Tristetraprolin Binds to the 3'-Untranslated Region of Cyclooxygenase-2 mRNA

A POLYADENYLATION VARIANT IN A CANCER CELL LINE LACKS THE BINDING SITE*

Hitoshi SawaokaDagger §, Dan A. Dixon||, John A. OatesDagger §**, and Olivier BoutaudDagger DaggerDagger

From the Departments of Dagger  Pharmacology, § Medicine,  Surgery, and || Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6602

Received for publication, January 2, 2003, and in revised form, January 30, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In human colorectal adenocarcinoma cell lines, we found two major transcripts of cyclooxygenase-2, the full-length mRNA and a short polyadenylation variant (2577 kb) lacking the distal segment of the 3'-untranslated region. Tristetraprolin, an mRNA-binding protein that promotes message instability, was shown to bind the cyclooxygenase-2 mRNA in the region of the 3'-untranslated region between nucleotides 3125 and 3432 and to reduce levels of the full-length mRNA. During cell growth and confluence, the expression of tristetraprolin mRNA was inversely correlated with that of the full-length cyclooxygenase-2 transcript, and transfection of tristetraprolin into HCA-7 cells reduced the level of full-length cyclooxygenase-2 mRNA. However, the truncated transcript escaped tristetraprolin binding and downregulation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cyclooxygenases (prostaglandin H2 synthases, EC 1.14.99.1) are enzymes that catalyze conversion of arachidonic acid to prostaglandin H2. Two isozymes have been described: COX-11 is constitutively expressed, whereas COX-2 is mitogen-inducible (1, 2). COX-2 is the product of an immediate-early gene (3) that is induced by growth factors and cytokines (4, 5) and is related to cell proliferation (6, 7). Levels of COX-2 expression are increased in 85-90% of human colorectal adenocarcinomas (8). COX-2 is also expressed in gastric cancer (9) as well as in esophageal carcinoma (10), pancreatic carcinoma (11), prostate carcinoma (12), lung cancer (13), and mammary cancer (14). The use of nonsteroidal anti-inflammatory drugs is associated with reduced mortality from colorectal cancer (15, 16). COX-2-derived prostaglandin can promote the multistep sequence of events that lead to colon cancer (17, 18). Constitutive expression of COX-2 leads to increased metastatic potential of colon cancer cells (19) and regulates angiogenesis in colon tumors (20). Moreover, COX-2-selective inhibitors have been shown to reduce tumor growth in vivo by inhibiting angiogenesis (21, 22) and by inducing apoptosis (23). As a result of altered regulation of its expression, COX-2 is overexpressed in a number of cancer cell lines, and its expression in the human colorectal carcinoma cell line HCA-7 is among the highest.

In HCA-7 cells, we found and characterized two major COX-2 transcripts. One is the full-length mRNA (4465 nt), and the second is a 2577-nt polyadenylation variant in which the terminal 1888 nt of the 3'-untranslated region (UTR) are not present. During cell growth and confluence, the levels of the full-length mRNA declined, whereas those of the truncated mRNA increased. Truncated COX-2 mRNAs characterized as polyadenylation variants have been described previously in other cell types (2, 24-26). Ristimaki et al. (24) provided evidence that deletion of the distal portion of the 3'-UTR in a polyadenylation variant of COX-2 mRNA confers increased stability to the mRNA. Because we found disparate regulation of the levels of the two COX-2 transcripts with different 3'-UTRs in HCA-7 cells and in the context of increasing evidence that the 3'-UTR of a number of mRNAs can regulate their stability, we considered the hypothesis that the distal 1888 nt of the 3'-UTR of COX-2 mRNA could participate in post-transcriptional regulation of message abundance.

Post-transcriptional mechanisms have been shown to play an important role in the regulation of COX-2 expression during carcinogenesis (27). p38 mitogen-activated protein kinase increases COX-2 mRNA stability (28-30), and inhibition of the kinase by anti-inflammatory glucocorticoids such as dexamethasone leads to a decrease in COX-2 mRNA stability (31).

The 3'-UTRs of some cytokines (e.g. TNF-alpha and granulocyte/macrophage colony-stimulating factor) and proto-oncogenes (e.g. c-fos and c-myc) contribute importantly to the post-transcriptional regulation of message abundance. These 3'-UTRs contain AU-rich elements (AREs) that are the binding sites for proteins that interact with the 3'-UTR to regulate mRNA de-adenylation and decay (32), and the 3'-UTR of COX-2 contains many AREs. A number of ARE-binding proteins have been identified, including HuR (33), AUF1/heterogeneous nuclear ribonucleoprotein D (34, 35), TIA-1 (36), and tristetraprolin (TTP) (37). Binding of some of these proteins to AREs in the 3'-UTR can enhance stability of the message, and HuR has been shown to stabilize COX-2 mRNA (38). Alternatively, proteins that bind to a 3'-UTR can accelerate mRNA degradation, as is the case with TTP (37, 39, 40). Because our findings suggested destabilization of COX-2 mRNA by a protein binding to the distal 1888 nt of the 3'-UTR, we explored the possibility that this protein might be TTP.

TTP (also known as TIS11 and Nup475) (41, 42) is a product of an "immediate-early gene." TTP-deficient mice have a severe inflammatory syndrome (43) and show increased stability of the mRNAs for TNF-alpha and granulocyte/macrophage colony-stimulating factor (44, 45). TTP binds to AREs from RNAs coding for immediate-early genes such as c-fos, interleukin-3, and TNF-alpha (39, 44, 46, 47) and confers instability to these messages. TTP also has a physiological role in the induction of apoptosis (48).

We report that TTP is expressed in HCA-7 cells, that its transcription is up-regulated with cell confluence, and that levels of its RNA vary inversely with those of the full-length COX-2 mRNA. TTP binds to the 3'-UTR of COX-2 mRNA in a region present in the full-length mRNA, but deleted from the truncated polyadenylation variant. Transfection of the TTP cDNA into HCA-7 cells leads to a decrease in the full-length COX-2 mRNA, but does not affect the 2577-nt mRNA.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines

The human colon cancer cell line HCA-7, which was established from a patient with well differentiated mucoid adenocarcinoma of the colon (49, 50), was kindly provided by Dr. Susan Kirkland (University of London). The moderately differentiated human colon cancer cell line Moser (50, 51) was provided by Dr. Harold Moses (Vanderbilt University). Human umbilical vein endothelial cells (HUVECs) were a generous gift from Dr. Douglas Vaughan (Vanderbilt University). HCA-7 cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (BioWhittaker, Inc., Walkersville, MD) and 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin B (Invitrogen). Moser cells were cultured in McCoy's 5A medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin B. HUVECs were grown in Medium 199 (Invitrogen) supplemented with 15% fetal calf serum, 25 µg/ml endothelial growth mitogen, 90 µg/ml heparin, 100 units/ml penicillin, 100 units/ml streptomycin, and 250 ng/ml amphotericin B. These cell lines were free of mycoplasma.

HCA-7 cells were grown at 37 °C to 50, 70, 90, and 100% confluence or overconfluence for 3 days. When indicated, HCA-7 cells at 50% confluence were stimulated with 10 ng/ml LPS in serum-free Dulbecco's modified Eagle's medium for 0, 1, 2, 4, or 8 h. When indicated, HUVECs were activated with IL-1alpha (1 ng/ml; Sigma) for 16 h as described previously (52).

Northern Blotting

Total RNA was extracted from cells using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). mRNA was purified from total RNA using a MicroPoly(A) Pure kit (Ambion Inc., Austin, TX). Total RNA (15 µg each) or mRNA (450 ng each) was denatured in 50% formamide and 6% formaldehyde and resolved by electrophoresis on a 1.0% formaldehyde-agarose gel in MOPS buffer. The RNA was subsequently transferred to a nylon membrane (Hybond-N+, Amersham Biosciences) and hybridized with 32P-labeled cDNAs from human COX-1, COX-2, TTP, and beta -actin. COX-2 cDNAs were obtained by PCR amplification of regions located on the end of each variant determined by 3'-rapid amplification of cDNA ends (RACE): A, nt 1846-2116; B, nt 2269-2525; C, nt 2578-2852; D, nt 2896-3097; E, nt 3135-3366; F, nt 3544-3834; G, nt 3927-4134; and H, nt 4204-4446. TTP cDNAs were obtained from American Type Culture Collection (Manassas, VA). cDNAs from human COX-1 (Oxford Biomedical Research, Inc., Oxford, MI) and COX-2, TTP, and beta -actin (Ambion Inc.) were labeled with 32P using a random prime labeling system (Rediprime, Amersham Biosciences). The blots were washed five times with 1× SSC with 1% SDS at room temperature and then exposed to x-ray film at -70 °C. RNA bands were quantified by densitometry using Scion Image (Scion Corp., Frederick, MD). Total RNA was extracted from three independent series of cells, and Northern blotting was performed twice with each RNA.

5'- and 3'-RACE

RACE was performed using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA). Total RNA was extracted from HCA-7 cells using TRI Reagent, and mRNA was purified from total RNA using the MicroPoly(A) Pure kit.

Primer Design-- The gene-specific primers were designed in exon 10 of COX-2 with the following criteria: 23-28 nt, 50-70% GC, and Tm > 65 °C. We chose exon 10 because it has the least homology to the COX-1 sequence.

First-strand cDNA Synthesis-- Poly(A)+ RNA (mRNA) from HCA-7 cells under confluent conditions (1 µg) was incubated with 5'- or 3'-cDNA synthesis primer and Moloney murine leukemia virus reverse transcriptase (Superscript II, Invitrogen) at 42 °C for 1.5 h.

RACE-- The 5'-RACE PCRs were performed using the gene-specific primer at position 1899 in exon 10 (GSP1, 5'-CTAGTCCGGAGCGGGAAGAACTTGCA) and the template-primer (SMART RACE cDNA amplification kit) and Advantage-GC 2 polymerase (Clontech) to amplify the GC-rich region. The 3'-RACE PCRs were performed using the gene-specific primer at position 1591 in exon 10 (GSP2, CTGTGGAGCTGTATCCTGCCCTTCTGGT) and the template-primer (SMART RACE cDNA amplification kit) and Advantage 2 polymerase (Clontech). The RACE products were resolved by electrophoresis on a 1.3% agarose gel. The bands of 5'- and 3'-RACE products were cut out from the gel and purified using a QIAquick gel extraction kit (QIAGEN, Hilden, Germany). The isolated fragments were cloned directly into a T/A cloning vector (pGEM-T-Easy vector system, Promega, Madison, WI). The identified clones were fully sequenced at the Vanderbilt-Ingram Cancer Center Sequencing Core Facility by the dideoxy chain termination method using an ABI 3700 automated DNA sequencer (Applied Biosystems, Foster City, CA).

Ribonuclease Protection Assay

The expression of each of the variants of COX-2 mRNA was evaluated by ribonuclease protection assay (RPA III kit, Ambion Inc.) with human cyclophilin mRNA as an internal standard following the manufacturer's protocol. To prepare the template for human COX-2 riboprobes, target fragments were amplified by PCR. Each template was designed to overlap the end of each of the variants determined by 3'-RACE (A, nt 1950-2341; B, nt 2322-2747; C, nt 2748-3169; D, nt 3168-3579; E, nt 3561-3988; F, nt 3816-4223; and G, nt 3989-4457). The sense primers were designed to contain a BamHI site at their 5'-ends, and the antisense primers were designed to contain the T3 promoter sequence at their 3'-ends to generate antisense RNA probes. The PCR fragments were resolved by electrophoresis, isolated using the QIAquick gel extraction kit, and ligated with the pGEM-T-Easy vector. To synthesize radiolabeled antisense riboprobes, the plasmids were linearized with BamHI (New England Biolabs Inc., Beverly, MA) and transcribed with T3 RNA polymerase (MAXIscript, Ambion Inc.) in the presence of 80 µCi of [alpha -32P]UTP at 37 °C for 1 h. The full-length radiolabeled riboprobes were purified by preparative electrophoresis on an 8 M urea and 5% acrylamide denaturing gel and eluted into 0.5 M ammonium acetate, 1 mM EDTA, and 0.2% SDS at 37 °C for 16 h. For ribonuclease protection assay, 10 µg of total RNA was hybridized with [alpha -32P]UTP-labeled antisense RNA probes complementary to human COX-2 mRNA or human cyclophilin mRNA (Ambion Inc.). After hybridization overnight at 45 °C and digestion with 7.5 units of RNase T1 (Ambion Inc.), the protected mRNA fragments were denatured and then resolved on an 8 M urea and 5% acrylamide gel. The gels were exposed to x-ray film at -70 °C. COX-2 mRNA abundance in each sample was normalized to the abundance of constitutively expressed cyclophilin mRNA to control for RNA loading.

Western Blotting

Following RNA extraction, the proteins were extracted using TRI Reagent. The protein extract was denatured at 70 °C in the presence of NuPAGETM lithium dodecyl sulfate loading buffer and loaded on a 10% acrylamide NuPAGETM BisTris gel from Novex (San Diego, CA). The proteins were separated by electrophoresis using the NuPAGETM MES running buffer from Novex. At the end of the electrophoresis, the proteins were transferred onto polyvinylidene difluoride membranes at 30 V for 1 h. The nonspecific sites were blocked with 5% dry skim milk in Tris-buffered saline with Tween 20 (Sigma), and the blot was incubated with anti-human COX-2 antibody or anti-human beta -actin antibody (both from Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After incubation with the secondary antibody coupled to horseradish peroxidase (1:2000 dilution; Santa Cruz Biotechnology) for 1 h at room temperature, the proteins were detected with luminous ECL reagent (Amersham Biosciences).

COX-2 Activity Assay

COX-2 activity in HCA-7 cells was evaluated by measurement of prostaglandin E2. [2H8]Arachidonic acid (Sigma) was added to fresh serum-free Dulbecco's modified Eagle's medium at concentration of 20.0 µM. After 15 min at 37 °C, the medium was removed. [2H4]Prostaglandin E2 (2 ng) was added to the samples as an internal standard. Prostaglandins were isolated and derivatized for analysis by gas chromatography/negative ion chemical ionization/mass spectrometry, monitoring selected ions, as described previously (53). The signal for the internal standard ([2H4]prostaglandin E2) is m/z 528. To account for the deuterium-protium exchange at C-12 of [2H7]prostaglandin E2, summation of the signals obtained at m/z 530, 531, and m/z 532 was performed (54).

RNA-Protein Cross-linking and Immunoprecipitation

The interactions between TTP protein and COX-2 RNA were evaluated by RNA-protein cross-linking and immunoprecipitation. To prepare the template for human COX-2 sense riboprobes, target fragments were amplified by PCR. Each template was designed to correspond to the end of each variant determined by 3'-RACE: nt 1941-2211, 2189-2599, 2581-2858, 2842-3151, 3125-3432, 3409-3822, 3801-4225, and 4207-4446. The sense primers were designed to contain the T3 promoter at their 5'-ends, and the antisense primers were designed to contain BamHI sequence at their 3'-ends to generate sense RNA probes. The PCR fragments were resolved by electrophoresis, isolated using the QIAquick gel extraction kit, and ligated with the pGEM-T-Easy vector. To synthesize radiolabeled sense riboprobes, the plasmids were linearized with BamHI and transcribed with T3 RNA polymerase (MAXIscript) in the presence of 80 µCi of [alpha -32P]UTP at 37 °C for 1 h. Cytoplasmic cell lysates were prepared from HCA-7 cells grown in T-75 flasks at 37 °C to 50, 70, 90, and 100% confluence or overconfluence for 3 days. They were washed twice with phosphate-buffered saline, and 2 ml of lysis buffer (25 mM Tris-HCl (pH 7.5) and 0.5% Nonidet P-40) was added. The cells were frozen at -70 °C. The thawed cells were scraped from the plate, vortexed shortly, and centrifuged at 14,000 × g for 10 min. The supernatant was assayed for protein concentration and keep frozen at -70 °C.

UV cross-linking experiments were performed as described previously (55). In brief, the cell lysates (10 µg) were incubated with 1 × 105 cpm of sense RNA probe in RNA binding buffer (20 mM HEPES (pH 7.5), 3 mM MgCl2, 40 mM KCl, 1 mM dithiothreitol, and 5% glycerol) in a total volume of 40 µl. The reaction mixtures were UV-irradiated in 96-well plates using a Stratalinker 9600 (Stratagene) for 5 min and then incubated with 10 µg of RNase A and 5 units of RNase T1 for 30 min at 37 °C. The samples were analyzed by SDS-PAGE, and the radioactivity associated with proteins was visualized by autoradiography.

Immunoprecipitation was performed using 200 µl of UV-cross-linked samples. Three affinity-purified goat polyclonal antibodies specific for TTP (Santa Cruz Biotechnology) were used: G-20, raised against the amino terminus of the mouse protein; P-20, raised against the carboxyl terminus of the human protein; and N-18, raised against the amino terminus of the human protein. The antibodies (2 µg) were added to the samples in immunoprecipitation buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol, and 10% glycerol) and incubated for 5 h at 4 °C with agitation. Then, 20 µl of protein G PLUS-agarose (Santa Cruz Biotechnology) was added to the mixplus the samples were incubated overnight at 4 °C with agitation. The pellets were collected by centrifugation at 2500 × g for 2 min and washed four times with phosphate-buffered saline. The immunoprecipitates were analyzed by SDS-PAGE, and the radioactivity associated with the proteins was visualized by autoradiography.

TTP Transfection

The pcDNA3.1-FLAG-TTP expression construct (under the control of the cytomegalovirus promoter) containing the human TTP coding region (nucleotides 10-990) was a generous gift from Dr. William Rigby (Dartmouth University) (56). Transient transfection of HCA-7 cells was performed using Cellfectin (Invitrogen) following the manufacturer's instructions. Briefly, 50% confluent HCA-7 cells in T-75 flasks were washed with Opti-MEM I reduced serum medium (Invitrogen). The TTP DNA (7.5 µg) in 500 µl of Opti-MEM I and Cellfectin (15 µl) in 500 µl of Opti-MEM were combined and added to the cells. After 18 h, the DNA-containing medium was replaced with Dulbecco's modified Eagle's medium with 10% fetal calf serum and incubated for 6 h at 37 °C. RNA and proteins were extracted from the cells using TRI Reagent.

Statistical Analysis

The data are presented as means ± S.E. and were compared by unpaired t test or by one-way ANOVA followed by Fisher's PLSD procedure. The criterion for statistical significance was p < 0.05.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

COX-2 Transcripts in HCA-7 and Moser Colon Cancer Cells and HUVECs-- Northern blot analysis of mRNA from HCA-7 cells using a COX-2-specific probe showed two bands migrating at ~4.5 and 2.6 kb (Fig. 1A). Interestingly, the smaller species was the more abundant in confluent HCA-7 cells. By comparison, the longest species at 4.5 kb was the main transcript in Moser cells (Fig. 1B) (50, 51). HUVECs grown under standard conditions did not express COX-2. However, after activation by IL-1alpha , HUVECs also expressed two transcripts of COX-2, the main one being 4.5 kb long. Because full-length COX-1 mRNA is 2.5 kb long, we investigated whether the small transcripts in HCA-7 and Moser cells were due to cross-reactivity of our probe with COX-1 RNA by analyzing the same total RNA by Northern blotting using a probe specific for COX-1. As shown in Fig. 1C, a band at ~2.5 kb was observed in HUVECs grown under normal conditions or after activation with IL-1alpha . On the contrary, both HCA-7 and Moser cells did not express COX-1. This indicates that the small transcript of 2.5 kb present in HCA-7 is specific to COX-2.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 1.   Expression of COX-1 and COX-2 RNAs in HUVECs, HUVECs activated with IL-1alpha , and HCA-7 and Moser cells upon Northern blot analysis. HCA-7 and Moser cells and HUVECs were grown as described under "Experimental Procedures." HUVECs were activated with or without 1 ng/ml IL-1alpha for 16 h. Cells were harvested at confluence, total RNA was extracted, and mRNA was purified on a poly(A) affinity column. 450 ng of mRNA (A) or 15 µg of total RNA (B and C) was used for Northern blot analysis. Membranes were hybridized with COX-2-specific (A and B) or COX-1-specific (C) probes. The lower panels show Northern blots for beta -actin as a control of equal loading of RNA.

Cloning of the Variants of COX-2 mRNA in HCA-7 Cells by 5'- and 3'-RACE-- We performed 5'- and 3'-RACE using the gene-specific primer located in the coding region of exon 10 because it has the least homology to COX-1. Analysis of HCA-7 mRNA by 5'-RACE generated a lone product at 1.9 kb, indicating no splice variant in this portion of the coding region. On the other hand, analysis of HCA-7 mRNA by 3'-RACE generated nine products at 2.9, 2.6, 2.3, 1.8, 1.6, 1.3, 1.0, 0.9, and 0.6 kb. Each product was subcloned in a T/A cloning vector and sequenced. The results of sequencing were compared with the published sequence of the human COX-2 gene (57). The 5'-RACE product started at -2 and ended at the gene-specific primer for 5'-RACE located in exon 10 (position 1899). The 3'-RACE products started at the gene-specific primer for 3'-RACE located in exon 10 (position 1591) and ended with a poly(A) tail starting at positions 4465, 4208, 3902, 3408, 3109, 2871, 2577, 2443, and 2187.

Characterization of the Small RNA Transcript-- To characterize the two transcripts observed by Northern blot analysis, we designed probes specific for each COX-2 3'-RACE product (A, nt 1846-2116; B, nt 2269-2525; C, nt 2578-2852; D, nt 2896-3097; E, nt 3135-3366; F, nt 3544-3834; G, nt 3927-4134; and H, nt 4204-4446). Total RNA and mRNA from HCA-7 cells were transferred to nylon membranes and examined by Northern blot analysis using these probes. Probes A and B recognized the short mRNA species, indicating two possible polyadenylation variants at 2443 and 2577 bp (Fig. 2A). Probe C-H recognized only the long mRNA transcript, identifying it as the full-length transcript at 4465 bp (Fig. 2A). To differentiate between the two polyadenylation sites at 2443 and 2577 bp, we analyzed the total RNA by ribonuclease protection assay. Antisense RNA probes ~400 nucleotides long were synthesized to span either side of the different polyadenylation sites (A, nt 1950-2341; B, nt 2322-2747; C, nt 2748-3169; D, nt 3168-3579; E, nt 3561-3988; F, nt 3816-4223; and G, nt 3989-4457). Hybridization with probe B generated two fragments corresponding to the fully protected probe (426 nt) and to the partially protected probe (256 nt) (Fig. 2B). This indicates that the antisense probe B hybridizes with an mRNA that corresponds to the COX-2 sequence through nt 2577. The 3'-terminal sequence of the polyadenylated 2577-nt mRNA determined from sequencing the 3'-RACE product is depicted in Fig. 2C, indicating the polyadenylation signal located 19 nt proximal to nt 2577. The five adenines at nt 2573-2577 are present in the COX-2 3'-UTR sequence. The generation of the fully protected fragments when probes C-G were used confirmed the identity of the long transcript as the full-length mRNA at 4465 nt (Fig. 2B).


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 2.   Characterization of the two COX-2 transcripts. A, Northern blots of COX-2 RNA. mRNA (450 ng each) was hybridized with each COX-2 probe as described under "Experimental Procedures." Probes A-H used for each panel are indicated (A, nt 1846-2116; B, nt 2269-2525; C, nt 2578-2852; D, nt 2896-3097; E, nt 3135-3366; F, nt 3544-3834; G, nt 3927-4134; and H, nt 4204-4446). The signals corresponding to the long and short COX-2 mRNAs are indicated by the arrowheads. B, RNase protection assay of COX-2 RNA. Antisense RNA probes ~400 nucleotides long were synthesized. Total RNA (10 µg) was hybridized with [alpha -32P]UTP-labeled antisense RNA probes designed to span either side of the putative polyadenylation site of interest (A, nt 1950-2341; B, nt 2322-2747; C, nt 2748-3169; D, nt 3168-3579; E, nt 3561-3988; F, nt 3816-4223; and G, nt 3989-4457). After digestion with RNase T1, the protected mRNA fragments were denatured and separated on polyacrylamide gel. Results obtained with probes B and G are represented. Lanes 1, protected fragments; lanes 2, negative control using yeast RNA; lanes 3, full-length probe without RNase digestion. The lengths of the protected fragments are indicated next to the arrowheads in nucleotides. The lengths of the corresponding transcripts are indicated in parentheses. C, sequence of the 52 final nucleotides of 2577-nt mRNA. The non-canonical polyadenylation signal is underlined. The polyadenylation tail is indicated in boldface. The end of the sequence matching the COX-2 cDNA at position 2577 is indicated.

COX-2 and TTP mRNAs in HCA-7 Cells Related to Cell Confluence-- HCA-7 cells were harvested at 50, 70, 90, and 100 confluence or at 3 days post-confluence. Total RNA extracted from cells was analyzed by Northern blotting with probe B (nt 2269-2525), and the presence of COX-2 protein was analyzed by Western blotting. Northern blot analysis revealed that, before confluence, the 4465-nt COX-2 mRNA was the main transcript in HCA-7 cells; the level of this variant was then reduced as the cells became overconfluent. The 2577-nt COX-2 mRNA represented a less abundant species before confluence, but its level was increased after confluence (Fig. 3A). Densitometric analysis of the Northern blots revealed that the differences between the values obtained before and after confluence for both transcripts were statistically significant as ascertained by ANOVA followed by Fisher's PLSD procedure (Fig. 3B).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   Relative abundance of the two major COX-2 mRNAs and TTP mRNA under different conditions of confluence. HCA-7 cells were harvested at 50, 70, 90, and 100% confluence or at 3 days post-confluence. Total RNA (15 µg each) was analyzed by Northern blotting. A, Northern blots for COX-2, TTP, and beta -actin are represented. Lanes 1-4, 50, 70, 90, and 100% confluence, respectively; lane 5, 3 days post-confluence. B, RNA bands for 4.5-kb (black-square) and 2.6-kb () COX-2 were quantified by densitometry and corrected by the amount of beta -actin in the same sample by one-way ANOVA followed by Fisher's PLSD procedure. The asterisks indicate statistical significance compared with 50% confluence (*, p < 0.05; **, p < 0.01; n = 3).C, RNA bands for TTP were integrated, corrected by the amount of beta -actin in the same sample, and compared by one-way ANOVA followed by Fisher's PLSD procedure. The asterisks indicate statistical significance (p < 0.01; n = 3) between every condition and each other.

The expression of TTP was also examined by Northern blotting using total RNA from HCA-7 cells. The analysis revealed that TTP was expressed in HCA-7 cells and that its transcription was increased with cell confluence. The densitometric analysis of Northern blots revealed that the differences between the values obtained before and after confluence were statistically significant as ascertained by ANOVA followed by Fisher's PLSD procedure (Fig. 3C). Interestingly, the increase in TTP transcription correlated with the decrease in the full-length COX-2 transcript, consistent with a possible role for TTP in the post-transcriptional regulation of COX-2. These results also indicate that these two COX-2 transcripts are differentially regulated.

Analysis of COX-2 protein expression by Western blotting revealed that, at the stage of confluence when the full-length RNA was down-regulated, the amount of protein produced was increased (Fig. 4, A and B). Analysis of the cyclooxygenase activity showed that production of prostaglandin E2 increased as the cells grew from 50% to overconfluence and while levels of the full-length transcript decreased (Fig. 4C). These results suggest that the short COX-2 polyadenylation variant is translationally competent.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Catalytic activity and expression of COX-2 in HCA-7 cells as a function of confluence. HCA-7 cells were harvested at 50% confluence or at 3 days post-confluence. A, proteins (50 µg) were analyzed by Western blotting after electrophoresis on a 10% polyacrylamide gel under denaturing conditions. B, the amount of protein was integrated and corrected by the amount of beta -actin in the same sample. The asterisk indicates statistical significance compared with 50% confluence (p < 0.01; n = 4). C, [2H8]arachidonic acid (20 µM) was added to the cells in fresh serum-free medium. After 15 min of incubation at 37 °C, the medium was harvested, and prostaglandin E2 (PGE2) was analyzed by gas chromatography/mass spectrometry. The asterisk indicates statistical significance compared with 50% confluence (p < 0.01; n = 4).

Binding of TTP Protein to COX-2 mRNAs-- To characterize the interactions between TTP protein and COX-2 mRNAs, we assessed whether TTP expressed in HCA-7 cells at 50% confluence and overconfluent for 3 days would bind to the COX-2 3'-UTR. We performed UV cross-linking of radiolabeled sense RNA probes spanning the COX-2 3'-UTR to soluble proteins from HCA-7 cells. Each probe was designed to hybridize with COX-2 RNA at the end of each variant determined by 3'-RACE: nt 1941-2211, 2189-2599, 2581-2858, 2842-3151, 3125-3432, 3409-3822, 3801-4225, and 4207-4446. As shown in Fig. 5A, numerous proteins that were bound to the different RNA probes were detected. Because only the full-length COX-2 transcript was down-regulated with confluence, we focused our attention on proteins that bound to the probes located distal to nucleotide 2577 and that were up-regulated between 50% confluence and 3 days post-confluence. Several proteins fulfilled these criteria, but one prominent protein binding between nucleotides 3125 and 3432 also had an apparent molecular mass of 43 kDa, which corresponds to human TTP. To determine whether TTP can bind this region of the full-length COX-2 mRNA, we performed another cross-linking experiment in which TTP was immunoprecipitated using three different antibodies specific for TTP after the UV cross-linking step. The immunoprecipitates were then analyzed by SDS-PAGE to detect bound TTP. As shown in Fig. 5B, only one band was detected with a molecular mass of 43 kDa and was strongly up-regulated with confluence. These results demonstrate that TTP protein expressed in HCA-7 cells can functionally bind to COX-2 RNA between positions 3125 and 3432. 


View larger version (92K):
[in this window]
[in a new window]
 
Fig. 5.   Analysis of TTP protein and COX-2 RNA interactions by RNA-protein cross-linking and immunoprecipitation. Human COX-2 sense riboprobes labeled with [alpha -32P]UTP were designed to bracket the end of each variant determined by 3'-RACE: nt 1941-2211, 2189-2599, 2581-2858, 2842-3151, 3125-3432, 3409-3822, 3801-4225, and 4207-4446. Cytoplasmic cell lysates were prepared from HCA-7 cells harvested at 50% confluence or at 3 days post-confluence. A, shown are the results from RNA-protein cross-linking experiments. The cell lysates (10 µg) were incubated with 1 × 105 cpm of sense RNA probe, UV-irradiated for 5 min, and then incubated with RNase. The samples were analyzed by SDS-PAGE, and the radioactive proteins were visualized by autoradiography. Lanes 1, 50% confluence; lanes 2, 3 days post-confluence. B, for immunoprecipitation, the three different TTP antibodies (Ab) were added to the UV-cross-linked samples, and the mixture was incubated for 5 h at 4 °C. Then, protein G PLUS-agarose was added to the mixture, and the samples were incubated overnight at 4 °C. The pellets were collected by centrifugation and washed. The immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. The results using probe 3125-3432 are represented. Lanes 1, 50% confluence; lanes 2, 3 days post-confluence.

Effect of TTP Transfection on COX-2 mRNA Stability-- To determine the effect of TTP expression on COX-2 post-transcriptional regulation, we transfected 50% confluent HCA-7 cells with a plasmid containing the human FLAG-tagged TTP coding region (nucleotides 10-990) regulated by the cytomegalovirus promoter. We examined COX-2 mRNA in the cells after transient transfection with this plasmid (Fig. 6). Northern blot analysis confirmed TTP transcription in cells transfected with the plasmid coding for the human protein. In the same time, we observed a statistically significant decrease in the amount of 4465-nt COX-2 mRNA in cells transfected with TTP compared with mock transfection. By comparison, the 2577-nt COX-2 mRNA was not decreased under the same conditions. As TTP transfection had no effect on the truncated polyadenylation variant, these data suggest that the reduction in the levels of the full-length COX-2 mRNA results from a destabilizing effect of TTP that is targeted to the long transcript containing the identified TTP-binding site.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 6.   Transcription of COX-2 and TTP in HCA-7 cells after transfection with human TTP. HCA-7 cells at 50% confluence were transiently transfected with the pcDNA3.1-FLAG-TTP expression construct using Cellfectin. RNA was extracted from the cells using TRI reagent. A, Northern blot analysis for COX-2, TTP, and beta -actin. Lane 1, mock transfection; lane 2, TTP transfection. B, RNA bands for 4.5- and 2.6-kb COX-2 and TTP were quantified by densitometry and corrected by the amount of beta -actin in the same samples. The asterisks indicate statistical significance (p < 0.05; n = 3) with mock transfection. The black and white bars indicate mock transfection and TTP transfection, respectively.

Inhibition of gene transcription with actinomycin D has been used to assess the rates of decay of some mRNAs. However, this approach is not possible for assessing the rate of mRNA decay if its regulation is influenced by an inducible transactivating protein because actinomycin D will arrest the transcription of the transactivator (24). As expected, we found that the levels of TTP mRNA fell rapidly after actinomycin D treatment (data not shown).

Correlation of TTP and COX-2 Transcription in HCA-7 Cells-- HCA-7 cells at 50% confluence were stimulated with 10 ng/ml LPS for 0, 1, 2, 4, or 8 h. Total RNA extracted from the cells was analyzed by Northern blotting using probe B (nt 2269-2525) (Fig. 7). TTP RNA was examined by Northern blotting following stimulation with LPS. The results indicate that LPS induced TTP transcription, with the maximum effect reached at 1 h (Fig. 7). The level of RNA was already decreased at 2 h and was back to the basal level at 4 h. This transient induction of TTP transcription is consistent with previous findings (39, 41). The expression of both COX-2 transcripts was seen with LPS stimulation, with the maximum induction reached after 1 h. The 2577-nt mRNA of COX-2 was still elevated at 4 h. On the other hand, the 4465-nt mRNA of COX-2 was only sustained until 2 h and then rapidly decreased; it was back to the basal level at 4 h. The greater reduction of the 4465-nt mRNA at 4 h is significant at the p < 0.05 level.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 7.   Relative abundance of the two major COX-2 mRNAs and TTP mRNA upon LPS stimulation. HCA-7 cells at 50% confluence were incubated with LPS (10 ng/ml) for 0, 1, 2, 4, and 8 h. Total RNA (15 µg each) was analyzed by Northern blotting for COX-2 and TTP transcription. A, the results of Northern blotting for COX-2, TTP, and beta -actin after induction with LPS are represented. Lane 1, 0 h of incubation; lane 2, 1 h; lane 3, 2 h; lane 4, 4 h; lane 5, 8 h. B, RNA bands for 4.5-kb (black-square) and 2.6-kb () COX-2 and TTP (black-triangle) were quantified by densitometry, corrected by the amount of beta -actin in the same sample, and compared by one-way ANOVA followed by Fisher's PLSD procedure. The asterisks indicate statistical significance compared with 0 h (p < 0.05; n = 3).

As an immediate-early gene, the induction of COX-2 transcription by LPS is rapid and brief. Because both the 4465- and 2577-nt COX-2 mRNAs derive from transcription of the COX-2 gene, termination of transcription after the brief burst elicited by LPS leads to concurrent arrest of formation of both the full-length and variant transcripts. Thus, a difference in the rate of decline in the concentration of the two COX-2 mRNAs reflects a difference in the rate of their degradation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This report provides evidence that TTP binds to the 3'-UTR of COX-2 mRNA. The investigations leading to this finding were informed by the finding that a polyadenylation variant of COX-2 mRNA is regulated differently from the full-length COX-2 transcript.

We found two major transcripts of COX-2 in the human colorectal adenocarcinoma cell lines HCA-7 and Moser. This is of particular interest because of the importance of COX-2 in the development of colorectal adenocarcinoma and because of the role COX-2 overexpression has in growth and tumorigenicity in the HCA-7 and Moser cell lines (50, 51). Accordingly, we fully characterized these two COX-2 transcripts. The larger was found to be the full-length mRNA, 4465 nt in length. The smaller is 2577 nt in size and represents a polyadenylation variant that lacks the distal segment 2578-4465 of the 3'-UTR (24, 25). It is polyadenylated downstream from the non-canonical polyadenylation signal, AUUAAA (58).

The consequences of deletion of the distal 1888 nt of the 3'-UTR may be considered in the context of evidence that the 3'-UTR participates in the regulation of the stability of a number of mRNA species, including that of COX-2 itself. The 3'-UTR of COX-2 mRNA contains 22 copies of a conserved ARE, the AUUUA pentamer. This pentamer, frequently located in or near a U-rich region, has been associated with the regulation of mRNA stability (57) in a number of mRNAs, including those of proto-oncogenes (59, 60) and cytokines (37). Recently, Dixon et al. (55) reported the importance of AREs in the post-transcriptional regulation of COX-2 mRNA. A number of ARE-binding proteins have been identified, including HuR (33), AUF1/heterogeneous nuclear ribonucleoprotein D (34, 35), TIA-1 (36), and tristetraprolin (37). Binding of these proteins to AREs in the 3'-UTR can exert either positive or negative effects on stability, translation, and subcellular localization of the mRNA. For example, HuR binds to the ARE present in the proximal segment of the COX-2 3'-UTR to enhance stability of the mRNA (38); both transcripts of COX-2 contain the HuR-binding site. By contrast, AREs present at the distal end of the 3'-UTR proto-oncogenes have been shown to participate in destabilization of the mRNA. For example, truncation of 61 bases containing AREs that are 24 nucleotides before the poly(A)+ addition signal in the myc proto-oncogene mRNA increases the half-life of the mRNA by at least 5-fold (59).

The evidence that AREs in the 3'-UTR region can influence the stability and translation of mRNA and the presence of 15 AREs in the deleted sequence between bases 2578 and 4465 provided the basis for the hypothesis that the regulation of the stability of the 2577-nt COX-2 mRNA could be different from that of the full-length transcript. As one approach toward assessing whether lack of sequence 2578-4465 altered the content of COX-2 mRNA or COX-2 expression and activity, we determined the effect of confluence of the cells on the amount of each of the mRNA species as well as COX-2 protein and prostaglandin production. One of the characteristics of cancer cells is the loss of contact inhibition. The HCA-7 cell line shares this characteristic, and cells continue to grow for several days after reaching confluence. We observed that, as the cell culture progressed from 50% confluence to a post-confluent state, the ratio of 4465-nt to 2577-nt mRNA decreased. This change in the ratio resulted from both a decrease in the abundance of 4465-nt mRNA and an increase in the abundance of 2577-nt mRNA. One explanation for these results is that confluence is associated with the synthesis of a cellular factor that signals the rapid degradation of the mRNA by binding to region 2578-4465 of the 3'-UTR. Despite the reduction in the level of the full-length transcript, COX-2 protein and activity were increased, indicating that the 2577-nt mRNA is translationally competent.

The TTP gene is a member of the immediate-early gene family (41, 61). The TTP protein binds to AREs and promotes destabilization of mRNAs of oncogenes and cytokines such as TNF-alpha (37, 39, 40). Because of the similarities between the 3'-UTRs of TNF-alpha and COX-2, we hypothesized that TTP could be responsible for full-length COX-2 mRNA down-regulation in HCA-7 cells. We demonstrated that the TTP gene was transcribed in HCA-7 cells and that its transcription was up-regulated with confluence. Its up-regulation correlated with the down-regulation of 4.5-kb COX-2 message.

To further characterize the differential regulation of the two COX-2 messages, we analyzed the proteins binding to the COX-2 3'-UTR. We observed that several proteins bound to the 3'-UTR distal to nucleotide 2577. Among these proteins, four were up-regulated with confluence, and one of these migrated on SDS-polyacrylamide gel at ~45 kDa. We addressed the question of whether that protein was TTP by immunoprecipitating the RNA-protein complexes cross-linked by UV, employing three different antibodies specific for TTP. The results demonstrate that TTP bound to the COX-2 3'-UTR between nucleotides 3125 and 3432. Analysis of the nucleotide sequence of COX-2 message in this region showed an ARE starting at nucleotide 3369 with the consensus sequence UAUUUA. Recently, Worthington et al. (47) showed that addition of Us on each side of the consensus sequence AUUUA increased the affinity of the ARE for TTP. Although the highest affinity was reached with the palindromic sequence UUAUUUAUU, they showed that TTP had a higher affinity for UAUUUAU than for AUUUA. Because the UAUUUA at nt 3369 is the only ARE in that region of the COX-2 3'-UTR, it is likely that TTP binds to the COX-2 3'-UTR by association with the ARE present between nucleotides 3369 and 3374.

Binding of TTP to ARE-rich 3'-UTRs has resulted in accelerated degradation of the mRNA. To determine whether TTP increases the removal of the full-length COX-2 mRNA, we transfected HCA-7 cells at 50% confluence with a plasmid coding for the human TTP. The levels of the 4.5-kb COX-2 mRNA were significantly reduced after the transfection. The absence of any change in the levels of the 2.6-kb message lacking the TTP-binding site provides evidence that TTP did not alter transcription, as the polyadenylation variant and full-length mRNAs both derive from a common transcription process (24). This finding is consistent with the hypothesis that the transactivating protein TTP binds to the distal portion of the 3'-UTR of COX-2 mRNA and facilitates its degradation.

We then examined the post-transcriptional regulation of the 4.5-kb mRNA in a pathophysiological model in which TTP levels are increased (44, 62). LPS induced a short burst of transcription of both TTP and COX-2; and in this TTP-induced environment, the removal of the COX-2 transcript containing the TTP-binding site was accelerated relative to that of the concurrently transcribed mRNA with a truncated 3'-UTR lacking the TTP-binding domain.

In conclusion, the transactivating protein tristetraprolin binds to the 3'-UTR of COX-2 mRNA in the region between nucleotides 3125 and 3432. This segment of the 3'-UTR contains the ARE UAUUUA, beginning at nucleotide 3369. Transfection of the TTP gene demonstrated that TTP can decrease the level of the full-length COX-2 mRNA, but it does not affect the truncated 2577-nt polyadenylation variant that lacks the TTP-binding site. This polyadenylation variant, which escapes down-regulation by TTP, is prominent in a colon cancer cell line. We also hypothesize that TTP up-regulation at confluence provides evidence for a role of the protein in the mechanism by which cells down-regulate specific mRNAs in the process of contact inhibition.

    ACKNOWLEDGEMENTS

We thank Elizabeth Shipp for excellent technical assistance and Joseph Covington for providing the HUVECs. We thank Dr. Ronald Emeson for valuable advice.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants CA68485, CA77839, and GM15431 and by a grant from Merck-Frosst Canada & Co.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** Thomas F. Frist, Sr. Professor of Medicine.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University, 514 RRB, 23rd Ave. S. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-7398; Fax: 615-322-4707; E-mail: olivier.boutaud@vanderbilt.edu.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M300016200

    ABBREVIATIONS

The abbreviations used are: COX, cyclooxygenase; nt, nucleotide(s); UTR, untranslated region; TNF-alpha , tumor necrosis factor-alpha ; ARE, AU-rich element; TTP, tristetraprolin; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharide; IL-1alpha , interleukin-1alpha ; MOPS, 3-(N-morpholino)propanesulfonic acid; RACE, rapid amplification of cDNA ends; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 2-(N-morpholino)ethanesulfonic acid; ANOVA, analysis of variance.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Hla, T., and Neilson, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7384-7388[Abstract]
2. O'Banion, M. K., Winn, V. D., and Young, D. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4888-4892[Abstract]
3. DuBois, R. N., Tsujii, M., Bishop, P., Awad, J. A., Makita, K., and Lanahan, A. (1994) Am. J. Physiol. 266, G822-G827[Medline] [Order article via Infotrieve]
4. DuBois, R. N., Awad, J., Morrow, J., Roberts, L. J., II, and Bishop, P. R. (1994) J. Clin. Invest. 93, 493-498[Medline] [Order article via Infotrieve]
5. Ristimaki, A., Garfinkel, S., Wessendorf, J., Maciag, T., and Hla, T. (1994) J. Biol. Chem. 269, 11769-11775[Abstract/Free Full Text]
6. Martinez, J., Sanchez, T., and Moreno, J. J. (1997) Am. J. Physiol. 273, C1466-C1471[Medline] [Order article via Infotrieve]
7. Sawaoka, H., Tsuji, S., Tsujii, M., Gunawan, E. S., Nakama, A., Takei, Y., Nagano, K., Matsui, H., Kawano, S., and Hori, M. (1997) J. Clin. Gastroenterol. 25, S105-S110[CrossRef][Medline] [Order article via Infotrieve]
8. Eberhart, C. E., Coffey, R. J., Radhika, A., Giardiello, F. M., Ferrenbach, S., and DuBois, R. N. (1994) Gastroenterology 107, 1183-1188[Medline] [Order article via Infotrieve]
9. Ristimaki, A., Honkanen, N., Jankala, H., Sipponen, P., and Harkonen, M. (1997) Cancer Res. 57, 1276-1280[Abstract]
10. Zimmermann, K. C., Sarbia, M., Weber, A. A., Borchard, F., Gabbert, H. E., and Schror, K. (1999) Cancer Res. 59, 198-204[Abstract/Free Full Text]
11. Molina, M. A., Sitja-Arnau, M., Lemoine, M. G., Frazier, M. L., and Sinicrope, F. A. (1999) Cancer Res. 59, 4356-4362[Abstract/Free Full Text]
12. Yoshimura, R., Sano, H., Masuda, C., Kawamura, M., Tsubouchi, Y., Chargui, J., Yoshimura, N., Hla, T., and Wada, S. (2000) Cancer (Phila.) 89, 589-596[CrossRef]
13. Wolff, H., Saukkonen, K., Anttila, S., Karjalainen, A., Vainio, H., and Ristimaki, A. (1998) Cancer Res. 58, 4997-5001[Abstract]
14. Soslow, R. A., Dannenberg, A. J., Rush, D., Woerner, B. M., Khan, K. N., Masferrer, J., and Koki, A. T. (2000) Cancer (Phila.) 89, 2637-2645[CrossRef]
15. Thun, M. J., Namboodiri, M. M., Calle, E. E., Flanders, W. D., and Heath, C. W., Jr. (1993) Cancer Res. 53, 1322-1327[Abstract]
16. Thun, M. J., Namboodiri, M. M., and Heath, C. W., Jr. (1991) N. Engl. J. Med. 325, 1593-1596[Abstract]
17. Giardiello, F. M., Hamilton, S. R., Krush, A. J., Piantadosi, S., Hylind, L. M., Celano, P., Booker, S. V., Robinson, C. R., and Offerhaus, G. J. (1993) N. Engl. J. Med. 328, 1313-1316[Abstract/Free Full Text]
18. Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trzaskos, J. M., Evans, J. F., and Taketo, M. M. (1996) Cell 87, 803-809[Medline] [Order article via Infotrieve]
19. Tsujii, M., Kawano, S., and DuBois, R. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3336-3340[Abstract/Free Full Text]
20. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois, R. N. (1998) Cell 93, 705-716[Medline] [Order article via Infotrieve]
21. Sawaoka, H., Tsuji, S., Tsujii, M., Gunawan, E. S., Kawai, N., Sasaki, Y., Hori, M., and Kawano, S. (1999) Prostaglandins Leukotrienes Essent. Fatty Acids 61, 315-322[CrossRef][Medline] [Order article via Infotrieve]
22. Masferrer, J. L., Leahy, K. M., Koki, A. T., Zweifel, B. S., Settle, S. L., Woerner, B. M., Edwards, D. A., Flickinger, A. G., Moore, R. J., and Seibert, K. (2000) Cancer Res. 60, 1306-1311[Abstract/Free Full Text]
23. Sawaoka, H., Kawano, S., Tsuji, S., Tsujii, M., Gunawan, E. S., Takei, Y., Nagano, K., and Hori, M. (1998) Am. J. Physiol. 274, G1061-G1067[Medline] [Order article via Infotrieve]
24. Ristimaki, A., Narko, K., and Hla, T. (1996) Biochem. J. 318, 325-331[Medline] [Order article via Infotrieve]
25. Newton, R., Seybold, J., Liu, S. F., and Barnes, P. J. (1997) Biochem. Biophys. Res. Commun. 234, 85-89[CrossRef][Medline] [Order article via Infotrieve]
26. Kawaguchi, H., Pilbeam, C. C., Gronowicz, G., Abreu, C., Fletcher, B. S., Herschman, H. R., Raisz, L. G., and Hurley, M. M. (1995) J. Clin. Invest. 96, 923-930[Medline] [Order article via Infotrieve]
27. Zhang, Z., Sheng, H., Shao, J., Beauchamp, R. D., and DuBois, R. N. (2000) Neoplasia 2, 523-530[CrossRef][Medline] [Order article via Infotrieve]
28. Ridley, S. H., Dean, J. L., Sarsfield, S. J., Brook, M., Clark, A. R., and Saklatvala, J. (1998) FEBS Lett. 439, 75-80[CrossRef][Medline] [Order article via Infotrieve]
29. Dean, J. L., Brook, M., Clark, A. R., and Saklatvala, J. (1999) J. Biol. Chem. 274, 264-269[Abstract/Free Full Text]
30. Jang, B. C., Sanchez, T., Schaefers, H. J., Trifan, O. C., Liu, C. H., Creminon, C., Huang, C. K., and Hla, T. (2000) J. Biol. Chem. 275, 39507-39515[Abstract/Free Full Text]
31. Lasa, M., Brook, M., Saklatvala, J., and Clark, A. R. (2001) Mol. Cell. Biol. 21, 771-780[Abstract/Free Full Text]
32. Xu, N., Chen, C. Y., and Shyu, A. B. (1997) Mol. Cell. Biol. 17, 4611-4621[Abstract]
33. Peng, S. S., Chen, C. Y., Xu, N., and Shyu, A. B. (1998) EMBO J. 17, 3461-3470[Abstract/Free Full Text]
34. Buzby, J. S., Brewer, G., and Nugent, D. J. (1999) J. Biol. Chem. 274, 33973-33978[Abstract/Free Full Text]
35. Zhang, W., Wagner, B. J., Ehrenman, K., Schaefer, A. W., DeMaria, C. T., Crater, D., DeHaven, K., Long, L., and Brewer, G. (1993) Mol. Cell. Biol. 13, 7652-7665[Abstract]
36. Piecyk, M., Wax, S., Beck, A. R., Kedersha, N., Gupta, M., Maritim, B., Chen, S., Gueydan, C., Kruys, V., Streuli, M., and Anderson, P. (2000) EMBO J. 19, 4154-4163[Abstract/Free Full Text]
37. Lai, W. S., Carballo, E., Strum, J. R., Kennington, E. A., Phillips, R. S., and Blackshear, P. J. (1999) Mol. Cell. Biol. 19, 4311-4323[Abstract/Free Full Text]
38. Dixon, D. A., Tolley, N. D., King, P. H., Nabors, L. B., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2001) J. Clin. Invest. 108, 1657-1665[Abstract/Free Full Text]
39. Raghavan, A., Robison, R. L., McNabb, J., Miller, C. R., Williams, D. A., and Bohjanen, P. R. (2001) J. Biol. Chem. 276, 47958-47965[Abstract/Free Full Text]
40. Lai, W. S., and Blackshear, P. J. (2001) J. Biol. Chem. 276, 23144-23154[Abstract/Free Full Text]
41. DuBois, R. N., McLane, M. W., Ryder, K., Lau, L. F., and Nathans, D. (1990) J. Biol. Chem. 265, 19185-19191[Abstract/Free Full Text]
42. Varnum, B. C., Lim, R. W., Sukhatme, V. P., and Herschman, H. R. (1989) Oncogene 4, 119-120[Medline] [Order article via Infotrieve]
43. Taylor, G. A., Carballo, E., Lee, D. M., Lai, W. S., Thompson, M. J., Patel, D. D., Schenkman, D. I., Gilkeson, G. S., Broxmeyer, H. E., Haynes, B. F., and Blackshear, P. J. (1996) Immunity 4, 445-454[Medline] [Order article via Infotrieve]
44. Carballo, E., Lai, W. S., and Blackshear, P. J. (1998) Science 281, 1001-1005[Abstract/Free Full Text]
45. Carballo, E., Lai, W. S., and Blackshear, P. J. (2000) Blood 95, 1891-1899[Abstract/Free Full Text]
46. Mahtani, K. R., Brook, M., Dean, J. L., Sully, G., Saklatvala, J., and Clark, A. R. (2001) Mol. Cell. Biol. 21, 6461-6469[Abstract/Free Full Text]
47. Worthington, M. T., Pelo, J. W., Sachedina, M. A., Applegate, J. L., Arseneau, K. O., and Pizarro, T. T. (2002) J. Biol. Chem. 277, 48558-48564[Abstract/Free Full Text]
48. Johnson, B. A., and Blackwell, T. K. (2002) Oncogene 21, 4237-4246[CrossRef][Medline] [Order article via Infotrieve]
49. Kirkland, S. C. (1985) Cancer Res. 45, 3790-3795[Abstract]
50. Shao, J., Sheng, H., Inoue, H., Morrow, J. D., and DuBois, R. N. (2000) J. Biol. Chem. 275, 33951-33956[Abstract/Free Full Text]
51. Brattain, M. G., Brattain, D. E., Fine, W. D., Khaled, F. M., Marks, M. E., Kimball, P. M., Arcolano, L. A., and Danbury, B. H. (1981) Oncodev. Biol. Med. 2, 355-366[Medline] [Order article via Infotrieve]
52. Boutaud, O., Aronoff, D. M., Richardson, J. H., Marnett, L. J., and Oates, J. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7130-7135[Abstract/Free Full Text]
53. Boutaud, O., Brame, C. J., Salomon, R. G., Roberts, L. J., II, and Oates, J. A. (1999) Biochemistry 38, 9389-9396[CrossRef][Medline] [Order article via Infotrieve]
54. Roberts, L. J., II, Lewis, R. A., Oates, J. A., and Austen, K. F. (1979) Biochim. Biophys. Acta 575, 185-192[Medline] [Order article via Infotrieve]
55. Dixon, D. A., Kaplan, C. D., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2000) J. Biol. Chem. 275, 11750-11757[Abstract/Free Full Text]
56. Brooks, S. A., Connolly, J. E., Diegel, R. J., Fava, R. A., and Rigby, W. F. (2002) Arthritis Rheum. 46, 1362-1370[CrossRef][Medline] [Order article via Infotrieve]
57. Appleby, S. B., Ristimaki, A., Neilson, K., Narko, K., and Hla, T. (1994) Biochem. J. 302, 723-727[Medline] [Order article via Infotrieve]
58. Wahle, E., and Keller, W. (1992) Annu. Rev. Biochem. 61, 419-440[CrossRef][Medline] [Order article via Infotrieve]
59. Aghib, D. F., Bishop, J. M., Ottolenghi, S., Guerrasio, A., Serra, A., and Saglio, G. (1990) Oncogene 5, 707-711[Medline] [Order article via Infotrieve]
60. Raymond, V., Atwater, J. A., and Verma, I. M. (1989) Oncogene Res. 5, 1-12[Medline] [Order article via Infotrieve]
61. Lai, W. S., Stumpo, D. J., and Blackshear, P. J. (1990) J. Biol. Chem. 265, 16556-16563[Abstract/Free Full Text]
62. Carballo, E., Gilkeson, G. S., and Blackshear, P. J. (1997) J. Clin. Invest. 100, 986-995[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.