 mRNA are expressed as a relative percentage prior to addition of actinomycin D. Bars, S.D.

FIG. 5. The AU-rich element of the COX-2 3-UTR is important for docetaxel-mediated stabilization of COX-2 mRNA.
A, various deletions of the 1455-nucleotide COX-2 3Ј-UTR (open bars) were fused to the reporter gene luciferase (black bars) to create expression constructs containing the luciferase cDNA fused to the full-length COX-2 3Ј-UTR (Lucϩ3Ј-UTR), the COX-2 AU-rich element (LucϩARE), the AU-rich element deleted from the full-length 3Ј-UTR (LucOEARE), or luciferase without a 3Ј-UTR (LucOE3Ј-UTR). The filled circles represent AU-rich sequences, AUUUA, contained within the 3Ј-UTR. B, 184B5/ HER cells were transfected with 1.8 g of a series of human COX-2 3Ј-UTR deletion constructs ligated to luciferase (Lucϩ3Ј-UTR, open bar; LucϩARE, black bar; and LucOEARE, speckled bar) and 0.2 g of pSV␤gal. After transfection, cells were treated with vehicle (control) or 10 M docetaxel. Reporter activities were measured in cellular extract 7 h later. Luciferase activity represents data that have been normalized with ␤-galactosidase. Columns, means; bars, S.D.; n ϭ 6. C, Northern blot analysis. Total cellular RNA was isolated from cells transfected with Lucϩ3Ј-UTR (lanes 1 and 2) or LucOEARE (lanes 3 and 4) and treated with vehicle (lanes 1 and 3) or 10 M docetaxel (lanes 2 and 4) for 7 h. The blot was hybridized with probes that recognized luciferase mRNA and 18 S rRNA. D, 5 g of cytoplasmic lysate protein was incubated with a 32 P-labeled in vitro transcribed oligonucleotide containing the ARE of COX-2.  (lanes 9, 11, 13, and 15) or cells treated with 10 M docetaxel (lanes 10, 12, 14, and 16). In lanes 9 -16, the following oligonucleotides from the COX-2 3Ј-UTR were used for binding studies: 400 -500 bp (lanes 9 and 10), 500 -600 bp (lanes 11 and 12), 700 -800 bp In lanes 1-3, cytosolic lysate protein was incubated with a 32 P-labeled in vitro transcribed oligonucleotide containing the ARE of COX-2. Lanes 4 and 5 represent cytosolic lysate from docetaxel-treated cells incubated with a 32 P-labeled in vitro transcribed oligonucleotide containing a non-ARE region of the 3Ј-UTR (300 -390 bp). In lane 5 the lysate was incubated with 2 l of anti-HuR antibody. A and B, RNA-protein complexes were subjected to electrophoresis as described under "Experimental Procedures." C, in lanes 2-4, cells were transfected with 0.9 g of Lucϩ3Ј-UTR construct and 0.2 g of pSV␤gal. In lane 4, cells also received 0.9 g of antisense (AS) HuR. The total amount of DNA in each of the three reactions was kept constant at 2 g by using corresponding empty expression vector. Following transfection, cells were treated with vehicle (lane 2) or 10 M docetaxel for 8 h (lanes 3 and 4). Lane 1 represents a standard for HuR. Cellular lysate protein (100 g/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. The immunoblot was probed with antibodies to HuR and ␤-actin. D, cells were transfected with 0.9 g of a human COX-2 3Ј-UTR (Lucϩ3Ј-UTR) construct ligated to luciferase and 0.2 g of pSV␤gal. Bar labeled AS HuR represent cells that received 0.9 g of antisense (AS) HuR. The total amount of DNA in each reaction was kept constant at 2 g by using corresponding empty expression vectors. Cells were treated with vehicle (control) or 10 M docetaxel for 8 h. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. E, total cellular RNA was isolated from cells transfected and treated as described in D. Lane 1, vehicle; lane 2, docetaxel; lane 3, docetaxel plus antisense HuR. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized luciferase mRNA and 18 S rRNA.
( lanes 13 and 14), and 900 -1000 bp (lanes 15 and 16). All treatments were for 6 h. In lanes 1-16, 5 g of cytoplasmic lysate protein was used for binding studies. D and E, RNA-protein complexes were subjected to electrophoresis as described under "Experimental Procedures." DISCUSSION COX-2 is regulated by both transcriptional and post-transcriptional mechanisms. Here we have shown that taxanes induce COX-2 expression by both stimulating transcription and stabilizing mRNA. Docetaxel stimulated COX-2 transcription via the PKC 3 MAPK 3 AP-1 pathway. Previously, we showed that other microtubule-interfering agents including paclitaxel activated COX-2 transcription by a similar mechanism (38).
To the best of our knowledge, the observation that both docetaxel and paclitaxel stabilized COX-2 mRNA represents the first evidence that taxanes can alter mRNA stability. Several recent studies (45)(46)(47)54) have shown that an ARE within the 3Ј-UTR of COX-2 mRNA is important for regulating message stability. Consistent with these prior studies, we found that the ARE within the proximal 3Ј-UTR of COX-2 mRNA was responsible for the increase in COX-2 message stability mediated by taxanes. HuR, a member of the embryonic lethal abnormal vision family of RNA-binding proteins (55,56), has been identified as a trans-acting factor that stabilizes messages containing AREs. In fact, HuR was shown recently (46) to bind to the COX-2 ARE and decrease message turnover. The current results suggest that HuR is responsible for the increase in COX-2 message stability following treatment with taxanes. More specifically, treatment with taxanes increased the binding of HuR to the ARE of COX-2 3Ј-UTR. The functional sig- nificance of HuR was established because taxane-mediated induction of COX-2 3Ј-UTR activity was suppressed by overexpressing antisense HuR. HuR can stabilize mRNAs encoding a variety of other proteins implicated in inflammation and carcinogenesis including c-Fos, p21, vascular endothelial growth factor, and cyclins A and B (57)(58)(59). The results of the current experiments suggest that it will be worthwhile to determine whether taxanes regulate the expression of these or other AREcontaining messages.
It was important to elucidate the signaling mechanism(s) that mediated the induction of COX-2 by docetaxel and paclitaxel (Fig. 13). Treatment with taxanes caused a dose-dependent increase in PKC activity. Importantly, overexpression of a dominant negative form of PKC-␣ blocked taxane-mediated stimulation of COX-2 5Ј-UTR and 3Ј-UTR activity. This implies that PKC signaling is important for both the transcriptional and post-transcriptional regulation of COX-2. Notably, another recent study (60) also demonstrated a role for PKC in regulating COX-2 transcription and message stability. Taxane treatment also activated ERK1/2, JNK, and p38 MAPK; this can be explained at least in part by enhanced PKC signaling. There is considerable evidence that MAPKs are important for regulating both COX-2 transcription and mRNA stability. Less is known, however, about potential differences in the role of individual MAPKs in regulating these different control mechanisms. It was therefore of interest to determine whether the same MAPKs were required for taxane-mediated induction of COX-2 transcription versus increased message stability. Inhibitors of MAPK kinase, JNK, and p38 MAPK blocked the induction of COX-2 by docetaxel. Moreover, overexpression of dominant negatives for ERK1, JNK, or p38 MAPK suppressed the induction of COX-2 5Ј-promoter activity by taxane. These results suggest the involvement of multiple MAPKs in regulating  2 and 3). Cell lysate protein (100 g/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblot was probed for phospho-MK-2. B, cells were transfected with 0.9 g of human COX-2 3Ј-UTR construct and 0.2 g of pSV␤-gal. Bar labeled MAPKAKK-2 represents cells that received 0.9 g of expression vector for wild-type MAPKAPK-2, and the bar labeled as MAPKAKK-2 DN represents cells that received 0.9 g of expression vector for dominant negative MAPKAPK-2. The total amount of DNA in each reaction was kept constant at 2 g by using corresponding empty expression vectors. Cells were treated with vehicle (Control) or docetaxel (10 M) for 8 h. Luciferase activity represents data that have been normalized to ␤-galactosidase activity. Columns, means; bars, S.D.; n ϭ 6. C, total cellular RNA was isolated from cells transfected and treated as described in B. Lane  taxane-mediated induction of COX-2 transcription. In contrast, p38 MAPK was principally responsible for the observed increase in COX-2 mRNA stability (61). This conclusion is supported by several findings. First overexpression of a dominant negative form of p38 blocked taxane-induced stimulation of COX-2 3Ј-UTR activity. In contrast, overexpressing dominant negative forms of ERK1 and JNK failed to suppress taxanemediated activation of the COX-2 3Ј-UTR although they effectively inhibited the induction of COX-2 5Ј-promoter activity. Two other findings support a pivotal role for p38 MAPK in regulating COX-2 message stability. SB 202190, a selective inhibitor of p38 MAPK, blocked both taxane-mediated stimulation of COX-2 3Ј-UTR activity and the increase in HuR binding to the COX-2 3Ј-UTR. In addition to taxanes, various other agents (e.g. nocodazole and cytochalasin D) affect the cytoskeleton and stimulate p38 MAPK activity (38). Therefore, it is noteworthy that both nocodazole and cytochalasin D stimu-lated COX-2 3Ј-UTR activity. This finding supports the concept that the cytoskeleton is important for propagating signals that regulate mRNA stability in addition to gene transcription.
MK-2 mediates the effects of p38 MAPK on mRNA stability (62). Taxanes stimulated MK-2 activity; a dominant negative form of MK-2 blocked taxane-mediated induction of COX-2 3Ј-UTR activity. Hence, p38 stabilizes COX-2 mRNA via MK-2 activation (Fig. 13). The precise mechanism by which MK-2 modulates mRNA stability is not well understood. However, there are at least two possible mechanisms by which MK-2 can increase COX-2 mRNA stability. One potential downstream effector of MK-2 is HSP27, which is a substrate of MK-2 (54). Studies have shown that a phospho-mimetic mutant of HSP27 stabilizes ␤-globin-COX-2 3Ј-UTR mRNA in transfected cells (54). A second possibility is that another substrate of MK-2, heterogeneous nuclear ribonucleoprotein A0, is responsible for taxane-mediated stabilization of COX-2 mRNA. This possibility is suggested by the recent finding that lipopolysaccharidemediated stabilization of COX-2 mRNA stability is dependent on MK-2 activation of heterogeneous nuclear ribonucleoprotein A0 (63). Further work will be required to define the mechanism(s) by which stimulation of p38 MAPK leads to enhanced binding of HuR to the COX-2 3Ј-UTR resulting in increased message stability.
Previously, dexamethasone was reported to decrease COX-2 message stability (64). Dexamethasone inhibits p38 MAPK activity by inducing MAPK phosphatase-1 (65). Taxane-medi- Nocodazole stimulates the COX-2 3-UTR via the AUrich element. A, 5 g of cytoplasmic lysate protein from cells was incubated with a 32 P-labeled in vitro transcribed oligonucleotide containing the ARE of COX-2. Lane 1 represents cytosolic lysates from vehicle-treated cells; lane 2 represents cytosolic lysates from cells treated with 10 M nocodazole for 6 h. RNA-protein complexes were subjected to electrophoresis as described under "Experimental Procedures." B and C, cells were transfected with 1.8 g of a series of human COX-2 3Ј-UTR deletion constructs ligated to luciferase (Lucϩ3Ј-UTR, open bar; LucϩARE, black bar; and LucOEARE, speckled bar) and 0.2 g of pSV-␤gal. After transfection, cells were treated with vehicle (control) or 10 M nocodazole. Reporter activities were measured in cellular extract 7 h later. Luciferase activity represents data that have been normalized with ␤-galactosidase (B). Columns, means; bars, S.D.; n ϭ 6. C, total cellular RNA was isolated from transfected cells (lanes 1 and 4, Lucϩ3Ј-UTR; lanes 2 and 5, LucϩARE; lanes 3 and 6, LucOEARE). Lanes 1-3 represent cells that were treated with vehicle. Lanes 4 -6 represent cells that were treated with 10 M nocodazole. 10 g of RNA was added to each lane. The blot was hybridized with probes that recognized luciferase mRNA and 18 S rRNA. ated activation of the COX-2 3Ј-UTR and HuR binding was inhibited by dexamethasone. Hence, it will be of considerable interest to investigate whether this is a consequence of enhanced MAPK phosphatase-1 activity. Steroids are given to protect against the side effects of taxane treatment. In all likelihood, the ability of steroids to prevent HuR-induced stabilization of ARE-containing mRNAs including COX-2 contributes to the anti-inflammatory properties of these agents.
Previously, we postulated that taxane-mediated induction of COX-2 in tumors might decrease the efficacy of this form of chemotherapy (38). Accordingly, we suggested that coadministration of a selective COX-2 inhibitor might increase the efficacy of taxane-based chemotherapy. In support of this notion, a selective COX-2 inhibitor was found recently to enhance the efficacy of docetaxel in the treatment of experimental lung cancer (39). Although taxanes are effective anti-tumor agents, tumor resistance remains a common problem. In this study, we show for the first time that taxanes enhance COX-2 message stability by augmenting HuR binding. Undoubtedly, other mRNAs that are regulated by HuR will also be stabilized. This mechanism could contribute to tumor resistance. As newer agents are developed that can impact on this mechanism, e.g. selective p38 MAPK inhibitors, it will be important to consider evaluating them in combination with taxane-based chemotherapy regimens.