Introduction

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PGHS is the enzyme that converts arachidonic acid to prostanoids. Expression of the inducible isoform of this enzyme, PGHS-2, is highly regulated by inflammatory cytokines and growth factors (DeWitt and Meade, 1993; Evett et al., 1993; Fletcher et al., 1992; Ristimaki et al., 1994). We have previously shown that TNF- increases PGHS-2 mRNA accumulation in fibrosarcoma MCA-101 cells and that transcriptional and post-transcriptional mechanisms contribute to the increase in PGHS-2 mRNA accumulation induced by this cytokine (Mahboubi et al., 1997, in press). Moreover, PGE₂ production in response to TNF- is mainly dependent on PGHS-2 and is regulated by protein tyrosine kinases and phosphatases (Mahboubi et al., 1997, in press). The importance of serine-threonine protein phosphatases in TNF- signal transduction pathways is suggested by the similarity in the pattern of phosphorylation of cellular proteins induced by TNF- and OA, a noncompetitive inhibitor of PP2A and PP1 serine-threonine phosphatases (Guy et al., 1992; Fujiki and Suganuma, 1994). Moreover, Guy et al. reported that a serine/threonine phosphatase of primary human fibroblasts is inactivated by TNF- and OA (Guy et al., 1993). OA acid inhibits PP1 about 50 to 100 times less than PP2A, leading to an increase in the phosphorylation of many cellular proteins (Guy et al., 1992), and induces expression of mRNA for many genes by enhancing gene transcription and/or increasing stability of mRNA (Cao et al., 1992; Xia et al., 1993; Kim et al., 1990; Pshenichkin and Wise, 1995). The contribution of serine-threonine phosphatases to the expression of PGHS-2 has not been determined.

Increased protein phosphorylation by OA activates several transcription factors, including NF-B (Menon et al., 1995; Rieckmann et al., 1992; Suzuki et al., 1994; Menon et al., 1993), AP-1 (Rieckmann et al., 1992; Thevenin et al., 1991), Sp1 (Vlach et al., 1995) and CRE (Wadzinski et al., 1993), all of which are regulated by phosphorylation. NF-B is a transcription factor that regulates the expression of a variety of genes and exists in the cytoplasm of resting cells as a heterodimer composed of two polypeptides of 50 kDa (p50) and 65 kDa (p65), which are associated with a cytoplasmic inhibitory protein, IB- (Siebenlist et al., 1994). Upon stimulation with a variety of inducers, including TNF- (Miyamoto et al., 1994; Finco et al., 1994; Beg et al., 1993) and OA (Menon et al., 1995; Rieckmann et al., 1992; Suzuki et al., 1994; Menon et al., 1993), IB- is phosphorylated and subsequently degraded, which makes possible the translocation of NF-B to the nucleus, where it binds to B consensus sequences and initiates gene transcription. A binding site for NF-B has been identified within the PGHS-2 promoter (Tazawa et al., 1994; Sirois et al., 1993). Yamamoto et al. (1995) demonstrated a role of NF-B in the induction of PGHS-2 by TNF- in MC3T3-E1 cells. NF-B also appears to contribute to TNF--mediated induction of PGHS-2 in MCA-101 cells (K. Mahboubi and N. R. Ferreri, unpublished observations).

Post-transcriptional mechanisms may be important in the regulation of PGHS-2. The 3-untranslated region of the mouse PGHS-2 mRNA has multiple adenosine-uridine (AU) repeats responsible for shortening the half-life of PGHS-2 mRNA (Kujubu et al., 1991). Consequently, increasing PGHS-2 mRNA stability may result in the accumulation of mRNA. For instance, IL-1 (Srivastava et al., 1994) and TNF- (Mahboubi et al., 1997, in press) increase PGHS-2 mRNA half-life in renal mesangial and fibrosarcoma MCA-101 cells, respectively. The factors involved in PGHS-2 mRNA stabilization or degradation have not been characterized. However, Srivastava et al. showed that IL-1 induces phosphorylation of cytosolic factors that may extend PGHS-2 mRNA half-life by binding to AU-rich regions of this mRNA (Srivastava et al., 1994).

In the present study, we evaluated the importance of serine-threonine phosphatases in the regulation of PGHS-2 in MCA-101 cells. Inhibition of serine-threonine phosphatases by OA results in PGHS-2-dependent formation of PGE₂ via NF-B-independent transcriptional and post-transcriptional mechanisms. These data suggest that subsets of transcription factors may differentially contribute to regulation of PGHS-2 gene transcription, depending on the cell type and activation signals.

	Materials and Methods

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Cell lines and reagents. MCA-101 (a kind gift from Dr. Nicholas Restifo, NCI) is a fibrosarcoma cell line of B6 origin that was generated in 8-week-old female C57BL/6n (B6) mice by i.m. injection of 0.1% 3-MCA in sesame seed oil. Tumor cell lines were passaged in B6 mice (Restifo et al., 1992). Tumors were harvested from mice, digested and maintained in monolayer culture in media consisting of RPMI 1640, 10% heat-inactivated fetal calf serum (Gemini, Calabasas, CA), 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml). Jurkat T cells, a human T cell leukemia cell line, was a kind gift from Dr. Nancy Ruddle, Yale University. Recombinant mouse TNF- was purchased from Genzyme (Boston, MA). Sodium orthovanadate, MTT and ACD were from Sigma Chemical Co. (St. Louis, MO). OA and calyculin A were purchased from LC Lab (Woburn, MA). NS-398 was purchased from Biomol (Plymouth Meeting, PA). The PGHS-2 cDNA probe was obtained from Oxford (Oxford, MI).

Measurement of PGE₂. Confluent, quiescent MCA-101 cells were incubated in the absence or presence of OA in media containing 0.5% serum for 24 hr, after which the supernates were assayed for PGE₂ by enzyme-linked immunoassay (Oxford, MI) (Farman et al., 1987). Briefly, 50 µl of diluted media and 50 µl of HRP-conjugated PGE₂ were added for 1 hr to wells of a 96-well plate that had previously been coated with anti-PGE₂ antibody. After incubation, substrate for HRP was added to each well for 30 min, and the reaction was stopped by addition of 1 N HCl. Quantitation was achieved by measuring absorbance at 450 nm. Protein concentration in each well was determined by the Bradford method.

Western blot analysis of PGHS-2. After the appropriate treatment with OA, the media were removed and cells washed twice with ice-cold PBS. Cells were harvested and centrifuged at 600 × g for 4 min in the cold room. The pellet was lysed using RIPA buffer (PBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 µg/ml leupeptin, 1 mM sodium orthovanadate) for 30 min on ice. The lysate was centrifuged at 10,000 × g for 20 min at 4°C. Protein concentrations of the supernatant were determined using a detergent-compatible Bio-Rad protein assay kit. Then 20 µg of cell lysate was dissolved in an equal volume of 2X SDS-PAGE (SDS-polyacrylamide gel electrophoresis) sample buffer (100 mM Tris-Cl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and boiled for 3 min. The proteins in the cell lysate were separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. Nonspecific sites on the membrane were blocked by incubating the membrane in blocking solution containing 3% nonfat dry milk in TBST at room temperature for 30 min. Membranes were immunoblotted with a rabbit anti-PGHS-2 polyclonal antibody (Cayman, Ann Arbor, MI) for 1 hr at room temperature. Membranes were washed with TBST and incubated with HRP-conjugated goat-anti rabbit antisera (Santa Cruz, CA) for 30 min at room temperature. Membranes were washed, and PGHS-2 protein was detected by the ECL system (Amersham, Arlington Heights, IL).

Western blot analysis of IB-. Cell lysates and gel electrophoresis were prepared as indicated above. After blocking, membranes were immunoblotted with rabbit anti-human IB- antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 1 µg/ml in blocking solution for 45 min and washed two times for 7 min with TBST. Membranes were incubated with HRP-conjugated goat anti-rabbit antisera (Santa Cruz Biotechnology, Santa Cruz, CA) and diluted in blocking solution for 30 min at room temperature. Membranes were washed with TBST, and IB- protein was detected by the ECL system.

Total RNA isolation and Northern blot analysis. Confluent, quiescent cells were incubated in the absence or presence of OA in media containing 0.5% serum. After various incubation periods, media were removed and the cell monolayers washed twice with ice-cold PBS. Total cellular RNA was isolated by lysing the cells in guanidine isothiocyanate sodium citrate buffer and extracting RNA with ethanol as described previously (Kamdar and Evans, 1992). Then 10 µg of RNA were electrophoresed in a 1% agarose/formaldehyde gel in 1X MOPS (3-[N-morpholino]propane sulfonic acid) as running buffer. RNA was transferred to a nylon membrane (Genescreen, Dupont-New England Nuclear, Boston, MA) and hybridized to a random-primed ³²P-labeled cDNA probe in buffer containing 50% formamide, 10% dextran sulfate, 0.2% polyvinylpyrrolidone, 0.2% ficoll, 0.2% bovine serum albumin, 1.0 M NaCl, 1.0% SDS, 0.05 M Tris, pH 7.5 and 0.1% sodium phosphate at 42°C for 24 hr. After hybridization, the membrane was washed with 2X SSC (standard sodium citrate), 1.0% SDS at 65°C for 1 hr and with 0.1% SSC at 25°C for 1 hr. Then the probed blots were exposed at 70°C to XAR-5 X-ray film (Eastman Kodak, Rochester, NY).

Nuclear extraction and EMSA. Nuclear protein extracts were prepared following the method of Schreiber et al. (1989). Cells were washed twice in ice-cold PBS and then harvested in buffer A (20 mM HEPES, pH 8.0, 0.32 M sucrose, 2.0 mM CaCl₂, 2.0 mM MgCl₂, 0.1 mM EDTA, 0.5% Nonidet P-40, 1.0 mM DTT (dithiothreitol), 0.25 mM PMSF, 1 µg/ml leupeptin), and nuclei were pelleted by centrifugation at 1500 × g for 5 min at 4°C. Pelleted nuclei were resuspended in 50 µl of buffer B (20 mM HEPES, pH 8.0, 25% glycerol, 0.42 M NaCl, 2 mM MgCl₂, 0.2 mM EDTA, 1.0 mM DTT, 0.25 mM PMSF, 1 µg/ml leupeptin). Nuclei pellets were gently mixed and incubated on ice for 15 min. Nuclear debris was removed by centrifugation for 15 min at 10,000 × g, and nuclear protein concentration was measured by the Bradford method.

Cytotoxicity assay. MCA-101 cells were cultured in 96-well plates (2.5 × 10⁵ per well) overnight. The following day, media were removed, and new media containing various drugs or media were added for 4 hr. At the end of the incubation periods, the viability of the cells was determined by assaying their metabolic capacity using MTT (Mosmann, 1983). Briefly, MTT at 1 mg/ml was added to all the wells. After a 2-hr incubation at 37°C/5% CO₂ in the presence of MTT, acidified isopropanol was added to each well, and the plates were read using a test wavelength of 570 nm and a reference wavelength of 630 nm.

	Results

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OA increases PGE₂ synthesis via PGHS-2. OA was used to investigate whether intracellular serine-threonine phosphatases regulate the expression of PGHS-2. Our first approach was to determine the effects of OA on PGE₂ synthesis. Confluent, quiescent cells were incubated with media containing 0.5% serum (control) or 100 nM OA for 24 hr. OA at a concentration of 100 nM was selected because it inhibited approximately 80% of total phosphatase activity in MCA-101 cell lysates (data not shown). Treatment with OA significantly increased PGE₂ synthesis (fig. 1) and did not affect cell viability, as determined by MTT assay and trypan blue exclusion. We determined whether the OA-induced PGE₂ production was associated with increased PGHS-2 activity in experiments in which MCA-101 cells were incubated with OA in the presence of NS-398, a selective inhibitor of PGHS-2 (Futaki et al., 1994; Copeland et al., 1994). NS-398 (0.01 µM) completely abolished the OA-mediated PGE₂ production but had no effect on PGE₂ production by unstimulated cells (fig. 1). Thus, OA-induced PGE₂ synthesis may have occurred via a PGHS-2-dependent mechanism.

View larger version (K): [in this window] [in a new window]   Fig. 1.   NS-398 inhibits OA-mediated PGE2 production by MCA-101 cells. Confluent cells were cultured for 18 hr in 0.5% serum and then incubated with media alone (control), NS-398 (0.01 µM), OA (100 nM) or OA and NS-398 for 24 hr. The amount of PGE2 released into the media was quantitated by enzyme-linked immunoassay. Data are expressed as mean ± S.E. of triplicate determinations. **P < .01 vs. control. ++P < .01 vs. OA.

Time- and dose-dependent increase in PGHS-2 protein expression. Confluent, quiescent cells were incubated with media in the presence or absence of 100 nM OA for various times. After incubation with OA, cells were lysed and analyzed for PGHS-2 protein by Western blot analysis using a specific PGHS-2 antibody. As shown previously (Mahboubi et al., 1997, in press), low levels of PGHS-2 protein were detected in unstimulated cells (fig. 2A). After challenge with OA, increased levels of PGHS-2 protein were observed at approximately 4 hr and remained elevated up to 24 hr (fig. 2A). Dose-response experiments with OA revealed that 10 nM OA significantly induced PGHS-2 protein expression and that 0.1 nM OA did not increase PGHS-2 protein levels (fig. 2B). OA at 10 nM and 0.1 nM inhibited phosphatase activity in MCA-101 cell lysates by approximately 60% and 50%, respectively (data not shown). Expression of PGHS-2 was maximal in response to 50 nM OA (fig. 2B).

View larger version (K): [in this window] [in a new window]   Fig. 2.   Western blot analysis of PGHS-2 protein levels. MCA-101 cells were cultured for 18 hr in 0.5% serum and then exposed to media (control; panels A and B), media containing OA (100 nM) for various times (panel A) or media containing various concentrations of OA for 24 hr (panel B). After incubations, cell lysates were separated on a 10% SDS gel, and PGHS-2 protein was detected with an anti-PGHS-2 antibody. Purified PGHS-2 protein was used as a positive control (panel A). These figures are representative of three similar experiments.

OA increases PGHS-2 mRNA accumulation in a time-dependent manner. We investigated whether accumulation of PGHS-2 mRNA could contribute to the augmentation of PGE₂ synthesis and PGHS-2 protein expression observed after stimulation of MCA-101 cells with OA. Confluent, quiescent cells were incubated with OA, and total RNA was isolated at zero (control), 1, 5, 8, 12, and 24 hr after the addition of OA (100 nM). PGHS-2 mRNA (4.2 kb) was detected in unstimulated cells; mRNA accumulation was similar at each of the time-points tested (fig. 3A). PGHS-2 mRNA accumulation was evident within 1 hr after stimulation with OA, increased dramatically up to 12 hr and was followed by a slight reduction at 24 hr (fig. 3A). Calyculin A, another inhibitor of PP1 and PP2A, increased PGHS-2 mRNA accumulation in MCA-101 cells (fig. 3B).

View larger version (K): [in this window] [in a new window]   Fig. 3.   Effects of OA and calyculin A on PGHS-2 mRNA accumulation. Confluent, quiescent MCA-101 cells were incubated with media (control), with media containing OA (100 nM) for various times (panel A) or with calyculin A (25 nM) for 3 hr (panel B). After incubation, total RNA was isolated, and PGHS-2 mRNA levels were detected by Northern blot analysis using a specific 32P-labeled cDNA probe for PGHS-2. RNA quantity and integrity were verified by ethidium bromide staining of 28S and 18S ribosomal RNA. These figures are representative of three similar experiments.

OA increases PGHS-2 mRNA accumulation by transcriptional and post-transcriptional mechanisms. The enhancement of PGHS-2 mRNA accumulation after OA treatment may be explained by enhanced gene transcription and/or stabilization of message. To test the first possibility, we arrested transcription with ACD treatment before the addition of OA and assessed PGHS-2 mRNA levels by Northern blot analysis. In the presence of ACD, OA-mediated PGHS-2 mRNA accumulation was significantly reduced (fig. 4). These results indicate that induction of PGHS-2 mRNA in response to OA was due, at least in part, to transcriptional regulation of the PGHS-2 gene. We also determined whether OA could increase PGHS-2 mRNA by prolonging message half-life. To determine PGHS-2 mRNA half-life in response to OA, MCA-101 cells were challenged with 100 nM OA (5 hr), and transcription was subsequently arrested by addition of 1.0 µM ACD. Cells were harvested at different times after the addition of ACD to assess mRNA half-life. The levels of PGHS-2 mRNA induced by OA decreased as a function of time in the presence of ACD (fig. 5). Because ACD blocks gene transcription, the amount of mRNA remaining at the various time-points is an index of mRNA half-life. PGHS-2 mRNA half-life of OA-stimulated cells was approximately 4 to 5 hr (fig. 5). We previously showed that the half-life of PGHS-2 mRNA in phorbol 12-myristate 13-acetate-stimulated MCA-101 cells is approximately 1 hr (Mahboubi et al., 1997, in press). These data suggest that OA-dependent enhancement of the steady-state levels of PGHS-2 mRNA is associated with transcriptional activation and stabilization of the message.

View larger version (K): [in this window] [in a new window]   Fig. 4.   ACD inhibits OA-induced PGHS-2 mRNA accumulation in MCA-101 cells. Confluent, quiescent cells were preincubated with ACD (1.0 µM) for 30 min before the addition of 100 nM OA for 5 hr. After incubation, total RNA was isolated, and PGHS-2 mRNA levels were assessed by Northern blot analysis using a specific 32P-labeled cDNA probe for PGHS-2 (upper panel). RNA quantity and integrity were verified by ethidium bromide staining of 28S and 18S ribosomal RNA (bottom panel). This figure is representative of three similar experiments.

View larger version (K): [in this window] [in a new window]   Fig. 5.   Effects of OA on the decay curve for PGHS-2 mRNA accumulation. Confluent, quiescent MCA-101 cells were challenged with 100 nM OA for 5 hr, and transcription was subsequently inhibited by the addition of 1.0 µM ACD. Cells were harvested at different time-points, and PGHS-2 mRNA levels were determined by Northern blot analysis. PGHS-2 mRNA was quantified by scanning densitometry, using -actin as an internal control. This figure is representative of three similar experiments.

Effects of OA on NF-B activation in MCA-101 cells. OA activates NF-B in a variety of cell types, including Jurkat T cells and fibroblasts (Menon et al., 1995; Suzuki et al., 1994; Menon et al., 1993; Sun et al., 1995). We evaluated the effects of OA on NF-B activation in MCA-101 cells. Jurkat cells (positive control cell line) and MCA-101 cells were stimulated with 100 nM OA for 1, 2, or 4 hr. After incubation, cells were lysed, and NF-B activity was measured by EMSA. TNF- is a potent activator of NF-B in a wide variety of cell types (Miyamoto et al., 1994; Finco et al., 1994; Beg et al., 1993; Henkel et al., 1993), so nuclear extracts from TNF--stimulated MCA-101 cells were used as a positive control for NF-B activation. OA and TNF- increased NF-B DNA binding activity in Jurkat T cells (fig. 6). In contrast, NF-B binding activity was detected in nuclear extracts prepared from TNF-, but not OA-stimulated MCA-101 cells (fig. 6). Moreover, no NF-B binding activity was detected in MCA-101 nuclear extracts after challenge with 500 nM OA for a range of different time periods (data not shown). These results suggest that OA does not activate NF-B in MCA-101 cells.

View larger version (K): [in this window] [in a new window]   Fig. 6.   NF-B activation in MCA-101 cells and Jurkat T cells. Cells were incubated with media (control) or with media containing either 1 nM TNF- or 100 nM OA. Nuclear extracts were prepared at the indicated times. EMSA was performed to detect activated NF-B complex in the nuclear extracts, as described in "Materials and Methods." The asterisk denotes a nonspecific complex; the specific binding complex is indicated by the NF-B label. This figure is representative of three similar experiments.

OA does not stimulate IB- degradation in MCA-101 cells. Activation of NF-B from cytoplasmic pools should be associated with proteolytic degradation of the inhibitory IB-, according to known mechanisms of NF-B activation. To confirm that OA does not activate NF-B in MCA-101 cells, we determined its effects on IB- protein degradation. Confluent, quiescent MCA-101 cells were incubated with media (control) or 100 nM OA for various times. After incubation, cells were lysed, and IB- protein was assessed by Western blot analysis using a specific IB- protein antibody. IB- protein was present in unstimulated cells (control) (fig. 7A). There was no change in IB- protein levels after treatment with OA (fig. 7A). On the other hand, IB- protein levels were slightly or completely reduced, respectively, after a 5- or 10-min stimulation with TNF- (fig. 7B). Complete degradation of IB- protein, after a 15-min treatment with TNF-, resulted in the activation of NF-B and the translocation of NF-B heterodimer to the nucleus (fig. 7B; fig. 6). After a 30-min stimulation with TNF-, IB- protein was again detected, because of its NF-B-dependent synthesis. OA causes IB- protein degradation in Jurkat cells (fig. 7C) and induces translocation of NF-B to the nucleus (fig. 6). These results are in agreement with the data obtained by EMSA and indicate that OA does not induce degradation of IB- protein, a prerequisite for activation of NF-B.

View larger version (K): [in this window] [in a new window]   Fig. 7.   Effects of OA and TNF- on IB- protein levels in MCA-101 cells. MCA-101 cells were incubated with media (control; panel A and B), with media containing 100 nM OA (panel A) or with media containing 1 nM TNF- (panel B) for various times. Jurkat cells were stimulated with media (control) or with media containing 100 nM OA for various times (panel C). After incubations, cell lysates were separated on a 10% SDS gel, and IB- protein was detected by Western blot analysis. These figures are representative of three similar experiments.

	Discussion

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We demonstrated that the inhibition of PP1 and PP2A by OA increased PGHS-2-dependent PGE₂ production, PGHS-2 protein expression, PGHS-2 gene transcription and PGHS-2 mRNA stability. These data are the first to show that inhibition of serine-threonine phosphatases up-regulates the expression of PGHS-2. Moreover, inhibition of PP1 and PP2A is not sufficient to activate NF-B in MCA-101 cells and therefore increases PGHS-2 by an NF-B-independent mechanism.

Treatment of MCA-101 cells with 100 nM OA, a dose that inhibited phosphatase activity in vitro by approximately 80% (data not shown), markedly increased PGE₂ synthesis by these cells. Ohuchi et al. (1989) showed that OA increased PGE₂ production in macrophages, an effect that was inhibited in the presence of cycloheximide, which suggests that protein synthesis was needed for the stimulation of arachidonic acid metabolism (Ohuchi et al., 1989). These authors did not determine the relative contribution of PGHS-1 and PGHS-2 in OA-induced PGE₂ synthesis. OA failed to increase PGE₂ synthesis in the presence of NS-398, a result that indicates a possible role for PGHS-2 in the OA-mediated increase in PGE₂ synthesis in MCA-101 cells. Interestingly, OA increased PGHS-2 protein synthesis and PGHS-2 mRNA levels without affecting PGHS-1 protein levels in MCA-101 cells (data not shown). Induction of PGHS-2 mRNA by OA was inhibited in the presence of ACD, which indicates that OA increases PGHS-2 mRNA by activating transcription of the PGHS-2 gene. These results suggest that inhibition of PP1 and PP2A may be important in regulating PGHS-2 gene transcription in MCA-101 cells. Because NF-B was not activated by OA in MCA-101 cells, a role for this transcription factor in OA-induced PGHS-2 gene transcription in these cells seems unlikely. However, TNF- caused degradation of IB- protein and activation of NF-B and has previously been linked to the increase in PGHS-2 mRNA in MCA-101 cells (Mahboubi et al., 1997, in press). NF-B is involved in the TNF--dependent induction of PGHS-2 in MC3T3-E1 cells (Yamamoto et al., 1995). Thus, although NF-B may be involved in the TNF--mediated increase in PGHS-2 in MCA-101 cells, it is not required for the OA-mediated increase in PGHS-2 mRNA accumulation. OA has been shown to activate Sp1 (Vlach et al., 1995) and CRE (Wadzinski et al., 1993), two transcription factors that are present in the PGHS-2 promoter (Tazawa et al., 1994). CRE was shown to act as positive regulatory element for PGHS-2 gene transcription (Xie et al., 1994; Inoue et al., 1994). Activation of these transcription factors by OA may be responsible for OA-induced PGHS-2 gene transcription in MCA-101 cells. The effects of OA on the activation of CRE and Sp1 and other transcription factors, which may play a role in regulating transcription of PGHS-2, remain to be determined.

The 3-untranslated region of the mouse PGHS-2 mRNA has multiple AU repeats responsible for shortening the half-life of many unstable mRNAs (Kujubu et al., 1991; Malter, 1989). The stability of AU-rich mRNAs is regulated by the activity of proteins designated AUBF. Phosphorylation of AUBF results in its activation and binding to labile mRNAs and the formation of a complex that is resistant to degradation (Malter and Hong, 1991). Previously, we have shown that TNF- increases PGHS-2 mRNA half-life in MCA-101 cells (Mahboubi et al., 1997, in press). Stephens et al. (1992) demonstrated that OA and TNF- up-regulate AUBF activity in 3T3-L1 preadipocytes cells, which subsequently results in stabilization of glucose transporter mRNA for in these cells. As shown in the present study, OA enhances PGHS-2 mRNA stability, which suggests that an increase in total serine-threonine phosphorylation by OA decreases the rate of degradation of PGHS-2 mRNA and contributes to its accumulation. Therefore, increased activity of an unidentified AUBF by OA and TNF- may increase PGHS-2 mRNA stability in MCA-101 cells.

OA activates NF-B in several cell types, including Jurkat T cells (Suzuki et al., 1994; Sun et al., 1995). However, OA did not activate NF-B or induce degradation of IB- in MCA-101 cells. Taken together, these data suggest that inhibition of serine-threonine phosphatases by OA is not sufficient for the release of NF-B from its inhibitor, IB-, in MCA-101 cells. Serine phosphorylation of IB-, an event that is essential for its degradation (Miyamoto et al., 1994), is achieved by an increase in serine-threonine kinase activity and/or inhibition of serine-threonine phosphatase activity. However, inhibition of phosphatases increases IB- phosphorylation only if kinases are activated. Therefore, it is possible that the serine-threonine kinase responsible for phosphorylation of IB- is not active in unstimulated MCA-101 cells. Interestingly, previous reports have suggested that OA-induced activation of NF-B is dependent on the endogenous redox status of cells. For instance, Menon and co-workers (1993) found that OA was unable to activate NF-B in primary fibroblasts and that oxidizing agents made possible the induction of NF-B by OA in these cells. Thus the inability of OA to activate NF-B in MCA-101 cells may be due to high intracellular levels of antioxidant molecules. In contrast, OA-mediated NF-B activation in Jurkat cells, which are highly susceptible to changes in the intracellular redox state, may have been due to low intracellular levels of antioxidants (Droge et al., 1994).

In this study, we illustrated the importance of serine-threonine phosphorylation to the regulation of PGHS-2 mRNA expression. We previously showed that signal transduction via protein tyrosine kinase(s) and protein tyrosine phosphatase(s) is required for TNF--mediated increases in PGHS-2 mRNA accumulation in MCA-101 cells (Mahboubi et al., 1997, in press). Thus more than one signaling pathway may initiate PGHS-2 gene transcription in MCA-101 cells. Moreover, Guy et al. suggested that TNF- signal transduction may involve the tyrosine phosphorylation of PP2A, an event that causes inactivation of PP2A (Guy et al., 1995; Guy et al., 1993; Chen et al., 1992). Therefore, it is also possible that inhibition of PP2A by TNF- is an important event in the TNF--mediated increase in PGHS-2 mRNA accumulation in MCA-101 cells.